2005 a comparative study of arsenic methylation in a plant

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A comparative study of arsenic methylation in aplant, yeast and bacteriumJiehua WuUniversity of Wollongong

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Recommended CitationWu, Jiehua, A comparative study of arsenic methylation in a plant, yeast and bacterium, Doctor of Philosophy thesis, School ofBiological Sciences, University of Wollongong, 2005. http://ro.uow.edu.au/theses/1914

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A Comparative Study of Arsenic Methylation

in a Plant, Yeast and Bacterium

A thesis submitted in partial fulfilment of

the requirements for the award of the degree

Doctor of Philosophy

From

THE UNIVERSITY OF WOLLONGONG

By

Jiehua Wu, M.Sc.

School of Biological Sciences

2005

II

Declaration

I, Jiehua Wu, declare that this thesis is submitted in partial

fulfilment of the requirements for the award of Doctor of Philosophy,

in the School of Biological Sciences, University of Wollongong. It

does not include any material previously published by another

person except where due reference is made in the text. The

experimental work described in this thesis is original and wholly my

own work. The document has not been submitted for a degree in any

other academic institution.

Jiehua Wu

Acknowledgments

III

Acknowledgments

In completing this thesis, I can’t keep from thanking many people. First of all, I

convey my thanks to supervisors Associate Prof. Ross McC. Lilley and Dr. Ren

Zhang without whom this project could not have happened. It is impossible to

express the gratitude I feel for their expert guidance and supervision. I am greatly

indebted for their patient, encouragement, support and belief that I could see the

project through to the end.

Special thanks must be expressed to Ross Lilley and his wife Xi Hui. Ross provided

the job opportunity as technical officer, which supported me financially. The

kindness and care from Ross and Xi Hui let me feel my home in Australia.

I extend my thanks to James Petrie. During the research with yeast and bacteria,

James offered me assistance within some experiment processes, touching cell

growth and collection, purification of extracts of cells, and extraction of methylated

products. I found enlightenment in his smart ideas.

I am particularly grateful to the labmates of Ross’ and Ren’s labs for support and

assistance during the long, and sometimes stressful, period of research. In no

particular order, I thank Michael Kelly, Lixia Liu, Chenwei Shu, Yanzhe Wu, You

Wang, Qian Wang, Donna Harmon and Sean Isbester. I have always found these

people to be friendly, helpful and willing to provide advice.

I am grateful to all the members of the School of Biological Sciences for their help

and friendship. Special thanks must be given to Jan Fragiacomo and Mari Dwarte

for their administrative assistance. I often think of Jan after her retirement.

My great thanks to Jin Gao (Geoffrey), a former Ph.D. student of Department of

Acknowledgments

IV

Chemistry, although we met mostly on the trains between Sydney and Wollongong

on the way to University, his kindness, intelligence and humour brought me

happiness, enlightened and encouraged me to complete this thesis. But a detestable

sin took his life just four days before the last Christmas day. I lost my best friend,

Geoffrey, forever.

The help and support from my Mother, Father and Brother are always what I rely on,

even if they live far away in China. I am greatly grateful to my family.

Finally, I would like to dedicate this thesis to my dear Mother. I can never forget the

tears in my mother’s eyes at the Shanghai airport when she saw me off for Australia,

an unknown world to us at that time. Her words still linger in my ears.

Unfortunately, she could not wait for my graduation, even seeing me again.

However, now I believe she is looking at me from the heaven, smiling and happy

with my completion of my Ph.D. thesis. Thanks for her blessing.

Table of Contents

V

TABLE OF CONTENTS

Title Page I

Declaration II

Acknowledgments III

Table of Contents V

Publication out of this thesis so far XII

Keywords and Abbreviations XIII

Abstract XIV

Chapter I. INTRODUCTION: REVIEW OF THE LITERATURE 1

1.1. Arsenic, Arsenical Forms and Structures

1.1.1. Arsenic physical and chemical properties 2

1.1.2. Formation and structures of arsenicals 3

1.1.3. Arsenate and arsenite 4

1.2. Arsenic Species in the Environment

1.2.1. Arsenic distribution 7

1.2.1.1. The arsenic cycle 7

1.2.1.2. Background arsenic in soils 8

1.2.1.3. Arsenic in aquatic systems 10

1.2.1.4. Arsenic mobilization 11

1.2.2. Application of arsenic 15

1.2.3. Pollution of arsenic 17

1.2.3.1. Arsenic in arsenic-contaminated soils 17

1.2.3.2. Arsenic contamination in water 19

Table of Contents

VI

1.2.3.3. Arsenic contamination in Australia 20

1.2.4. Arsenic bioavailablity and uptake by organisms 23

1.2.5. Toxicity of arsenic to organisms 27

1.3. Natural Resistance and Tolerance Mechanisms for Arsenic

1.3.1. Arsenic transport out of cells and into the vacuole 30

1.3.1.1. ars operons in bacteria 30

1.3.1.2. The families of arsenic-resistance transporters in yeast 34

1.3.1.3. Arsenic resistance in mammalian cells 36

1.3.1.4. Arsenic transport in plants 36

1.3.2. Arsenic chelation 37

1.3.2.1. Roles of glutathione in chelation 37

1.3.2.2. Roles of arsenic-binding protein in animals 37

1.3.2.3. Roles of peptides in chelation of arsenic in plants (phytochelation) 39

1.3.3. Arsenic metabolism and organo-arsenical synthesis 42

1.3.3.1. Biomethylation 42

1.3.3.1.1. Methylation of arsenic by bacteria 43

1.3.3.1.2. Methylation of arsenic by yeast and other fungi 45

1.3.3.1.3. Methylation of arsenic by fresh water algae 47

1.3.3.1.4. Methylation of arsenic by mammals 47

1.3.3.1.5. Methylation of arsenic by terrestrial plants 48

1.3.3.2. Organo-arsenical synthesis by marine organisms 49

1.4. Enzymology of Arsenic Biomethylation

1.4.1. Enzymes catalyzing reduction of arsenic compounds 52

1.4.1.1. Arsenate reductase 53

1.4.1.2. Methylarsonate reductase 54

1.4.2. Enzymes catalyzing methyl group transfers to

arsenic compounds 55

Table of Contents

VII

1.4.2.1. Arsenite methyltransferase 55

1.4.2.2. Methylarsinate methyltransferase 56

1.4.2.3. S-adenosyl-methionine:arsenic(III) methyltransferase 57

1.5. Strategies for Arsenic Bioremediation of Arsenic Pollution

1.5.1. Strategies for the removal of toxic metals by plants 59

Phytoextraction 59

Rhizofiltration 59

Phytostabilization 60

1.5.2. Removal of arsenic from soils 60

1.5.2.1. Remediation of arsenic-contaminated soils 60

1.5.2.2. Phytoremediation of arsenic 62

1.6. Project aims and experimental approach 63

Chapter II. MATERIALS AND METHODS 64

2.1. Materials

2.1.1. Plants and growth conditions 65

2.1.2. Yeast and growth conditions 65

2.1.3. E. coli cells and growth conditions 66

2.1.4. Reagents 67

2.2. Methods

2.2.1. Preparation of cell extracts 68

2.2.1.1. Yeast and E. coli 68

2.2.1.2. Plant tissues 68

2.2.2. Arsenic methyltransferase assay 69

Table of Contents

VIII

2.2.3. Extraction and determination of methylated arsenic compounds 69

2.2.4. Separation of methylated metabolites 70

2.2.5. Preparation and determination of authentic MMA and DMA 70

2.2.6. Expression and purification of the yeast proteins YHR209w and YER175c 71

2.2.7. Digestion of the yeast GST fusion proteins 71

Chapter III. METHYLATION OF ARSENIC IN VITRO BY CELL EXTRACTS FROM BENTGRASS (AGROSTIS TENUIS) 72

3.1. Introduction 73

3.2. Results

3.2.1. Validation of extraction process 75

3.2.2. Effect of arsenate in the culture media on Agrostis tenuis 76

3.2.2.1. Effect of arsenate concentration in culture Media on methylation by leaf extracts 76

3.2.2.2. Arsenate tolerance in Agrostis tenuis 77

3.2.2.3. Time-course analysis of Arsenite Methylation, and separation of products 80

3.2.2.4. Methylation of arsenate 82

3.2.2.5. Methylation by roots 85

3.3. Discussion 86

Chapter IV. INDUCTION OF ARSENIC METHYLATION IN THE YEAST SACCHAROMYCES CEREVISIAE AND THE BACTERIUM E. COLI 90


4.2. Results

4.2.1. Effect of arsenate and arsenite concentration in culture media on the growth of S.cerevisiae strains 93

Table of Contents

IX

4.2.2. Effect of arsenate in the culture media on methylation by cell extracts from wild-type BY4741 and the arsenic -sensitive YPR201w 94

4.2.2.1. Time-course analysis of arsenite methylation by cell extracts from BY4741 94

4.2.2.2. DMA methylation in yeast BY4741 exposed in arsenate 97

4.2.2.3. Time-course analysis of arsenite methylation by cell extracts from YPR201w 98

4.2.3. Effect of arsenate and arsenite concentration in culture media on the growth of E. coli strains 100 4.2.4. Effect of arsenate in the culture media on methylation by cell extracts from wild-type W3110 and the arsΔ strain AW3110 101

4.2.4.1. Time-course analysis of arsenite methylation by cell extracts from W3110 101

4.2.4.2. Time-course analysis of arsenite methylation by cell extracts from AW3110 103

4.2.5. Optimal pH for arsenic methylation in yeast and E. coli 105

4.3. Discussion

4.3.1. Effects of arsenic on the growth of the wild types and mutants of yeast and E.coli 106

4.3.2. Arsenic methylation to products further than DMA

occurs in S. cerevisiae and E. coli 108

4.3.3. The deletions of arc3 and ars genes affect arsenic methylation 109

4.3.4. Arsenate induction of methylation in yeast and E. coli 110 4.3.5. The induciblity of arsenic methylation by acute

concentrations of arsenate 112

4.3.6. The possible source of the methylation background 113

Table of Contents

X

Chapter V. IDENTIFICATION OF ARSENIC METHYLTRANSFERASES GENES IN SACCHAROMYCES CEREVISIAE 114


5.2. Results

5.2.1. Preliminary search of possible genes for arsenic methyltransferases by genome database searching 116

5.2.2. Growth response of the yeast knock-out mutants to

arsenate 118 5.2.3. Arsenite methylation assays by cell extracts from 10 knock-out mutants 120 5.2.4. Expression and digestion of fusion proteins of

GST-YHR209w and GST-YER175c 124

5.2.5. Arsenite methylation activities of fusion proteins GST-YHR209w, GST-YER175c and their thrombin

digestion products 127 5.2.6. Test for MMA methyltransferase activities of digested

proteins GST-YHR209w and GST-YER175c 129 5.2.7. Homology search for arsenite methyltransferase

sequences in yeast genome 130

5.3. Discussion 5.3.1. Arsenate has little effects on the growth of the mutant

strains 134

5.3.2. Further enzyme activity screen 135 5.3.3. Protein YHR209w: an arsenite methyltransferase 137 5.3.4. Protein YHR209w: a typical S-AdoMet-dependent

methyltransferase 138 5.3.5. Comparison of YHR209w with known arsenic

methyltransferases from other organisms 139 5.3.6. Yeast - a promising study bridge between plants

and animals 140

Table of Contents

XI

Chapter VI. FINAL DISCUSSIONS AND CONCLUSIONS 141

6.1. Uptake and reduction of arsenate by plants and microorganisms 143

6.2. Methylation of arsenic by plants and microorganisms 143

6.3. Induction of arsenic methylation by arsenate and its implication 145

6.4. Molecular studies of arsenic methylation and application 147

References 149

Publication out of this thesis so far

XII

Publication out of this thesis so far

Wu JH, Zhang R and Lilley RM (2002) The methylation of arsenic in vitro by cell

extracts from bentgrass (Agrostis tenuis): effect of acute exposure of plants to

arsenate. Functional Plant Biology 29: 1-8

Keywords and Abbreviations

XIII

Keywords Agrostis tenuis, Saccharomyces cerevisiae, E. coli, arsenic methylation,

monomethylarsonate, dimethylarsinate, arsenite methyltransferase, monomethylarsenite

methyltransferase, arsenate reduction, plant, yeast

Abbreviations 3H-AdoMet: S-[3H-methyl] adenosyl-L-methionine

APL: acute promyelocytic leukaemia

DMA, DMAV: dimethylarsinate

DMAA, DMAIII: dimethylarsinic acid

dpm: disintegrations per minute

DTT: dithiothreitol

ExClones: Yeast expression clones

GMM: glucose minimal medium

GSH: glutathione

GST: glutathione-S-transferase

GYP medium: 2% glucose, 1% yeast extract and 1% peptone medium

iAsIII, AsIII: arsenite

iAsV, AsV: arsenate

ICP-MS: inductively coupled plasm-mass spectrometry

Km: constant of Michaelis-Menten kinetics

MMA, MMAV: monomethylarsonate, monosodium acid methane arsonate

MMAA, MMAIII: monomethylarsonic acid

ORF: open reading frame

PC: phytochelatin

MOPS: 3-[N-morpholino]propanesulphonic acid

PAGE: polyacrylamide gel electrophoresis

ppm: parts per million

pptn: precipitation

TMA: trimethylarsine

TMAO: trimethylarsine oxide

Abstract

XIV

Abstract

Arsenic methylation is the common pathway of arsenic metabolism in most organisms.

Compared with mammalian tissues, information is scant on the enzymes responsible for

arsenic methylation in plants and microorganisms. The research in this thesis investigated

the arsenic methylation activities in an arsenic-resistant plant species (Agrostis tenuis

Sibth) and two common microorganisms: budding yeast (S. cerevisiae) and E. coli.

Plants were grown in complete nutrient media with arsenate (135 to 538 μM) for 3 days

before harvesting. Methylation activity was determined in leaf and root tissue extracts

using an in vitro assay based on the methyl group transferred from S-[3H-methyl]

adenosyl-L-methionine (3H-AdoMet) with either arsenite or arsenate as substrate.

Arsenite methylation activity was low in leaf extracts from plants not exposed to arsenate,

but was greatly enhanced after acute exposure, with the induced methylation activity

greatest in extracts from plants exposed to 269 µM arsenate. Monomethylarsonate

(MMA) was the predominant early product, but over longer assay times, dimethylarsinate

(DMA) accumulated at the rate of 660 amol.mg protein-1.min-1 to levels exceeding MMA.

With arsenate as substrate, methylation activity was much lower than with arsenite,

implying that arsenite is the preferred substrate for methylation. Methylation assays with

root extract generated no DMA, however, small amounts of MMA were formed with

arsenite as substrate. In contrast to leaves, the methylation activity did not increase in root

extracts from plants exposed to arsenate. These findings suggest that arsenate in the plant

growth medium was taken up by the roots and converted to arsenite before methylation

proceeded in the leaves, accompanied by induction of arsenic methyltransferase activities.

A comparative study was made of the enzymic methylation of arsenic in yeast and E.

coli. Research using yeast offers an approach to the information on genes coding for the

enzymes responsible for arsenic methylation. Studies investigated the arsenic

methylation activities extractable from yeast Saccharomyces cerevisiae BY4741and its

Abstract

XV

arc3 (arsenite transporter gene) knock-out mutant YPR201w, and from bacterium E.

coli W3110 and its ars (arsenic resistance operon) deletion mutant AW3110. These

strains were grown in complete media with arsenate for 20 hours (yeast) or 4 hours (E.

coli) before harvesting. Methylation activity was determined in cell extracts using the in

vitro assay with either arsenite or dimethylarsinate (DMA) as substrate. Arsenite

methylation activity was detectable in the extracts from all four strains even when not

exposed to arsenate and the activities increased over time. With the BY4741 strain,

monomethylarsonate (MMA) was the predominant early product, but over longer assay

times, DMA accumulated at the rate of 4.1 amol.mg protein-1.min-1 to levels exceeding

MMA. Longer assay times were required for extracts from YPR201w to produce

substantial amounts of MMA and DMA, implying that the deletion of acr3 affects arsenic

methylation. After acute exposure to arsenate, the methylation activity was highest at 15

minutes with both strains. DMA was produced at the rate of 33.4 amol. mg protein-1.min-1

in BY4741 during a 15 minute assay. The amounts of MMA and DMA in the reaction

mixtures decreased after 15 minutes. This phenomenon implied further methylation,

which was confirmed by DMA-dependent arsenic methylation assays. The findings

suggest that yeast possesses a constitutive arsenic methyltransferase activity, but that

exposure to arsenate can stimulate additional higher methylation activity. However, for E.

coli wild type W3110, arsenite and MMA methyltransferase activities were not affected

by arsenate in the culture medium. Searching for the gene(s) encoding for arsenic methyltransferases, thirteen genes were

chosen from the S. cerevisiae genome database and knockout mutants for each of the 13

genes were obtained. Arsenic methylation by partially purified extracts from these

mutants was investigated by the in vitro methylation assay with arsenite as substrate.

The yeast knockout mutants were grown in the GYP complete medium without arsenic

present. Compared with the other knockout mutants, the mutant lacking functional gene

YHR209w showed low rates of formation of MMA and DMA, while the mutant of gene

YER175c, which is known to code for a trans-aconitate methyltransferase, formed little

MMA but substantial amounts of DMA. Fusion genes of GST-YHR209w and

Abstract

XVI

GST-YER175c were highly expressed in yeast stain EJ758. The fusion proteins

produced were treated with thrombin to release the target protein from GST. The

products of GST-YHR209w with thrombin treatment at 19°C exhibited arsenite

methyltransferase activity but were not able to methylate MMA. It is suggested that

protein YHR209w is probably responsible for the methyl group transfer to arsenite but

not to MMA. This differs from S-adenosyl-L-methionine:arsenic(III) methyltransferase

in rat liver cells, which catalyses both arsenite and MMA. Protein YHR209w is an

S-adenosyl-methionine-dependent methyltransferase and contains all four typical

motifs that have been found in other methyltransferases.

Chapter I Introduction: Review of the Literature

1

Chapter I

Introduction: Review of the Literature


2

1.1. Arsenic, Arsenical Forms and Structures

1.1.1. Physical and chemical properties of arsenic

Arsenic and its compounds were known long before it was clearly recognized as a

chemical element. In the 4th century BC, Aristotle wrote of a substance called

sandarache, now believed to have been the mineral realgar, a sulphide of arsenic. The

first clearly authentic report of the free substance was made in 1649 by Johann

Schroeder, a German pharmacist. He prepared arsenic by heating its oxide with

charcoal. By the 18th century, arsenic was well known as a unique semimetal.

Arsenic, a chemical element in the nitrogen family (Group V of the periodic table),

exists in both grey and yellow crystalline forms. Although some forms of arsenic are

metal-like, the element is best classified as a nonmetal. Grey arsenic, which is more

stable and more common than the softer yellow form, is metallic, very brittle, tarnishes

in air, and with low thermal and electrical conductivity. Table 1.1 lists some basic

properties of this element. Other forms have been reported but are not well

characterized, including especially the yellow form, which may consist of As4

molecules analogous to white phosphorus, P4.

Table: 1.1. Arsenic element properties:

Atomic number 33 Atomic weight 74.9216

Melting point (grey form) 814°C (1,497°F) at 36 atmospheres pressure

Density (grey form) 5.73 g/ml at 14°C (57°F) (yellow form) 2.03 g/ml at 18°C (64°F)

Oxidation states -3, +3, +5 Electron configuration 2-8-18-5 or 1s22s22p63s2 3p63d104s24p3

The outer-shell similarity of arsenic and phosphorus suggests that arsenic can form

three covalent bonds per atom with an additional lone pair of electrons. The oxidative

state of arsenic should be either +3 or -3 depending on its electronegativity and that of

the elements with which it is combined.


3

Free arsenic is stable in dry air, but in moist air it tends to become coated with a black

oxide. Sublimed arsenic vapour readily burns in air to form arsenious oxide. The free

element is essentially unaffected by water, bases, or nonoxidizing acids, but it can be

oxidized by nitric acid to the +5 state. Arsenic can be attacked by halogens or sulfur and

then combines directly with many metals forming arsenides.

1.1.2. Formation and structures of arsenicals

The abundance of arsenic in the Earth's crust is about five grams per ton. The element is

widely distributed. Because arsenic has a range of oxidation states from -3 to +5 (-3, 0,

+3, +5), it can form a wide variety of compounds and this generates the complexity of

its chemistry in the environment. Only a small amount exists in the native state

(90-98% purity). Most is combined in more than 150 different minerals, as sulfides,

arsenides, sulfoarsenides, and arsenites. FeAsS (Mispickel, or arsenopyrite), is among

the most common of arsenic-bearing minerals; others are As4S4 (realgar), As2S3

(orpiment), FeAs2 (loellingite), and Cu3AsS4 (enargite). Their solubilities are less than

0.05mg/L (Gupta and Chen, 1978). Most commercial arsenic is recovered as a

by-product of the smelting of copper, lead, cobalt, and gold ores.

Among the most important commercial compounds are the oxides, the principal forms

of which are arsenious oxide (As4O6) and arsenic pentoxide (As2O5). Arsenic pentoxide

is formed by the action of an oxidizing agent (e.g., nitric acid) on arsenious oxide.

Arsine (AsH3), a colourless, extremely toxic gas composed of arsenic and hydrogen, is

another familiar arsenic compound. The gas, also called arsenic hydride, is produced by

the hydrolysis of metal arsenides and by the reduction by metals of arsenic compounds

in acidic solutions. Transformed by bacterial and fungal microorganisms, volatile

dimethylarsine and trimethylarsine can be produced from dimethylarsinic acid

(Cacodylic acid).


4

The major arsenic species found in environmental and clinical samples, which are

water-soluble or lipid-soluble, are arsenate [AsO(OH)3, (AsV)], arsenite [As(OH)3,

(AsIII)], arsenious acids (H3AsO3, H2AsO3-, HAsO3

2-), arsenic acids (H3AsO4, H2AsO4-,

HAsO42-), monomethylarsonic acid [CH3AsO(OH)2, MMAAV; monomethylarsonate

(MMA)], dimethylarsinic acid [(CH3)2AsO(OH), DMAAV; dimethylarsinate (DMA)],

arsenobetaine (AB) and arsenocholine (AC)( Andreae 1978; 1979; 1986; Cullen and

Reimer 1989). There are other identified arsenic species with arsenic-carbon bonds

including trimethylarsine oxide, tetramethylarsonium ion, arsenolipid and arsenosugars

in marine organisms. The structures of some of the above named arsenicals are shown

in Figure 1.1.

Actually, no matter where arsenic species are, in soil or water systems, they are subject

both chemically and biologically to oxidation and reduction (Walsh and Keeney, 1975).

Arsenicals are of interest because the various species exhibit the differing levels of

toxicity to organisms.

1.1.3. Arsenate and arsenite

Arsenate (AsV, AsO43 -) has four oxygen (O) atoms arranged at the corners of a

tetrahedron about a central arsenic (As) atom. Each arsenate tetrahedron has a net

electric charge of -3, which is neutralized by large, positively charged metal ions (e.g.,

calcium, manganese, or ferrous iron) outside the tetrahedron. (Unlike the similar

tetrahedra of silicate, which link to form chains, sheets, rings, or frameworks, arsenate

tetrahedra are insular.) Arsenate closely resembles phosphate (Pi) in structure and

reactivity. In the presence of arsenate during glycolysis, arsenate uncouples oxidation

and phosphorylation by forming a highly labile acyl arsenate (Stryer, 1995).


5

Figure 1.1. The structures of some relevant arsenic compounds.

Arsenobetaine

CH2COO-+CH3

CH3 As

CH3

Arsenocholine

CH2CH2OH+CH3

CH3 As

CH3

Tetramethylarsonium ion

+CH3

CH3 CH3As

CH3

+3

Arsenite As(III)

As

O-

O- O-

Arsenosuger g

CH3 CH2As

CH3

CH3

O

OHOH

OCH2CHOHCH2 OSO3-

Arsenolipid

O CH2As

CH3

CH3

O

OHOH

OCH2CHOHCH2OPOCH2CHCH2-R

R

O-

O-

Arsenosugars a-f

f: R

NH2

N

NN

N

a: R-OCH2CHOHCH2OH

c: R-OCH2CHOHCH2SO3-

d: R-OCH2CHOHCH2OSO3--

-e: R-OCH2CHCH2SO3

NH2

-

O CH2As

CH3

CH3

RO

OHOH

b: R-OCH2CHOHCH2OPOCH2CHOHCH2OH

-O

-O

Trimethylarsine oxide

As

O

CH3

CH3 CH3

HO

Methylarsonic acid(MMA)

OHAs

O

CH3

Dimethylarsinic acid(DMAA, cacodylic acid)

OHAs

O

CH3

CH3

As+5

O

O-

O- O-

Arsenate As(V)


6

Arsenite (AsIII, AsO33-, AsO2

-), the reduced state arsenic oxide, is much more toxic,

soluble and mobile than the oxidized state arsenate (Deuel and Swoboda, 1972).

Arsenite readily forms adducts with thiols. Therefore, it inhibits enzymes by reacting

with two sulfhydryl groups (-SH) within a protein, forming a stable ring structure that is

not easily ruptured by monothiols. But arsenite is not reactive with amino groups or

organics having reduced nitrogen constituents. On the contrary, arsenate reacts with

nitrogen groups such as amines, but not sulphydryl groups.

The oxidation of arsenite by iodine, in aqueous solution proceeds only in the presence

of sodium hydrogen carbonate that provides a strong alkaline environment making

iodine unstable: AsO3

3- + I 2 + 2OH- AsO43- + 2I- + H2O

In acidic solution, the reverse reaction occurs, and arsenate is reduced to arsenite.


7

1.2. Arsenic Species in the Environment

1.2.1. Arsenic distribution 1.2.1.1. The arsenic cycle

Arsenic is a ubiquitous element, ranking 20th in abundance in the earth’s crust. The

translocation and transformation of arsenic species is due to natural phenomena such as

weathering, volcanic activity, water dissolution and biological activity, in addition to

anthropogenic activity. The flux of arsenic is within and through the global reservoirs

of the lithosphere, atmosphere, pedosphere, hydrosphere, biosphere and anthrosphere.

Using the information from many research workers, Bhumbla and Keefer constructed a

simplified and comprehensive cycle of arsenic in the environment (Bhumbla and

Keefer, 1994; Figure 1.2). The exchange of large amounts of arsenic between water and

soils, the atmosphere and biota is emphasised.

Figure 1.2. The cyclic transfer of arsenic in environment (Bhumbla and Keefer, 1994).

Atmosphere: Volatiles

Biota: Animals, Humans, Plants, Microbes

Fresh water, Oceans

Soils, Rocks, Sediments

Agriculture: Pesticides, Fertilizers

Non-agriculture: Fossil Fuels, Industrial wastes, Municipal wastes

Mining, Smelting, Volcanoes


8

1.2.1.2. Arsenic in soils

The amount of arsenic in natural uncontaminated soils varies from areas, rock type,

moisture and depth.

In the Earth’s crust, the total arsenic amount is estimated to be 40.1 Eg, based on

concentrations in rock material. The normal concentration range of arsenic in the

continental crust is 1.5-3.0 mg/kg (Wedepohl, 1995). The annual natural arsenic

liberation from the lithosphere into the exogenic cycle is 17.2 Gg by terrestrial volcanic

exhalations and eruptions, in addition to 4.9 Gg per year from submarine vulcanism.

The total input to the lithosphere can be calculated as 84.6-142.2 Gg per year by the

sedimentation into marine basins and crustal subduction (Matschullat, 2000). Typical

amounts of arsenic in soils varied from 5 – 6mg/kg in Austria (Aichberger and Hofer,

1989; Horvath and Moller, 1980), 11mg/kg in the Netherlands (Wiersma et al., 1985)

and Canada (Maclean and Langille, 1981). Higher concentration of up to 300 – 5,000

ppm (mg/kg) in African soils associated with gold deposits or reefs (Woolson, 1983),

4,600 mg/kg in the soils near mineralized veins in British Columbia, Canada (Warren et

al., 1964) have been also recorded.

Limestone soils have more arsenic than those derived from nonlimestone material

(Aichberger and Hofer, 1989). Elevated arsenic concentrations of 250 mg/kg have been

reported in soils derived from shales and granites (Colbourn et al., 1975), and 100 – 200

mg/kg in Australian soils derived from quartzite (Fergus, 1955).

Soil moisture, affected by seasonal changes, is a factor in determining arsenic

concentration (Oatey and Thornton, 1984). Total arsenic in natural soils was evenly

distributed with depth (Aichberger and Hofer, 1989).

Inorganic arsenate and arsenite are the main species in the soil. Organic dimethylarsinic

acid (cacodylic acid) is also common. However, the proportions of many forms of

arsenic depend on oxidation, reduction, adsorption, dissolution, precipitation and


9

volatilization of arsenic reactions which commonly occur in soil, usually associated

with microorganisms such as bacteria and fungi (Figure 1.3). Thus, the proportion of

bound and mobile arsenic can vary widely. To assess the potential mobilization of

arsenic, the dissolved organic carbon concentration in the soil solution should be

known, besides the element content (mobile or acid-soluble), soil pH, redox conditions

and soil characteristics describing the soil potential for arsenic adsorption (clay, oxides,

cation exchange capacity) (Kalbitz and Wennrich, 1998). In addition to soil mineralogy

and organic matter, Al, Fe and Mn influence sorption and retention of arsenic (Hingston

et al., 1974). In soils only arsenic solid is As2S3. Arsenic usually forms solids with Fe,

Al, Ca, Mg and Ni (Gupta and Chen, 1978).

Bacteria

oxidation

As2S3FeAsO4

pptn.

Fungi oxidationAsS2-

Trimethylarsine(Volat ile)

As CH3CH3

CH3

Bacteria Bacteria

+ S2-

oxidation

reduction

+ Fe(adsorp. pptn.)

Dimethylarsine(Volatile)

As

H

CH3CH3

Arsenate

H2AsO4 pH 6-7-HAsO4 pH 8-9

2-

Arsenite

AsO2-

Methylarsonic acid(MMA)

As+5HO O H

O

CH3

Dimethylarsinic acid(DMAA, cacodylic acid)

CH3 As O H

O

CH3

+5

Figure 1.3. Arsenical transformations in soils (Bhumbla and Keefer, 1994). pptn: precipitation.

In sediments, maximal abundance of redox-active elements such as As and Fe is

generally found close to the sediment-water interface. Soluble arsenic concentrations

are usually controlled by redox condition, pH, biological activity, and adsorption

reactions but not by solubility equilibria (Walsh and Keeney, 1975). Arsenic is more

soluble and mobile within reduced environments that produce arsenite. The profile of

arsenic abundance may be partly attributed to metal(loid)-transforming bacteria.


10

1.2.1.3. Arsenic in aqueous systems

Arsenic absorbed on soil particles was positively correlated with arsenic content in the

associated waters (Alam and Sattar, 2000). In aqueous systems, arsenic exhibits anionic

behaviour (Figure 1.4). pE-pH diagrams for arsenic-water and arsenic-sulphate-water

systems at 25oC have been described by Cherry et al. (1979). Eh, the thermodynamic

redox potential, is another parameter which like pH, describes geochemical systems. At

high Eh values, pentavalent arsenic exists as H3AsO4, H2AsO4-, HAsO4

2- and AsO43-; in

contrast, at low Eh, trivalent arsenic as H3AsO3, H2AsO3-, HAsO3

2- and AsO33- is

present with AsS2- (Ferguson and Davis, 1972). In aerobic waters (oxidative conditions)

arsenic acid (H3AsO4) predominates only at extremely low pH(<2); with a pH range of

2-11, it is replaced by H2AsO4- and HAsO4

2-. Arsenious acid (H3AsO3) appears at lower

pH (pH5-9) and under mildly reduced conditions, but it is replaced by H2AsO3- as the

pH increases (pH 9-12). Only when the pH exceeds 12 does HAsO32- appear. At low pE

in the presence of sulphide, HAsS2 can form; arsine, arsine derivatives and free arsenic

metal can occur under extreme reducing conditions (Scow, 1981).

Figure 1.4. A typical cycle of arsenic in a stratifield lake (Ferguson and Davis, 1972).

