CHAPTER 1

INTRODUCTION

1

INTRODUCTION

CHEMICAL SPECIATION

Chemical speciation is an important aspect of environmental

analysis and research. Bioavailability and toxicity of an element

depend on its species. Metal ions are complexed in fresh water, sea

water, polluted water and bio-system with naturally occurring ligands

and chelating agents such as humic substances, sulfide and

pesticides. For example cobalt, nickel and copper are predominantly

organically complexed in seawater and bio-system. It is estimated that

the coordination compounds entering environment are about 5-10

million kinds, most of them being contained in aquatic environment,

including water and sediment. These metal coordination interactions

would affect the biogeochemical pathways taken by the metal, such as

its bioavailability and toxicity to aquatic organisms, and adsorption/

desorption reactions on suspended materials.

International Union of Pure and Applied Chemistry (IUPAC)

defined “speciation” as the distribution of an element amongst the

defined chemical species in a system. A chemical species is a specific

form of an element which attributes to its isotopic composition,

electronic or oxidation state, and/or complex or molecular structure.1

The speciation determines the behavior of trace elements in a system,

and in the human organism speciation has a profound effect on

bioavailability, distribution and toxicity. Speciation may also exploit

certain issues in medical sciences such as therapeutic, diagnostic and

2

investigative uses of trace metals. Several examples could be

portrayed at each level of structure, affecting bioavailability, toxicity

and clinical usage.2, 3 Here the focus is on several biological principles

that contribute to the importance of speciation, such as metabolic

kinetics, barrier transport, and metal–metal interactions.

Various conformations, excited states, or transient forms of an

element, its coordinated atoms, and the molecules of which they are

part constitute unique species. Nevertheless, analytical methodology

and practical considerations dictate that the speciation analysis of a

system yields a profile of sufficiently different and measurable species

for the desired level of understanding of the system’s behavior.

Various aspects of structure contribute to identifiable species for a

particular element, and may differ in importance depending on the

reasons for which a speciation analysis is undertaken.1 Speciation

scheme suggesting the possible distribution of metal ions in the

environment is given in Fig. 1.1.

3

Fig. 1.1: Speciation scheme for the distribution of metal ions in the environment

Mobilization and Transport of Metal Complexes

Thousands of organic compounds are produced every year by

industrialized countries. These compounds as well as their by-

products eventually find their way into the environment where they

can undergo biological, oxidation-reduction or photochemical

reactions. It is expected, therefore, that some of these organic

compounds have appropriate functional groups which could mobilize

and transport certain metals by complex formation. It is estimated

that the number of these potential ligands will increase up to 1 to 2%

each year as a consequence of the development of new products.

Roughly one-third of the elements in the periodic table can react with

organic ligands. The elements of most concern are abundant,

extracted and used in various chemical industries. They will perturb

biogeochemical cycles and eventually be exposed to humans.

4

Complexes are formed by some of the essential elements such

as cobalt, nickel, copper, zinc, calcium, magnesium and heavier

elements like arsenic, selenium, cadmium, indium, antimony,

tellurium, mercury, thallium, lead, and bismuth. These elements and

their complexes are mobilized and transported in air, surface waters,

in sediments and in the soil, thereby significantly perturbing the

environment. Moreover, their intakes by humans via food, water, and

particulates in the air have caused health concerns.4 It is important to

determine the nature of these complexes experimentally or deduce

from the published data.

The bioavailability and toxicity of these complexes are critically

dependent on their thermodynamic and kinetic stability. Identifying

these species (i.e., speciation) and obtaining reasonable estimates of

their thermodynamic and kinetic stabilities are the problems that

continue to plague environmentalists and toxicologists.

Catabolism of amino acid

Amino acids are the building blocks or acid pools for the

synthesis of proteins. Proteins mediate a wide range of functions such

as, storage and transport, coordinated motion, mechanical support,

immune protection, excitability and control of growth and

differentiation. Amino acids (Fig. 1.2) are combined in various ways to

form several intermediates inside the body and more than 40,000

proteins known so far.5 If essential amino acids which cannot be

synthesized by body in sufficient quantities to satisfy the nutritional

5

requirements for good health, they must be supplemented through the

diet.

In the process of amino acid degradation, the amino groups are

removed by transamination or oxidative deamination. The carbon

skeletons of amino acids are degraded by twenty different pathways.