The arsenic behaviour of dissolution-precipitation was once examined in a

contaminated stream which was differentiated as surface water (oxidative and slightly

basic), shallow hypo-oxidative zone water (pH 6-7, 0-3mg/L of dissolved oxygen) and

adjacent ground water (anoxic and acidic). Arsenic and iron were enriched in the

HAsO2 HAsO42-

H2AsO4- HAsO2

eg. FeAsO4

As2S3

AsS2-

(CH3) 3As

S2-

Oxidation pH 8-9

pH 6-7

Reduction

ads. precip.

precip. Reduction

Methylation

Adsorption

Epilimnion

Thermocline

Hypolimnion

Sediments


11

hypo-oxidative zone with arsenic in the form of arsenite. In the oxidative surface water,

20% of the total dissolved arsenic was found to occur in its oxidative form arsenate.

Upon burial and reduction of ferric oxyhydroxides in the streambed, sediment-bound

arsenic is transferred into the dissolved phase as arsenite, and it is subsequently

released into the surface water, where it does not immediately re-oxidize (Nagorski and

Moore, 1999). Unlike arsenic in aqueous systems, increased mobility of arsenic after

liming of Fe oxide minerals is a pH-dependence of sorption rather than a consequence

of dissolution-precipitation (Jones et al., 1997).

However, in both soil and water systems, both redox potential and pH control the

speciation and solubility of arsenic.

1.2.1.4. Arsenic mobility in the environment

Arsenic mobility and transport in the environment are strongly influenced by arsenic's

associations with solid phases in soil and sediment. Increased mobilization of arsenic

from contaminated soils and sediments under anaerobic conditions is of great concern

because water-borne arsenic can migrate into pristine areas, endangering aquatic

organisms and people. In addition, the volatilisation of arsenicals is the major way that

arsenic moves up from ground to atmosphere, and thus tends to cause air pollution. The

identification of important release mechanisms may assist in designing safe and

effective remediation strategies.

The mobility of arsenic commonly increases as reducing conditions are established

within sediments or flooded soils. Although the reduction of arsenic increases its

solubility at neutral pH, hydrous ferric oxides strongly sorb both arsenate and arsenite.

Thus, in the presence of excess hydrous ferric oxides, reductive dissolution of iron may

be the dominant mechanism by which arsenic is released into solution (Cummings et al.,

1999). Arsenic is associated with ferric oxyhydroxides when released from sediments

containing iron arsenate, a solid-phase surrogate for sedimentary arsenic. Arsenate is

more strongly sorbed than arsenite (Klarup, 1997). Reduction of arsenate to arsenite


12

accompanied iron arsenate dissolution, suggesting that dissimilatory arsenic reduction

may contribute to arsenic flux from anoxic sediments in the most arsenic-contaminated

region (Ahmann et al., 1997).

Experimental results and thermodynamic models showed that arsenic mobility could be

divided into three redox zones (Meng et al., 2001):

a) an adsorption zone at pE > 0, which is characterized by strong adsorption of

arsenate on ferric oxyhydroxide;

b) a mobilization (transition) zone at -4.0 < pE < 0, where arsenic is released because

of reduction of ferric oxyhydroxide to ferrous iron and arsenate to arsenite;

c) a reductive fixation zone at pE < -4.0, where arsenic is immobilized by pyrite and

other reduced solid phases. (see Section 1.2.1.3. for the definition of pE)

Hydrous oxides of Mn also affect arsenic surface reactions (Hingston et al., 1974).

Addition of trivalent arsenic as HAsO2 to MnO2- coated sediments also caused

oxidation to pentavalent arsenic and adsorption onto the surfaces of the oxides. Iron

oxides adsorb arsenic more strongly than do manganese oxides (Oscarson et al., 1981).

Rates of arsenic mobilization during reduction in soils are highly dependent on oxide

surface area and arsenic surface coverage (Jones et al., 2000).

Retardation of arsenic movement in soils is affected by the concentration of phosphate

present from fertilizers or wastes disposed on land, but not by Cl-, NO3-, or SO42-.

Phosphate substantially suppressed arsenic adsorption, but the degree of suppression

varied from soil to soil (Livesey and Huang, 1981). Phosphate (H2PO4-) can effectively

compete with arsenic for adsorption sites, particularly aluminum and iron oxide

surfaces (Pierce, 1981). About 60% of the adsorbed arsenate and 70% of the adsorbed

arsenite were displaced by H2PO4- in a solution of 10-6M phosphate (Thanabalasingam

and Pickering, 1986). Increasing levels of manure application to soils resulted in a

greater loss of arsenic from arsenic contaminated soils [30% > 15% > 5% (w/w)

manure]. The rates of arsenic loss were correlated with the levels of manure


13

amendment (Edvantoro et al., 2004).

Since it forms anions in solution, arsenic does not form complexes with simple anions

like Cl- and SO42- as do cationic metals. Rather, anionic arsenic complexes behave like

ligands in water. Arsenic forms bonds with organic sulphur, nitrogen and carbon.

Trivalent arsenical reacts with sulphur and sulphydryl groups such as cystine, organic

dithiols, proteins and enzymes, but it does not react with amine groups or organic

compounds with reduced nitrogen constituents. On the other hand, pentavalent

arsenical reacts with reduced nitrogen groups such as amines, but not sulphydryl groups.

Carbon forms organoarsenicals with both the trivalent and the pentavalent forms

(Reimers, undated). Methylation increases the water solubility of arsenic, especially of

pentavalent arsenicals. The complexation of trivalent and pentavalent arsenic by

dissolved organic matter in natural environments prevents sorption and coprecipitation

with solid-phase organic and inorganic compounds; essentially, it increases the

mobility of arsenic in aquatic systems and in the soil (Callahan et al., 1979).

Microbial activity affects the mobilization and speciation of arsenic in soil. Studies in

arsenic-contaminated sediments indicated that a direct microbial arsenic-mobilizing

activity exists in the sediments. Arsenic mobility can be enhanced by the activity of

dissimilatory iron-reducing bacteria in the absence of arsenic reduction (Cummings et

al., 1999). Microorganisms catalyzed rapid dissolution of arsenic from iron arsenate,

mobilizing 20-28% of the present arsenic. A native arsenate-reducing microorganism

Strain MIT-13 is a strong arsenic-transforming agent native to the arsenic-contaminated

sediments, dissolving 38% of the arsenic present (Ahmann et al., 1997).

Bacteria capable of either oxidizing arsenite or reducing arsenate coexist and are

ubiquitous in soil environments, suggesting that the relative abundance and metabolic

activity of specific microbial populations plays an important role in the speciation of

inorganic arsenic in soil pore waters (Macur et al., 2001). Microbial reduction of

arsenate in arsenic-contaminated soils may occur under aerobic conditions over


14

relatively short time scales resulting in enhanced arsenic mobilization (Macur et al.,

2001). In anaerobic conditions, arsenite can be mobilized by dissimilatory reduction of

adsorbed arsenate. Oxyanions of arsenic can be used in microbial anaerobic respiration

as terminal electron accepters. Iron-oxidizing bacterium Sulfurospirillum barnesii is

capable of anaerobic growth using ferric iron or arsenate as electron accepters when it

was adsorbed onto the surface of the common soil mineral ferrihydrite by a variety of

mechanisms (e.g., coprecipitation, presorption). Reduction of FeIII in ferrihydrite to

soluble FeII also occurred, but dissolution of ferrihydrite was not required for adsorbed

arsenate reduction to be achieved. This was illustrated by bacterial reduction of

arsenate coprecipitated with aluminum hydroxide, a mineral that does not undergo

reductive dissolution (Zobrist et al., 2000).

Microbes were also able to carry out reactions that resulted in changes in the speciation

of arsenicals in soil. Under both aerobic and anaerobic conditions, the transformation of

arsenate, the soil dominating species, to arsenite, monomethylarsonic acid,

dimethylarsinic acid and to volatile trimethylarsine was less than 0.5%, of which the

production of trimethylarsine represented 0.02-0.3% (Turpeinen et al., 2002).

Formation of dissolved methylated arsenic species by microbes was low (< 0.1%)

during the 5-day incubation (Turpeinen et al., 1999). Augmentation by arsenic

methylating fungi (Penicillium sp. and Ulocladium sp.) was able to enhance arsine

evolution rates in field contaminated soils (Edvantoro et al., 2004). Three fungi

(Scopulariopsis koningii, Fomitopsis pinicola and Penicillium gladioli) were identified

to methylate arsenic to form pentavalent trimethylarsenic species, precursors to volatile

trimethylarsine (Lehr et al., 2003). However, the 'life-time' of arsines in air was short

and they were rapidly converted back to water-soluble species, arsenate and trimethyl

arsine oxide (Turpeinen et al., 2002).


15

1.2.2. Application of Arsenic

Historically, as early as 2000 BC, arsenic played an ambivalent role for human in

respect to its application for healing purposes and for use as poisons in murder and

suicide. Arsenic-bearing minerals were mined by the early Chinese, Indian, Greek, and

Egyptian civilisations already (Azcue and Nriagu, 1994).

The following summarises the range of arsenic applications (National Occupational

Health and Safety Commission, Australia, 1989).

• Mining and processing: Arsenic is a constituent of many alloys, including those of

copper, lead, zinc, silver and germanium. It is alloyed with lead in making lead shot,

lead-based bearing metals and battery grids etc. Arsenic is often removed as an

impurity during the smelting of copper, lead and zinc.

• Ammunition factories: It is used to harden and improve flight characteristics of

projectiles. Arsine was used as a military poison gas.

• Electronics industries: Arsine is used in semiconductor production as a doping

agent. Arsenide used for manufactory lasers, converts electrical energy into

coherent light.

• Glass making: Arsenic trioxide and pentoxide serve as decolourizers to produce

clear glass, free from the green stain of iron impurity.

• Chemistry industries: Dyes and colours, drying agent for cotton.

• Wood preservation: Wood fibres are impregnated under pressure with copper

chrome arsenate.

• Herbicide: Monosodium methyl arsenate, disodium methyl arsenate and sodium

arsenate are used extensively in agriculture.

• Pesticide: Lead arsenate (PbHAsO4) is used to some extent in horticulture. Arsenic

trioxide is used in termite control.

• Disinfectants: arsenic acid (H3AsO4) and its salts as lead arsenate (PbHAsO4) and

calcium arsenate [Ca3(AsO4)2] are useful for sterilizing soils.

• Hide preservation: Arsenic trioxide and sodium arsenite are used as hide

preservatives.


16

• Food additive: Derivatives of phenylarsenic acid are added to fowl and pig feed.

• Medicine: Arsenic trioxide is effective in treating acute promyelocytic leukemia.

Several complex organic compounds of arsenic have been employed in the

treatment of certain diseases, such as amebic dysentery, caused by microorganisms.

Arsenicals as medicines gained importance in the beginning of the last century as the

primary mode of treating syphilis. An arsenical preparation called Fowler's solution,

initially used by Folkner and Scott in 1931, was used in the treatment of chronic

myeloid leukaemia until the introduction of busulphan in 1953. In the 1970s, arsenic

trioxide was found to be extremely effective in treating acute promyelocytic leukaemia

(APL) in China. Since then, numerous in vitro and in vivo studies have confirmed this

observation (Mathews et al., 2001). Over the last ten years, great improvements have

been achieved in the treatment of acute promyelocytic leukaemia. Arsenic trioxide can

induce complete remission in most APL patients refractory to all-trans retinoic acid and

chemotherapy. It acts mainly by inducing apoptosis of APL cells (Fenaux et al., 2001).

However, As2O3 may be significantly or even fatally toxic at doses currently used (0.1

mg/kg per day intravenously) because it caused sudden death among patients

(Westervelt et al., 2001). Low-dose As2O3 (0.08 mg/kg) had the same effect as the

conventional dosage. The mechanism of low-dose arsenic seemed to primarily induce

differentiation of APL cells (Shen et al. 2001).

In addition to arsenic trioxide (As2O3) treatment of acute promyelocytic leukemia,

As2O3 is also promising as a novel and safe anticancer agent for treatment of solid

cancers. In human androgen-independent prostate cancer cell lines, high concentrations

of As2O3 induced apoptosis and low concentrations inhibited growth. As2O3 activated

p38, JNK, and caspase-3 in a dose-dependent manner. In mice, in vivo tumor growth

inhibition was demonstrated in orthotopic and metastatic lesions with no signs of

toxicity (Maeda et al., 2001).


17

1.2.3. Pollution by arsenic

Arsenic is an environmental pollutant as a result of mining and industrial uses. Arsenic

has also found widespread use in agriculture and industry to control a variety of insect

and fungicidal pests in the 1940s through the 1970s. Most of these uses have been

discontinued, but residues from such activities, together with the ongoing generation of

arsenic wastes from the smelting of various ores, have left a legacy of a large number of

arsenic-contaminated sites. In Australia, many arsenic-contaminated sheep and cattle

dips, where As-containing pesticides were used in the past to kill ticks, are scattered

through rural areas. The locations of many of these sites are poorly recorded.

1.2.3.1. Arsenic in arsenic-contaminated sediments and soils

In general, arsenic levels increase or remain about the same with increasing soil profile

depths. Total arsenic was significantly related to soil texture; soils with higher clay

content having higher arsenic contents. Arsenic level is weakly associated with organic

matter and inversely related to soil pH.

The widespread arsenic heavy contamination of soils in southwest England results from

mineralisation and mining activity. The scale of arsenic dispersion by natural and

anthropogenic processes is such that 722 km2 of land contains concentrations of arsenic

in excess of 110 µg g-1, more than twice the maximum that might be expected in a

normal soil (Mitchell and Barr, 1995). An arsenic concentration gradient in the mining

area indicated that the local environment was contaminated by mining activities. In

Brazil, very high arsenic concentrations were even found in some ore waste. The

sediments nearby had a high degree of contamination (up to 347 µg g-1), decreasing

toward water (Magalhaes et al., 2001). The soils surrounding wood-preserving plants,

are often contaminated with highly caustic solution containing 5% arsenite.

Contaminated soil resulted in groundwater plumes that contained up to 25 mg L-1

arsenic, with arsenate being the predominant species (Moore et al., 2000).

In many states in U.S.A., background concentrations of soil arsenic have been used as


18

an alternative soil cleanup criterion. An upper baseline concentration of 6.21 mg As

kg-1 was estimated for undisturbed soils (n = 267) compared to 7.63 mg As/kg for

disturbed soils (n = 181) (Chen, et al., 2001). The vertical distribution of arsenic was

examined in six contaminated orchard soils in the State of Washington, USA. Most of

arsenic was restricted to the upper 40 cm of soil. In all cases, there were lower arsenic

concentrations at the soil surface than deeper in the profile. Absolute soil enrichment

with arsenic occurred to depths between 45 and > 120 cm. At 120 cm, arsenic

concentrations ranged from 0.07 to 0.63 mmol kg-1 (5.25-47.25 mg kg-1) (Peryea and

Creger, 1994).

Using a soil-water-sediment mesocosm, Ruokolainen et al. (2000) studied leaching and

runoff of arsenic from the contaminated soil of an old wood impregnation plant, and its

fate in a recipient freshwater ecosystem. Of the total arsenic load, 7.5% remained in the

water, 44% settled down to the shallow (water depth 5-30 cm) sediment zone, and

48.5% to the deeper (water depth 80 cm) sediment zone.

The translocation of arsenic to the above-ground portion of plants can appreciably raise

the concentrations of arsenic associated with unwashed herbage. In numerous copper

mine sites located at high altitudes in equatorial regions, unusually high concentrations

of arsenic occur in leaves of some plant species (e.g. up to 1.43 mg As kg-1 dry wt in

Bidens cynapiifolia) (Bech et al., 1997). In Ladder brake fern (Pteris vittata L.), soil

concentrations of arsenic of 50-100 mg kg-1 stimulated the plant growth, arsenic uptake

and translocation (Tu and Ma, 2002). The high bioaccumulation ratios of fern also

reflected both soil and air sources of pollution (Amonooneizer et al., 1996). The

consumption of soil-contaminated grass by cattle can be a major pathway of arsenic to

the livestock, with up to 97% of the total dry matter intake of arsenic via ingested soil.

Cattle on contaminated land may ingest up to 31 times more arsenic than livestock

grazing uncontaminated pastures (Abrahams and Thornton, 1994). The edible

mushroom Laccaria amethystine is known to accumulate arsenic. The total arsenic

concentration in two samples of the mushroom was 23 and 77 µg As g-1 (dry weight)


19

when growing in uncontaminated soils and 1,420 µg As g-1 from arsenic-contaminated

soils (containing 500-800 µg As g-1). Of the total arsenic in this mushroom,

dimethylarsinic acid accounted for 68-74%, methylarsonic acid for 0.3-2.9%,

trimethylarsine oxide for 0.6-2.0% and arsenic acid for 0.1-6.1% (Larsen et al., 1998).

1.2.3.2. Arsenic contamination in water

Arsenic bound to soil was positively correlated with arsenic content in waters (Alam

and Sattar, 2000). Relating to geological and geochemical features, arsenic in water

may be derived from naturally-occurring arsenic-rich minerals and rocks, the burning

of arsenic-rich coal, and from sulfide mineralization and waster materials resulting

from mining and smelting activities. In certain countries, such as India, China, Chile,

the United States of America and Germany, there are numerous cases of arsenic in

ground water in concentrations, exceeding the WHO maximum (0.01 mg L-1).

In Germany, the wetland soils of the Mulde River in the industrial district of

Bitterfeld-Wolfen are highly contaminated with heavy metals and arsenic. The

acid-soluble concentration (mostly equal to the total content) of arsenic in soil was up

to 265 mg kg-1. In the Bengal basin of Bangladesh, the mean arsenic concentrations in

groundwater in the Char Ruppur (0.253 mg As L-1), Rajarampur (1.955 mg As L-1) and

Shamta areas (0.996 mg As L-1) greatly exceed the WHO recommended value (Islam et

al., 2000).

A typical example of arsenic contamination in India caused by pesticide production in a

heavily populated area，is in South Calcutta. Manufacture of copper acetoarsenite

[(Cu(CH3COO)2)3Cu(AsO2), (Paris-Green)], an arsenical pesticide has been in

progress for the past 25 years. Another industry in Calcutta was producing 20-30 tons

of copper acetoarsenite per year and was discharging most of the effluent without

proper treatment in an open land just outside the boundary of the factory. Soil around

the effluent dumping point of the factory was exceptionally contaminated, with arsenic


20

concentrations of 20.1-35.5g kg-1. Arsenic and copper concentrations in bore-hole soils

collected up to a depth of 24.4 m at the effluent dumping point, decreased with depth.

Arsenous acid, arsenic acid, methylarsonic acid (MA) and dimethylarsinic acid (DMA)

were detected in bore-hole soils up to a depth of 1.37 m, after which only inorganic

arsenical compounds were present. Sparingly soluble Paris-Green cumulatively

deposited in the waste disposal site is decomposed by micro-organisms to water-soluble

forms which percolated to underground aquifers along with rain water through the

discharge zone (Chatterjee and Mukherjee, 1999). Due to the high porosity of the soil,

more than 7000 people living around the discharge point were exposed to arsenic

contaminated water. Higher internal arsenic concentrations were found in these people

than in the normal population (Chakraborti et al., 1998).

In Eastern Finland, to assess the transportation of chlorophenols and chromated copper

arsenate from an old contaminated sawmill site to the surrounding water system, the

concentrations of these compounds in selected sediment samples were analyzed. Of all

the wood preservatives studied, only arsenic was found to bioaccumulate in the

prevailing conditions, reaching a tissue concentration of 362 µg g-1 dry weight in

organisms exposed for 28 days to river sediment. High concentration of arsenic in

oligochaeta tissue was related to high concentrations of arsenic in the surrounding pore

water (Lyytikainen et al., 2001).

1.2.3.3. Arsenic contamination in Australia

Arsenic contamination of soils and crops due to inadvertent addition of impurities in

agricultural fertilizers, application of pesticides and mining has occurred in Australia.

Arsenic was used in agricultural chemicals during the twentieth century. Arsenic

remains in soil from usage prior to 1985 for the control of sheep lice (Bovicola ovis),

flystrike (Morcombe, 1996) and cattle ticks. Soils surrounding sheep and cattle dips in

Australia are known to be highly contaminated with arsenic and are potentially of major

concern to the environment and human health. Soils of 11 dip sites in northern New


21

South Wales (NSW) have been studied. The results revealed arsenic contamination (up

to 3,542 mg As kg-1 soil) in the surface soil (0-10 cm) and considerable movement of

arsenic down through the soils at 20 to 40 cm with concentrations ranging from 57 to

2,282 mg As kg-1 soil. At one particular site, an arsenic concentration was found to

exceed 14,000 mg As kg-1 soil at a depth of 40 to 45 cm. The bulk of the contaminant

arsenic at those sites seemed to be associated with amorphous soil Fe and Al minerals.

When soils from different sites and depths were analysed by a sequential fractionation

scheme based on a soil phosphorus fractionation, soil arsenic was separated into six

fractions with (i) anion exchange resin, (ii) NaHCO3, (iii) NaOH, (iv) NaOH following

sonication, (v) HCl and (vi) HCl/HNO3. Most sites were found to contain substantial

concentrations of arsenic in the two most labile fractions (resin extractable and

NaHCO3 extractable), indicating the high potential for phytotoxicity and leaching

(Mclaren et al., 1998).

At the Mole River mine, northern New South Wales, there was an arsenic oxide

treatment plant for mining and processing of arsenopyrite ore operated in the

1920-1930s, where left abandoned mine workings and waste dumps (2.6-26.6% arsenic

by weight). At the mine, highly contaminated soils (about 12 km2) have extreme arsenic

(mean 0.93 % by weight) compared with background soils (mean 8 ppm arsenic). In the

surrounding regions, estimated to be 60 km2, contaminated soils have a mean arsenic

concentration of 55 ppm. Hydromorphic dispersion from the dissolution of arsenic-rich

particulates, erosion of arsenic-rich particulates from the dumps, and atmospheric

fall-out from processing plant emissions enriched the soils with arsenic. After rainfall,

water soluble, arsenic-bearing mineral salts (pharmacolite, arsenolite, krautite) were

formed, affecting surface waters. The erosion and collapse of waste-dump material into

local creeks, seepages and ephemeral surface runoff, together with the erosion and

transport of contaminated soil into the local drainage system caused stream sediments

within a radius of 2 km of the mine to contain 62 ppm to 27.5 % arsenic by weight

compared with the mean background of 23 ppm arsenic. In addition, water samples

from a mineshaft and waste-dump seepages had the lowest pH (4.1) and highest arsenic


22

values (up to 13.9 mg L-1), and contained algal blooms of Klebsormidium sp.

Bioaccumulation of arsenic and phytotoxicity to lower plants has been observed in the

mine area. Several metal-tolerant plant species (Angophora floribunda, Cassinia laevis,

Chrysocephalum apiculatum, Cymbopogon refractus, Cynodon dactylon, Juncus

subsecundus and Poa sieberiana) colonised the periphery of the contaminated site. It

was also found that the flow of the Mole River diluted the arsenic-rich drainage waters

and lowered arsenic concentrations to background values (mean 0.0086 mg L-1 arsenic)

within 2.5 km downstream (Ashley and Lottermoser, 1999).


23

1.2.4. Arsenic bioavailability and uptake by organisms

Arsenic concentration in water, equivalent to its bioavailability, is governed more by

the particular arsenic species than by the total amount of arsenic present. Arsenic

oxidation-reduction and biochemical methylation reactions, precipitation-dissolution,

and adsorption-desorption control mobilization, bioavailability and bioaccumulation of

arsenic in the environment. The propensity for plants to accumulate and translocate

arsenic to edible and harvested parts depends largely on soil type and climatic factors,

plant genotype and agronomic management (McLauphlin et al., 1999).

The practice of adding lime to remediate contaminated soils has the potential to

mobilize arsenic, due to the pH dependence of arsenic sorption reactions on oxide

minerals and layer silicates. Increased mobility of arsenic with liming appears to be

consistent with the pH-dependence of sorption reactions of As on Fe oxide minerals

rather than dissolution-precipitation reactions involving arsenic (Jones et al., 1997).

Some bacteria possess two distinct phosphate transport pathways: one system takes up

both phosphate and arsenic at similar rate, whereas the other is highly specific for

phosphate and transports arsenate poorly (Willsky and Malamy, 1980).

A recent study on yeast Saccharomyces cerevisiae showed that undissociated arsenite

(pKa 9.2) was transported across the plasma membrane via a glycerol transporting

channel (Andrew et al., 2003). Glycerol competed with arsenite for transport in a

dose-dependent manner, indicating that arsenite and glycerol uptake mechanisms were

the same. Arsenate transport was unaffected by glycerol, confirming that arsenate and

arsenite are taken up into cells by different mechanisms. Antimonite (Sb2S3, SbS2-), an

arsenite analogue that is transported into S. cerevisiae cells by aquaporins, also

competed with arsenite transport in a dose-dependent manner, providing further

evidence that arsenite is transported into rice roots via glycerol-transporting channels.

Mercury (Hg2+) inhibited both arsenite and arsenate uptake, suggesting that inhibition


24

of influx was due to general cellular stress rather than the specific action of Hg2+ on

aquaporins (Andrew et al., 2003).

Arsenic uptake and transport in plants is species-specific. Although arsenic

concentration influenced both arsenic availability and toxicity, the arsenic chemical

form present in the nutrient solution primarily determined arsenic phytoavailability that

followed the trend DMA << MMA<AsV < AsIII in marsh grasses (Spartina patens and

Spartina alterniflora) (Carbonellbarrachina, et al., 1998). In rice, the trend was DMA

<AsV <MMA < AsIII (Oryza saliva L.) (Marin et al., 1992). However, arsenic

concentrations in root and shoot significantly increased with increasing arsenic

application rates, regardless of the arsenic chemical form (Carbonellbarrachina, et al.,

1998). MMA treatments caused the highest arsenic accumulation in both roots and

shoots, and showed a higher uptake rate than the other arsenic compounds. Inner root

arsenic concentrations were, in general, within the normal range for arsenic contents in

food crops but root skin arsenic levels were close or above the maximum threshold set

for arsenic content in edible fruit, crops and vegetables (Carbonell-Barrachina et al.,

1999).

A study of vegetables showed that lettuce and radish had much higher survival than

tomato and bean treated with arsenic. All four plant species effectively accumulated

and translocated arsenic. Tomato and bean plants contained arsenic mainly in the roots,

and little was translocated to the fruits. Radish roots accumulated less arsenic compared

to the leaves, whereas lettuce roots and leaves accumulated similar concentrations of

arsenic. Lettuce leaves and radish roots accumulated significantly more arsenic than

bean and tomato fruits (Cobb, et al., 2000). With carrot plants, the soil-to-carrot uptake

rate (bioavailability) of arsenic was 0.47 +/- 0.06 (standard deviation) % of the arsenic

content in the soil (Helgesen and Larsen, 1998).

Arsenite had more inhibitory effects on crop yield than arsenate. But only minor

differences between concentrations of arsenite and arsenate were observed in crops.


25

Soil arsenic concentration of 50 and 250 mg kg-1 resulted in significant yield reduction

and marked increase of arsenic concentration in ryegrass (Lolium perenne L) and barley

(Hordeum vulgare L) and these concentrations could be considered as critical for both

crops. The application of arsenic-containing fertilizer had little or no effect on crop

yield. The application of additional phosphorus to a fertiliser treatment (containing 3 g

As kg-1 fertiliser) only slightly increased crop yields but increased arsenic

concentration in ryegrass markedly (Jiang and Singh, 1994). Application of arsenate

and MMA significantly increased root, shoot and total dry matter production at low

arsenic application (Carbonell et al., 1998). This positive plant growth response may be

linked with phosphorus nutrition (Carbonellbarrachina et al., 1998). Phosphorus uptake

by canola (Brassica napus L.) seedlings tended to decrease as soil arsenic increased, but

Pi concentration in tissue was not affected. A decrease in shoot P:As ratios in plants

indicated competition between these ions (Cox and Kovar, 2001).

The uptake of arsenic has also been studied in a range of animals. Intake and

accumulation of arsenic by wild rodents living in arsenic-contaminated habitats varied

between species, sexes and age classes. Adult and juvenile wood mice and bank voles

(free-living small mammals), accumulated similar amounts of arsenic on

arsenic-contaminated mine sites and the extent of accumulation depended upon the

level of habitat contamination. Wood mice and bank voles in the same sites had similar

concentrations of arsenic in their stomach contents and accumulated comparable

residues in the liver, kidney and whole body. Female bank voles, but not wood mice,

had significantly higher stomach content and liver arsenic concentrations than males.

Arsenic concentrations in the stomach contents and body tissues did not vary with age.

The bioaccumulation factor (ratio of arsenic concentration in whole body to that in the

diet) in wood mice was not significantly different to that in bank voles and was 0.69 for

the two species combined, indicating that arsenic was not bioconcentrated in these

rodents (Erry et al., 2000).

DMA is taken up by red blood cells (RBCs) in the form of dimethylarsinous (DMAIII)


26

acids and exits as dimethylarsinic (DMAV). The influx and efflux are dependent on the

animal species. Although DMAV was excluded by red blood cells of all the animal

species, DMAIII was taken efficiently in the order: rat > hamster > human. Red blood

cells of mice took DMAIII up less efficiently and with a different pattern from the

former three animals. Although DMAIII taken up by rat red blood cells was retained, it

was exited as DMAV from hamster red blood cells. The influx of DMAIII and efflux of

DMAV was much slower in human red blood cells than rat and hamster ones. The

uptake of DMAIII by red blood cells was inhibited when glutathione was oxidized by

diamide. DMAIII taken up by red blood cells is retained through the formation of a

complex with protein(s) specific to animal species and the arsenic leaves the red blood

cells after being oxidized to DMAV (Shiobara et al., 2001).


27

1.2.5. Toxicity of arsenic to organisms

With respect to global human health hazards, arsenic is one of the most severe

environmental toxic elements. Arsenic-contaminated environments adversely affect

both the chemistry and microbiology of the underlying arsenic-saturated zone

(Brunning et al., 1994). Arsenic is the focus of public attention due to the health

problems of hundreds of thousands of people caused by arsenic-contaminated

groundwater. These problems include increasing risk of skin, lung and bladder cancers.

The acute toxicity of arsenic differs between its chemical forms. In general, among the

arsenic compounds in the environment, arsenite is ten times more toxic than arsenate

and seventy times more than the methylated species, DMA and MMA (Squibb and

Fowler, 1983). DMA and MMA are moderately toxic, whereas arsenobetaine and

arsenocholine are virtually non-toxic (Cannon et al., 1979). The toxic effects of arsenic

vary between different parts of the plant. Growth of the shoot was more affected by

arsenate than that of the roots in P. sativum L. (Paivoke and Simola, 2001).

Monomethylarsonous acid (MMAIII) and dimethylarsinic acid (DMAIII) contain arsenic

in the trivalent oxidation state. These forms are more cytotoxic, more genotoxic, and

more potent inhibitors of the activities of some enzymes both in vivo and in vitro than

are inorganic arsenicals in the trivalent oxidation state (Petrick et al., 2001). Hence, it is

reasonable to regard the methylation of arsenic, in this case, as a pathway for its

activation, not as a mode of detoxification.

Dimethylarsinic acid (DMAIII) induces an organ-specific lesion - single strand breaks

in DNA - in mice, rats and human lung cells in vitro. This damage is due mainly to the

peroxyl radical of DMA and production of active oxygen species by pulmonary tissues.

DMA acts as a promotor of urinary bladder, kidney, liver and thyroid gland cancers in

rats and of lung tumors in mice. DMA is a complete carcinogen and plays a role in the

carcinogenesis of inorganic arsenic (Kenyon and Hughes, 2001). DMA has been used

as a herbicide (cacodylic acid). Farmers exposed to arsenic pesticides are at risk of skin


28

cancer, mostly morbus Bowen (carcinoma in situ), multiple basal cell carcinomas and

squamous cell carcinomas (Spiewak, 2001).

Arsenite is not significantly mutagenic at non-toxic concentrations, but acts as a

cocarcinogen with a second (genotoxic) agent. The comutagenic effect of arsenite is

due to inhibition of DNA repair, but no specific repair enzyme has been found to be

sensitive to low (<1 µM) concentrations of arsenite. Low (non-toxic) exposure to

arsenite enhances positive growth signaling. The absence of normal p53 functioning,

along with increased positive growth signaling in the presence of DNA damage may

result in defective DNA repair and account for the comutagenic effects of arsenite (Vogt

and Rossman, 2001; Rossman et al., 2001).

The mechanism of arsenite toxicity is believed to be due to the ability of arsenite to bind

protein thiols. Glutathione is the most abundant cellular thiol, and both glutathione and

glutathione-related enzymes are important antioxidants that play an important role in

the detoxification of arsenic and other carcinogens. However, no evidence has been

found for a direct interaction of arsenic with the glutathione-related enzymes,

glutathione reductase, bovine glutathione peroxidase, or equine glutathione

S-transferase (GST) that use glutathione. None of these enzymes are inhibited or

activated by physiologically relevant concentrations of AsV, AsIII, AsIII (GS)3, or

CH3AsIII (MMA) (Chouchane and Snow, 2001).

Arsenic trioxide has significant cytotoxic effects, and induces of a number of stress

genes in human liver carcinoma cells. Co-exposure to atrazine strongly potentiates

arsenic trioxide-induced cytotoxicity and transcriptional activation of stress genes in

transformed human hepatocytes (Tchounwou et al., 2001).

Gallium arsenide (GaAs) causes various toxic effects in animals, including pulmonary

diseases. GaAs is used in various semiconductor products, although its toxicity is not

completely understood. Exposure to GaAs suspension and AsCl3 solution induced


29

cytotoxicity of alveolar macrophages. Enzyme release assays and morphological

findings indicated damage to macrophages. Because the release is thought to be

associated with the cytoskeleton, exposure to GaAs impairs their motor function. Thus

the cytotoxicity caused cytostructural changes and cell death (Okada et al., 2001).

Multiple exposure to GaAs may produce an adverse effect on the haematopoietic, renal

and immune system, and also decreases blood glutathione contents. Fortunately,

sodium 2,3-dimercaptopropane I-sulfonate (DMPS) is an effective chelating drug for

reversing most of the GaAs induced immunological alterations and reducing tissue

arsenic burden (Flora and Kumar, 1996).

Besides its tumorigenic potential, arsenic has been shown to be genotoxic in a wide

variety of different experiment-designed starts and biological endpoints. In vitro,

arsenic was shown to induce chromosomal mutagenicity including micronuclei,

chromosome aberrations, and sister chromatid exchanges. Arsenic mainly acts in a

clastogenic manner but also has an aneugenic potential. The potential of arsenic to

induce point mutations is very low in bacterial and mammalian cell systems. However,

in combined exposure with point mutagens in vitro, arsenic was shown to enhance the

frequency of chemical mutations in a synergistic manner. In vivo, arsenic was shown to

induce chromosome aberrations and micronuclei in mice. After long-term exposure to

arsenic-contaminated drinking water, elevated frequencies of DNA lesions (e.g.

micronuclei and chromosome aberrations) were also found in the great majority of

human subjects. It is not clear by which mechanism the genotoxicity of arsenic is

mediated, although the data available indicate that arsenic acts indirectly on DNA, i.e.

via mechanisms like interference of regulation of DNA repair or integrity. Because of

the indirect mode of action, the genotoxicity of arsenic may underlie a sublinear

dose-response relationship (Gebel, 2001).


30

1.3. Natural Resistance and Tolerance Mechanisms for Arsenic Three general mechanisms of resistance to arsenic toxicity have been recognised.

These are enhanced transport of inorganic arsenicals out of the cell, binding of arsenic

by specific chelators, and conversion of arsenicals to less- or non-toxic forms.

1.3.1. Transport of arsenic out of cells and into the vacuole

1.3.1.1. ars operons in bacteria

In prokaryotes, arsenic detoxification is accomplished by export systems that are both

chromosomal and plasmid-borne operon-encoded. Arsenic is transported out of the

cytosol of cells. Operons which encode arsenic resistance have been found in multicopy

plasmids from both gram-positive and gram-negative bacteria (Novick and Roth, 1968;

Hedges and Baumberg, 1973). The ars operons from E. coli plasmid R773 (Rosen, et al.,

1993) and R46 (Kuroda et al., 1997) contains five genes, named arsR, arsD, arsA, arsB

and arsC. In contrast, the ars operons from the gram-positive Staphylococcus aureus

plasmid pI258 (Ji and Silver, 1992) and Staphylococcus xylosus plasmid pSX267

(Rosenstein et al., 1992) consist of only three genes, arsR, arsB and arsC. An arsenic

resistance operon very closely homologous to that of E. coli plasmid R773 occurs on

the E. coli chromosome (GenBank accession U00039: locus ECOUW76). This

chromosomal operon also is without arsA and arsD genes, containing only arsR, arsB

and arsC (Carlin et al., 1995; Figure 1.5). The ArsR (Wu and Rosen, 1991) and arsD genes (Wu and Rosen, 1993) encode

regulatory proteins. The arsA and arsB encode the actual resistance, a pump that

extrudes arsenite. The last gene of the operon, arsC, encodes a reductase that catalyzes

the conversion of arsenate to arsenite.


31

Figure 1.5. Bacterial ars operons. In the top line the five genes of the ars operon of plasmid R773 are indicated by large arrows showing the direction of transcription starting with the promoter pars. Genes are indicated with the intergenic spaces as single lines. The genes of homologous ars operons of plasmid R46, the E. coli chromosomal operon, and staphylococcal plasmids pI258 and pSX267 are aligned below. The similarities of the gene products to the R773 proteins are given as % identity. (Copied from Xu et al., 1998)

A. Metalloregulatory proteins The arsR gene encodes in all operons a dimeric

trans-acting repressor protein (Ji and Silver, 1992). As a metal-repressive repressor

protein that negatively regulates the transcription of a metal efflux operon, each

member of the ArsR family should have at least three domains, a metal binding domain,

a DNA binding domain and a dimerization domain. In the ArsR sequence

ELC32VC34DLC37, residues Cys32, Cys34 and Cys37 are ligands for AsIII, although

Cys37 is not required for induction and not conserved in all ArsR proteins (Shi et al.,

1996). ArsR protein binds to a DNA region of imperfect dyad symmetry just upstream

of the ars promoter -35 site. A putative helix-turn-helix (HTH) region from residues 38

to 54 in ArsR has the DNA-binding motif (Shi, et al. 1994). The core sequence of ArsR

from residues 8-90 contains the capabilities for dimerization, repression and metal

recognition (Xu and Rosen, 1997). Xu et al. (1998) hypothesized that the derepression

would start with arsenite binding to the thiolates Cys32 and Cys34. The formation of

As-S bonds distorts the first putative helix of the DNA binding domain so that ArsR

protein can not interact with the operator site.

pars

E. coli

pI258

87% 85% 89% 92% 92%

75% 90% 94%

29% 56% 12%

29% 56% 12%

arsR arsD arsA arsB arsC

R46

R773

pSX267


32

In addition to arsR, the E.coli R773 and R46 ars operon, each has a second regulatory

gene, arsD. The ArsD protein binds to the same site on the ars promoter element as the

ArsR protein but with affinity two orders of magnitude lower (Chen and Rosen, 1997).

As result of this lower affinity, exposure only to high levels of arsenite can cause

dissociation of ArsD, affecting further expression of the ars genes and increased the

synthesis of the Ars extrusion pump. With ArsR controlling the basal level of

expression and ArsD controlling maximal expression, the two repressors form a

homeostatic regulatory circuit that maintains the level of ars expression within a

narrow and necessary range, perhaps to prevent overexpression of ArsB which is toxic

in high amounts.

B. The Ars pump Plasmid-mediated arsenic resistance in E. coli is ascribed to the

operation of an ATP-driven efflux system that pumps arsenite out of the cells (Dey and

Rosen, 1995). This pump is composed of two types of subunits, the products of arsA

and arsB genes in the R773 operon (Dey et al., 1994).

The E.coli arsA gene encodes the ATPase subunit as inferred initially from significant

homology with other ATP-binding proteins, although this homology is only within the

ATP-binding regions but not in the entire protein sequence (Chen et al., 1986). ArsA

protein has two homologous halves (a dimer: A1 and A2) which interact with each

other, each containing the signature sequence of an ATP-binding site. Both binding

sites are necessary for resistance and ATPase activity (Kaur and Rosen, 1993).

Although ArsA protein tightly binds to the ArsB membrane protein, soluble ArsA

forms a homodimer which is the active form, and exhibits arsenite or

antimonite-stimulated ATPase activity (Rosen et al., 1988). The Cys113, Cys172 and

Cys422 (Bhattacharjee and Rosen, 1996), His148 and His453 (Zhou et al., 2000) in

ArsA sequence are each involved in the metallostimulation of the ArsA ATPase.

Although the AsIII-thiol structure in ArsA resembles that in ArsR, the two are clearly

evolutionarily unrelated. Because there is the three-gene arsenic resistance system

which has arsD and arsA missing, the As-thiol structures involved in metalloregulation


33

of ArsR have evolved independently more than once.

The E.coli ArsB is a 429-amino acid integral inner membrane protein with 12

transmembrane spanning α-helices (Wu et al., 1992), which act as the membrane

anchor for ATP subunits of ArsA. However, Ars B is more similar to secondary carrier

proteins than a pump subunit. It can function without ArsA, such as in the

staphylococcal ars operons (arsRBC) with only three genes (Ji and Rosen, 1992). In the

absence of ArsA ATPase, ArsB catalyzes arsenite transport coupled to the membrane

potential, requiring electrochemical energy, hence it is a secondary active transporter

(Dey and Rosen, 1995). When arsA and arsB were co-expressed, the system required

chemical energy and dramatically increased the level of arsenite resistance, suggesting

an interaction between these two proteins (Bröer et al., 1993). It also reflects the fact

that the ATP-driven pump is a more efficient mechanism, forming larger standing

concentration gradients than the secondary carrier. It is reasonable to expect that,

during evolution, an arsRBC operon would have appeared first, following by capture of

an ancestral arsA gene to develop the more efficient ArsA-ArsB pump (Rosen, 1999).

C. Arsenate reductase The arsC gene in both types of ars operons encodes a

soluble protein, acting as an arsenate reductase which converts intracellular arsenate to

arsenite that is then pumped out of the cells by the Ars pump. ArsC from S. aureus has

131 amino acid residues and that from E. coli has 141, with less than 20% identical

amino acids. Thus these two ArsCs belong to two unrelated enzyme families. However,

the two ArsCs possess the same function to reduce arsenate both in vivo and in vitro.

They differ in their energy coupling systems. S. aureus ArsC is coupled to thioredoxin

as its electron source (Ji et al., 1994), while E. coli ArsC is coupled to glutaredoxin

(Gladysheva et al., 1994) which utilises a different substrate (glutathione) from

thioredoxin. In addition, ArsC from S.aureus has a higher affinity for arsenate than that

of E. coli.


34

1.3.1.2. The families of arsenic-resistance transporters in yeast

A. ACR family The ACR (arsenic compounds resistance) family was identified

originally in the chromosome of the bacterium Bacillus subtilis. There are two distinct

ars operons found in B. subtilis. One is typical arsRB with an unlinked arsC gene

(Beloin et al., 1997). Another is atypical with four genes yqcJKLM corresponding to

arsRDBC in which yqcJ is a typical arsR and yqcM encodes an ArsC homolog (Sato

and Kobayashi, 1998). Although the yqcK gene shares no homologs with arsD and

YqcL (a membrane protein) is unrelated to ArsB, this operon actually confers resistance

to arsenate and arsenite.

ACR3, encoding a homolog of YqcL, has been found to be required for arsenite

resistance in the yeast, Saccharomyces cerevisiae. A contiguous three-gene cluster

(ACR1, ACR2 and ACR3) located on chromosome XVI has been isolated from S.

cerevisiae (Bobrowicz et al., 1997). ACR1 encodes a putative transcriptional regulator

that shares no common features with either of the bacterial ArsR or ArsD but is

structurally related to the transcriptional regulatory proteins encoded by the YAP1 and

YAP2 genes from S. cerevisiae. The yAP1and yAP2 proteins are involved in multidrug

and oxidative stress resistance (Balzi and Goffeau, 1994). The Acr2 protein has been

proven to have arsenate reductase activity (Mukhopadhyay and Rosen, 1998;

Mukhopadhyay et al., 2000). ACR3 encodes a plasma membrane arsenic transporter

that shows high similarity to the membrane protein YqcL and weak similarity to the

ArsB protein in S. aureus. ArsB (429 a.a., 45.6KD) has 12 membrane-spanning

segments, while in comparison, Acr3p (404 a.a., 45.8 KD) has only ten. Acr3p is

arsenite specific, whereas ArsB confers resistance to both arsenite and arsenate.

Because of their striking similarities, the yeast ACR gene cluster and the bacterial

arsRBC operon seem to carry out the same biological role of arsenic detoxification.

However, unlike bacteria, the ACR1 and ACR2 genes are expressed in the same

direction and ACR3 in the opposite (Figure 1.6) (Bobrowicz et al., 1997).


35

ACR1 ACR2 ACR3

arsR arsB arsC Figure 1.6. Organization of the ACR gene cluster in Saccharomyces cerevisiae and the ars operon in Staphylococcus aureus. B. MRP family The multidrug resistance-associated protein (MRP) is a member of

the ATP-binding cassette (ABC) transporter superfamily. In addition to providing

resistance to many drugs, human MRP1 also confers resistance to arsenite (Cole et al.,

1994), by functioning as an arsenite-triglutathione [As(GS)3] carrier (Zaman et al.,

1995). In S. cerevisiae, Ycf1p (yeast cadmium factor protein), is 63% identical to

human MRP1, and has been shown to be a vacuolar glutathione S-conjugate pump with

a broad range of substrate specificity (Li et al., 1996). Ycf1p confers cadmium

resistance in S.cerevisiae by catalyzing sequestration of Cd(GS)2 in the vacuole (Li et

al., 1997) and provides the second pathway for removal of arsenite by vacuolar

sequestration of As(GS)3 from yeast cytosol. This arsenite-resistance pathway is

parallel to and independent of Acr3p. Disruption of YCF1 or ACR3 resulted in

equivalent sensitivity to both arsenite and arsenate. Double disruption of both YCF1

and ACR3 resulted in additive hypersensitivity to arsenicals. Arsenate is reduced by

Acr2p to arsenite that can be transported by either the YCF1 or ACR3 systems. Ycf1 has

been shown to catalyze ATP-coupled Cd(GS)2 transport into the vacuole. Using

radiolabelled arsenite, 73As(GS)3 was shown to be accumulated on vacuolar

membranes even in ACR3-disrupted cells but not with the YCF1-disrupted strain

(Ghosh et al., 1999). Thus, Ycf1 is a vacuolar ATP-couple pump for accumulation of

glutathione conjugates of arsenite in the vacuoles. Unlike Acr3p, which is specific for

AsIII, Ycf1 nonspecifically transports of GS-conjugates of various metals including

AsIII, SbIII, CdIII and HgII (Ghosh et al., 1999).

ars in S. aureus

ACR in S. cerevisia


36

1.3.1.3. Arsenic resistance in mammalian cells

Long-term exposure to low levels of arsenite induces malignant transformation in

cultured rat liver epithelial cells. Microarray analysis of these chronically

arsenic-exposed cells showed increased expression of the genes encoding the

glutathione S-transferase Pi (GST-Pi), multidrug resistance-associated protein genes

(MRP1/MRP2, which encode the transporter Mrp1/Mrp2) and the multidrug resistance

gene (MDR1, which encodes the transporter P-glycoprotein). Resistance of arsenic is

accompanied by reduced cellular arsenic accumulation, suggesting a mechanistic basis

for reduced arsenic sensitivity (Liu et al., 2001).

1.3.1.4. Arsenic transport and tolerance in plants

Organic arsenicals and arsenite were the most phytotoxic species when effects on plant

growth were considered. Upon absorption, arsenite, arsenate and MMA were mainly

accumulated in the root system, while DMA was readily translocated to the shoot

(Marin et al., 1992). MMA and DMA showed a higher upward translocation than

arsenite and arsenate, contributing to the greater phytotoxicity and lower dry matter

productions of these organic treatments (Carbonell-Barrachina et al., 1999). More

information about arsenic bioavailability to plants can be referred to section 1.2.4.

The highest concentrations of arsenic were recorded in old leaves of plants on mine

waste (Porter and Peterson, 1975). However, Pickering et al. (2000) recently found that

after arsenate uptake by the roots, most of the arsenic was stored in plants as

AsIII-tris-thiolate complex with the majority in roots. Only a small fraction of arsenic

was exported to the shoot as the oxyanions arsenate and arsenite. Some of plants,

including Highland grass (Agrostis tenuis S.) (Porter and Peterson, 1975) and Ladder

brake fern (Pteris vittata L.) (Ma et al., 2001) were found to tolerate and accumulate

high concentration of arsenic.


37

1.3.2. Arsenic chelation

1.3.2.1. Roles of glutathione in chelation

There is evidence that both in vitro and in vivo glutathione (GSH) functions as a

reductant (Thomas, 1993). A disulfide redox couple with GSH drives the reduction of

arsenate to arsenite in vitro and further, forms arsenotriglutathione

[tri(γ-glutamylcysteinylglycinyl)-trithioarsenite], via the following reactions:

The formation of arsenotriglutathione provides trivalent arsenic that is much more

active than pentavalents. This glutathione conjugate of arsenite can be sequestered to

the vacuole of yeast cytosol. Therefore, glutathione has a dual role in response to

arsenic stress, one as reductant, the other as substrate for production of glutathione

conjugate and the glutathione derivative, phytochelatin.

1.3.2.2. Roles of arsenic-binding proteins in animals

The detoxification of inorganic arsenic in mammals involves several biotransformation

processes including arsenic-protein binding in tissue and blood, methylation of

inorganic arsenic to MMA and DMA, and excretion of MMA and DMA into the urine

(Thompson, 1993). By exposure to 0.1 M CuCl at pH, more than 90% of protein-bound

arsenicals are liberated from binding sites on liver cytosolic proteins, permitting more

accurate estimates of the pattern and extent of arsenic transformation in vitro and in

vivo (Styblo et al., 1996b). In mammalian cells, arsenite-specific binding sites on

cytosolic proteins were determined to be 67% of the total (specific and non-specific)

number of possible binding sites. Rabbit liver cytosolic proteins bind AsIII similarly in

vitro and in vivo (Bogdan et al., 1994). This protein binding may decrease the in situ

toxicity of inorganic arsenic by decreasing its metabolic availability until enzymatic

methylation occurs. The binding of arsenite to tissue protein has been proposed as an

additional or perhaps the first step in the detoxification of inorganic arsenic prior to

H3AsVO4 + 2 GSH H3AsIIIO3 + H2O + GS-SG

H3AsIIIO3 + 3 GSH AsIII(SG)3 + 3 H2O

(1)

(2)


38

methylation (Vahter et al., 1982). Major supporting evidence is that animals deficient in

methylatransferases do not excrete methylated arsenical metabolites in the urine after

ingesting arsenite or arsenate. In those animals, the binding of arsenite to protein is an

important detoxification pathway for inorganic arsenic (Vahter and Marafante, 1985).

There have been some other examples of proteins binding or sequestering heavy metals

(such as lead) with a resultant decrease in toxicity (Fowler and DuVal, 1991).

The extent to which methylated arsenicals are bound by protein in vivo is under dispute.

Zhang et al. (1998) found that only inorganic arsenic species are bound to serum

proteins, while Styblo et al. (1995b) found that both inorganic and methylated

arsenicals were bound to proteins in rat liver cytosol and in mouse liver and kidney.

Absorbed arsenate is rapidly reduced in the blood to arsenite, which implies increased

toxicity. The intermediate methylated arsenic metabolite, MMAIII, is highly toxic. The

authors presented evidence of specificity for the binding of MMA and DMA to

cytosolic proteins, and showed that the protein-bound MMAIII was a substrate for the

dimethylation reaction. Trivalent methylated arsenicals have been demonstrated to be

more avidly bound to cytosolic protein than the pentavalents (Styblo and Thomas,

1995).

Arsenite is the main form of arsenic bound to tissues (Vahter and Marafante, 1983).

Experimental data suggested that arsenic binding occurred mainly at dithiol or vicinal

thiol groups on proteins and not at single thiol groups. Interestingly, although

glutathione might be expected to compete with the cysteinyl groups of proteins for

arsenite, studies of Vahter et al. (1982) indicated that despite high endogenous

glutathione levels in the cytosol, the binding of arsenite to specific cytosolic proteins

still occurred. Thus, another functional group besides cysteine may be involved in the

cytosolic proteins that preferably bind arsenite (Bogdan et al., 1994).


39

1.3.2.3. Roles of peptides in chelation of arsenic in plants (Phytochelation)

Phytochelatins (PCs) are heavy-metal-binding peptides derived from glutathione, with

general structure (γ-glutamate-cysteine)n-glycine (n=2-11). PCs are an elaboration of

the tripeptide glutathione, for which n=1. PC biosynthesis involves the enzyme PC

synthase which transpeptidates of the γ-glutamyl-cysteinyl dipeptides from GSH onto a

second GSH to form PC (n=2), or onto an existing PC molecule to produce an oligomer

(n>2) (Grill, et al., 1989). The gene encoding PC synthase has been identified in

Arabidopsis, Triticum aestivum, and Schizosaccharomyces pombe (Clemens et al.,

1999; Ha et al., 1999).

Since the immobilized metals are less toxic than the free ions, PCs has been considered

as a part of the detoxifying mechanism of plants (Grill et al., 1985), algae (Gekeler et

al., 1988) and some fungi (Kondo et al., 1985). PC production was involved in

detoxification process of Cd (De Knecht et al., 1992) and Cu (Schat and Kalff, 1992)

but not responsible for metal tolerance. However, later studies of arsenate tolerance

illustrated significant differences from those of other metals (Meharg, 1994). A role for

PCs in the detoxification of arsenate was first suggested by the induction of PC

formation by arsenate (Grill et al., 1987), and supported by evidence of As-SH

complexes found both in vivo and in vitro (Scott et al., 1993). It has also been shown

that Arabidopsis and Schizosaccharomyces pombe mutants lacking the ability to

synthesize PCs were much more sensitive to arsenate than the wild types (Ha et al.,

1999).

Chelation by PCs provides a mechanism to autoregulate the PC biosynthesis. This PC

biosynthesis was found to terminate if the activating metal ions were chelated either by

the PCs formed or by addition of another metal chelator, such as EDTA (Loeffler et al.,

1989). After the PC biosynthesis was terminated, degradation of PCs became apparent

(De Knecht et al., 1995). The high PC concentrations in plants chronically exposed to

arsenate (compared to those in the short-term exposure) suggest that the breakdown of

arsenic-induced PCs is very slow. This was confirmed by the degradation experiment.


40

As the result, the continuous accumulation of PCs occurred throughout longer periods

of arsenic exposure (Sneller et al., 1999).

Arsenic not only induces phytochelatin synthesis, it also stimulates the production of

the PC precursor GSH (Li and Chou, 1992). It has been suggest that phytochelatin

production could itself cause oxidative stress due to GSH depletion (De Vos et al.,

1992). Upon exposure to arsenate, the pool of GSH would be rapidly depleted as result

of phytochelatin production. In non-tolerant plants, rapid arsenate influx to plant cells

resulted in phytochelatin production, glutathione depletion and lipid peroxidation. This

process would also occur in tolerant plants. However, in arsenate-tolerant Holcus

lanatus L., significantly higher concentrations of phytochelatin were accumulated

compared with non-tolerant plants at equivalent levels of stress. This accumulation

resulted from decreased rates of arsenate uptake in arsenate-tolerant plants by adaptive

suppression of the high-affinity phosphate uptake system (Hartley-Whitaker, et al.,

2001a). The adaptive suppression reduced their burden of arsenic toxicity, allowing

maintenance of their constitutive functions, detoxifying arsenate through phytochelatin

and cellular processes to continue without inhibition (Hartley-Whitaker et al., 2001b).

The production of arsenic-phytochelatin complexes and detoxification of arsenic by the

induced phytochelatins have been unequivocally demonstrated through purification of

phytochelatins by electrospray ionization mass spectroscopy (ESI-MS) (Schmöger et

al., 2000) and x-ray absorption spectroscopy (Pickering, et al., 2000). The identity of

the arsenic-induced phytochelatin showed that an approximately 3:1 ratio of the

sulfhydryl groups from phytochelatins to arsenic is compatible with reported

arsenic-glutathione complexes. The biochemical fate of arsenic taken up by Indian

mustard (Brassica juncea) was investigated by Pickering et al. (2000). Arsenate was

taken up by roots possibly via the phosphate transport mechanism. A small fraction of

arsenate was transported to the shoot via the xylem as the oxyanions arsenate and

arsenite, and then stored as an arsenite-tris-thiolate complex. The majority of the

arsenic remained in the roots as an arsenite-tris-thiolate complex, which is


41

indistinguishable from that found in the shoots and from arsenite-tris-glutathione. The

thiolate donors are thus probably either glutathione or phytochelatins (Pickering et al.,

2000).

Phytochelatins have been identified in plants, algae and some microorganisms,

including Schizosaccharomyces pombe, and may also be expressed in some animal

species since a similar gene was identified in the nematode, Caenorhabditis elegans

(Ha et al., 1999). Cellular response and resistance mechanisms to arsenic trioxide

(As2O3), arsenite (NaAsO2) and arsenate (Na2HAsO4) in porcine endothelial cells were

studied recently. Responding to the oxidative stress by trivalent arsenic compounds, the

GST activity in porcine endothelial cells increased, and thus the intracellular GSH level

was elevated. The results implied that these GSHs might be used to conjugate arsenic in

porcine endothelial cells and facilitate arsenic efflux (Yeh et al., 2002).


42

1.3.3. Arsenic metabolism and organo-arsenical synthesis

1.3.3.1. Biomethylation

Biomethylation of arsenic is very widespread, occurring not only in microorganisms

(bacteria and fungi) but also in algae, higher plants, animals and humans, forming both

volatile (e.g., methylarsines) and nonvolatile (e.g., MMA and DMA) compounds,

involving the oxidation-reduction cycling between trivalent and pentavalent states.

Elucidation of arsenite biotransformation might enable a better understanding,

prevention, and treatment of chronic arsenic poisoning, a major environmental health

problem.

There is a significant difference in the level and chemical forms of arsenic in terrestrial

and marine organisms. In land creatures, the arsenic level is typically about 1µg As/g

(dry weight) or less, whereas for marine organisms the value ranged from several µg

As/g to more than 100 µg As/g (Lunde, 1977; Andreae and Froelich, 1984).

The separation and identification of volatile arsines in gases is relatively

straightforward, but the identification of the nonvolatile compounds is much more

difficult. However, the nonvolatile arsenical metabolites may be converted to volatile

arsines by hydride generation, reacting with a reducing reagent such as sodium

borohydride. At pH below 4, arsenite is reduced to arsine, AsH3. Other

oxyacids-methylarsonate, dimethylarsinate and trimethylarsine are oxidized and yield

monomethylarsine, dimetylarsine and trimethylarsine respectively (Cullen et al., 1994b;

1995). Thus, nonvolatile arsenicals can be partially identified as mono-, di-or trimethyl

species and detected after they are converted to corresponding arsines. Hydride

generation can be coupled with separation instruments such as gas

chromatography-mass spectrometer (MS) then using atomic absorption spectrometric

(AAS) detection that can reach very low detection limit, parts per billion (Andrewes et

al., 2001). High-pressure liquid chromatography-inductively coupled plasma- MS

(HPLC-ICP-MS) is probably the most powerful separation tool for analysis of arsenical


43

compounds (Granchinho et al., 2001). Non-volatile arsenic species can also be

analyzed by thin-layer chromatography (Styblo et al., 1995a) or ion-exchanged

chromatography (Zakharyan et al., 1995a).

1.3.3.1.1. Methylation of arsenic by bacteria

It was not until 1971 that arsenic methylation by bacteria was demonstrated. The initial

observation of alkylararsine synthesis was made in Methanobacterium bryantii. The

reaction mixture containing cell extracts and whole cells of M. bryantii, hydrogen, ATP,

arsenate and methylcobalamin, reduced and methylated arsenate to dimethylarsine

under anaerobic condition. ATP and hydrogen were found to be essential.

Methylcobalamin, but not the usual donor, S-adenosyl-methionine (AdoMet), was the

methyl donor of choice (McBride and Wolfe, 1971). It is believed that the

biomethylation of arsenic by methylcobalamin occurs in anaerobic organisms (Ridley

et al., 1977).

Arsenic metabolism was studied in five bacteria (Proteus sp., Escherichia coli,

Flavobacterium sp., Corynebacterium sp. and Pseudomonas sp.) grown in the presence

of sodium arsenate. All five bacteria transformed arsenate to arsenite and produced

dimethylarsine. The Pseudomonas sp. formed all three of the methylated arsines

(Shariatpanahi et al., 1981). Following a 48-hour exposure to 10 and 100 mg/L of

arsenate, all five species showed an average accumulation of 3.0 and 25 µg/g total

arsenic, respectively. Most of the arsenic accumulated by the cells was found in a

protein fraction (Shariatpanahi et al., 1982). The bacteria Flavobacterium sp.,

Pseudomonas sp. and other five species (Achromobacter sp., Aeromonas sp.,

Alcaligenes sp., Enterobacter sp. and Nocardia sp.) were found to converted arsenate to

methylarsonate, and then produced mono- and dimethylarsine. Only Aeromonas sp. and

Nocardia sp. produced trimethylarsine from methylarsonate. Methylation and

demethylation occurred in the bacteria. Nocardia sp. was the only bacterium that

formed all metabolites of arsenate including three methylarsines from methylarsonate

as substrate (Shariatpanati et al., 1983; Table 1.2).


44

Three arsenic-tolerant bacteria (Klebsiella oxytoca, Xanthomonas sp. and

Pseudomonas putida), isolated from a contaminated culture of the algae Chlorella sp.

(Maeda et al., 1990), could tolerate arsenic as high as 1000 mg arsenate/L. Most of the

arsenic in the cells, and that excreted, was inorganic. The cells also contained

nonvolatile trimethylarsenic species and excreted mono-, di- and trimethylarsenic

compounds (MMA, DMA and TMA). The bacteria bioaccumulated methylated

arsenicals, and demethylated these species as well (Maeda et al., 1992).

Table 1.2. Metabolites of arsenate in bacteria (Shariatpanahi et al., 1981; 1982; 1983)

Bacterium Species

Metabolites of Arsenate arsenite Mono-

methyl- arsine

Mono- methylarsonate

Di- methyl- arsine

Di- methylarsinate

Tri- methyl- arsine

Tri- methyl-

arsine oxide Corynebacterium sp. √ √ √ Escherichia coli √ √ √ Flavobacterium sp. √ √ √ Proteus sp. √ √ √ Pseudomonas sp. √ √ √ √ √ √ Achromobacter sp. √ √ √ √ √ Aeromonas sp. √ √ √ √ √ √ Alcaligenes sp. √ √ √ √ √ Enterobacter sp. √ √ √ √ √ Nocardia sp. √ √ √ √ √ √ √

Kuroda et al. (2001) studied E. coli isolated from rats which had been fed

dimethylarsinate for 6 months. These isolated E.coli strains converted dimethylarsinate

into two nonvolatile, unidentified compounds, and converted trimentylarsine oxide into

a further, nonvolatile, unidentified compound. The results also showed that cysteine but

not GSH was required for dimethylarsinate metabolism. It strongly suggested that the

reductant required for arsenic metabolism in these intestinal bacteria may differ from

that for mammals, and that the bacteria may play a possible role in mammalian arsenic

metabolism. More studies of arsenic metabolism in E. coli are required to understand

the fate of arsenic in mammals.


45

1.3.3.1.2. Methylation of arsenic by fungi

Based on studies with trimethylarsine gas, 1945, Challenger and his associates

suggested a mechanism for arsenic methylation (Challenger, 1945). Following later

research, S-adenosyl-methionine (AdoMet) was demonstrated the methyl donor

(Challenger, 1954), and confirmed by Cullen et al. (1977). A modified version (Figure

1.7) of the Challenger mechanism was proposed by Cullen and associates for the

conversion of arsenate to trimethylarsine (Cullen et al., 1984).

Figure 1.7. Challenger mechanism for arsenic methylation (a modified version, Cullen et al., 1984). The top line shows the pentavalent AsV intermediates. The bottom line shows the trivalent AsIII intermediates. R: indicates the reduction reactions; M: indicates the methylation steps by AdoMet.

Cullen et al. studied arsenic metabolism in cell extracts of the fungus Candida

humicola (later reclassified as Cryptococcus humiculus). They found C. humiculus

converted 74As-arsenate to arsenite, methylarsonate, dimethylarsinate and

trimethylarsine oxide. In addition, products from 14C-DMA including 14C-MMA and 14C-TMA indicated that demethylation also occurred in the fungus (Cullen et al., 1979).