These pathways converge to form only five products, all of which enter

the citric acid cycle for undergoing complete oxidation to CO2 and

H2O (Fig. 1.3).

The amino acid catabolism has a profound influence on the

biosystems. An elevated level of NH4+ in the blood (hyper

ammonemia) leads to mental retardation. The synthesis of urea in the

liver is the major route for removal of +4NH . In terrestrial vertebrates,

urea is synthesized in the urea cycle. This series of reactions was

proposed by Krebs et al.6 before the elucidation of the citric acid cycle.

The following equation represents the net reaction in the urea cycle.

NH4++CO2+4ATP+Aspartate → Urea+Fumarate+4ADP+4Pi

6

Fig. 1.2: Classification of amino acids.

Amino acids

Sources are meat, milk, fish, poultry and legumes

Essential

Isoleucine, Leucine, Lysine,

Methionine, Phenylalanine,

Threonine, Tryptophan, Valine

Alanine, Asparagine, Aspartic acid, Cystien, Glutamic acid, Glutamine,

Glycine, Proline, Selenocysteine, Serine, Tyrosine, Arginine, Histidine, Ornithine,

Tarunine

Non-essential

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Fig. 1.3: Interaction between the urea cycle and the tricarboxylic acid cycle.

8

L-Aspartic acid

L-Aspartic acid (Asp) is a non-essential amino acid (Fig. 1.4)

with the molecular formula C4H7NO4, the structural formula

HOOCCH(NH2)CH2COOH and the molecular mass 133.1 g mol-1. It

was first discovered in 1827 by Plisson, synthesized by boiling

asparagine (discovered in 1806) with a base.7 Other names of Asp

are aminosuccinic acid, asparagic acid, asparaginic acid and 2-

aminobutanedioic acid.

Fig. 1.4: Structure of L-aspartic acid

It is primarily used in the body's metabolic processes. Cellular

energy is directly dependent upon this critical amino acid. The

responsibility for the increased stamina in neurological and muscular

activities lies with sufficient levels of Asp. Chronic fatigue is a

condition that may be directly linked to lower levels of aspartic

acid.8 Inadequate levels of aspartic acid have also been linked to

irregularities of certain chemicals in the brain, largely

serotonin, whose decreased level is a primary contributor to

depression in all age groups. A proper balance ensures proper

functioning and balance of the brain and its associated processes.

9

Asp has also been used in various clinical applications, showing

promise in persons suffering from opiate addiction.9

Asp is significant in the removal of excess ammonia and other

toxins from the blood stream. It assists in the proper functioning of

carriers for genetic information-RNA and DNA. It is equally important

in antibody synthesis/immune system support, production of

immunoglobulin, cell-functioning, and the movement of minerals

across intestinal linings into blood and cellular structures located

throughout the body.

The most bioavailable sources of aspartic acid include portions

of dairy, meat and sprouting seeds (cottonseed meal, spirulina,

sunflower seed flour, sesame flour, soya beans and peanut flour).

Deficiency

There has been no evidence suggesting deficiency of aspartic

acid in human biology. Symptoms of deficiency are predicted to be

fatigue, depression, autoimmune disorders and ammonia toxicity in

the body.

Toxicity

Aspartame is a dipeptide composed of the amino acids aspartic

acid and phenylalanine. Upon human ingestion aspartame is quickly

metabolized into aspartic acid, phenylalanine, and also the toxic wood

alcohol, methanol. Aside from the toxic properties of methanol,

aspartic acid and phenylalanine are not completely benevolent. If one

10

is not predisposed to ridding the body of both of these amino acids

quickly, build-up may occur leading to alterations in brain chemistry.

Excessive consumption of aspartic acid and phenylalanine leads

to their increased level in the brain. Neurological damage may occur

due to over-stimulus of the cerebral cortex. The neurotransmitter

serotonin may also be adversely affected due to over consumption of

these two amino acids. Depletion of serotonin can lead to conditions

associated with such a disproportionate change in brain serotonin

levels, including fatigue, headache, sleep, eating disorders and in

extreme cases, depression.