In later experiments, the nonvolatile arsenicals of C. humiculus and Scopulariopsis

brevicaulis were identified in their culture media. With methylaronate as substrate, both

C. humiculus and S. brevicaulis produced lower levels of dimethylarsinate and

trimethylarsine oxide, with slow increases over 4 weeks. The arsenic metabolism of S.

brevicaulis was much slower than C. humiculus (Cullen et al., 1994a). This is the first

report of trimethylarsine oxide as an end product metabolite for the two fungi.

Apparently these two organisms tolerated low levels of trimethylarsine oxide, and

further conversion to volatile trimethylarsine for transport out of cells might not be

necessary. The reaction rates suggested a possible sequential transfer of two methyl

Arsenate Methylarsonate Dimethylarsinate Trimethylarsine oxide

Arsenite Methylarsonite Dimethylarsinite Trimethylarsine

R R R R M M M

AsV

AsIII


46

groups from AdoMet to methylarsonate and forming directly trimethylarsine oxide

(TMAO). The methylation of arsenic by microorganisms is found more complex than

the initial Challenger mechanism. Hence, an extended model for arsenic methylation

involving both cell and medium was proposed by Cullen et al. (1994a; Figure 1.8).

Figure 1.8. Expended version of the Chanllenger mechanism for arsenic methylation (Cullen et al., 1994a). This version, proposed for C. humiculus, indicates roles for components both inside of the cells and in the culture medium. (A) Phosphate transport system; (B) thiols and/or dithiols; (C) active transport system; (D) active/passive transport; (E) passive diffusion.

MEDIUM

Arsenate

Arsenite

Arsenate

TMAO TMAO

DMA

CELLS

2CH3CH3

DMAMMAArsenite

A B C

D E

E

Arsenite Arsenite

2e, CH3- 2e, CH3

-

4e- 2e-


47

1.3.3.1.3. Methylation of arsenic by freshwater algae

The study with alga Chlorella vulgaris revealed that C. vulgaris takes up not only

inorganic arsenic compounds but also methylated arsenic compounds. The

methylarsenic compounds taken up are further biomethylated but not demethylated.

The dimethylarsenic compound seems to be the most stable arsenical form in the algal

cells (Maeda et al., 1992). However, in some other freshwater algae, biotransformation

of arsenite to MMA, DMA and TMAO was found, but no volatile arsine or

methylarsines were detected (Baker et al., 1983b).

1.3.3.1.4. Methylation of arsenic by mammals

Examination of arsenic compounds in the urine after administration of arsenate or

arsenite to animals or humans has indicated that these arsenic species are

biotransformed via methylation in most mammals. In urine and faeces of animals and

humans exposed to inorganic arsenic, dimethylarsinate (DMA) is the predominant

metabolite. Smaller amounts of methylarsonate (MMA), arsenate and arsenite are

excreted in urine (Hughes et al., 1994). In hamsters, a trimethylated arsenic species is

also excreted in urine after inorganic arsenic treatment (Yamauchi and Yamamura,

1985).

Arsenic methylation in mammals follows the Challenger mechanism (Figure 1.7). The

availability of methyl group donors affects the capacity for arsenic methylation in vivo

(Marafante and Vahter, 1984). AdoMet was the likely donor of methyl group for the

oxidative methylation of arsenic. In vitro, methylarsonate production from arsenite by

cell extracts was not dependent on addition of exogenous AdoMet to assay system.

However, addition of 0.1 mM AdoMet maximized dimethylarsinate production (Styblo

et al., 1996a).

GSH-dependent reduction of arsenicals from the pentavalent to trivalent states has been

demonstrated in aqueous solution and in intact rabbit erythrocytes (Scott et al., 1993).

Because both inorganic and organic AsIII species are biologically active by forming


48

complex with GSH (Delnomdedieu et al., 1993; Styblo and Thomas, 1995), omission

of glutathione from the assay system nearly abolished the methylation of arsenite.

Addition of exogenous glutathione to the assay system (up to 20 mM) decreased protein

binding of arsenic and increased the production of methylarsonate and dimethylarsinate

(Styblo et al., 1996a).

Despite the ubiquity of arsenic methylation, some animals are now known not to

biomethylate arsenic. These include several monkeys (marmoset, squirrel and

tamarind), chimpanzees and guinea pigs. Vahter et al. observed that the marmoset did

not methylated arsenite (Vahter et al., 1982). Later research work showed that the rat,

hamster, rabbit, pigeon and many other Old World animals had arsenite and MMA

methyltransferase activities in their livers, whereas New World animals such as the

marmoset, tamarin, squirrel monkey and guinea pig were deficient in these enzymes.

The chimpanzee is the only Old World animal found so far which does not appear to

methylate inorganic arsenic (Vahter et al., 1995). This biochemical peculiarity of

lacking of arsenic methyltransferases might have been evolutionarily beneficial to

animals native to certain regions, such as South America, where Chagas’s disease

(trypanosomal disease, caused by Trypanosoma cruzi) was endemic. Because the levels

of arsenite in the blood and liver were high enough to kill T. cruzi, those animals

deficient in or totally lacking arsenite methyltransferase could accumulate adequate

arsenite and thus selectively survived (Aposhian, 1997).

A number of studies have confirmed that methyl derivatives of arsenic appear to be less

toxic than the parent compound (Squibb and Fowler, 1983) (Section 1.2.5). In addition,

since methylation tends to reduce the retention of inorganic arsenic in tissue and thus

increases excretion rates, the methylation process can be considered to be a

“detoxification” mechanism (Gebel, 2002).

1.3.3.1.5. Methylation of arsenic by terrestrial plants

Arsenic is available to plants mainly in the form of arsenate. Arsenate is taken up via


49

the phosphate transport mechanism (Asher and Ready, 1979) and is readily reduced in

the plant to arsenite. Only under conditions of phosphate deficiency was the arsenite

methylated, with most methylation activity apparent in the leaves (Nissen and Benson,

1982). In plants, while dimethylarsinic acid (cacodylic acid, DMA) predominated in

the leaves (Marin et al., 1993), the roots accumulated relatively more of the singly

methylated methanearsonic acid (Nissen and Benson, 1982). Although some arsenic

hyper-tolerance and hyper-accumulation plants, including Highland grass (Agrostis

tenuis S.) (Porter and Peterson, 1975) and Ladder brake fern (Pteris vittata L.) (Ma et al.

2001) have been found, little information about arsenic methylation in terrestrial plants

has been reported.

1.3.3.2. Organo-arsenical synthesis in marine organisms

A large part of the arsenic in marine organism is in organic forms whose chemical

structures in many cases have been found, whereas very few chemical species of

arsenic in fresh water organisms have been found. With the intermediates being isolated

and identified, the proposed pathways of biosynthesis of the organo-arsenical

compounds in marine system are being confirmed.

In seawater, arsenic is in predominantly inorganic forms of arsenate and arsenite.

Marine algae are exposed to arsenate at about 2µg As/L (Johnson and Pilson, 1972).

Following its transport into the algal cells, arsenate is methylated and further converted

to organoarsenic compounds. Most of the arsenic in algae is bound to carbohydrate

molecules (ribosides). These organoarsenic compounds are referred to collectively as

arsenosugars (Figure 1.1). Arsenosugars are also known as dimethylarsinyl-ribosides

(trialkylarsinyl compounds, R3As--O). Among over 25 arsenic species occurring in

marine systems, a total of 15 arsenosugars have been reported as naturally-occurring

constituents of marine algae (Francesconi and Edmonds, 1998). The formation of

arsenosugars has been proposed, involving methylation and alkylation of trivalent

arsenic compounds by AdoMet, following the pathway initial proposed by Challenger

for conversion of arsenate to trimethylarsine (Figure 1.8). Neither arsenobetaine, nor


50

arsenocholine, nor tetramethylarsonium ion has been detected in algae or seawater.

Although the arsenosugars in algae are thought to be detoxification end products, they

also been proposed as probable precursors of other arsenic compounds found in marine

animals (Francesconi and Edmonds, 1994). It is possibly because these compounds can

participate in cellular redox reactions, being readily reduced to their respective arsines.

In contrast, only traces of trimethylarsonio-ribosides (tetraalkylarsonio compounds) are

present in algae because they are fully substituted and chemically unreactive

(Francesconi and Edmonds, 1994). However, this hypothesis awaits confirmation.

After isolation from the western rock lobster (Panulirus cygnus) and identification by

x-ray crystallographic analysis (Edmonds and Francesconi, 1977), arsenobetaine was

revealed to be the major arsenic compound in a large range of marine animals

(Francesconi and Edmonds, 1993). There is no evidence that aquatic animals are able to

synthesize arsenobetaine de novo from arsenate. All evidence suggests that marine

animals accumulate arsenobetaine primarily from their food. The source of

arsenobetaine in animals is likely to be arsenic-containing ribosides in algae. However,

arsenic-containing ribosides appear unable to be converted to arsenobetaine directly by

animals. This conversion is possibly mediated by microbial metabolism through an

intermediate stage (Francesconi and Edmonds, 1994). Arsenocholine is likely to be the

immediate precursor to arsenobetaine in marine animals, and readily transformed to

arsenobetaine. Arsenocholine has been identified in dogfish muscle using

HPLC-ICP-MS (Beauchemin et al., 1988). Its chromatographic properties are similar

to tetramethylarsonium ion, which may lead to misidentification (Shibata and Morita,

1989). Arsenobetaine was reported to break down to trimethylarsine oxide microbially

(Hanaoka et al., 1992). This can explain why the levels of trimethylarsine oxide are

higher in frozen fish than in fresh fish (Norin et al., 1995).

Toxicological studies confirmed that arsenobetaine is nontoxic in organisms (Sabbioni

et al., 1991). Francesconi and Edmonds’ data (1998) indicated that arsenobetaine is

highly bioavailable to marine animals. The concentrations (mg kg-1 wet wt.) of arsenic


51

accumulated in mussels (Mytilus edulis) after exposure to various arsenic compounds

exhibited the trend as follows: arsenobetaine >> arsenocholine >> tetramethylarsonium

ion >> arsenite > dimethylsinate = methylarsonate > trimethylarsine oxide > arsenate. It

appears that organo-arsenicals are much less toxic than the inorganic parent. Uptake

and accumulation of arsenic by marine animals are highly dependent on the form of

arsenic.


52

1.4. Enzymology of Arsenic Biomethylation

The enzymology of arsenic biomethylation is complicated by the several oxidation

states for arsenic and the fact that only small amounts of the arsenic compounds are

present in biological specimens. As proposed by Challenger (1945), arsenic

methylation occurs via alternating reduction of pentavalent arsenic to trivalent and

addition of a methyl group. Although the complete sequence of events has not been

elucidated, the chemical intermediates and reactions in the metabolism of arsenate are

assumed to be generally the same in microorganisms and animals. In animals the major

urinary metabolite is dimethylarsinate, of which only a very small amount is further

methylated. Except for arsenate reduction, most reactions of the arsenic methylation

pathway are enzymatic in the mammalian system. However, in bacteria and yeast, the

reactions tend to proceed to methylarsines or trimethylarsine oxide.

1.4.1. Enzymes catalyzing reduction of arsenic compounds

For all arsenic metabolic pathways in living organisms, pentavalent arsenicals must be

reduced to trivalent before methylation (Marafante et al., 1985a). Trivalent arsenicals

are preferred substrates for methylation reactions and the reduction of arsenic from

pentavalent to trivalent states may be a critical step in the control of the rate of

metabolism of arsenic (Styblo et al., 1995b). Experimental studies have shown that a

major part of the absorbed arsenate is rapidly reduced to arsenite, probably mainly in

the blood (Vahter and Marafante, 1985; Marafante et al., 1985b). Hepatocytes take up

arsenite from the blood plasma much more readily than arsenate. At physiological pH,

arsenite is present mainly in undissociated form (pKa 9.23), which facilitates passage

of cellular membranes, while arsenate is in ionized form (Lerman et al., 1983). Because

arsenite is more toxic but more active than arsenate, this initial step in the

biotransformation of arsenate may be regarded as a bioactivation. Much of the formed

arsenite is distributed to the tissues where it is methylated to MMA and DMA

(Marafante et al., 1985b).


53

1.4.1.1. Arsenate reductase

GSH can reduce arsenate to arsenite nonenzymatically in vitro (Scott et al., 1993;

Delnomdedieu et al., 1994a). However, the genes for arsenate reductase are present in

the ars operon in bacteria and the ACR family in yeast, and confer resistance to arsenic

in microorganisms (Section 1.3.3.1.). Arsenate reductases from E. coli and S. aureus

utilize glutaredoxin and thioredoxin respectively as reductants rather than GSH which

is usually postulated to play a role in biomethylation. arsC encodes arsenate reductase

that reduces arsenate in cells to arsenite, which is then transported from the cells via an

ATP-dependent pump (ArsA-ArsB). The ATPase activity of the ArsA protein is

activated by arsenite but not by arsenate (Silver et al., 1993), hence arsenate reduction

is the initial step. Since arsenite is removed from the cells, arsenate reduction is part of

a detoxification mechanism in bacterial systems, as in yeast. In the yeast S. cerevisiae,

the ACR2 gene encodes an arsenate reductase.

The secondary structure of E. coli arsenate reductase was determined by use of

multidimensional, multinuclear magnetic resonance (Stevens et al., 1999). It is a α/β

protein, containing large regions of extensive mobility at the active site. (Other

information about the arsenate reductases has been described in 1.3.1.1.)

An arsenate reductase has been partially purified from human liver by DEAE-Sephacel

and Sephacryl-200 chromatography, with specific activity of 0.9 unit/mg protein

(Radabaugh and Aposhian, 2000). Its molecular mass is 71.8kD. The arsenate

reductase required a thiol and a heat stable cofactor for activity. The thiol requirement

can be satisfied by dithiols such as dithiothreitol (DTT) but not by glutathione,

thioredoxin or reduced lipoic acid under the experiment conditions. The nature of the

heat stable cofactor is not yet established. This cofactor is less than 3kD, but is not an

EDTA chelatable divalent cation (Zn2+, Cu2+, Ca2+, Mn2+ or Mg2+). The reductase is

specific for arsenate of which the concentration necessary to saturate the active site of

the enzyme appears to be about 300 µM. The arsenate reductase does not reduce


54

monomethylarsonic acid. The isolation and characterization of this arsenate reductase

demonstrated that the reduction of arsenate to arsenite is enzymatically catalyzed in

human cells, as arsenate reduction in microorganisms. Non enzymatic chemical

reduction of arsenate by glutathione apparently plays a minor role.

1.4.1.2. Methylarsonate reductase

The first product of arsenite methylation is the pentavalent MMA, which has to be

reduced again for further methylation. MMAV reductase activity has been detected in

rabbit liver (Zakharyan and Aposhian, 1999) and male hamster tissues

(Sampayo-Reyes et al., 2000). Zakharyan and Aposhian (1999) reported that a MMAV

reductase has been partially purified from rabbit liver by using DEAE-cellulose,

carboxymethlcellulose, and red dye ligand chromatography. The Km and Vmax values

of the MMAV reductase were 2.16 x 10-3 M and 10.3 µmol h-1 mg-1 protein. The enzyme

appeared to be saturated with MMAV at approximately 15 mM. DMAV and arsenate

could also be substrates for MMAV reductase and be reduced to trivalent species but

with much higher Km and lower Vmax (Zakharyan and Aposhian, 1999).

The Km values of rabbit liver arsenite methyltransferase for arsenite and MMAIII

methyltransferase for MMAIII are 5.5 x 10-6 M and 9.2 x 10-6 M, respectively

(Zakharyan et al., 1999). In comparison, the Km value of this MMAV reductase

(2.16x10-3 M) is much higher, suggesting that the MMAV reductase is the rate-limiting

enzyme of inorganic arsenite biotransformation pathway.

MMA reductase has an absolute requirement for GSH. Neither DTT nor L-cysteine

alone satisfied this requirement, but in the presence of 5mM GSH, DTT exhibited

stimulatory activity. At pH below the physiological pH (pH ≤ 6.0), GSH carried out this

reduction nonenzymatically, but this GSH reduction in the absence of the enzyme was

at a much slower rate at pH 7.5. Above pH 7.5, the rate of reduction without this

enzyme rapidly fell (Zakharyan and Aposhian, 1999).


55

1.4.2. Enzymes catalyzing methyl group transfers to arsenic compounds

The methyltransferase activities involved in arsenic biomethylation from arsenate to

dimethylarsinate have been mainly investigated in mammals. There are no reports of

characterization of specific arsenic methyltransferases from bacteria, fungi or plants.

1.4.2.1. Arsenite methyltransferase

Arsenite methyltransferase has been studied in the liver, kidney, testis and lung from

mice (Healy, et al., 1998) and rat (Styblo et al., 1996a); partially purified from and

characterized in the rabbit (Zakharyan et al., 1995a) and rhesus monkey liver

(Zakharyan et al., 1995b). Rat red blood cells have a very high affinity for DMA that

retards the excretion of this arsenic metabolite and makes the rat a questionable model

for arsenic toxicity in humans (Aposhian, 1997).

Early studies with purified arsenite methyltransferases (by gel filtration and

DEAE-cellulose chromatography) outlined many features. The methylation of arsenite

by rabbit liver arsenite methyltransferase required AdoMet and was sharply pH

dependent (Zakharyan et al., 1995b). Studies on mice and rabbits have shown that

chemical inhibition of the AdoMet-dependent methylation by periodate-oxidized

adenosine results in a marked decrease in the methylation of arsenic (Marafante et al.,

1985). Later, in vitro incubations of rat liver preparations with trivalent arsenic have

confirmed that AdoMet is the main methyl donor in arsenic methylation (Styblo et al.,

1995b). The optimal pH for the arsenite methyltransferase is pH 8.2 (from rabbit liver)

and pH 7.6 (from hamster liver) (Wildfang et al., 1998). The enzyme appears to be very

specific for arsenite and the substrate saturation concentration is 50 µM of arsenite

(Zakharyan et al., 1995b). The Km of arsenite methyltransferase from rabbit liver is 5.5

x 10-6 M, 3.2 x 10-6 M from Chang human Hepatocytes and 1.79 x 10-6 M from hamster

liver (Wildfang, et al., 1998). MMAIII is a noncompetitive inhibitor of arsenite

methyltransferase (Zakharyan, et al., 1999). There is not an absolute requirement for

GSH, since DTT, cysteine and β-mercaptoethanol are effective in place of GSH.


56

L-cysteine is more active than GSH as substrate for the arsenite methyltransferase in

vitro, while the reverse is true for the MMA methyltransferase (Zakharyan et al.,

1995b). Two arsenic methyltransferases have been molecularly cloned which will be

described in section 1.4.2.3.

1.4.2.2. Methylarsonite methyltransferase

Methylarsonite (MMAIII) methyltransferase was partially purified from rabbit, Chang

human hepatocytes (Zakharyan et al., 1999) and hamster liver (Wildfang et al., 1998),

copurifying arsenite methyltransferase activity.

The methylation by MMAIII methyltransferase also required AdoMet. Its optimal pH is

8.0. Arsenite is a noncompetitive inhibitor of MMAIII methyltransferase (Zakharyan et

al., 1999) that is specific for MMAIII. The substrate saturation concentration is 1,000

µM of MMAIII. The Km for MMAIII of MMAIII methyltransferase from rabbit liver is

9.2x10-6 M, 3.0x10-6 M from Chang human Hepatocytes (Zakharyan et al., 1995b) and

7.98 x10-4 M from hamster liver (Wildfang et al., 1998). In rabbit and hamster liver, the

values of Km of MMAIII methyltransferase for MMAIII are larger than that of arsenite

methyltransferase for arsenite. It suggests that MMA may be produced at a higher rate

than it can be subsequently methylated to DMA, thereby allowing MMA to accumulate

and be excreted in these two species. Considering the very high affinity for DMA of rat

red cells, which thus retards the excretion of DMA and results in higher toxicity,

accumulation and excretion of MMA may decrease arsenic toxicity.

In addition, while GSH is more active than L-cysteine, a dithiol (reduced lipoic acid or

dithiothreitol) appears to be more active than GSH in satisfying the thiol requirement of

MMAIII methyltransferase (reduced lipoic acid > DTT > GSH > L-cysteine) (Wildfang

et al., 1998).

There is no evidence to suggest that the arsenite methyltransferase and MMAIII

methyltransferase are two different proteins. Even if the two activities are on one


57

protein, the differing substrates and kinetic properties of MMAIII methyltransferase and

arsenite methyltransferase suggest that there are two different activity sites on the same

protein responsible for the two methyltransferase activities.

There is no observation of the induction of arsenite or MMAIII methyltransferase

activities in mammals under the experimental conditions (Healy et al., 1998).

Subchronic exposure to arsenate in drinking water (2.5 ppm) did not increase the

amount of methylated species in the urine of mice (Ughes and Thompson, 1996).

1.4.2.3. S-adenosyl-methionine:arsenic(III) methyltransferase

After the information about arsenic methyltransferases obtained from rabbit liver,

hamster liver and Chang human hepatocytes, a novel S-adenosyl-

methionine:arsenic(III) methyltransferase recently was purified from rat liver cytosol

and the gene sequence for this enzyme was published (Lin, et al., 2002). This arsenite

methyltransferase is a 41KD protein with 369 amino acids which was purified by a

combination of acid treatment of cytosol, chromatofocusing, and affinity

chromatography on an S-adenosyl-L-homocysteine-Sepharose gel column (Lin et al.,

2002). This methyltransferase exhibits both arsenite and MMAIII methyltransferase

activities. The Km of its MMAIII methyltransferase is 2.5 x 10-7 M, indicating higher

affinity of the enzyme for MMAIII than MMAIII methyltransferase purified from rabbit

and hamster liver cytosol. Concentrations of MMAIII greater than 5 µM inhibited the

production of dimethylated arsenicals. The activity of the purified

S-adenosyl-methionine:arsenic(III) methyltransferase was dependent on the

concentration of AdoMet in the reaction mixture. In absence of AdoMet, methylated

arsenicals were not detected in reaction mixtures. Maximal conversion of arsenic to

methylated products occurred at 50 µM AdoMet and the yield fell at higher AdoMet

concentrations. The arsenic methylating activity of the enzyme is also pH-dependent.

Substantial activity was found between pH 7.5 and 10 with peak activity at pH 9.5,

different from the values reported before with the purified rabbit (pH 8.0-8.2) and

hamster liver cytosol (pH 7.6-8.0) enzymes.


58

The S-adenosyl-methionine:arsenic(III) methyltransferase mRNA was detected in

many rat tissues, including liver and kidney. This is consistent with the detection of

both mono-methylated and bimethylated arsenicals in livers and kidneys of

arsenite-treated rats (Kitchin et al., 1999). The amino acid sequence of

S-adenosyl-methionine:arsenic(III) methyltransferase contained several motifs that

have been found conserved in other methyltransferases (Kagan and Clarke, 1994). This

methyltransferase has motif I, post-motif I and motif II but not motif III. Alignment of

the amino acid sequences of S-adenosyl-methionine:arsenic(III) methyltransferase and

human Cyt19 suggested a close relationship between these two gene products.

Very recently, Wang et al. (2004) identified a gene arsM in a haloarchaeon

Halobacterium sp. strain NRC-1. The arsM gene is located less than 1 kb from the

arsADRC cluster, as an apparent operon with an upstream arsR homolog (arsR2). The

ArsM protein is a putative arsenite(III)-methyltransferase, with 60% amino acid

similarity to the S-adenosylmethionine-dependent methyltransferase in

Magnetospirillum magnetotacticum (ZP_00054798). The arsM△ knockout mutant

showed sensitivity to arsenite, suggesting that arsM is responsible for arsenite

methylation in the bacterium.


59

1.5. Strategies for Arsenic Bioremediation of Arsenic Pollution

1.5.1. Strategies for the removal of toxic metals by plants

Pollution of the biosphere with toxic metals has accelerated dramatically since the

beginning of the industrial revolution and is a major environmental problem. All plants

have the ability to accumulate heavy metals (Fe, Mn, Zn, Cu, Mg and Mo) essential for

their growth and development from soil and water. Some plants even have the ability to

accumulate heavy metals and metalloids (Cd, Cr, Pb, Co, Ag, Se, Hg, As and Sb) which

have no known biological function (Raskin et al., 1994). However, excessive

accumulation of these elements is toxic to most plants. Tolerant and hyperaccumulating

plants have been reported (Brooks et al., 1979; 1978; 1981; Reeves and Brooks, 1983;

Banuelos and Meeks, 1990). The basic idea that plants can be used for environmental

remediation has a long history. Three subsets of the technology for cleanup of

hazardous wastes are applicable to toxic metal remediation (Salt et al., 1995).

Phytoextraction Phytoextraction is the use of metal-accumulating plants to remove toxic metals from

soil. The optimum plants for the phytoextraction are those not only have ability to

tolerate and accumulate high levels of heavy metals in their harvestable parts but also

have a rapid growth rate and the potential to produce a high biomass in the field.

Rhizofiltration Rhizofiltration is the use of plant roots to remove toxic metals from polluted waters.

The ideal plant for rhizofiltration has the ability to remove toxic metals from solution

over extended periods of time by its rapidly growing roots. Rhizofiltration is effective

and economically compelling for low concentrations of contaminants in large volumes

of water, particularly effective for radionuclides when used in combination with a

microorganism-based bioremediation strategy. Mechanisms of toxic metal removal by

plant roots differ for various metals:

1. Surface sorption, a combination of physical and chemical processes such as chelation,


60

ion exchange and specific adsorption. It does not require biological activity and even

occurs with by dead roots.

2. Biological processes, including intracellular uptake, vacuolar deposition and

translocation to the shoots.

3. Root-mediated precipitation, involving a release of root exudates. Cell walls of roots

exposed to metals accumulate large amounts of insoluble metal complexes.

Phytostabilization Phytostabilization is the use of plants to decrease the bioavailability of toxic metals in

soils. Due to the toxic effects of heavy metals, polluted soils usually lack vegetation

cover, and are subject to erosion and leaching which spreads pollutants further in the

environment. Phytostabilizing plants must tolerate high levels of heavy metals and

immobilize them in the soil via root uptake, precipitation or reduction, and have low

shoot accumulation of heavy metals to avoid harvest and treatment of hazardous waste

in shoots.

1.5.2. Removal of arsenic from soils

1.5.2.1 Remediation of arsenic-contaminated soils

Current engineering approaches for arsenic-contaminated soils include soil leaching

and washing, physical stabilization, and chemical amendments. Generally, these

methods are expensive and disruptive to the environment.

Solidification of arsenic in soils

Solidification/stabilisation with cement and/or lime proved to be an efficient method to

lower the amount of arsenic leached from the soil to below the norm of 1mg L-1. The

results of a semi-dynamic leach test indicated that arsenic was released very slowly

from the solidified material. Over the period of the leach test only 0.10% of the arsenic

initially present was leached (Dutre et al., 1998).


61

Chemical amendments Arsenic-contaminated soils have been successfully treated using fixation methods

whereby chemicals are added to prevent arsenic mobilization. FeII treatment was used

for remediation of arsenic-contaminated soils by decreasing water-soluble arsenic

concentrations. Due to the property of the oxidation and subsequent precipitation of

ferrous ions (Section 1.2.1.4.), iron-oxidizing bacteria have been applied in the removal

of ferrous iron and arsenic from groundwaters. Iron oxides are efficient adsorbents in

arsenic removal processes, in which arsenate is more efficiently sorbed onto iron oxides

than arsenite (Katsoyiannis and Zouboulis, 2004). Moore et al. treated the

arsenic-contaminated soils with Fe:As molar ratio of 2 to lower the arsenic water

quality limit to <50 µg L-1. Sequential extraction of Fe-treated soils illustrated that

arsenic was relatively stable (Moore et al., 2000). Voigt et al. (1996) used a fixation

process by the addition of ferrous sulfate and water followed by Ca(OH)2, Portland

cement and water. The intent of the first step was to form insoluble Fe arsenate, the

second to bind the soil. The particle-size distribution of the untreated soil grains

containing arsenic was bimodal, with 70% of the arsenic occurring in two size fractions:

38% in the 0.25-0.5 mm fraction mostly as the mineral hoernesite [Mg-3(AsO4)2.

8H2O], and 32% in the size fraction less than 5 µm. From sequential extractions,

arsenic in the finest fraction is inferred to be present in an oxyanionic form adsorbed on

mineral surfaces. Arsenic fixation occurs through precipitation of an unknown As-Fe

phase or by incorporation during Fe oxide precipitation aided by immobilization by a

cement coating (Voigt et al., 1996). These methods comprise the cost competitive

technology, which can find application in treatment of groundwaters with elevated

concentrations of iron and arsenic.


62

1.5.2.2. Phytoremediation of arsenic

The phytoremediation most likely to successfully remediate arsenic-contaminated soils

is probably phytoextraction, which requires the plants used to be tolerant to arsenic and

have a high rate of biomass accumulation. The plant must accumulate a high

concentration of arsenic, produce sufficient biomass, and allow practical harvesting of

arsenic-rich biomass.

The availability and phytotoxicity of arsenic affect the concentration of this element

that can be tolerated and the rate of plant biomass production. Arsenic is a nonessential

element for plants. Because of its similarity to phosphate but lower reaction ability, at

high concentrations, arsenic interferes with plant metabolic processes and inhibits

growth, usually leading to death.

The arsenic chemical form present in the nutrient solution primarily determines its

phytoavailability (see Section 1.2.4.). The different location and phytotoxicity of

arsenite, arsenate, MMA and DMA, contributing to dry matter production should be

considered when plants are used for phytoremediation of arsenic contaminated soils.

The higher upward translocation in plants of MMA and DMA (Carbonell-Barrachina et

al., 1999a) suggests that arsenic methylation is probably advantageous to collect

arsenic in soils by harvest shoots of plants.

Studies of arsenic metabolism will provide more information for improving the ability

of plants to take up arsenic, translocate arsenic to the aboveground biomass and

produce more biomass, making it applicable and effective to phytoremediating

arsenic-contaminated soils.


63

1.6. Project Aims and Experimental Approach

The aim of this project was, firstly, to determine whether methylation of arsenic occurs

in the plant Agrostis tenuis, which is known to accumulate large amounts of arsenic

(Section 1.3.3.1.5). The metabolic pathway of arsenic methylation is well- established

in animal cells (Section 1.3.3.1.4) including recent information on the kinetics (Section

1.4) and gene coding for the enzymes involved (Section 1.4.2.3). There were no reports

of arsenic methylation in higher plants other than under conditions of severe nutrient

deficiency (Section 1.3.3.1.5).

After establishing the presence of arsenic biomethylation in A. tenuis, the second aim of

the project was to investigate arsenic biomethylation in yeast S. cerevisiae and E. coli,

two common microorganisms. Although methylated arsenicals were known to present

in yeast and E. coli, the biomethylation process had not been characterized (Section

1.4.2). It would be interesting to compare the arsenic biomethylation between different

living organisms.

Thirdly, based on the system established with yeast, screening of arsenic

methyltransferase genes using yeast mutants will be carried out, taking the advantage of

the available whole set genetic mutants resource. The expressed product of any

preliminarily identified gene in yeast S. cerevisiae will be tested and verified for

arsenite methylation activity in an in vitro assay system.

Chapter II. Materials and Methods

64

Chapter II

Materials and Methods


65

2.1. Materials 2.1.1. Plants and growth conditions

Seeds of Agrostis tenuis Sibth. (highland bentgrass) were provided by Wrightson Seeds

(Seven Hills, NSW, Australia – note that Agrostis tenuis Sibth. is synonymous with

Agrostis capillaris L.). Seeds were germinated in small pots containing liquid culture

medium, with nylon mesh covering the surface of the liquid. Seeds (6,000-8,000 per pot)

were distributed on the mesh and grown for 4 weeks under fluorescent light (140 µmol.

m-2. s-1) at 25oC. The culture medium contained macronutrients (2.5 mM KNO3, 1 mM

MgSO4, 1 mM KH2PO4, and 1.5 mM Ca(NO3)2) and micronutrients (25 μM Na2EDTA,

25 µM FeCl3, 88 µM H3BO3, 16 µM MnCl2, 2.5 µM ZnSO4 and 1.6 µM CuCl2), with

final pH ranging from 5.5-6.0. Sodium arsenate was added to the culture medium of

some pots 3 days before harvesting the leaves and roots. The remaining pots provided

arsenate-free control plants.

For investigation of the chronic effects of 50 ppm arsenate on the long-term growth of A.

tenuis, three pots (A, B and C) were prepared. Each pot contained 180 seeds of A. tenuis

grown on mesh covered by growth medium. Numbers of plants were counted from day

21 after seeding. Pot A was treated by 50 ppm of arsenate for 3days from day 28 to day

31. Pot B was treated by 50 ppm of arsenate from day 28 till day 64. Pot C, as control,

was not treated by arsenate during whole period.

2.1.2. Yeast and growth conditions

Wild-type yeast strain BY4741 (Y0000), two ACR knockout mutant strains (YPR201w

and YPR200c) and 13 putative methyltransferase knockout mutant strains were

obtained from EUROSCARF, Germany (Table 2.1). The strains were first grown at

30oC for 2 days or more if necessary on GYP solid medium, containing 5 g Yeast

Extract, 10 g Bacto Peptone, 20 g Glucose and 20 g agar per litre. For later assay, a

single colony was inoculated to 5 ml of liquid GYP medium and shaken (180rpm) at

30oC overnight. Three ml of this culture was transferred to 300 ml of liquid GYP


66

medium and growth continued until the culture reached an O.D.600 between 0.9 and 1.1.

Sodium arsenate was then added to the liquid culture medium, followed by a further 20

hours growth. An identical procedure was used for control cultures with the exception

that sodium arsenate was omitted.

Table 2.1. Genotypes of yeast strains

Strain Genotype Wild type BY4741 Mat a; his3∆1; leu2∆0; met15∆0; ura3∆0

ACR Mutants

YPR201w BY4741; Mat a; his3∆1; leu2∆0; met15∆0; ura3∆0; YPR201w::kanMX4 YPR200c BY4741; Mat a; his3∆1; leu2∆0; met15∆0; ura3∆0; YPR200c::kanMX4

Knock-out Mutants: 1 YDL201w BY4741; Mat a; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YDL201w::kanMX4 2 YIL064w BY4741; Mat a; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YIL064w::kanMX4 3 YNL092w BY4741; Mat a; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YNL092w::kanMX4 4 YPL157w BY4741; Mat a; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YPL157w::kanMX4 5 YHL039w BY4741; Mat a; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YHL039w::kanMX4 6 YBR261c BY4741; Mat a; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YBR261c::kanMX4 7 YBR271w BY4741; Mat a; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YBR271w::kanMX4 8 YGL136c BY4741; Mat a; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YGL136c::kanMX4 9 YHR209w BY4741; Mat a; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YHR209w::kanMX4

10 YNL063w BY4741; Mat a; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YNL063w::kanMX4 11 YDR140w BY4741; Mat a; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YDR140w::kanMX4 12 YDR316w BY4741; Mat a; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YDR316w::kanMX4 13 YER175c BY4741; Mat a; his3Δ1; leu2Δ0; met15Δ0; ura3Δ0; YER175c::kanMX4

2.1.3. E. coli cells and growth conditions

Strains of W3110 [K12 F2 IN(rrnD-rrnE)] and AW3110 (W3110 ∆ars::cam) (Carlin et

al., 1995) were generously provided by Dr. Barry P. Rosen. Cells were incubated on LB

medium (1 L contains 5 g Yeast Extract, 10 g Bacto Tryptone, 10 g NaCl and 20 g agar

for solid medium) at 37oC. A single colony of E. coli was inoculated to 5 ml of LB

media and shaken overnight. Then 5 ml of overnight culture was transferred to 500 ml

of LB media and growth continued to O.D.600 of about 1.0, followed by arsenate

addition to the test culture, and an extended 4 hours growth.


67

2.1.4. Reagents

Sodium arsenite (NaAsO2, 98%) and sodium arsenate (Na2HAsO4.7H2O) were

purchased from Sigma- Aldrich (Milwaukee, WI, USA). Monomethyl arsonate (MMA,

as monosodium acid methane arsonate, 98.0%) and dimethy arsonate (DMA, as

dimethylarsinic acid, 98.0%) were purchased from Chem Service, Inc. (West Chester,

PA, USA). S-[3H-methyl] adenosyl-L-methionine (3H-AdoMet, 68.5 Ci/mmol) was

purchased from DuPont-NEN Life Science Products. All chemicals were analytical

reagent grade or of highest available quality.

Specific activity of 3H-AdoMet in DPM/mol is calculated as:

68.5 x10-3 x 3.7 x 1016 x 60 DPM/mol = 1.52 x 1017 DPM/mol


68

2.2. Methods

2.2.1. Preparation of cell extracts

2.2.1.1. Yeast and E. coli

Yeast or E.coli cells after culture were collected at 4oC by centrifuge at 5,000 rpm

(4,420 g) for 15 min. Cells were washed with dH2O twice, then immediately

resuspended in 15 ml of 1xPBS buffer (pH7.4) containing 0.5 mM

phenylmethylsulfonyl fluoride (PMSF). Resuspended cells were used immediately or

stored at -80oC. Cells were broken by French Press at 69 MPa (10,000 lb/in2) in the cold

room (4oC), and then centrifuged at 27,200 g for 30 minutes at 4oC. The proteins in the

supernanant were fractionated by ammonium sulphate precipitation. The precipitate

that appeared at 55-80% saturation was dissolved in 1.0 ml of buffer A (10 mM MOPS,

0.5 mM reduced glutathione, 0.5 mM phenylmethylsulfonyl fluoride, adjust to pH7.2

with NaOH) and desalted using a Sephadex G-25 fine column (volume, 10 ml)

equilibrated with buffer A at 4oC. Elution was at a rate of 1.1-1.5 ml/min. Fractions (1.5

ml) were collected and those containing the peak of enzyme activity were used

immediately for all studies unless otherwise stated. Protein concentration was

determined by Coomassie reagent (Sigma) using bovine serum albumin as standard.

2.2.1.2. Plant tissue

Shoots from several hundred plants or roots from about thousand plants were collected,

immediately snap frozen, and ground in liquid nitrogen. The frozen powder was

homogenised in buffer (1/1, w/v). The homogenization buffer consisted of 10 mM

MOPS, 250 mM sucrose, 0.5 mM reduced glutathione, and 0.2 mM

phenylmethylsulfonyl fluoride (adjust to pH7.2 with NaOH). The homogenate was

centrifuged at 27,200 g for 20 minutes at 4oC. The supernanant was used immediately

or stored at -80 oC. The supernanant was treated by two alternative procedures. In one

case, the supernanant was centrifuged at 125,000 g for 60 min at 4oC to obtain crude


69

cytosolic extract for direct enzyme assays. In the other, the proteins in the supernanant

were fractionated by ammonium sulphate precipitation. The precipitate that appeared at

55-80% saturation was dissolved in 1.5 ml of buffer A and desalted using the same

procedure as for the yeast and E. coli extracts (Section 2.2.1.1).

2.2.2. Arsenic methyltransferase assay

The arsenic methyltransferase activity of each preparation was measured in 250 μl

reaction mixture containing 100 mM MOPS, 1mM MgCl2, 4 mM reduced glutathione,

20 µM NaAsO2 or Na2AsO4, (adjusted to pH7.2 with NaOH) and 100 pmol (1.05 µCi)

of carrier-free 3H-AdoMet. For MMA methyltransferase assay, the substrate was

monosodium acid methane arsonate (MMA) and 1mM DTT in reaction mixtures. The

reaction was initiated by the addition of either crude cell extract (85-130 µg protein) or

fractionated extract (320-430 µg protein from plants, 6.0-7.5 mg protein from yeast and

E. coli), or purified fusion proteins (200 µg; see later). The mixture was incubated for

up to 60 min at 25oC for plants, 30oC for yeast and 37oC for E. coli. The reaction was

terminated by the addition of 750 µl 12M HCl and either extracted immediately or

stored at -20oC.

Addition of 10 mM EDTA inhibited formation of both mono-and di-methylated

metabolites, suggesting that an endogenous divalent cation might be involved in

enzymatic methylation of arsenic (Styblo, et al., 1996a). Therefore during our studies,

divalent cations in assay systems may have effects on the arsenic methylation.

2.2.3. Extraction and determination of methylated arsenic compounds

The principle of the extraction is that, in the presence of HCl and KI, arsenicals exist as

halides that are soluble in chloroform (Zakharyan et al., 1995a; Fitchett et al., 1975;

Chappell et al., 1995). Samples from terminated reaction mixtures (250 ml) were

placed in capped polypropylene tubes with 10 µl of 40% KI, 20 µl of 1.5% potassium

dichromate, 750 µl of 12 M HCl and 750 µl of chloroform. The contents were vortexed

for 3 min and then centrifuged at 1,500 g for 3 min. The upper, acidic aqueous phase,


70

containing 3H-AdoMet was discarded. The lower chloroform phase was washed twice

with 250 µl of H2O, 5 µl of 40% KI and 750 µl of 12 M HCl, vortexing, centrifuging

and discarding the upper acidic aqueous phase. The methylated arsenic compounds in

chloroform phase were back-extracted by adding 1 ml of H2O, vortexing for 3 min and

centrifuging at 1,500 g for 4 min. The radioactivity in samples of the aqueous

back-extract was measured by liquid scintillation counting and the remainder was

applied to a cation exchange column.

2.2.4. Separation of methylated metabolite

The aqueous back-extract was fractionated on small columns of cation exchange resin

under conditions where MMA elutes in HCl and DMA in a subsequent NaOH elution

(Tam et al., 1978; Zakharyan et al., 1995). A chromatography column (2 ml volume,

DOWEX 50WX4-200R cation exchange resin 100-200 mesh hydrogen form, Aldrich)

was washed successively with 30ml 500 mM HCl, sufficient water to raise the pH of

the effluent 5.5, 30 ml 500 mM NaOH, sufficient water to return the pH of effluent to

5.5, 30 ml 500 mM HCl and 50 ml 50 mM HCl. Samples (1ml) of the aqueous

back-extract or solutions of authentic arsenic compounds were applied to the column

which was then eluted with 6ml 50 mM HCl followed by 14 ml 500 mM NaOH.

Fractions (1ml) of the elutant were collected, and for radioactivity determination,

mixed with 9ml liquid scintillation cocktail overnight before liquid scintillation

counting. As to calculation of the methylated products, the amount of MMA was the

sum of the radioactivities in fractions 2 to 5 which included the recovered MMA from

the extraction procedure for methylated metabolites, and that of DMA was in Fraction

12 to 20.

2.2.5. Preparation and determination of authentic MMA and DMA

In order to detect the distribution MMA and DMA in the fractions of the cation

exchange column, 10 µmol of authentic MMA and 5 µmol of DMA in the volume of 1

ml was extracted separately as described in Section 2.2.3. The extraction was applied to

the cation exchange column detailed above in Section 2.2.4. The presence of the


71

arsenicals in fractions was determined by elemental arsenic analysis using inductively

coupled plasma mass spectrometry (ICP-MS, Leco Renaissance).

2.2.6. Expression and purification of the yeast proteins YHR209w and YER175c

The glutathione S-transferase (GST)-YHR209w and GST-YER175c fusion proteins

were expressed in strain EJ758 [MAT a his3∆-200 leu2-3,112 ura3-52 pep4::URA3]

(ResGen Invitrogen Coporation). A colony of each yeast strain was inoculated to 50 ml

GMM (1 L contains 1.7 g yeast nitrogen base, 5 g ammonia sulphate, 20 g Glucose, 50

mg histidine) at 30°C for two days. A 50 ml aliquot of the 2-day culture was then added

to 550 ml of the same medium and the culture was incubated at 30°C until the O.D.600

was 0.8. Expression was induced by addition of CuSO4 to 0.5 mM. After 15 h induction,

the cells were washed in 300 ml of dH2O twice and 60 ml of ice-cold PBS (140 mM

NaCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, 2.7 mM KCl, pH 7.3), resuspended in 10 ml

of PBS with 20 µM leupeptin and ruptured using two passes on a French Press at 69

MPa (10,000 lb/ in2). The lysate was centrifuged for 30 min at 27,200 g (17,000 rpm) at

4°C. The supernatant was added to a column of 5 ml of GSH–Sepharose 4B (Pharmacia

Biotech) with a speed of 160 µl /min. The column was then washed by 10 column

volumes (50 ml) of 1xPBS. Five column volumes (25 ml) of glutathione elution buffer

(50 mM Tris-HCl, 10 mM glutathione, pH 8.0) was applied to the column while 1.4 ml

of each fraction was collected. Pellets were stored at -80oC for further analysis.

2.2.7. Digestion of the yeast GST fusion proteins

Thrombin (1u/µl) was used in the digestion of the yeast fusion proteins GST-YHR209w

and GST-YER175c. The amount of 10 u of thrombin was applied to 500 µg of GST

fusion protein. The mixture was incubated in 19oC for 2.5 hours or 0oC for overnight.

The digested mixture was used immediately in assays or stored at -80oC.

Chapter III. Methylation of Arsenic in vitro by Cell Extracts from Bentgrass (Agrostis tenuis)

72

Chapter III

Methylation of Arsenic in vitro by

Cell Extracts from Bentgrass (Agrostis tenuis)


73

3.1. Introduction

Arsenic enters living organisms by a variety of pathways. Arsenic is ingested by

animals (Aposhian, 1997) and metabolised by bacteria and fungi (Cullen and Reimer,

1989). Marine organisms in tropical oceans face an environment where the

concentration of inorganic arsenate may be comparable to that of phosphate (Knowles

and Benson, 1983a). Higher plants growing in soils with elevated inorganic arsenic

levels take up arsenic via the roots, often mediated by mycorrhizae (Sharples et al.,

2000). Some higher plants appear to have achieved a degree of resistance to elevated

arsenic concentrations in the soil by reduced uptake, while others actually accumulate

arsenic (Bhumbla and Keefer, 1994). Soils with naturally elevated levels of arsenic,

resulting from outcropping of arsenic-rich mineral ores, have probably provided

environments where arsenic-resistant plants have evolved (Ernst, 2000). Porter and

Peterson (1975) studied the arsenic levels in plants growing on mine waster and

reported extraordinarily high foliar concentrations, exceeding 0.1% arsenic for several

species. Similar accumulation by a fern (Pteris Vittata L.) was also reported (Ma et al.,

2001). The mechanism whereby plants take up, metabolise and store arsenic from the

soil is of interest for the possible development of plants capable of remedying soils

contaminated by this toxic element.

The arsenic methylation mechanism commences with the reduction of arsenate to

arsenite followed by methylation to form MMA, reduction of MMA to methyl arsonous

acid, then methylation to DMA. This conversion has been observed in bacteria and

fungi (Cullen and Reimer, 1989), algae (Fujiwara et al., 2000), and Old World animals

(Aphosian, 1997), but has not been confirmed in plants (Section 1.3.3.1). It is also

unclear whether the methylation occurs in shoots, roots or both.

Four enzyme activities catalyse the conversion of arsenate to DMA (Section 1.4). These

enzymes have been purified and characterized from mammalian tissue as follows:


74

arsenate reductase (Radabaugh and Aposhian, 2000), arsenite methyltransferase (Healy

et al., 1998), MMA reductase (Zakharyan and Aposhian, 1999) and monomethyl

arsonate methyltrasferase (Zakharyan et al., 1999). Terrestrial higher plants seem to be

much less active in arsenic methylation than Old World animals. Nissen and Benson

(1982) studied the uptake of arsenate by a range of higher plants including corn, melon,

pea and tomato. They found that substantial amounts of methylated arsenicals were

detected only when plants were grown under severe nitrogen- and phosphorus-deficient

conditions. While only small amount of DMA was formed in the roots, it was the

predominant product in the leaves. Indian mustard plants grown in complete nutrient

media took up arsenate, but X-ray absorption spectroscopy revealed no significant

accumulation of MMA or DMA (Pickering et al., 2000).

There is a view that arsenic methylation is not a major route for detoxification in

healthy terrestrial higher plants (Schmöger et al., 2000). However, the scenario in

plants that accumulate arsenic from the soil remains unclear. If the methylation

pathway from arsenate to DMA is not part of an effective detoxification mechanism,

then these reactions should be suppressed in arsenic accumulators. In this thesis, this

proposition was tested by examining the arsenic-methylating activities in cell extracts

from the leaves and roots of bentgrass (Agrostis tenuis Sibth.). Both of the methylation

of arsenite and arsenate in an in vitro assay system containing cell extracts of A. tenuis, 3H-AdoMet and glutathione were determined.

Highland grass (Agrostis tenuis S.) has synonyms or other Latin names as Agrostis

capillaris L. and Agrostis vulgaris With. It is also known as its common names:

common bentgrass, brown top, sand couch, Rhode Island bent grass, colonial bentgrass

(from The Global Compendium of Weeds). Porter and Peterson (1975) studied

Highland grass growing on arsenic-contaminated soil and found that it could

accumulate in excess of 2g kg-1 dry weight arsenic. Because of its high ability to

accumulate arsenic, Highland grass was used as one of the materials in this thesis.


75

3.2. Results

3.2.1. Validation of extraction and separation processes

When reaction mixtures containing 3H-AdoMet, but without plant extract were

subjected to the standard extraction procedure for methylated arsenic compounds

(Section 2.2.3), the radioactivity recovered was not more than a few disintegrations per

minute (dpm) above background, demonstrating that carry-over of 3H-AdoMet was

negligible. Authentic MMA and DMA were extracted and separated by the standard

procedure (Figure 3.1). The recovery of MMA during elution with 50 mM HCl was

90.7% and of DMA during elution with 500 mM NaOH was 49.9%.

Figure 3.1. Extraction of authentic MMA and DMA. Authentic MMA and DMA were extracted separately by standard extraction procedures, and fractionated by column chromatography (Section 2.2.4). The concentration (ppm) of total elemental arsenic was measured in each fraction by ICP-MS.

0 5 10 15 20

0

100

200

300

400

500

MMADMA

HCl NaOHEluant

0

100

200

300

400

500

Fraction number


76

3.2.2. Effect of arsenate in the culture media on Agrostis tenuis

3.2.2.1. Effect of arsenate concentration in culture media on methylation by leaf

extracts

Arsenate was added to the culture media of some A. tenuis culture pots 3 days before

harvesting to achieve a range of concentrations (135, 269, 403 and 538 µM, equivalent

to 0, 25, 50, 75 and 100 parts per million (ppm) arsenic, respectively). The crude

extracts from arsenate treated and untreated control plants were assayed directly for

arsenic methyltransferase activity with arsenite as substrate for 60 minutes (Table 3.1).

When plants were grown without arsenate and the extracts were assayed in the absence

of arsenite, appreciable methylation (43.1 dpm, which we term ‘background’) was

observed (Table 3.1). For extracts from these plants, the addition of arsenite to the assay

medium inhibited this background methylation by 54% to 23.4 dpm (Table 3.1). With

the assumption that arsenite exerted the same degree of inhibition of background

methylation in all assays, the background methylation by extracts from arsenate-treated

plants was calculated by applying this factor to the extracted radioactivity measured in

the absence of arsenite as the substrate. The additional radioactivity due to arsenite

methylation was determined by subtracting this calculated background from the

radioactivity extracted from assays in the presence of arsenite. The additional

radioactivity due to arsenite methylation is also expressed as a proportion of total

extracted radioactivity. The methylation of arsenite by extracts of plants grown with

arsenate increased with the concentration of arsenate in the growth media, attaining

apogee at 269 µM (50 ppm) arsenate and thereafter declined at higher concentrations.


77

Table 3.1. Effect of arsenate concentration in the plant culture media on methylation of arsenite by crude cytosolic extracts of Agrostis tenuis

Concentration of Arsenate in

Growth Medium

Radioactivity of Methylated

Fraction (DPM) No arsenite

substrate


Fraction (DPM)

plus arsenite

Calculated Background

in presence of. arsenite (DPM)


Fraction (DPM) plus arsenite

minus background

Proportion not due to

background (%)

(ppm) (µM) 0.0 0.0 43.1 23.4 23.4 0.0 0.0 25 134.4 97.5 74.2 52.9 21.3 28.7 50 268.8 36.0 98.4 19.5 78.9 80.13 75 403.2 61.1 69.3 33.2 361 52.13 100 537.6 31.6 24.0 17.2 6.8 28.51

3.2.2.2. Arsenate tolerance in Agrostis tenuis

In order to investigate the chronic effects of 269 µM (50 ppm) arsenate on the

long-term growth of A. tenuis, three pots (A, B and C) were prepared. Each pot

contained 180 seeds, and was grown for 28 days. Arsenate was added to the culture

media of two pots (A and B) to final concentration of 50 ppm. Three days after arsenate

addition, the media were changed again with no arsenate in pot A and 50 ppm arsenate

in pot B. No arsenate was added to pot C at any time. The number of living plants in

each pot was counted during the experiment.

All three pots of A. tenuis displayed similar rates of germination of about one third. The

plants in pot C (no arsenate) grew well after germination with green leaves through the

64-day experiment. After addition of arsenate to media pot A and pot B, the plant

numbers decreased about 12% between days 28 and 31 (Figure 3.2). The growth of

both shoots and roots was remarkably inhibited (Figure 3.3).

The number of the plants in pot A decreased only slightly after the arsenic-free growth

media was restored at day 31. Some of leaves of the plants in pot A turned green-yellow

in colour after arsenate treatment, but most of the leaves were still green and strong

although the colour was lighter than the controls. Many leaves turned darker green after

50 days, suggesting that the plants had recovered.


78

Figure 3.2. Effect of arsenate in growth medium on survival of A. tenuis. A. tenuis plants were grown for 64 days with or without arsenate presence. The numbers of the surviving plants were recorded during the growth (Section 2.1.1). The plant numbers in pot B declined continuously after arsenate addition at 28 days, in

contrast to those in pot A. All plants in pot B were smaller and shorter. Many younger

leaves were yellow-white, whereas older leaves remained green. From 42 days after

planting, the surviving plants in pot B seemed to be stabilized. Most leaves in pot B

were white. Only 4-5 plants had leaves with light green colour. By 50 days, the leaves

grew wider and longer although still yellow. By 64 days after planting, more colour

(yellow and green) had return and the plants appeared to adjust to the presence of 50

ppm arsenate in the medium (Figure 3.3)

15 20 25 30 35 40 45 50 55 60 65 70

60

70

80

90

100

110

C: No iAsVA: 50 ppm iAsV for 3daysB: 50 ppm iAsV

Days

Days21283137425064

C (%)100.0

98.496.896.896.893.691.9

A (%)100.0

95.183.682.080.378.777.1

B (%)100.0

97.285.978.970.470.467.6

Addition of arsenate

Removal of arsenate


79

Figure 3.3. Plants of Agrostis tenuis at day 64. A: treated by 50 ppm of arsenate for 3 days from day 28 to day 31; B: treated by 50 ppm of arsenate from day 28 to day 64; C (control): arsenate-free during whole period.

A B C

C

B

A


80

3.2.2.3. Time-course analysis of arsenite methylation, and separation of products

Arsenate was added to the culture media of some A. tenuis culture pots 3 days before

harvesting to achieve a concentration of 269 µM. Partially purified extracts were

prepared from leaves of arsenate-treated and untreated control plants. The methylation

of arsenite by each extract was examined during assay reaction times of 15, 30 and 60

minutes, after which the radioactive products were separated by column

chromatography (Section 2.2.4). Background radioactivity was determined in assays

with arsenite omitted, as before.

When leaf extracts from plants grown in the absence of arsenate were assayed without

arsenite as substrate, the background methylated product co-eluted with MMA and

contained no DMA (Figure 3.4 and Figure 3.5). However, if arsenate was added to the

growth medium 3 days before harvest, a small amount of DMA was detectable in the

background after 60 minutes incubation (Figure 3.4c and Figure 3.5). When arsenite

was included in assays, an appreciable amount of DMA was present at 30 minutes

incubation assays containing extracts from plants not exposed to arsenate (Figure 3.4b).

The presence of arsenate in the plant growth medium stimulated MMA formation

somewhat, but much greater amount of DMA were formed (Figure 3.5). The amount of

DMA extracted increased sharply from 15 to 30 minutes, and further by 60 minutes.

The apparent rate of synthesis of DMA between 15 and 30 minutes was 660 amol.mg

protein-1.min-1, based on the sum of the radioactivity in fractions 13 to 19 (Figure 3.4),

the specific activity of 3H-AdoMet, and the recovery of DMA in the extraction

procedure for methylated metabolites. In these experiments, the maximum

incorporation of added 3H-AdoMet label into products was 0.05%.


81

Figure 3.4. Methylation of arsenite by partially purified extracts from leaves of A. tenuis. Samples were taken at the times indicated from methylation assay mixtures containing partially purified shoot extracts with arsenite as substrate. Some plants were exposed to arsenate in growth medium as indicated. Methylated arsenicals were extracted and fractionated by column chromatography (Section 2.2.4). The total 3H-methyl radioactivity in each fraction is shown.

a. Incubation Time: 15 min

0 5 10 15 20

0

50

100

150

+arsenite -arsenite +arsenite

-arsenate

+arsenate+arsenate

Grow th medium Assay medium

-arsenite

Fraction Number

-arsenate

b. Incubation Time: 30 min

0 5 10 15 20

0

100

200

300

400

Fraction Number

c. Incubation Time: 60 min

0 5 10 15 20

0

100

200

300

400

Fraction Number


82

Figure 3.5. Methylated products of arsenite methylation by partially purified extracts from leaves of A. tenuis. Some plants were exposed to arsenate in growth medium as indicated. The total 3H-methyl radioactivity in the fractions corresponding to MMA (fraction 2-5) and to DMA (fraction 13-19) are shown at 15, 30 and 60 minutes incubation for each assay condition in Figure 3.4.

3.2.2.4. Methylation of arsenate

Plants were exposed to 269 µM 3 days before harvesting and preparation of partially

purified leaf extracts. Methylation assays were performed using arsenate as

methylation substrate instead of arsenite, with conditions otherwise identical to those

mentioned previously. Methylated products were detected (Figure 3.6 and 3.7) at much

lower levels than with arsenite substrate (Figure 3.4 and 3.5). The amounts of MMA

and DMA were similar to the backgrounds when the extracts were incubated for 30

minutes. However, when the assay was extended to 60 minutes, appreciable amounts of

MMA and DMA were formed by the extracts from plants exposed to arsenate (Figure

3.6 and 3.7). Much less DMA was formed than when arsenite was the substrate (Figure

3.5). A minor peak, evident in fractions 9 and 10 (Figure 3.4 and 3.6) suggested the

MMA

0 10 20 30 40 50 60

0

100

200

300

400

500

600

700

800

900

(-arsenate, -arsenite) (-arsenate, +arsenite) (+arsenate, -arsenite) (+arsenate, +arsenite)

Assay Time (minutes)

DMA

0 10 20 30 40 50 60

0

100

200

300

400

500

600

700

800

900



83

formation of a third, unidentified methylated product with both arsenite and arsenate as

assay substrates.

Figure 3.6. Methylation of arsenate by partially purified extracts from leaves of A. tenuis. Experiments performed in an identical manner to those of Figure 3.4, except that arsenate was the assay substrate instead of arsenite. The 3H-methyl radioactivity in each fraction is shown after 30 and 60 minutes incubation for extracts from plants exposed to arsenate, with arsenate as substrate in the methylation assay.

a. Incubation Time: 30 min

0 5 10 15 20

0

20

40

60

80

100

120

+ arsenate- arsenate+ arsenate

- arsenate+ arsenate+ arsenate


Fraction Number

b. Incubation Time: 60 min

0 5 10 15 20

0

20

40

60

80

100

120

Fraction Number


84

Figure 3.7. Methylated products of arsenite methylation by partially purified extracts from leaves of A. tenuis. The total 3H-methyl radioactivity in the fractions corresponding to MMA (fraction 2-5) and to DMA (fraction 13-19) are shown at 30 and 60 minutes incubation for each assay condition in Figure 3.6.

MMA

0 10 20 30 40 50 60

0

100

200

300

400

500

600

700

800

900

- arsenate + arsenate - arsenate + arsenate

- arsenate- arsenate+ arsenate

+ arsenate



DMA

0 10 20 30 40 50 60

0

100

200

300

400

500

600

700

800

900



85

3.2.2.5. Methylation by roots

Plants were exposed to 269 µM arsenate 3 days before harvesting, following which

partially purified roots extracts were prepared. Methylation assays were performed

using arsenite as the methylation substrate. Extracts prepared from roots of A. tenuis

catalysed insignificant methylation of arsenite (Figure 3.8), unlike leaf extracts. After

30 minutes incubation in vitro, there was no appreciable amount of DMA formation. A

small amount of methylated product, co-eluting with MMA, was formed in assay

mixtures lacking arsenite but containing extract from plants grown in the presence of

arsenate. As with leaf extracts, such product was reduced when arsenite was present in

the assay, but prior exposure of the plants to arsenate did not increase methylation

greatly, nor did it yield DMA.

Figure 3.8. Methylation of arsenite by partially purified extracts from roots of A. tenuis. Samples were taken at 30 minutes, from methylation assay mixtures containing root extracts with arsenite as substrate. Methylated arsenicals were extracted and fractionated by column chromatography (Section 2.2.4). The 3H-methyl radioactivity in each fraction is shown.

Root extract. Time: 30 min

0 5 10 15 20

0

50

100

+ arsenite- arsenite+ arsenite

- arsenate+ arsenate+ arsenate


Fraction Number


86

3.3. Discussion

There is no doubt that arsenate is harmful to plant growth. During the first 3 days

arsenate was added to the growth medium at 50 ppm, about 12% of A. tenuis shoots

died (Figure 3.2). After the arsenate was removed, the plants recovered quickly. The

plant survived stablised (Figure 3.2), so that about 80% of the plants kept growing

strongly, similar to the controls (Figure 3.3). When arsenate remained in the growth

medium, the percentage of survival continued to fall. After 22 days in arsenate, 70% of

the A. tenuis shoots still survived (Figure 3.2) and most of them continued to grow

although both their leaves and roots were shorter (Figure 3.3). The resistance of this

plant species is known (Section 3.1) and validate by my experiments which enabled the

later arsenic methylation study.

A large increase in MMA and DMA formation was observed in the leaves upon addition

of arsenate to the growth medium of the plants 3 days before harvesting. The control

assays, performed in the absence of arsenite substrate, confirmed that such increases

were not artefacts stemming from carry-over in the extract of protein-bound arsenic

compounds accumulated by the plants grown in arsenate. For these controls, the extent

of methylation by extracts from plants exposed to arsenate was not generally greater

than from plants grown in arsenate-free medium (Table 3.1). Moreover, the formation

of DMA was largely dependent on the presence of arsenite in the assay (Figure 3.4 and

3.5), and the amount of methylated products increased with assay duration. The results

of these controls also negate the possibility that bacteria in the plant culture medium

took up arsenate and then passed some form of arsenic to the plants, as does the fact that

little MMA and no DMA were formed in methylation assays with root extracts (Figure

3.8).

When extracts from plants grown in arsenate-free media were assayed in the absence of

arsenite, low amounts of radioactivity were extracted which was not carry-over of


87

3H-AdoMet (Section 3.2). This was true for both crude (Table 3.1) and partially purified

extracts (Figure 3.4). The source of this background methylation was not determined.

We speculate that it may have included methylation of traces of arsenic present

originally in the seeds and carried over in the crude extract, or the methylation of

compounds other than arsenite. In assays with extracts from these plants, the presence

of 50 µM arsenite in the assay reduced the apparent background methylation, perhaps

by a competitive effect on a constitutive arsenic methyltransferase. Arsenite, in the

concentration range of 10-40 µM, has been shown to inhibit the production of MMA

and DMA in hepatocytes from rats and humans (Styblo et al., 1999). Alternatively, it is

possible that some of the background methylation involved methyltransferases specific

for substrates other than arsenite, but subject to inhibition by arsenite. Compounds that

could be subjected to methylation to produce methylated compounds that resemble

MMA include phosphite, which may occur in plants (Schink and Friedrich, 2000). The

traces of arsenic accumulated from normal soil environments are probably detoxified

by a combination of the constitutive arsenic methyltransferase activity and chelation.

Exposure of plants to arsenate did not stimulate methylation activity in root extracts

(Figure 3.8), although background methylation was evident. This was in sharp contrast

to the large increase in methylation activity of extracts from the upper parts of the plants,

implying that arsenate is not methylated immediately following uptake by the roots.

This phenomenon accords with the findings of Pickering et al (2000) that after uptake,

arsenate is reduced to arsenite in the root and then bound to a peptide thiolate.

The results demonstrated clearly that cell extracts from shoots of A. tenuis were capable

of catalysing the methylation of arsenite by 3H-AdoMet. Nissen and Benson (1982)

showed that MMA and DMA were produced from arsenate in the leaves of tomato

plants, but only under conditions of severe nutrient deficiency. Our present findings, to

our knowledge, are the first to demonstrate in vitro arsenite methyltransferase activities

in a higher plant.


88

The extent of arsenic methylation by shoot extracts varied with the concentration of

arsenate in the growth medium, with greatest activity at 269 µM arsenate (Table 3.1).

High concentrations of arsenate probably began to overwhelm the defences of the plant

against environmental arsenic during this 3-d exposure. Arsenic methylation activity

then decreased presumably reflecting the onset of poisoning of the plant.

The induction of methyltransferase activities by arsenate in A. tenuis contrasts with the

characteristics of these enzymes in mammalian cells, which do not exhibit any

induction (Healy et al., 1998). From the rate of DMA accumulation, we estimated the

methyltransferase activity in the leaf extracts of A. tenuis to be approximately 0.66

fmol.mg protein-1.min-1 (at 25oC), which is comparable to the activity of 3.7-24.0

fmol.mg protein-1.min-1 in mammalian cells at 37oC, estimated from the rate of MMA

accumulation (Healy et al., 1998). These findings are compatible with the generally

accepted pathway for the methylation of arsenicals, where trivalent arsenite and

methylarsonous acid are the substrates for methylation, provided reduction of

pentavalent arsenicals precedes their methylation. Whereas the amount of MMA

formed in assays did not increase greatly after 15min, 30min was required for

substantial amounts of DMA to appear (Figure 3.4). DMA formation was delayed even

further when arsenate was the substrate (Figure 3.6). By comparing the time-course of

MMA formation from arsenite (Figure 3.5) and arsenate (Figure 3.7), it was clear that

arsenite is the preferred substrate. It is concluded that reduction of arsenate to trivalent

arsenite occurred in the reaction mixtures, but was relatively slow. This reduction may

have been a non-enzymic reaction with the glutathione present (Radabaugh and

Aposhian, 1999), or catalysed by an arsenate reductase. The thiol requirement of

arsenate reductase from human liver is satisfied by dithiothreitol, but not glutathione

(Radabaugh and Aposhian, 1999). Hence, if an enzyme in the plant extracts we used

was responsible for the low rates of arsenate reduction, this enzyme is likely to have a

thiol requirement different from the enzyme of human liver.

Our in vitro results, when viewed along with the known characteristics of arsenic

uptake by other plants, suggest the following scenario for arsenic metabolism in A.

tenuis. Arsenate in the growth medium is taken up by the roots through the symplastic

pathway, perhaps by a plasmalemma-located transporter for phosphate (Pickering et al.,


89

2000). In the cytosol of root cells, arsenate is reduced to arsenite, which forms a

complex with the thiolate of glutathione or a phytochelatin (Pickering et al., 2000).

Such complex is of low stability (Schmöger et al., 2000), but if transport of the

arsenite-peptide complex across the tonoplast into the vacuole occurs, it would be more

stable in the vacuole since this provides a more acidic environment. Ghosh et al. (1999)

showed that, in yeast, a transporter in the tonoplast with sequence identity to the

cadmium-factor gene YCF1, removes arsenite from the cytosol and sequesters it in the

vacuole as a glutathione conjugate. A. tenuis is known to accumulate large amounts of

arsenic in the foliage (Porter and Peterson, 1975). The form of arsenic and the cellular

pathway of transport are uncertain, but are likely to be as the arsenite conjugate via the

xylem, respectively. On reaching the cytosol of leaf cells, arsenite dissociating from the

weak complex is methylated to DMA by the enzyme activities, as demonstrated in the

leaf and root extracts. The concurrence of arsenite binding and methylation was evident

in the in vitro experiments of Styblo and Thomas (1997) using rat liver extract. The

methylation assays performed here were in the presence of 4mM reduced glutathione,

affirming the ability of the arsenite methyltransferase to compete successfully for

substrate with this arsenite-sequestering agent.

Our in vitro findings demonstrated that DMA as the major end product in A. tenuis in

vivo, consistent with the pattern of arsenic metabolites found in nutrient-deficient

tomato plants (Nissen and Benson, 1982). However, many questions concerning

arsenic uptake by plants in general, and A. tenuis in particular, remain unanswered.

Isolation and full characterization of the plant enzymes for arsenic metabolism is

required. Furthermore, the whole plant studies are needed to confirm the major

products, and to establish whether any further metabolism to organic arsenicals more

complex than DMA occurs in A. tenuis. At the physiological level, the uptake and

transport of arsenic and the intracellular location of accumulated products in A. tenuis

needs to be characterised for both acute and chronic exposure to arsenate. It is

concluded that, for the plant A. tenuis, the uptake or sensing of arsenate in the growth

medium by the roots leads to the induction, in the leaves, of methyltransferase activity

arsenite and methylarsonite. The roots remained free of this activity. It is not known

whether this phenomenon is a special property of arsenate-accumulating plants such as

A. tenuis, or a characteristic more general to higher plants under particular conditions

such as nutrient deficiency.

Chapter IV. Induction of Arsenic Methylation in the Yeast Saccharomyces cerevisiae and the Bacterium E. coli

90

Chapter IV

Induction of Arsenic Methylation in

the Yeast Saccharomyces cerevisiae and the Bacterium E. coli


91

4.1. Introduction

As reviewed in Chapter I, two general mechanisms of resistance to arsenic toxicity

have been recognised in microorganisms. The first is a variety of export systems and

the second is methylation.

The mechanism of arsenic methylation commences with arsenate reduction to arsenite

and arsenite methylation to monomethylarsonic acid then to dimethylarsinic acid. This

enzymatic process has been confirmed in plant (Chapter III) and mammalian cells

(Zakharyan et al., 1995a) but not yet in yeast to bacteria. Arsenite methyltransferase

enzymes from rabbit liver (Bogdan et al., 1994) and Chang human liver hepatocytes

(Zakharyan et al., 1999) have been partially purified and their Michaelis-Menten

kinetics characterised. The activities of these enzymes in mammalian cells do not

exhibit any induction by arsenic compounds. In contrast, the enzymatic methylation of

arsenite has been found to be inducible by arsenate in the plant A. tenuis (Chapter III).

The objective of this study was to investigate the enzymatic processes of arsenic

methylation and the effects of arsenate on arsenic methyltransferase activity in the yeast

S. cerevisiae and the bacterium E. coli, as representatives of eukaryote and prokaryote

microorganisms. In addition, comparative studies were to be conducted using wild

types of yeast and E. coli and mutants of both, whose acr and ars genes were knocked

out. This would reveal the effect, if any, of the pumping systems on the methylation of

arsenicals in the microbes. With the full genome data and a rich resource of gene

mutants available for these two models, the results should enable the further

identification of the equivalent genes of arsenic methyltransferases in these

microorganisms, plants and other organisms.

Since arsenic-methylating microorganisms are not sensitive to changes in pH and

methylate arsenic in the pH range 3.5-7.5 (Baker et al., 1983a), in my experiments with


92

yeast S. cerevisiae and bacterium E. coli, pH was not considered to be a major

parameter for cell growth. Most of the methylation assays were carried out at

physiological neutral pH. However, after information on the effect of pH on

S-adenosyl-L-methionine:arsenic(III) methyltransferase in rat liver was reported (Lin

et al., 2002), my experiments were extended to determine the optimal pH for arsenic

methylation in S. cerevisiae and E. coli.


93

4.2. Results 4.2.1. Effects of arsenate and arsenite in the culture media on the growth of different S. cerevisiae strains To test obtained yeast strains BY4741 (wild type), YPR201w (∆arc3) and YPR200c

(∆arc2) (Section 2.1.2), cultures of each strain were spotted to GYP-medium plates

containing a range of concentration of arsenate from 0 to 20 mM, and arsenite of 0 to

3.0 mM and incubated at 30oC for two days.

Figure 4.1. Effects of arsenate (A) and arsenite (B) concentration in the culture media on arc2

-deficient stain YPR200c (a), arc3-deficient strain YPR201w (b) and wild-type strain BY4741(c). Each spot initially contained 2x105 cells in 5 µl of culture.

B. Arsenite (mM)

a b c

0.1

0

0.2

0.3

0.4

0.5

1.0

1.5

A. Arsenate (mM)

0

0.1

0.2

0.3

0.4

0.5

1.0

2.0

3.0

4.0

5.0

a b c


94

On the arsenate plates (Figure 4.1A), BY4741 grew well in 1.0 mM arsenate, with

progressive inhibition up to 3.0 mM arsenate. At 4 mM arsenate and higher, no colony

formation of this yeast strain was observed. YPR200c was more sensitive with

inhibition of growth evident at 1.0 mM arsenate. YPR201w was sensitive to 0.1 mM

arsenate and its colony couldn’t be seen in 0.4 mM arsenate.

Both BY4741 and YPR200c survived in 0.5 mM arsenite. But YPR201w was more

sensitive, barely surviving in 0.2 mM arsenite. At 1.5 mM arsenite, no colony of all

three strains could be observed (Figure 4.1B). The resistance profiles were consistent

with the gene types of the strains.

4.2.2. Effect of arsenate in the culture media on methylation by cell extracts from yeast wild-type BY4741 and the arsenic-sensitive YPR201w 4.2.2.1. Time-course analysis of arsenite methylation by cell extracts from BY4741 Growth of the yeast strain BY4741 was significantly inhibited by 2 mM arsenate

(Section 4.2.1), and this concentration was chosen to investigate the effect of arsenate

on arsenite methylation in yeast. Partially purified extracts were prepared by

ammonium sulphate precipitation (Section 2.2.1.1) from BY4741 grown with and

without 2 mM arsenate. The methylation of arsenite by the partially purified extracts

(5.7-6.0 mg protein per assay) was examined at different times up to 60 minutes, after

which the radioactive products were separated by column chromatography (Section

2.2.4.). The background radioactivity was determined by methylation assays with

arsenite omitted.

When BY4741 cells were grown without arsenate in the growth medium and their

extracts were assayed in the absence of arsenite as substrate, two major background

products were found, which co-eluted with MMA and DMA in the same manner as with

Agrostis tenuis (Figure 3.4, Chapter III). When arsenite was included in the methylation

assays of extracts from BY4741 not exposed to arsenate, amounts of MMA and DMA


95

substantially above the background were present, as the reaction time increased (Figure

4.2A). The radioactivity (dpm) of MMA above background peaked at 30-min then fell

slightly. In contrast, the rising radioactivity (dpm) of DMA kept rising up to 60 min.

The apparent rates of synthesis of MMA or DMA during each reaction incubation time

is based on the sum of the radioactivities in fractions 2 to 5 for MMA and fractions 14 to

17 for DMA, the specific activity of 3H-AdoMet, and the recovery of DMA in the

extraction procedure. The maximum rates of synthesis of MMA (19.7 amol.mg

protein-1.min-1) and DMA (7.8 amol.mg protein-1.min-1) were between 15 and 30 min

(Figure 4.2A).

The presence of arsenate in the growth medium enhanced the production of MMA and

DMA (Figure 4.2B). The amounts of MMA and DMA both peaked at 15 min, but then

decreased between 15 and 60 min. The maximum rate of synthesis of MMA (37.1

amol.mg protein-1.min-1) and DMA (33.4 amol.mg protein-1.min-1) were between 6 - 15

min (Figure 4.2B).


96

Figure 4.2. Formation of methylated arsenicals (MMA and DMA) in assay mixtures containing cell extracts of yeast BY4741 (5.7 – 6.0 mg protein/assay), cultured in growth medium without (A) or with (B) 2 mM arsenate.

0 20 40 60

0

200

400

600

800

1000

MMADMA

Assay time (minutes)

0 20 40 60

0

200

400

600

800

1000


Assay time (min)MMA153060DMA153060

I.

531.91136.3963.6

92.4927.4671.4

II.

552.71426.11157.5

97.41145.01112.8

III.

20.8289.8193.9

5.0217.6441.4


I.

206.1233.9

1202.2924.1773.1

765.6682.3856.9519.5353.8

II.

240.9256.8

1614.31190.1834.0

1006.71031.61754.3927.0384.3

III.

34.822.9

412.1266.0

60.9

241.1349.3897.4407.5

30.5

A. -iAsV

B. +iAsV

I.Radioactivity of background (minusarsenite) (dpm)II.Radioactivity of methylated products (plusarsenite) (dpm)III.Radioactivity of methylated arsenicals(II.-I.) (dpm)


97

4.2.2.2. Methylation of DMA in yeast BY4741 exposed to arsenate In order to investigate whether further methylation of DMA was occurring in yeast, the

60-min arsenic methylation assay by cell extracts from BY4741 grown with 2.0 mM

arsenate was repeated using DMA in comparison with arsenite as substrate. Total

methylated products were extracted without separation and the radioactivity was

determined. In the presence of arsenite or DMA, the radioactivity of methylated

products was substantially above background (Table 4.1), indicating that DMA was

further methylated. The methylation of DMA was 83% of that of arsenite methylation.

Table 4.1. Total methylated products formed in 60-min assay mixtures with arsenite or DMA as substrate, containing cell extracts (3 mg protein) from BY4741grown in the culture media GYP with 2.0 mM arsenate.

Substrate (20 µM)

Radioactivity (dpm) Experiments Methylated Products

(Average+SEM) Methylated Arsenicals 1. 2. 3.

No arsenic 618.3 621.7 635.5 625.2+9.1 -

Arsenite 984.1 922.3 - 953.2+43.7 328.0

DMA 728.3 806.6 997.9 862.3+118.1 273.1


98

4.2.2.3. Time-course analysis of arsenite methylation by cell extracts from the yeast YPR201w

YPR201w is a yeast cell line lacking the gene acr3 that codes for the membrane pump

to exclude arsenite. Its growth was partially inhibited by 0.1 mM arsenate (Figure 4.1B).

The effect of this concentration of arsenate on arsenite methylation by cell extracts

from YPR201w was determined. The yeast was grown in GYP media with or without

0.1 mM arsenate. Partially purified extracts (Section 2.2.1.1) from YPR201w were

prepared and used in amounts containing 7.4-7.5 mg of protein during assay reaction

times of 15, 30 and 60 minutes. The procedures for the methylation assays and the

separation of the radioactive products were the same as those used for wild type

BY4741 (Section 4.2.2.1).

When extracts from yeast YPR201w, grown without arsenate in the GYP growth

medium, were assayed in the absence of arsenite as substrate, the background

methylated products co-eluted with MMA and DMA as with yeast BY4741 and A.

tenuis. When arsenite was included in methylation assays containing these extracts,

after 15 min of incubation, the radioactivities eluting from the column in the MMA and

DMA fractions were slightly below background (Figure 4.3A). After 30 min reaction,

sufficient DMA appeared to balance the background, but MMA remained below the

background. After 60 min reaction time, much greater amounts of MMA and DMA had

been formed well above the background. The maximum rates of synthesis of MMA (7.7

amol.mg protein-1.min-1) and DMA (8.2 amol.mg protein-1.min-1) occurred between 30

and 60 min (Figure 4.3A). The presence of 0.1 mM arsenate in the yeast growth medium altered greatly the pattern

of methylated product formation when extracts from YPR201w were assayed (Figure

4.3B). MMA and DMA were formed over the first 15 min at rates of 7.5 and 2.3

amol.mg protein-1.min-1 respectively. But over longer assay times, the amounts of

MMA and DMA present in the reaction mixtures fell to near zero (Figure 4.3B).


99

Figure 4.3. Formation of methylated arsenicals (MMA and DMA) in assay mixtures

containing cell extracts of yeast mutant YPR201w (7.4-7.5 mg protein/assay), cultured in

growth medium without (A) or with (B) 0.1 mM arsenate.

0 20 40 60

-200

-100

0

100

200

300

400

500

600

MMADMA


0 20 40 60

-200

-100

0

100

200

300

400

500

600


A. -iAsV

B. +iAsV


I.

846.6681.1733.6

231.4111.7104.9

II.

818.4659.2989.9

133.6113.1654.2

III.

-28.2-21.9256.3

-97.81.4

549.3


I.

641.2630.8651.0

188.788.9

117.6

II.

768.3667.8648.3

265.7116.0133.8

III.

127.137.0-2.7

77.027.116.2

I.Radioactivity of background (minusarsenite) (dpm)II.Radioactivity of methylated products (plusarsenite) (dpm)III.Radioactivity of methylated arsenicals(II.-I.) (dpm)


100

4.2.3. Effect of arsenate and arsenite concentration in culture media on the growth of E. coli strains

E. coli strains W3110 [K12 F2 IN(rrnD-rrnE)] and AW3110 (W3110 ∆arsRBC::cam)

were grown overnight in 5 ml of LB and then diluted 10 times with LB. The diluted

culture (5 µl) was spotted onto LB plates containing a range of concentrations of

arsenate from 0 to 10 mM, and 0 to 4.0 mM of arsenite. Significant growth inhibition

occurred at 8 mM arsenate and 1.5 mM arsenite for W3110, and at 0.5 mM arsenate and

0.1 mM arsenite for AW3110 (Figure 4.4).

Figure 4.4. Effect of arsenate (A) and arsenite (B) concentration in culture media on the

growth of E. coli W3110 and AW3110.

A. Arsenate (mM) W3110 AW3110

0

0.1

0.5

1.0

1.5

2.0

3.0

4.0

5.0

6.0

8.0

10.0

W3110 AW3110 B. Arsenite (mM)

0.5

1.0

2.0

1.5

2.5

0

0.1

0.2

0.4

0.3


101

4.2.4. Effect of arsenate in culture media on methylation by cell extracts from E. coli wild-type W3110 and the ∆ars strain AW3110 4.2.4.1. Time-course analysis of arsenite methylation by cell extracts from W3110 The growth of W3110 was partially inhibited by 6 mM arsenate and this concentration

of arsenate was adopted to study the effect of arsenate on arsenite methylation by

wild-type E.coli cell extracts. Partially purified extracts from W3110 grown with and

without 6 mM arsenate were prepared (Section 2.2.1.1). The methylation of arsenite by

the extracts (containing 6.0 mg of protein) was examined during assay reaction times of

15, 30 and 60 min, after which the radioactive products were separated by the same

procedures used for yeast (Section 2.2.3 and 2.2.4). The background radioactivity was

determined by methylation assays with arsenite omitted.

The amounts of MMA and DMA above background increased with reaction time up to

30 min. Longer incubation to 60 min resulted in declines of methylated products. The

apparent rates of synthesis of methylated products were 17.9 amol.mg protein-1 min-1

(MMA) and 8.8 amol.mg protein-1min-1 (DMA) during the first 30 min incubations

(Figure 4.5A).

When arsenate was present in the growth medium, a similar pattern of methylation was

observed (Figure 4.5B.). The rates of synthesis of both MMA and DMA were most

rapid during the first 15 min (27.3 amol. mg protein-1min-1 for MMA and 8.0 amol. mg

protein-1min-1 for DMA). The accumulation of MMA up to 30 min was a little higher

than that in absence of arsenate, whereas that of DMA was much lower (Figure 4.5A

and 4.5B). The rates of synthesis fell after 30 min with net loss of MMA and DMA

between 30 and 60 min (Figure 4.5B).


102


containing cell extracts of E. coli W3110 (6.0 mg protein/assay), cultured in growth medium

without (A) or with (B) 6 mM arsenate.

0 20 40 60

0

100

200

300

400

500

600

MMA

DMA


0 20 40 60

0

100

200

300

400

500

600


A. -iAsV

B. +iAsV


I.

399.5546.4576.3

670.9614.9630.0

II.

657.91037.3907.2

890.21095.9785.6

III.

258.4490.9330.9

219.3481.0155.6


I.

2148.52103.92171.5

335.0401.0328.4

II.

2522.62633.72359.2

501.0624.4428.0

III.

374.1529.8187.7

166.0223.499.6

I.Radioactivity of background (minus arsenite)(dpm)II.Radioactivity of methylated products (plusarsenite) (dpm)III.Radioactivity of methylated arsenicals(II.-I.) (dpm)


103

4.2.4.2. Time-course analysis of arsenite methylation by cell extracts from AW3110

The E. coli strain AW3110 has the ars operon deleted, impairing its ability to pump

arsenite out of the cell. A concentration of 0.5 mM arsenate was used to determine the

effects on arsenite methylation by cell extracts since it partially inhibited the growth

(Figure 4.4A). AW3110 was grown in LB media with or without 0.5 mM arsenate and

extracts were partially purified (Section 2.2.1.1). The methylation of arsenite was

determined (Figure 4.6) with 9 mg protein per assay. The procedures of methylation

assays and the separation and determination of the radioactive products were the same

as those with W3110 (Section 4.2.4.1).

When AW3110 was grown in normal LB media without arsenate, arsenite-dependent

formation of MMA proceeded after an initial lag at an approximately rate of 41.4

amol.mg protein-1min-1 at 30 min (Figure 4.6A). Unlike observed with the wild type E.

coli W3110 (Figure 4.5A), there was no fall in MMA or DMA levels after 30 min, and

much more MMA (at rate of 17.5 amol.mg protein-1min-1) but less DMA (at rate of 3.3

amol.mg protein-1min-1) were produced within AW3110.

Compared with the pattern of wild type W3110 cultured with arsenate (Figure 4.5B),

extracts from AW3110 cells grown in arsenate were less active in catalyzing

methylation of arsenite (Figure 4.6B). After an initial lag phase the MMA formed in

AW3110 reached gradually to a similar amount after 60 min, to that formed in W3110

at 30 min. However, the amount of DMA formed was much smaller.


104


containing cell extracts of E. coli mutant AW3110 (9 mg protein/assay), cultured in growth

medium without (A) or with (B) 0.5 mM arsenate.

0 20 40 60

-250

0

250

500

750

1000

1250

1500

1750

2000

MMADMA


0 20 40 60

-250

0

250

500

750

1000

1250

1500

1750

2000


A. -iAsV

B. +iAsV


I.

2217.01691.51373.8

90.984.8

600.1

II.

2320.82644.43046.5

116.080.3

868.1

III.

103.8952.9

1672.7

25.1-4.5

268.0


I.

2482.82122.91528.4

201.0198.452.6

II.

2386.62399.22037.0

197.3168.9100.8

III.

-96.2276.3508.6

-3.7-29.548.2

I.Radioactivity of background (minus arsenite)(dpm)II.Radioactivity of methylated products (plusarsenite) (dpm)III.Radioactivity of methylated arsenicals(II.-I.) (dpm)


105

4.2.5. Optimal pH for arsenic methylation in yeast and E. coli The previously described assays were all conducted at the physiological pH of 7.2.

Since the recent study by Lin et al. (2002) reported that the optimal pH was 9.5 for an

arsenite methyltransferase from rat liver, the optimal pH values for arsenic methylation

in yeast and E. coli were determined. Cells of yeast BY4741 and E. coli W3110 were

collected and resuspended in buffers with pH ranging from 5.5 to 10.0. The cells were

broken by French press and centrifuged at 27,200 g for 30 min at 4oC. The supernanants

were centrifuged at 125,000 g for 60 min at 4oC to obtain a crude cytosolic extract for

direct use in 60-min enzyme assays. Sodium arsenite was added as substrate and

arsenite omission was the control. The activity of arsenic methyltransferases per mg

extracted protein reached the highest at pH 9.2 in yeast and at pH 7.0 in E. coli extracts.

The maximum rate of formation of the methylated products of yeast was lower than that

of E. coli (Figure 4.7).

Figure 4.7. The effect of pH on arsenic methyltransferase activities by yeast BY4741 (A) and E.coli AW3110 (B).

A. yeast BY4741

7 8 9 10 11

0.0

2.5

5.0

7.5

10.0

pH

B. E. coli AW3110

5 6 7 8 9 10 11

0.0

2.5

5.0

7.5

10.0

pH


106

4.3. Discussion

4.3.1. Effects of arsenic on the growth of the wild types and mutants of yeast

and E. coli Differences in the effects of arsenate and arsenite on growth observed among the yeast

strains BY4741, YPR201w (∆arc3) and YPR200c (∆arc2) were obvious. In the highly

arsenite-sensitive YPR201w, the gene arc3 is knocked out, so that the cell has lost the

plasma membrane protein responsible for pumping arsenite from the cytosol to the

outside of the cell. The ability of wild type BY4741 to survive in arsenite

concentrations as high as 0.5 mM is attributed to the ability of this pump to maintain a

standing gradient of arsenite across the plasma membrane and hence a low internal

concentration. When YPR201w was treated with arsenate, the arsenate taken up by the

cell would be reduced to the more toxic arsenite by its active arsenate reductase, but the

arsenite could not be pumped out. As the result, the cells were nearly as sensitive to

arsenate as to arsenite in the medium. The cells of YPR201w presumably detoxify

arsenite mainly by chelation (with products perhaps transported to the vacuole) and by

methylation. This result (Figure 4.1) demonstrates that efflux of arsenite is the main

pathway for yeast to detoxify arsenic. Without the arsenite exporter, Acr3, the cell is

unable to keep the internal arsenite concentration low. The capacity for detoxification

by chelation and methylation is limited and is overcome by sufficient arsenite.

The mutant strain YPR200c was only slightly more sensitive to arsenite and arsenate

than the wild type BY4741, and much less sensitive than the YPR201w mutant.

YPR200c is defective in arc2, with loss of arsenate reductase activity. The three

detoxification pathways for arsenic, efflux, chelation and methylation, all require

arsenite as substrate (Tsai et al., 1997; Bogdan et al., 1994; Schmöger et al., 2000).

Hence deleting arsenate reductase is likely to impede all three of these pathways. Since

the arsenate transported into the YPR200c cell would not be reduced to the more toxic

arsenite, the cell’s tolerance to arsenate is higher than to arsenite on a concentration

basis. When arsenite was present in the growth media, the arsenate reductase defect had


107

a very small additional influence on YPR200c growth compared with the wild type

(Figure 4.1B). However, even if the chelation and methylation pathways in YPR201w

had not been affected by the deletion of arc3, it still exhibited more sensitivity to

arsenate than YPR200c (Figure 4.1A). Arsenate taken up by YPR201w cells is reduced

to arsenite, which is accumulated (see foregoing) and is toxic to cells. This agrees with

the general concept that arsenite is much more toxic than arsenate.

The ∆ar2 and ∆arc3 double mutant was not available to this study. Otherwise, this

double mutant could have been applied to determine the effects of arsenate and arsenite

on its growth. It could be predicted that the mutants’ sensitivity to arsenate would be

YPR201w > ∆ar2 and ∆arc3 double mutant > YPR200c > wild type BY4741, and their

sensitivity to arsenite would be ∆ar2 and ∆arc3 double mutant ≥ YPR201w >

YPR200c > wild type BY4741, because the double mutant loses the both abilities of

transforming arsenate to arsenite and pumping arsenite out. However, future

experiments are required to confirm this prediction.

A similar phenomenon was observed between the wild-type E. coli strain W3110 and

mutant AW3110 (∆ars), when grown on LB medium with a range of concentrations of

arsenite and arsenate (Figure 4.4). On account of the deletion of the ars operon,

AW3110 is expected to have lost the abilities to reduce arsenate and to pump arsenite

out of the cells, making AW3110 more sensitive to arsenate and arsenite than the wild

type W3110 (Carlin et al., 1995).

When comparing the sensitivity of yeast and E. coli to inorganic arsenic, the resistance

to arsenate is much higher in the E. coli mutant (1.0 mM) than in the yeast mutant (0.3

mM), ie. the E. coli mutant is more resistant to arsenate by a factor of 3.3. So it still has

some capability for dealing with arsenate that yeast does not have – perhaps does not

transport it into the cell so readily. Interestingly, the E. coli wild type is also 3.3 times

more resistant to arsenate than the yeast wild type, both wild types being 10 times more

resistant to arsenate than their respective mutants. For arsenite, the resistance of the


108

wild types of yeast and E. coli differ (1.5 mM for yeast BY4741, 3.0 mM for E. coli

W3110), but is similar in the mutants (0.2 mM). Assuming that both cell types take up

arsenite from the medium to a similar extent, perhaps, both mutants are left with the

same basic resources for resisting arsenite which they are unable to pump out of the cell.

Their similar sensitivity to arsenite implies that the pumping system is more important

to E. coli than yeast. Arsenite enters cells by diffusion and arsenate, perhaps, by a

plasmalemma-located transporter for phosphate (Pickering et al., 2000). The efficiency

of the processes from arsenate transport into cells, reduction to arsenite and then export

or inactivation allows cells to survive in even 10 mM arsenate in E. coli and 3 mM in

Yeast. When cells were exposed directly to arsenite, the inward diffusion overwhelmed

the export pumping of arsenite at concentrations exceeding 3 mM in the wild type E.

coli and 1.5 mM in yeast. Thus, E. coli has a more effective arsenite pumping system

and relies on this system much more than yeast which has other resistance mechanisms

such as chelation and methylation.

4.3.2. Arsenic methylation to products further than DMA occurs in S. cerevisiae

and E. coli If DMA were the end product of arsenite methylation, it would be accumulated.

Actually, both MMA and DMA decreased after incubation longer than 30-min of

extracts from wild-type yeast and the YPR201w mutant grown with arsenate. With

DMA in place of arsenite as substrate in the methylation assay, a great amount of

further methylated products were obtained (Table 4.1). Cullen et al. (1994a) reported

that trimethylarsine oxide (TMAO), formed form from DMA and MMA was the major

arsenic metabolite of the yeast Cryptococcus humiculus (Apiotrichum humicola).

Hence the major further methylated arsenical of DMA in our work may have been

TMAO. It is not known yet if any other methylated arsenicals were formed. TMAO and

other further methylated products may be detected when extracted without separation

(Table 4.1). However, these arsenic compounds were not recovered in the column

chromatography method used for separation of MMA and DMA (Section 2.2.4). There

is also the possibility of further metabolism in the assay mixtures to form volatile


109

arsenicals such as trimethyl arsine, which might be partly lost to the atmosphere

(Cullen et al., 1994a).

Having established (Section 4.2.2.2) that DMA is an intermediate rather than and

end-product in the methylation assays, it is clear that the time-course of their

concentration will be dependent on a number of enzyme activities. For example, DMA

formation from arsenite requires arsenite methyltransferase, MMA reductase and

monomethylarsonous acid methyltransferase. Further methylation of DMA requires

additional methyltransferases and reductases. If the relative activities of the

DMA-forming and DMA-methylating enzymes changes during the long 60 min

time-course of the assay, accumulation or loss of DMA could occur at different times.

The same applies to MMA. It is concluded that the DMA methylating activities are

more stable than the activities forming DMA, leading to a cessation of accumulation

and progressive loss after 20 to 30 min incubation.

In the arsenic methylation assays with extracts from the E. coli wild type strain W3110,

no matter whether there was arsenate in the bacteria growth medium or not, MMA and

DMA were accumulated to the peaks by 30-min incubations. After 30 min, both

amounts of MMA and DMA decreased faster in the presence of arsenate in the culture

medium than in its absence, implying that exposure to arsenate accelerated further

methylation after DMA (Figure 4.5). Further arsenic methylation after DMA has been

demonstrated in some bacteria (Section 1.3.3.1.1). The E. coli strains isolated from rats

could even convert dimethylarsinate and trimethylarsine to nonvolatile, unidentified

compounds (Kuroda et al., 2001). It is probable that further methylated products are

less toxic or/and much easier to be excluded from cells (eg. volatile), as observed in

yeast (Cullen et al., 1994a).

4.3.3. The deletions of arc3 and ars genes affect arsenic methylation Comparing the arsenic methylation profiles of the wild type yeast BY4741 and E. coli


110

W3110 with their ∆arc3 and ∆ars mutants (Figure 4.2 vs 4.3; 4.5 vs 4.6), a general

trend is the delay in the accumulation of MMA and DMA by the extracts from both the

yeast and E. coli mutants. This lag with time was more pronounced with DMA than

with MMA in E. coli. The reasons why the deletion of the arsenite exclusion systems

leads to this lag in accumulation are not known. It was also noticed that when arsenate

was not present in the growth media, the yeast and E. coli mutants exhibited higher

methylation activities than their wild type strains, respectively. Again it was more

pronounced in the case of E. coli (Figures 4.2A vs 4.3A; 4.5A vs 4.6A). However, the

increased arsenic methyltransferase activities were not found in the extracts from the

mutants that had been grown in media containing arsenate. If it was the opposite (i.e.

arsenate present in the media enhanced methyltransferase activities in the mutant

extracts more than the wild types), one could postulate that the increased

methyltransferase activities would have been due to the cells’ increased dependence on

the methylation pathway when the exclusion pump systems had been best.

In AW3110, arsenate in the culture medium inhibited the activities of arsenic

methyltransferase, resulting in much lower MMA and DMA production than those in

absence of arsenate. This confirms that the ars pump system is the major pathway to

eliminate arsenate. As to the methylation by extracts from AW3110, both MMA and

DMA increased after 60 min of incubation (Figure 4.6), whereas they decreased in the

wild type W3110 after 30 minutes (Figure 4.5). One of the probable reasons is that the

further methylation processes after MMA and DMA were affected by the ars operon

deletion.

4.3.4. Arsenate induction of methylation in yeast and E. coli Only 15 min were required for both BY4741 and YPR201w grown in the presence of

arsenate to accumulate substantial amounts of methylated arsenicals, whereas at least

30 minutes were required for MMA without arsenate. It seems that acute exposure of

yeast to arsenate stimulated arsenic methylation in yeast. Because arsenite in the cells


111

of YPR201w could not be pumped out of the cell and thus inhibited the activities of

enzymes, the amounts of methylated products in the assays with extracts from

YPR201w grown in the presence of arsenate were much less than that of BY4741. However, the general time course of the amounts of methylated arsenical products was

similar between W3110 and AW3110 grown with and without arsenate in growth

medium. Arsenate in culture medium seemed not to induce more arsenic

methyltransferase activity in E. coli cells as it did in yeast.


112

4.3.5. The inducibility of arsenic methylation by acute concentrations of arsenate

Some arsenic methylation was found with extracts of yeast BY4741 and YPR201w

grown in GYP media without arsenate. Thus, arsenite methyltransferase activity in

yeast seems to be only partially inducible, in comparison with these enzymes in

mammalian cells, which generally do not exhibit any induction (Healy et al., 1998). In

the arsenic-accumulating plant, A. tenuis, arsenate in the growth medium is required for

arsenite methylation and the enzyme activities are almost fully induced by arsenate

(Chapter III). No arsenic methylation was detected in some plants such as corn, a

monocot, and the dicots melon, pea and tomato grown in sufficient nutrients (Nissan

and Benson, 1982).

In yeast, the constitutive arsenic methylation is adequate for arsenic encountered in

normal environments. But when arsenic is present in acutely high concentrations, the

induced methyltransferase activities appear. It is not known how plants evolved

methylation induction, nor whether such induction only occurs in some arsenic-tolerant

plants. In the case of, Ladder brake fern, (Pteris vittata L.), concentrations of 50-100

mg As kg-1 in soils stimulated plant growth, arsenic uptake and translocation (Tu and

Ma, 2002). It will be worthwhile to investigate whether there is induction of arsenic

methyltransferase activity in the fern Ladder brake. Numerous unsuccessful attempts

were made to detect an inducible tolerance response to arsenite in a variety of human

cells. Human cells, like the arsenite hypersensitive Chinese hamster As/S27D cell line,

lack an inducible tolerance response. In contrast, wild-type Chinese hamster V79 cells

and their arsenite-resistant variants were found to have an arsenite- and

antimonite-inducible tolerance mechanism (Rossman et al., 1997). Although no

evidence of inducible arsenite methylation has been found in human cells, it is possible

that such induction exists in human cell types not yet tested.


113

4.3.6. The possible source of the methylation background

In all methylation assays, 20 µM arsenite was used which was much lower than the

concentration of arsenate in culture media (0.1 mM for YPR201w and 0.5 mM for

AW3110). The methylated arsenicals extracted from the assays should include the

arsenite reduced from any arsenate transported into the cells during growth. The

amounts of these in vivo methylated products were not detected. Considering 3H-AdoMet used in the in vitro assays, these backgrounds formed in vivo should not be

detectable in our experiments and thus did not affect the results.

When extracts from wild type strain BY4741 grown in arsenate-free GYP media were

assayed in the absence of arsenite, a small amount of radioactivity was extracted which

was not carry-over of 3H-AdoMet. A similar phenomenon was observed in Agrostis

tenuis (Section 3.2.2.3). Since there was no other resource of arsenic, it is likely that

this background methylation involves methyltransferases specific for substrates other

than arsenite, but subject to inhibition by arsenite. The methylated compounds in the

background resemble MMA and DMA, since they co-elute with MMA and DMA by

column chromatography. The inhibition by arsenite of the background methylation can

explain why the MMA and DMA formation from the extracts of YPR201w cultured

with 0.1 mM arsenate were negative (Figure 4.3B). This will occur whenever the

inhibition by arsenite exceeds the formation of methylated arsenic.

Chapter V. Identification of Arsenic Methyltransferase Genes in Saccharomyces cerevisae

114

Chapter V

Identification of Arsenic Methyltransferase Genes in

Saccharomyces cerevisiae


115

5.1. Introduction

Methylation of arsenic is obviously a common feature of the metabolism of this

metalloid in organisms ranging in complexity from microorganisms to plants and

animals, including humans (Cullen and Reimer, 1989; Fujiwara et al., 2000; Nissen

and Benson, 1982; Wu et al., 2002; Aposhian, 1997). The mechanism of arsenic

methylation includes arsenate reduction to arsenite and arsenite methylation to MMA,

which is then reduced and methylated to DMA. The methyl donor in bacteria is

methylcobalamin (McBride and Wolfe, 1971), whereas it is S-adenosylmethionine in

yeast, fungi and algae (Cullen and Reimer 1989). Arsenic methylation has been

confirmed to be an enzymatic process in plants (Chapter III), mammalian cells

(Zakharyan et al., 1995a; 1999), bacteria and yeast (Section 4.2). Partially purified

and kinetically characterized enzymes include arsenic methyltransferase [from rabbit

liver and Chang human liver hepatocytes (Zakharyan et al., 1999)], arsenate reductase

[from human liver (Radabaugh and Aposhian, 2000)] and MMAV reductase [from

rabbit liver (Zakharyan and Aposhian, 1999)]. The Km of rabbit liver

methyltransferase was 5.5 µM for arsenite and 9.2 µM for MMAIII.

Enzymatic arsenic methylation has been demonstrated in yeast and E. coli (Chapter

IV) but the genes responsible have not been identified. Taking advantage of the in

vitro assay system developed in the work reported in Chapter 4, yeast S. cerevisiae

genome database, and the availability of yeast knockout mutants and yeast expression

clones, a search has been made for the yeast arsenic methyltransferase genes. In

particular, genes with predicted S-adenosylmethionine-dependent methyltransferase

activities have been sought, and the outcome is reported in this chapter.

Thirteen putative methyltransferase genes were tested by the analysis of their

knockout mutants using in vitro enzymatic arsenic methylation. A large amount of a

yeast fusion protein could be obtained from overexpressing yeast clones (Yeast

ExClones), and used to determine the arsenic methyltransferase activities of the gene

product.


116

5.2. Results

5.2.1. Preliminary search for possible arsenic methyltransferase genes by genome database searching

The Saccharomyces cerevisiae genome database [http://www.yeastgenome.org/],

funded by the National Human Genome Research Institute at the US National

Institutes of Health, was used to search for putative methyltransferase genes in this

study.

At the beginning of this study, when “methyltransferase” was used as the searching

keyword, 36 genes were considered responsible for predicted or identified

methyltransferases, based on the Gene Ontology annotations (Table 5.1A, B).

Table 5.1. Methyltransferases (MTs) annotated in the S. cerevisiae genome database

A. Function assigned MTs

Genes Product Reference MET6 5-methyltetrahydropteroyltriglutamate-

homocysteine S-MT Smith et al. 1996

PPM1 C-terminal protein carboxyl MT Kalhor et al. 2001 SPB1 RNA MT Pintard et al. 2002b CTM1 [cytochrome c]-lysine N-MT Polevoda et al. 2000 COQ3 hexaprenyldihydroxybenzoate MT Clarke et al. 1991 SET2 histone-lysine N-MT Strahl, et al. 2002 MHT1, SAM4 homocysteine S-MT Thomas D et al. 2000 ABD1 mRNA (guanine-N7)-MT Schroeder et al. 2000 IME4 mRNA- MT Mizuno et al. 1998 RRP8 MT homolog ; nucleolar protein required for

efficient processing of pre-rRNA at site A2; Bousquet-Antonelli et al. 2000

OPI3 phosphatidyl-N-methylethanolamine N-MT Paltauf et al. 1992 CHO2 phosphatidylethanolamine N-MT Summers et al. 1988 STE14 protein-S-isoprenylcysteine O-MT Hrycyna and Clarke 1990 HMT1, RMT2 protein-arginine N-MT Green et al. 2002;

Niewmierzycka and Clarke 1999 PET56 rRNA (guanine-N1-)-MT Sirum-Connolly and Mason 1995 MRM2 rRNA (uridine-2’-O-)-MT Pintard et al. 2002a NCL1 tRNA (cytosine-5-)-MT Motorin and Grosjean 1999 TRM1 tRNA (guanine-N2-)-MT Ellis et al. 1986 TRM2, TRM7 tRNA MT Pintard et al. 2002b COQ5 ubiquinone biosynthesis MT Barkovich et al. 1997

B. Unknown function putative MTs

Genes Products Reference YDR140W, YDR316W, YHR209W, YNL063W

S-adenosylmethionine-dependent MT Niewmierzycka and Clarke 1999

YDL201W1 Good match to Conserved MT motifs 1. Alexandrov et al. 2002 YER175C2, YGL136C3, YIL064W, YNL092W, YPL157W4,5

Putative MTs 2. Cai et al. 2001; 3. Pintard et al. 2002; 4. Verheggen et al. 2002; 5. Mouaikel et al. 2002

YBR261C, YBR271W YHL039W

Unknown function proteins

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117

Among these 36 genes, 23 genes for 20 different methyltransferases had been

recognized. Some of these were regarded as putative rather than confirmed

methyltransferases; some had been found to function in some biological processes

(Table 5.1A).

The other thirteen genes with hypothetical open reading frames (ORFs) were chosen

among the 36 genes for this study (Table 5.1B). None of these had been assigned to

any specific methyltransferases. Among these 13 genes, there were four ORFs

(YHR209w, YNL063W, YDR140W and YDR316W) whose deduced protein

sequences have strong similarity to probable methyltransferases and match the

conserved methyltransferase motifs to bind S-adenosyl-methionine (Ado-Met)

(Niewmierzycka and Clarke, 1999).

Even though S-adenosylmethionine has been found to be the methyl donor of arsenic

methylation in yeast, fungi and algae (Cullen and Reimer, 1989), in order not to miss

any methyltransferases that possibly use other methyl donors or whose functions

could not be predicted by the protein sequences, other genes for putative

methyltransferases were considered. Nine genes whose functions were unknown but

possibly related to methyltransferase were found in the S. cerevisiae genome database

(Table 5.1B). After the start of this study, specific methyltransferase activities have

been reported by others for four ORFs (YDL201w, YER175c, YGL136c and

YPL157w) among these nine.

Mutant strains for the thirteen selected genes, based on wild type BY4741, were

obtained from EUROSCARF, Germany (Section 2.1.2). In each of these strains, an

ORF for a putative methyltransferase was knocked out.


118

5.2.2. Growth response of the yeast knock-out mutants to arsenate

Based on the hypothesis that yeast strains defective in arsenic methylation become

more sensitive to the toxic effects of arsenic, a range of arsenate concentrations from

0 to 5.0 mM were utilized to determine the arsenate sensitivity of the 13 yeast knock-

out mutants listed in Table 5.1. Cultures of BY4741 (wild type) and YPR201w (∆arc3)

were used as positive and negative controls. Spots of 2 x 105 cells of each strain were

applied to the plates and then incubated at 30oC for two days (Section 2.1.2).

YPL157w, YGL136c and YDR140w cells grew slowly on the complete culture

medium even without arsenic (Figure 5.1) and, notably, these three strains also grew

more slowly than the other strains in liquid culture media (data not shown). The other

ten strains grew similarly to the positive control BY4741 in GYP media and showed

similar responses to arsenate: growth was inhibited by arsenate concentrations

exceeding 3.0 mM and the cells died at concentrations of 4.0 mM and above. There

were no consistent differences in growth between any of these ten strains and the wild

type control on the arsenate plates. Conversely, the negative control YPR201w (∆arc3)

was sensitive to 0.1 mM arsenate and died in concentration of 0.3 mM and above as

observed previously (Section 4.2.1).


119

Position of 13 mutants on plates: YDL201w YIL064w YNL092w YPL157w

YHL039w YBR261c YBR271w YGL136c

YHR209w YNL063w YDR140w YDR316w

YER175c YIL064w Negative control: YPR201w

Positive control: BY4741

Figure 5.1. Effects of arsenate on 13 yeast knockout mutants Spots of YPL157w, YGL136c and YDR140w were larger to contain the same amount of cells because of slow growth in the liquid media.

0

0.5

3.0

0.1

1.0

4.0

0.3

2.0

5.0

Arsenate (mM)

Arsenate (mM)

Arsenate (mM)


120

5.2.3. Determination of arsenic methyltransferase activities in cell extracts

from knock-out mutants

The arsenate sensitivity assay did not reveal any elevated sensitivity mutants. Ten

mutant strains exhibited resistance to arsenate similar to the wild type BY4741. The

other three, YDR140w, YGL136c and YPL157w, grew slowly even in GYP media

without arsenate, requiring a further two days before they appeared on the arsenate

plates. These three slow-growing strains were omitted from further studies of the

activities of arsenic methyltransferases, since their knocked out genes might be related

to normal growth, rather than arsenic methylation.

Methyltransferases for arsenite methylation in yeast do not require arsenate induction

for expression (Chapter IV); therefore, each of the 10 mutant strains was grown in 350

ml of GYP complete media without arsenate. Partially purified extracts from each

strain were prepared (Section 2.2.1.1) and the methylation of arsenite by the extracts

containing 6 - 7.5 mg of protein was examined during assay reaction times of 60

minutes, after which the radioactive products were separated by column

chromatography (Section 2.2.4). The background radioactivity was determined by

methylation assays with arsenite omitted.

The extracts from the wild type strain BY4741 generated two main methylated

product peaks in the elution positions of MMA and DMA (Section 4.2.2.1). All the 10

mutant strains also generated these peaks in the same positions when arsenite was

absent and when present as substrate (Figure 5.2). The arsenite-dependent amounts of

MMA and DMA formation were estimated from the difference between the assays

with and without arsenite (Table 5.2). The proportions of arsenite-dependent to the

total methylated product at each elution position were also calculated (Table 5.2).

Apparent arsenite-dependent formation of MMA was negative in YER175c and

YHR209w. In the other 8 strains, arsenite stimulated MMA formation by 2.6% to

19.1%. In contrast, arsenite increased DMA formation in all 10 strains by 14.8 to

136.8%.


121

Table 5.2. Arsenite-dependent methylation products of cell extracts from 10 mutant strains

Strains MMA Peak (dpm)

DMA Peak (dpm)

% arsenite-dependent MMA DMA

BY4741(wt) 193.9 441.4 20.1 65.7 1.YDL201w 130.6 283.9 13.4 119.3 2.YIL064w 130.5 82.2 10.9 17.0 3.YNL092w 81.8 226.1 8.0 67.9 5.YHL039w 164.6 430.1 12.4 136.8 6.YBR261c 196.7 116.4 14.7 23.3 7.YBR271w 216 162.9 19.1 24.2 9.YHR209w -85.6 29.8 NV* 14.8 10.YNL063w 29.2 165.9 2.6 92.5 12.YDR316w 74.5 136.1 9.0 38.7 13.YER175c -29.4 198.7 NV* 77.7%

“*” denotes a negative value.


122

1. YDL201w

MMA DMA0

500

1000

1500

-arsenite +arsenite

2.YIL064w

MMA DMA0

500

1000

1500

3.YNL092w

MMA DMA0

500

1000

1500

5.YHL039w

MMA DMA0

500

1000

1500

6.YBR261c

MMA DMA0

500

1000

1500

7.YBR271w

MMA DMA0

500

1000

1500

9.YHR209w

MMA DMA0

500

1000

1500

10.YNL063w

MMA DMA0

500

1000

1500

13.YER175c

MMA DMA0

500

1000

1500

12.YDR316w

MMA DMA0

500

1000

1500

Figure 5.2. Methylated products of arsenite methylation by cell extracts from 10 mutant strains(From data of Table 5.2) Light columns indicate the background activity in the absence of arsenite in the assays.Dark columns indicate the activity in the presence of arsenite in the assays.


123

Methylation assays with extracts from YHR209w and YER175c were repeated in

triplicate data (Figure 5.3), giving results that confirmed the previous trend (Figure

5.2). Although arsenite-dependent MMA formation was absent, the production of

DMA was stimulated by arsenite, particularly for YER175c. Extracts from YNL063w

behaved similarly (Figure 5.2).

Table 5.3. Arsenite-dependent methylation products of cell extracts from YHR209w and YER175c

Standard error of mean (SEM) of three determinations is shown.

Strains MMA (dpm ±SEM)

DMA (dpm ±SEM)

% arsenite-dependent MMA DMA

9.YHR209w -2.3±74.0 57.4±40.0 -0.5±8.2 25.9±13.5 13.YER175c -11.5 ±40.2 334.4 ±195.7 -1.4 ±4.0 171.6 ±138.2

Figure 5.3. Methylated products by cell extractsYHR209w and YER175c (From data of Table 5.3) Light columns indicate the background activity in the absence of arsenite in the assays. Dark columns indicate the activity with the presence of arsenite in the assays.

0

500

1000

1500

9.YHR209w -SEM

- arsenite

+ arsenite

MMA DMA0

500

1000

1500

13.YER175c-SEM

MMA DMA


124

5.2.4. Expression and digestion of fusion proteins of GST-YHR209w and GST- YER175c

To further investigate whether the knocked-out putative methyltransferases in

YHR209w and YER175c have arsenic methyltransferase activities, two strains

harbouring pYEX4T-1/YHR209w-GST and pYEX4T-1/YER175c-GST [Yeast

ExClones, http://www.dbsr.duke.edu/yeast/Genome% 20Libraries/exclones.htm] were

obtained from ResGen (Invitrogen Corporation). Each Yeast Exclone is able to

express a full-length yeast open reading frame (ORF) fused to glutathione S-

transferase (GST) by the vector pYEX4T-1 (Figure 5.4A). The GST-fusion

expression is under the control of the CUP1 promoter and thus can be induced by

Cu2+. About 10 mg fusion protein/L media was obtained after 15 hours induction. The

two fusion proteins were separated and purified by affinity column chromatography (5

ml of GSH–Sepharose 4B, Pharmacia Biotech, Section 2.2.6), and then were used

directly in arsenic methylation assays.

Neither fusion proteins showed any arsenic methyltransferase activity (Section 5.2.5).

In order to determine whether the fused GST peptide caused loss of arsenic

methyltransferase activity, removal of the GST by partial thrombin digestion was

attempted. The fusion protein GST-YHR209w was treated with thrombin at 0oC and

19oC. The fusion protein GST-YER175c was digested by thrombin at 19oC. There are

four internal potential hydrolysis sites on protein YHR209w for thrombin (Figure 5.5).

The hydrolysis was temperature-sensitive (Figure 5.6). Both temperature treatments

resulted in 27 kD bands, corresponding to GST. Low-temperature treatment produced

fragments from 13.8 to 33.8 kD and around 60 kD. The main fragments distributed

around 34 kD protein YHR209w. The 19oC treatment gave much fewer but more

concentrated bands than the 0oC treatment. A major band of 36 kD from the 19oC

treatment corresponded to the complete YHR209w protein with a short N-terminal

extension (23 amino acids: NYLFDDEDTPPNPKKEIEFQLTT) of 2.8 kD between proteins

YHR209w and GST. The peptide of 23 a.a. was formed due to an insertion

(AACTATCTATTCGATGATGAAGATACCCCACCAAACCCAAAAAAAGAGATCGAATTCCAGC

TGACCACC) between GST and the ORF of YHR209w during the construction of the

plasmid (ftp://ftp.resgen.com/pub/yeastex/).

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125

Thrombin recognition site CTGGTTCCGCGTGGATCCAACTATCTATTCGATGATGAAGATACCCCACCAAACCCAAAAAAAGAGATCGAATTCCAGCT BamHI

GACCACC-ORF-ATGGCAATTCCCGGGGATCCGTCGACCTGCAGAGATCTATGAATCGTAGATACTGAAAAACCCCGCAAG

Thrombin recognition site

2.8 kD Figure 5.4. A. Structure of pYEX4T-1 Vector (Protocol # PT3046-5, 1998, CLONTECH

Laboratories, Inc. Version # PR81821; ftp://ftp.resgen.com/pub/yeastex/) Thrombin recognition site is shown. B. Structure of fusion protein.

MPKTSYLNKNFESAHYNNVRPSYPLSLVNEIMKFHKGTRKSLVDIGCGTGKATFVVEPYF KEVIGIDPSSAMLSIAEKETNERRLDKKIRFINAPGEDLSSIRPESVDMVISAEAIHWCNLER LFQQVSSILRSDGTFAFWFYIQPEFVDFPEALNVYYKYGWSKDYMGKYLNDNQREILLYG GEKLRSLLSDRFGDIEVTIYSPSDPNASTVTAENSQFLWRAAITLNQFKEFVKSWSIYTSWA RDNPSKPDIADIFINELKEICHCEDLNVPLKIEWSTFYYLCRKRE Figure 5.5. Predicted additional hydrolysis sites for thrombin in the protein sequence of

YHR209w

GST (27kD) Target protein

NYLFDDEDTPPNPKKEIEFQLTT

N C

A.

B.

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126

60

30

1510

2520

40

50

708090

100120160220

27KD

GST-YHR209wGST-YER175c

13.83K,14.11K, 15.25K,

19.58K

36KD 31.4KD, 32.8KD,33.32K, 33.785K

1 2 3 4 5 6

60

30

1510

2520

40

50

708090

100120160220

27KD

GST-YHR209wGST-YER175c

13.83K,14.11K, 15.25K,

19.58K

36KD 31.4KD, 32.8KD,33.32K, 33.785K

1 2 3 4 5 6 Figure 5.6. SDS-PAGE (4-12% NuPAGE Bis-Tris Gel/MOPS running Buffer, Invitrogen) of fusion

protein GST-YHR209w treated with and without thrombin at 0 ˚C and 19 ˚C. The fusion protein GST-YER175c and thrombin were used as controls.

Lane 1: Marker (BenchMark™ Protein Ladder, Invitrogen); Lane 2: Treated with thrombin at 19 ˚C for 2.5 hrs (6 µg); Lane 3: Treated with thrombin at 0 ˚C for 20 hrs (7 µg); Lane 4: GST-YHR209w without thrombin treatment (6 µg); Lane 5: GST-YER175c without thrombin treatment (6 µg); Lane 6: Thrombin (3 µg).


127

5.2.5. Arsenite methylation activities of fusion proteins GST-YHR209w, GST-

YER175c and their thrombin digestion products

The thrombin-treated and untreated fusion proteins of YHR209w-GST and YER175c-

GST were tested for arsenic methyltransferase activity. Each protein (400 µg) was

assayed with reaction times of 60 minutes, using arsenite as substrate. The substrate

was omitted for background controls. The radioactive products were extracted and

separated by column chromatography (Section 2.2.3 and 2.2.4).

Neither untreated fusion protein, YHR209w-GST or YER175c-GST, showed any

arsenite methyltransferase activity (Figure 5.7). After both HCl and NaOH elution, the

amounts of methylated products with arsenite as substrate were lower than with

arsenite omitted.

Figure 5.7. Methylation of arsenite by fusion proteins GST-YHR209w (A) and GST-YER175c (B). Methylated arsenicals were extracted and fractionated by column chromatography (Section 2.2.3 and 2.2.4). The 3H-methyl radioactivity in each elution is shown after 60 min incubation. Light column: the background in the absence of arsenite as substrate. Dark column: the methylated products in the presence of arsenite as substrate.

However, after YHR209w-GST was digested with thrombin, the methylated arsenical

MMA increased in the HCl elution and exceeded the background. The amount of

MMA formed by thrombin treatment at 19°C for 2.5 hours was more than that at 0°C

for 24 hours (Figure 5.8A, B.). No arsenite-dependent DMA formation was observed

after either thrombin digestion.

MMA DMA0

100

200

300

(- arsenite )

(+ arsenite )

A. GST-YHR209w

MMA DMA0

250

500

750 B. GST-YER175c


128

For the fusion protein YER175c-GST, no evidence for any arsenite-dependent

formation of methylated arsenicals was apparent (Figure 5.8C) after this fusion

protein was treated by thrombin at 19°C for 2.5 hours. Figure 5.8. Methylation in the absence and presence of arsenite by GST fusion proteins treated with thrombin. Methylated arsenicals were extracted and fractionated by column chromatography (Section 2.2.3 and 2.2.4). The 3H-methyl radioactivity in each elution is shown after 60 min incubation. A. Arsenite methylation by GST-YHR209w treated with thrombin at 19°C for 2.5 hrs; B. Arsenite methylation by GST-YHR209w treated with thrombin at 0°C for 20 hrs; C. Arsenite methylation by GST-YER175c treated with thrombin at 19°C for 2.5 hrs. Light column: the background in the absence of arsenite as substrate. Dark column: the methylated products in the presence of arsenite as substrate.

B. GST-YHR209w at 0 oC

MMA DMA0

100

200

300

C. GST-YER175c at 19 oC

MMA DMA200

250

300

350

A. GST-YHR209w at 19 oC

MMA DMA0

100

200

300

(- arsenite) (+arsenite)


129

5.2.6. Test for MMA methyltransferase activities of digested proteins GST-

YHR209w and GST-YER175c

To examine the possibility that monomethylarsonate (MMA) is a substrate for

methylation by the proteins YHR209w and YER175c, both fusion proteins GST-

YHR209w and GST-YER175c were treated with thrombin for 2.5 hours at 19°C and then

tested for methylation activity. The digested fusion proteins (200 µg) were assayed in

triplicate by incubation for 60 minutes in the presence and absence of MMA as substrate.

The methylated products were extracted in chloroform and radioactivities detected

directly in the scintillation counter (Section 2.2.3). The radioactivities of products for

both proteins were slightly lower with MMA as substrate than in the background controls

(Figure 5.9). No evidence for any MMA-dependent methylation was evident.

Figure 5.9. Methylation in the presence and absence of MMA by GST fusion proteins treated with thrombin at 19°C for 2.5 hrs. The assays were carried out in triplicate and standard error of mean (SEM) was shown.

0

100

200

300

400

500 (-MMA) (+MMA)

GST-YHR209w GST-YER175c


130

5.2.7. Homology search for arsenite methyltransferase sequences in yeast genome

After the experiment work reported in Chapter V was finished, two publications

identifying a mammalian (Lin et al., 2002) and a bacterial (Wang et al., 2004) arsenic

methyltransferases at the molecular level appeared.

Lin et al. (2002) reported a novel AdoMet:arsenic(III) methyltransferase from rat liver

cytosol (GenBankTM/ EBI Data Bank accession number(s) AF393243). A FASTA query

on S. cerevisiae sequences for this novel AdoMet:arsenic (III) methyltransferase (Length:

369 a.a., MW: 34.8 kD) was performed using the Saccharomyces Genome Database

(http://genome-www4.stanford.edu) (Table 5.4). This identified 16 proteins with higher

scores than YHR209w (Length: 292 a.a., MW: 33.8 kD). Three (including YIL064w) of

these sixteen proteins are of unknown function. A BLAST query was also performed

within the Saccharomyces Genome Database (Table 5.5). YHR209w was the fourth best

match with score 81 (33.6 bits) and P 0.042, just after other three known proteins HMT1,

ABP140 and COQ5. YIL064w also produced a high BLAST score but ranked behind

YHR209w. YIL064w had been selected for this study (Table 5.1).

A global alignment of protein YHR209w sequence (291 a.a.) and rat AdoMet:arsenic(III)

methyltransferase (369 a.a.) was carried out. The alignment result shows (Figure 5.10)

the typical motifs of S-AdoMet-dependent methyltransferase indicated by horizontal lines.

Interestingly, both proteins have a C-terminal cysteine-rich region in which C364, C365 and

C371 for rat arsenite methyltransferase compared with C269, C271 and C289 for YHR209w.

Wang et al. (2004) reported that ArsM in Halobacterium sp., is possibly responsible for

arsenite methylation (NCBI accession: NC_001869, protein ID: NP_046066.1). FASTA

and BLAST queries on S. cerevisiae sequences were performed using the Saccharomyces

Genome Database. YHR209w matches ArsM poorly (data not shown). The global

alignment (Figure 5.11), scores much lower than YHR209w vs. rat AdoMet:arsenic(III)

methyltransferase. No common methyltransferase motif was found in ArsM.

http://genome-www4.stanford.edu/�

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131

Table 5.4. FASTA search results for rat AdoMet:arsenic(III) methyltransferase

FASTA (3.47 Mar 2004): Query performed by the Saccharomyces Genome Database; Query Sequence: S-adenosylmethionine:arsenic (III) methyltransferase (Length: 369) Database: Translation of all Standard S. cerevisiae ORFs; 3024823 residues in 6699 sequences Expectation_n fit: rho(ln(x))= 4.5594+/-0.000952; mu = 12.6847+/- 0.056 mean_var = 64.4607+/-12.340, 0's: 0 Z-trim: 5 B-trim: 0 in 0/50 Lambda= 0.159745 Kolmogorov-Smirnov statistic: 0.0239 (N=29) at 46 function [optimized, BL50 matrix (15:-5)] ktup: 2, join: 37, opt: 25, open/ext: -10/-2, width: 16

The best score sequences: Length of Prot. (a.a.)

opt bits E(6699) Systematic

Name Standard Name

YOR239W ABP140 629 116 35.7 0.028 YML110C COQ5 308 112 345 0.031 YML008C ERG6 384 97 31.1 0.41 YIL064W YIL064W 258 95 30.5 0.42 YOL096C COQ3 313 93 30.1 0.66 YDR243C PRP28 589 91 29.9 1.4 YDR406W PDR15 1530 95 31.2 1.5 YCL024W KCC4 1038 91 30.1 2.2 YBR226C YBR226C 137 78 26.3 4 YPR035W GLN1 371 81 27.4 5.1 YJL125C GCD14 384 80 27.2 6.1 YOR216C RUD3 48.5 79 27.1 8.5 YCR047C BUD23 276 76 26.2 9.1 YBR248C HIS7 553 79 27.1 9.4 YBR240C THI2 451 78 26.8 9.5 YFL049W YFL049W 624 79 27.2 10 YHR209W YHR209W 292 74 25.7 13

Table 5.5. BLAST search results for rat AdoMet:arsenic(III) methyltransferase

BLASTP 2.0MP-WashU [07-Dec-2002] [sol8-ultra-ILP32F64 2002-12-07T21:43:03] Copyright (C) 1996-2002 Washington University, Saint Louis, Missouri USA. All Rights Reserved. Query Sequence: S-adenosylmethionine:arsenic (III) methyltransferase (Length: 369) Database: Translation of all Standard S. cerevisiae ORFs; 5878 sequences; 2,922,576 total letters.

Sequences producing High-scoring Segment Pairs: High Score Smallest Sum Probability P(N)

N

YBR034C HMT1 protein-arginine N-MT 88 0.0095 1 YOR239W ABP140 actin filament binding protein 87 0.027 1 YML110C COQ5 ubiquinone biosynthesis MT 82 0.036 1 YHR209W YHR209W 81 0.042 1 YML008C ERG6 delta(24)-sterol C-MT 78 0.13 1 YIL064W YIL064W 72 0.30 1 YML032C RAD52 DNA strand annealing activity and recombinase 53 0.59 3 YGR171C MSM1 methionine-tRNA ligase 69 0.90 1 YOL096C COQ3 hexaprenyldihydroxybenzoate MT 63 0.992 1 YOR216C RUD3 Golgi matrix protein 67 0.992 2 YLR205C HMX1 heme oxygenase 61 0.999 1 References: Gish, W. (1996-2002) http://blast.wustl.edu

MT: methyltransferase

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132


133

10 20 30

YHR209 M--PK-T-------SYL-N--KNFESA--HYNN--VRPS---YP--L--SL-VN--E-IM : :. . .: : :. :: . .: : :. : . :: : : .. rat_As MAAPRDAEIHKDVQNYYGNVLKT--SADLQ-TNACVTPAKGV-PEYIRKSLQ-NVHEEVI 10 20 30 40 50 40 50 60 70 YHR209 -KFH-------K---GTR-KSLVDIGCGTGK-ATFV----V-EPYFK-EVI-GIDPSSAM ... . . : : :.: :.:. .: : . : . : ::: : rat_As SRYYGCGLVVPEHLENCRI--L-DLGSGSGRDC-YVLSQLVGQ---KGH-ITGID----M 60 70 80 90 100 80 90 100 110 YHR209 LS-I-AE--K---E--TNERRLD-KK--IRFINAPGE-D-LS-S-I-RPESVDMVISAEA . . .: : : : :. . . . :.. :. . :. . : . :: :.::: rat_As -TKVQVEVAKAYLEYHT-EK-FGFQTPNVTFLH--GQIEMLAEAGIQK-ESYDIVIS--- 110 120 130 140 150 120 130 140 150 YHR209 IHWC--NL--ER---L---FQQVSSILR-SDGTFAFWF---Y--IQPEFV--DFPEALN- . : :: .. : .: : :. . : . . : : . : : :. .. . rat_As -N-CVINLVPDKQKVLREVYQ-V---LKYG-GEL-Y-FSDVYASL--E-VSEDI-KS-HK 160 170 180 190 200 160 170 180 190 YHR209 VYY-K-YG----WSKDYM-------GKY----L-N-DNQREIL-LNYGGEK-L-RSLLSD : . . : : :: . : . : . : :. . :. : : : .:.: rat_As VLWGECLGGALYW-KD-LAVIAKKIG-FCPPRLVTA-N---IITV--GN-KELER-VLGD 210 220 230 240 200 210 220 230 YHR209 -RFGDIEVT--IYS-P-SDPNAS-T--V-TA-----ENSQFLWRAAITL--N-QFKE--- :: . .: ... : ..: :. : .. :. . : : . : ::: rat_As CRF--VSATFRLFKLPKTEP-AGRCQVVYNGGIMGHEK-E-L----I-FDANFTFKEGEA 250 260 270 280 290 240 250 260 270 YHR209 FV---K-SWS-IY-TS-WARD---NP---S---KPD--IA--D-IFINELKEICHCE-D- : . . . : .: .:.: .: : :. . : : .: : : : rat_As -VEVDEET-AAILRNSRFAHDFLFTPVEASLLA-PQTKVIIRDP-F--KLAE----ESDK 300 310 320 330 340 280 290 YHR209 LNVPLK-I-EWST--FYYLC--RKRE-- .. : . : .: : ::. rat_As MK-P-RCAPE-GTGG----CCG-KRKSC 350 360

Figure 5.10. A global alignment of protein YHR209w sequence (291 a.a.) and the rat AdoMet:arsenic(III) methyltransferase (369 a.a.) [Version 2.0u. Myers and Miller, CABIOS (1989) 4:11-17] Scoring matrix was BLOSUM50. Gap penalties were -1/-2. These two sequences have 23.7% identity. Global alignment score was 615. The common methyltransferase motifs I, post-I, II and III of protein YHR209w, and the motifs I, post I, II of rat AdoMet:arsenic(III) methyltransferase are underlined.


134

10 20 YHR209 M----------PK--TSYLNKNFE--S--AH-Y------N--NV-R-P--------SYPL : :. :: .. .. : .. . : .. : : : : Ars_M MELWTHPTPAAPRLATSTRTR-WRRTSRCSQPWATTPGTNSSDASRTPTTASASATSKPQ 10 20 30 40 50 30 40 50 60 YHR209 SL---VNEIMK--------FHKGTRKSLVDIGCGTGKATFVVEPYF--K-----EV-IGI : . . . . .:.:: : : :.. .: : : :. .. Ars_M SASARARSVRRSPDCTPRAWSRGARK---DRGATTNRPR---RPKFCSKRSTTCEATMSN 60 70 80 90 100 110 70 80 90 100 110 120 YHR209 DPSSAMLSIAEKETNE-RRLDKKIRF--INAPGEDL-SSIRPESVDMVISAEAIHWCN-- : . .: .:... .: :.. .. :. : . :.: ... ..: .:... :. Ars_M D-NETM--VADRDPEETREMVRE-RYAGIATSGQDCCGDV---GLD--VSGDGGC-CSDE 120 130 140 150 160 130 140 150 160 YHR209 LE-----RL-FQ--QVSSILRSDGT-FAFWFYIQPE-FVDF-P-EA-LNVYYKYGWSKD- : :: .. .:.:. .::. ... .:. :. . : :. :.. :. : Ars_M TEASGSERLGYDADDVASV--ADGADLGLGCG-NPKAFAAMAPGETVLDLGSGAGF--DC 170 180 190 200 210 170 180 190 200 YHR209 YMG-KYLN-DNQREIL-LNYGGE---KLR-SLL-SD------RFGDIEVTIYSP-SDP-- ... . .. :.. .. ... : : : .. .: :.: :. . : .: Ars_M FLAAQEVGPDGH--VIGVDMTPEMISKARENVAKNDAENVEFRLG--EIG-HLPVADESV 220 230 240 250 260 270 210 220 230 240 250 YHR209 NA--ST-VT--AENSQ--F--LWRAAITLNQFKEFVKSWS--IYTS-WARDNPSKPD--- :. :. :. : ..: : .: . : . : . : . :. . : :: Ars_M NVVISNCVVNLAPEKQRVFDDTYR--V-LRPGGR-V-AISDVVQTAPFPDDVQMDPDSLT 280 290 300 310 320 260 270 280 YHR209 --IADI-FINELKEIC-HC--EDLNV-PLK------I-EWST---F-YYLC------RK- .: ...:: . . : ... : : : .:.. . :: :: Ars_M GCVAGASTVDDLKAMLDEAGFEAVEIAP-KDESTEFISDWDADRDLGEYLVSATIEARKP 330 340 350 360 370 380 290 YHR209 -RE- :. Ars_M ARDD 390

Figure 5.11. A global alignment of protein YHR209w sequence (291 a.a.) and ArsM, a putative AdoMet-dependent arsenite methyltransferase (391 a.a.) [Version 2.0u. Myers and Miller, CABIOS (1989) 4:11-17] Scoring matrix was BLOSUM50. Gap penalties were -4/-2. These two sequences have 19.8% identity. Global alignment score was 136. The common methyltransferase motif I, post-I, motif II and motif III of YHR209w are underlined.


135

5.3. Discussion

5.3.1. Arsenate has little effects on the growth of the mutant strains

Each of the 13 knockout yeast mutant strains had one of the putative methyltransferase

genes deleted. When applied to nutrient medium plates with a range of arsenate

concentrations, ten of the mutant strains exhibited arsenate resistance similar to the wild

type BY4741, and the other 3 of these mutant strains grew slower than the wild type

BY4741 even in nutrient medium without arsenate. YPR201w behaved as observed

previously and died on the plate containing 0.3 mM arsenate.

The slow growth of the three knockout strains (YDR140w, YGL136c and YPL157w) in

complete medium without any arsenate suggest these genes are important for yeast

normal growth but their nullity is not fatal, even in exposure to arsenate. Arsenic is

known not to be an essential element for yeast growth and low arsenic concentrations in

most environments are far from harmful and fatal. When the other pathways of arsenic

detoxification, such as efflux and thiolation, are intact in yeast, it is reasonable that

deficiency of the activities of arsenic methyltransferases would not have much effect on

the yeast normal growth. It is concluded that YDR140w, YGL136c and YPL157w are not

directly related to arsenite methylation.

Winzeler et al. (1999) have shown that the knockout mutant of YDR140w, whose

function is required for yeast viability on toxin exposure, is viable but exhibits K1 killer

toxin hypersensitivity. In this study, the YDR140w mutant didn’t show arsenic sensitivity

but slow growth in general. YGL136c is new known to be the gene for mitochondrial

rRNA (uridine-2'-O-)-methyltransferase (Pintard et al., 2002). Nullity of YGL136c

results in thermosensitive respiration and loss of mitochondrial DNA with high frequency

(Winzeler et al., 1999). YPL157w was recently reported as the gene for trimethyl-

guanosine synthase, which has RNA methyltransferase activity. The product of YPL157w

is involved in snRNA capping (Verheggen et al., 2002) and is located in the nucleolus

(Mouaikel et al., 2002).


136

Delection of the arsM gene coding for arsenite methyltransferase in a haloarchaeon

Halobacterium sp. Strain has been shown to result in a significantly increased sensitivity

of the mutant bacterial line to arsenic (Wang et al., 2004). By analogy, the screen based

on the grown sensitivity assay conducted in Section 5.2.7 would identify the arsenic

methyltransferase gene mutant of yeast. The fact that none of the mutants screened

exhibited such an elevated sensitivity to arsenic indicated that either an arsenic

methyltransferase null mutant was not among the screened or the assay was not sensitive

enough for yeast.

Considering arsenic methylation may not be a main pathway for arsenic detoxification or

the enzyme activity encoded by more than one gene in yeast, knocking out a single gene

would have little effect on yeast sensitivity to arsenate.

5.3.2. Further enzyme activity screen

The 10 mutants with low sensitivity to arsenate similar to BY4741 were subjected to the

methyltransferase activity assay. Eight out of the ten mutants showed enzymatic activities

of arsenic methylation (Figure 5.2). During the 60-min assay with extract from YDL201w,

there was an arsenite-dependent increase in MMA and accumulation of DMA (Table 5.2).

This result suggests that the deletion of the ORF of YDL201w had no effect on arsenic

methylation. Similar results were observed with YHL039w. Among the other strains,

three knockout mutants YIL064w, YBR261c and YBR271w formed more MMA than

DMA, whereas other three mutants YNL092w, YNL063w and YDR316w exhibited little

increase in MMA but high accumulation of DMA. Within these six strains, it seems that

arsenite is methylated to MMA by arsenite methyltransferase, with further reduction and

methylation by MMA methyltransferase to form DMA. The results suggest that in those

mutants with low DMA formation, MMA methyltransferase be partially inactivated.

Although knocking-out of ORFs led to only partial inhibition of arsenic methyltransferase

activities, arsenic methylation occurred in lower efficiency. These six genes are

considered unlikely to be the sought arsenic methyltransferase gene mutant(s). After the

assay was finished the identities for several of these genes have been worked out in other

laboratories and confirmed our results (see references for Table 5.1).


137

The characteristics of each of the 13 yeast knock-out mutants regarding growth and

arsenite methylation activity are summarized in Table 5.6.

Table 5.6. Summary of 13 mutant strains in the experiments of arsenate sensitivity and arsenite methylation assays

Mutant strains Standard Gene Name

Slow growth

High MMA and DMA

High MMA but Low DMA

Low MMA but high DMA

Arsenic MT (Yes or No)

Good match to Conserved MT motifs

YHR209W YNL063W √ No YDR140W FYV9 √ No YDR316W √ No

Putative MTs YIL064W √ No

Unknown function proteins

YBR261C √ No YBR271W √ No YHL039W √ No YNL092W √ No

Known proteins YDL201W TRM8 √ No YER175C TMT1 √ No YGL136C MRM2 √ No YPL157w TGS1 √ No

MT: methyltransferase

To our interest, YHR209w and YER175c mutants were found with no apparent arsenite

methyltransferase activity (Figure 5.2). Although the latter had a low MMA production

but a much higher DMA production, the YER175c protein had just been reported to be a

trans-aconitate methyltransferase (Cai et al., 2001). Mutant YER175c was used as a

control in this study. It has been known that the sequence of YHR209w has high

homology to that of YER175c, with the exception that YER175c protein lacks conserved

methyltransferase motifs (Niewmierzycka and Clarke, 1999). It seems likely that MMA

formed by YER175c mutant extracts was methylated to DMA quickly, resulting in rapid

DMA accumulation. Only one knockout mutant, YHR209w, remained to be identified.

Little MMA but substantial DMA was formed by extracts of YHR209w knockout mutant


138

(Table 5.2), suggesting that YHR209w may be an arsenite methyltransferase, converting

arsenite to MMA.


139

5.3.3. Protein YHR209w: an arsenite methyltransferase

In order to investigate the arsenic methyltransferase activity of protein YHR209w,

glutathione-S-transferase (GST) fusion protein system was applied to express YHR209w.

The fusion of GST to protein YHR209w and the tendency of GST to form dimers (Vargo

et al., 2004) may affect the correct folding and the activities of YHR209w. While the

expressed fusion protein GST-YHR209w did not show any arsenic methyltransferase

activity (Figure 5.7), after release of YHR209w by thrombin treatment (partial digestion),

arsenite-dependent MMA formation was detected in the assays (Figure 5.8). In

comparison, neither the intact GST-YER175c (fusion protein) nor its thrombin partial

hydrolysate exhibited any arsenite-dependent MMA production. As hydrolysis of the

fusion protein by thrombin was incomplete or formed truncated YHR209w protein, the

true yield of arsenite-dependent MMA on a protein mass basis could be much higher than

indicated by the results of Figure 5.8. Unlike the S-adenosyl-L-methionine:arsenic(III)

methyltransferase found in rat liver (Lin et al., 2002), which exhibits both arsenite

methyltransferase and MMA methyltransferase activities, YHR209w exhibited only

arsenite methyltransferase activity in our experiments. DMA was formed in arsenic

methylation assays by the partially purified cytosol extracts from YHR209w knockout

mutant (Table 5.2), whereas the protein YHR209w purified by affinity chromatography

produced no DMA with MMA as substrate (Figure 5.9), even with DTT in the assay

system. DTT existing in methylation reaction mixtures was once reported to stimulate

strongly the methylation of methylarsonous acid by the S-adenosyl-L-methionine:

arsenic(III) methyltransferase purified from rabbit liver (Zakharyan et al., 1999). This

might be due to that the cell extract from YHR209w knockout mutant contained non-

specific methyltransferases that transformed arsenite to MMA, the substrate for the MMA

methyltransferase existing in cytosol. These results suggest that YHR209w may be an

arsenite methyltransferase, transforming arsenite to MMA but not DMA. Furthermore,

arsenite and MMA methyltransferase activities are probably not on the same protein in

the yeast. The assumption that there is another gene product responsible for MMA

methyltransferase for further methylation from MMA to DMA in yeast waits to be

demonstrated. Enzymatic assays using directly expressed non-fusion YHR209w protein


140

would provide further evidence to its identity as an arsenite methyltransferase. However,

the time constrain of the present Ph.D. study has not allowed this further work.

5.3.4. Protein YHR209w: a typical S-AdoMet-dependent methyltransferase

YHR209w has been annotated to an S-adenosylmethionine-dependent methyltransferase

based on sequence features (Niewmierzycka and Clarke, 1999). The AdoMet binding

domain is located between residues 35 to 143 (Figure 5.12), which contains all four

typical motifs that have been found in other methyltransferases and where all intervals

between two motifs are close to the average length. Residues 42 to 50 (LVDIGCGTG)

in the sequence of YHR209w almost completely match the consensus sequence for motif

I of (V/I)(L/V)(D/E)(I/V)G(G/C)G(T/P)G which conforms to the general consensus for

motif I [hh(D/E)hGXGXG], where h is a hydrophobic amino acid, X is any amino acid,

and the position of glycine residues is strongly conserved (with frames) (Wu et al., 1992).

Motif I has been associated with the binding of AdoMet by the enzyme. An acidic residue

motif post-I (VIGID) that contributes to the binding of AdoMet and matches the general

consensus hhXhD has been found 12 residues to the C-terminal of motif I in YHR209w.

A sequence (ESVDMVIS) in YHR209w is partially matched to the motif II with the

consensus sequence (P/G)(Q/T)(F/Y/A) DA (I/V/Y)(F/I)(C/V/L) found in other

methyltransferases (Wu et al. 1992). The motif II in YHR209w is 37 residues N-terminal

to motif post-I. The consensus sequence for motif III (LL(R/K)PGG(R/I/L)(L/I)(L/F/I/V)

(I/L)) was identified in YHR209w as LRSDGTFAFW, 19 residues N-terminal to motif II.

Motif I and post-I interact directly with AdoMet and are regarded involved in the binding

of AdoMet by the enzyme. Motif II and motif III have been known to interact with each

other and with a portion of motif I to form the central portion of a β-sheet

(Niewmierzycka and Clarke, 1999). Complete digestion by thrombin treatment of fusion

protein GST-YHR209w will result in hydrolysis among the AdoMet binding motifs and

thus loss of arsenite methyltransferase activity. In addition, the three cysteine residues

that may be critically involved in the binding of arsenite locate at the C-terminal

sequence and their binding to arsenite may be affected by proximity to the fused GST

protein after folding.


141

35 MPKTSYLNKNFESAHYNNVRPSYPLSLVNEIMKFHKGTRKSLVDIGCGTGKATFVVEPYF I KEVIGIDPSSAMLSIAEKETNERRLDKKIRFINAPGEDLSSIRPESVDMVISAEAIHWCNLER

post-I 143 II LFQQVSSILRSDGTFAFWFYIQPEFVDFPEALNVYYKYGWSKDYMGKYLNDNQREILLYG

III GEKLRSLLSDRFGDIEVTIYSPSDPNASTVTAENSQFLWRAAITLNQFKEFVKSWSIYTSWA RDNPSKPDIADIFINELKEICHCEDLNVPLKIEWSTFYYLCRKRE

Figure 5.12. Hydrolysis sites of thrombin, methyltransferase motifs and Cys residues in YHR209w. Hydrolysis sites of thrombin in the sequence of YHR209w protein are indicated by vertical arrow. Positions for common methyltransferase motif I (I), post-I, motif II (II) and motif (III) are indicated by horizontal line. Letters with frames indicate the cysteine residues in the C-terminal sequence.

5.3.5. Comparison of YHR209w with known arsenic methyltransferases from other organisms

A rat S-AdoMet:arsenic(III) methyltransferase has been identified (Lin et al., 2002).

Recently, a bacterial gene arsM has also been reported to encode a putative arsenite

methyltransferase (ArsM) in Halobacterium sp. (Wang et al., 2004). Up to date, there has

not been any report about arsenic methyltransferases in yeast. A global alignment (Figure

5.11) suggested that there was no significant homology between the sequences of

YHR209w and ArsM. No common motifs of AdoMet-dependent methyltransferase was

found in ArsM (Figure 5.11), whereas both YHR209w and rat S-AdoMet:arsenic(III)

methyltransferase have the typical motifs (Figure 5.10 and 5.12). Furthermore, three

cysteine residues were found in both C-terminals of YHR209w and the rat S-

AdoMet:arsenic(III) methyltransferase but not in ArsM. It seems that YHR209w is

similar to rat S-AdoMet:arsenic(III) methyltransferase and far from bacterial ArsM.

However, it is difficult to identify any sequence signature for the arsenic

methyltransferases at this stage.


142

5.3.6. Yeast - a promising study bridge between plants and animals in arsenic methylation

Despite the controversy over whether methylation is a detoxification mechanism for

inorganic arsenic, the methylation of arsenicals is a common feature of the metabolism of

this metalloid in organisms ranging in complexity from microorganisms to humans

(Zakharyan et al., 1995a; Hall et al., 1997; Turpeinen et al., 1999). Arsenic methylation

is undoubtedly an enzymatic process (Chapter III; Zakharyan et al., 1995a). Arsenic

methyltransferase in mammals is produced without exposure to inorganic arsenic (Healy

et al. 1998). Yeast and E. coli also exhibited arsenic methyltransferase activities without

the presence of arsenate (Chapter IV), whereas those activities in the plant Agrostis tenuis

were absent unless induced by the presence of arsenate (Chapter III). In humans, a higher

percentage of methylated arsenic in urine has been found in individuals with arsenic-

induced skin lesions, including cancer, than in individuals without arsenic-induced

lesions (Hsueh et al., 1997; Yu et al., 2000). Exposure of the yeast Saccharomyces

cerevisiae to arsenate could achieve higher arsenic methyltransferase activities than

omission of arsenate (Chapter IV). Studies of mechanisms and polymorphisms of S-

adenosyl-L-methionine:arsenic(III) methyltransferase in mammals will help in

understanding the contribution of this factor to differences between individuals in

metabolism, retention, clearance and toxicity of arsenic. The study of arsenic

methyltransferase in plants has added dimension of defining its future possible role in

plants engineering to remediate and collect this element from polluted soils. Yeast

provides a bridge to study arsenic methylation in plants from knowledge obtained in

mammals. The complex multicellular structure of plants requires that attention will need

to be paid to transport and accumulation of arsenic in any potential accumulation plant.

However, the existence of both plasmalemma export and tonoplast vacuolar import

pumps in yeast has been demonstrated (Bobrowicz et al., 1997; Ghosh et al., 1999) and is

likely to be related to plants. Additional studies of the location of arsenic

methyltransferase and distribution of methylated arsenicals in yeast cells will be required

to determine the pathway of arsenicals transport and storage.

Chapter VI. Final Discussion and Conclusions

141

Chapter VI

Final Discussions and Conclusions


142

Arsenic is a ubiquitous element and many of its inorganic and organic compounds

account for the toxic and carcinogenic effects of this metalloid. Living organisms are

unavoidably exposed to arsenicals and have developed a variety of detoxification

mechanisms. The putative pathways of arsenic metabolism and translocation for

detoxification in cells are illustrated in Figure 6.1.

MMA

Vacuole

Ycf1p

_

_+

_

_

_

+

+

+

+

As(III)

Phytochelatins

As(III)

As(V)As-Binding

Proteins

DMA+ 3GSH

(Urine)

?

P

A

Y Y&B

a

b

As(III)

As(V)

TMAO

?

MMA&DMAAs (III), As (V)

Figure 6.1. A summary of metabolism pathways for arsenic in cells. a: glycerol-transporting channel; b: phosphate transport mechanism; A: animals; B: bacteria; P: plants; Y: yeast; GSH: glutathione; MMA: monomethylarsenicals; DMA: dimethylarsenicals. The thick arrows show reactions studied in this thesis.


143

6.1. Uptake and reduction of arsenate by plants and microorganisms

Arsenic is available to living organisms mainly in the form of arsenate. Plant roots take

up arsenate from the external medium, possibly via the phosphate transport mechanism.

Most arsenate taken up is stored in the roots as arsenite-tris-thiolate complexes (Section

1.3.2.3), and only a small proportion is thought to transport from roots to shoots as the

free oxyanions arsenate and arsenite. For plants, most of the internal cells are protected

from the environment by the epidermis with its cuticle. Unlike plants, yeast and

bacterial cells are equally exposed directly to arsenate in the culture media. Hence high

concentrations of arsenate cause much higher stress to unicellular microorganisms than

to plants such as A. tenuis as judged by the concentrations that cause toxic effects

(Section 3.2 and Section 4.2). Arsenate can be reduced to arsenite enzymatically by

arsenate reductase or nonenzymatically by glutathione. In the in vitro methylation assay

systems used in this thesis, glutathione was an electron donor for the reduction of

arsenate to arsenite and partially purified cell extracts were used. These cell extracts

contained the reductase activities that reduced pentavalent MMA to trivalent MMA.

Because trivalent arsenic is more toxic than pentavalent arsenic, the initial step in the

biotransformation of arsenate may be regarded as a dangerous but necessary activation.

The reduced product, arsenite, then undergoes further reactions such as protein binding,

methylation and chelation that result in its detoxification.

6.2. Methylation of arsenic by plants and microorganisms

Biomethylation is a common feature of arsenic metabolism in organisms ranging from

bacteria to humans (Figure 6.1). Before methylation, all pentavalent arsenicals must be

reduced to the corresponding trivalent forms. This was illustrated by the delay in

formation of monomethylarsonate when arsenate was supplied in place of arsenite as

substrate in arsenic methylation assays with cell extracts of the plant A. tenuis (Section

3.2.2.3 and 3.2.2.4). Arsenite is the substrate for arsenic methyltransferase and arsenate

must be reduced before methylation (Section 3.3). S-AdoMet is the methyl (-CH3+)


144

group donor for arsenic methylation reactions.

The activity of arsenite methyltransferase in yeast and E. coli have been characterized

in this study (Section 4.2). Arsenite is methylated to monomethylarsonate, then

dimethylarsinate. Dimethylarsinate can be further methylated to trimethylarsine oxide

and possibly to further methylated products in the yeast S. cerevisiae and bacterium E.

coli (Section 4.3).

It was shown that the enzymatic activity for arsenic methylation is present in the shoots

of the plant A. tenuis (Section 3.2), confirming Nissen and Benson’s report (1982) of

apparent arsenic methylation activity in leaves. Nissen and Benson (1982), and Marin,

et al. (1992) found that DMA was mostly in the shoots and MMA was mainly

accumulated in the roots. Because arsenite is methylated in shoots to MMA and then to

DMA, it seems that MMA is more mobile than DMA and can be translocated to roots

where further arsenic methylation probably does not occur, whereas DMA is inclined to

stay and accumulate in shoots as the leaves age. This results in the different distribution

of MMA and DMA in plants.

While arsenic methylation occurs in shoots of the plant A. tenuis, it is the roots that are

exposed to arsenic in the soil or growth medium. In view of the low rates of arsenic

methylation (Table 6.1) and the toxicity of the methylated arsenicals, methylation does

not seem to effectively detoxify the arsenic entering the cells. It seems that methylation

is not the only choice, nor the main pathway, for arsenic detoxification by organisms.

However, further methylation of di-methylated arsenic observed in yeast and bacteria

(Section 4.2) may reduce the toxicity of methylated arsenicals, and probably increases

their elimination by other pathways such as volatilization (Cullen et al., 1994a;

Edvantoro et al., 2004), although more study of this pathway and its mechanism is

required. It is unknown whether all methylated metabolites can be transported to the

vacuole in plants and fungi or how they are removed from cells.


145

6.3. Induction of arsenic methylation by arsenate and its implication

In the plant A. tenuis, arsenic methyltransferase activity is induced by arsenate (Section

3.3). Partial induction was also observed in the yeast S. cerevisiae (Section 4.2.2.1) but

does not occur in the bacterium E. coli (Section 4.2.4.1), nor has been reported in

animals (Healy et al., 1998). Without arsenate in the growth medium, the constitutive

arsenic methyltransferase activity is not higher than background in the plant A. tenuis,

but is measurable in the yeast S. cerevisiae, bacterium E. coli, and mouse cytosols. In

the presence of arsenate in the growth medium, significant arsenic methyltransferase

activity appeared in cell extracts of A. tenuis leaves, while an increase of the enzyme

activity was observed in S. cerevisiae. In contrast, no distinct difference in arsenic

methyltransferase activity was found between mice with or without arsenate

administration (Healy et al., 1998), neither was E. coli with or without arsenate in

growth medium (Section 4.2.4.1). A comparison of arsenic methylation in various

organisms is displayed in Table 6.1.

Table 6.1. Comparison of arsenic methylation in organisms

Nissen and Benson (1982) found that, on exposure of roots to low concentration of

arsenic (<0.05ppm), methylation and reduction to MMA and DMA were apparent only

in nitrogen and phosphate-deficient plants. It is most likely that, as an analogue of

phosphate, arsenic was taken up by those N- and P-deficient plants, resulting in the

rapid increase of arsenic concentration in the plants. The alternative possibility is that

Organisms Arsenic methylation

Methyltransferase Activity No arsenate in

growth medium

Induction by arsenate during

growth Plant

(A. tenuis) ++ low Yes

Yeast (S. cerevisiae)

++ + Partial

Bacterium (E. coli)

++ ++ No

Animals ++ ++ No


146

competition by inorganic phosphate for the phosphate transporters on the root cell

membrane impedes arsenate uptake. When adequate phosphate is present, the small

amount of arsenate taken up can be successfully detoxified by phytochelation, which is

thought to be the main detoxification mechanism (Pickering et al., 2000). When the

concentration of arsenate in the environment is much higher and greatly increases the

concentration of inorganic arsenic in plants, genes for arsenic methyltransferases are

activated, triggering methylation. The high constitutive arsenic methyltransferase

activity in bacteria and yeast is perhaps related to the greater possibility for

microorganisms to face environments containing high concentrations of arsenic.

The induction by arsenate of additional arsenic methyltransferase activity in the yeast S.

cerevisae occurs within the same cells. In contrast, for the plant A. tenuis, the uptake

and sensing of arsenate in the growth medium by the roots leads to the induction of

arsenic methyltransferase activities in the leaves but not in the roots. The tissue-specific

induction in plants implies that the induction process is more complicated in plants than

in yeast. In the leaf tissue of A. tenuis, the identity of the inducer of arsenic

methyltransferase is not known. The identity of the messenger that transmits the

induction signal from roots to leaves is of considerable interest.

In mammals, arsenic methylation accelerates excretion of arsenic and thus enables

removal of this element via the urine (Gebel, 2002). In comparison, in plants, the

methylated arsenical metabolites probably stay in the cells they were formed in and are

accumulated in the leaves (Nissen and Benson, 1982). Evolutionary pressure has led to

a constitutive arsenic methyltransferase activity that is high in animals and low in plants,

in order to enable immediate excretion of methylated arsenicals from animals. However,

in plants, arsenic methylation occurs in two known specific scenarios: severe nutrient

deficiency (Nissen and Benson, 1982) and hyper-accumulation of arsenic (Section 3.3

and 1.3.1.4). The induction of arsenic methyltransferase activity in plants may be a part

of the general response to stress caused by abnormally high uptake of arsenic. The

sequestration processes for methylated products in plants are unknown. There is


147

evidence that arsenic is stored in the vacuole (Pickering et al., 2000). In some plants,

methylated arsenicals are accumulated in leaves (Section 3.3 and 1.3.1.4). Abscission

and loss of dead leaves provides a means to eliminate arsenic (Marin et al., 1992).

Obviously, arsenic methylation is not the only major detoxification pathway for this

element in organisms. However, the methylation of arsenic is critical for controlling its

biological effects and its fate. Elucidation of this metabolic pathway can lead to a better

understanding of the processes that control the oxidative states of arsenic, the formation,

distribution, and clearance of methylated arsenicals, and the removal of these arsenicals

from the cellular environment.

6.4. Molecular studies of arsenic methylation and application

Molecular technology has provided powerful tools to researchers in almost all areas of

biology and advanced our understanding of arsenic methylation. So far, the genes for

arsenate reductases have been identified in many bacteria and the yeast S. cerevisiae,

although the protein sequence for a rat arsenate reductase has also already been

obtained (Section 1.4). Two genes for arsenic methyltransferases have been isolated

from rat and the bacterium Halobacterium sp. The rat methyltransferase has been

shown to take both arsenite and MMAIII as substrates while the specificity for the

bacterial ArsM is yet to be characterized. In this thesis, an arsenite methyltransferase

gene was identified in the yeast S. cerevisiae genome. This methyltransferase, which

contains the typical motifs of S-AdoMet-dependent methyltransferase, is responsible

only for the transfer of methyl groups from S-AdoMet to arsenite. More research is

required to study MMAIII methyltransferase and identify its gene in the yeast S.

cerevisiae and bacteria. Understanding the basis for the difference of the co-location or

separation of activities on proteins between yeast and animals may clarify the origin

and evolution of these methyltransferases. Yeast is a promising tool to further the study

arsenic methyltransferases in plants (Section 4.1), because up to date, the genes for

plant arsenate reductase and methyltransferases are unknown. The methylated products


148

extracted and isolated in this thesis were mainly monomethylarsonate and

dimethylarsinate. However, further methylation was also observed in yeast and bacteria.

The genes involved in these further methylation processes are to still be identified.

More research is required to confirm whether any further metabolism of

dimethylarsinate occurs in plants, especially arsenic-accumulating plants such as A.

tenuis. The mechanism by which the arsenite methyltransferase activities are induced,

either at the transcriptional or translational level remains to be elucidated.

The integration of novel information on the formation, fate, and actions of methylated

arsenicals in organisms will reduce some of the uncertainties in the risk assessment for

the use of arsenic and its compounds. This effort should help to develop safe, practical

and effective biotechnology to remediate sites polluted by this element, and protect

public health against the toxic and carcinogenic effects associated with exposure to

many forms of arsenic. Therefore, it is stimulating to note that a bacterial arsC gene has

been reported to enhance hyper arsenic accumulation in an engineered plant (Dhankher

et al., 2002). The highly methylated arsenicals in marine organisms are much less toxic

and even non-toxic (Section 1.3.3.2). The possibility of engineering the metabolic

pathways for arsenic methylation from marine phytoplankton into terrestrial plants

arouses great interest.

149

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2005 a comparative study of arsenic methylation in a plant

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