Aspartic acid, also a metabolite in the urea cycle, participates in

gluconeogenesis in the generation of glucose from non-sugar carbon

substrates like pyruvate, lactate, glycerol and glycogenic amino acids

(primarily alanine and glutamine). Aspartic acid carries reducing

equivalents in the malate-aspartate shuttle, which utilizes the ready

interconversion of aspartate and oxaloacetate, the latter being the

oxidized derivative of malic acid. Aspartic acid donates one nitrogen

atom in the biosynthesis of inositol, the precursor to the purine bases.

Aspartic acid in mammals is produced from oxaloacetate by

transamination. It plays an important role in citric acid cycle or Krebs

cycle, during which other amino acids and biochemicals, such as

asparagine, arginine, lysine, methionine, threonine and isoleucine are

synthesized. The conversion of aspartic acid to these amino acids

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begins with the reduction of aspartic acid to its semialdehyde

(HOOCCH(NH2)CH2CHO).10

Ethylenediamine

1, 2-Diaminoethane is more commonly known as ethylenediam-

ine (en) (Fig. 1.5). Other common synonyms include dimethylenediam-

ine, 1, 2-ethanediamine, 1, 2-ethylenediamine, β-aminoethylamine

and ethane-1, 2-diamine. Ethylenediamine is a colourless to yellowish

hygroscopic liquid with an ammonia-like odour. Its molecular weight

is 60.12. It is strongly alkaline (pH of 25% en in water is 11.9), highly

volatile, pungent material, which fumes profusely in air. It has a

melting point of 8.5 °C, a boiling point of 116 °C (at 101.3 KPa) and a

vapour pressure of 1.7 KPa at 25°C. Ethylenediamine is miscible with

water and alcohol. pKa1 and pKa2 (calculated) are 10.71 and 7.56,

respectively, indicating protonation at environmentally relevant pH.11

H2N NH2

Fig. 1.5: Structure of Ethylenediamine.

Ethylenediamine is manufactured by the reaction between

ammonia and 1, 2-dichloroethane. The reaction yields a mixture of

ethylenediamine and linear polyamines.

ClCH2CH2Cl + 4 NH3 → H2NCH2CH2NH2 + 2 NH4Cl Ethylenediamine is used as monodentate, bidentate or bridging

ligand. It is also used in the manufacture of EDTA, carbamate

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fungicides, surfactants and dyes. It is involved in the synthesis of

seven membered ring components with β-ketoesters resulting

secondary amines and β-enaminoesters.12 en plays an important role

in the synthesis of Schiff base compounds.13

Precursor to chelating agents, pharmaceuticals and agrichemicals

The most prominent derivative of ethylenediamine is EDTA,

which is derived from ethylenediamine via a Strecker synthesis

involving cyanide and formaldehyde. Hydroxyethylethylenediamine is

another commercially significant chelating agent. The salen ligands,

derived from the condensation of salicylaldehydes and ethylenedi-

amine, are popular chelating agents in the research

laboratory.14 Numerous bio-active compounds contain the -N-CH2-

CH2-N- linkage, including aminophylline and some anti-

histamines.15 Salts of ethylene bis(dithiocarbamate) are commercially

significant fungicides under the brand names Maneb, Mancozeb, Zineb

and Metiram. Some imidazoline -containing fungicides are derived

from ethylenediamine.

Specialized applications

1. As a solvent, it is miscible with polar solvents and is used to

solubilise proteins such as albumins and casein. It is also used

in certain electroplating baths.

2. As a corrosion inhibitor in paints and coolants.

3. Ethylenediamine dihydroiodide (EDDI) is added to animal feeds

as a source of iodine.

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4. Chemicals for color photography developing, binders, fabric-

softeners , adhesives, curing agents for epoxys and dyes

5. It is a common organic additive to the plant in vitro

culture Murashige and Skoog medium.

HEALTH HAZARD INFORMATION

Exposure to ethylenediamine can occur through inhalation,

ingestion and eye or skin contact and absorption through the skin.16

It is a corrosive liquid that is severely irritating to the skin and eyes.

Systemic toxicity from exposure to high vapor concentrations causes

damage to the kidneys, liver and lungs.17 The LD50 for acute oral

exposure in rats was reported to be in the range 0.5 g/kg and 1.16

g/kg.18 The dermal LD50 in rabbits is 0.73 g/kg.19 In rats fed with 0.5

g/kg/day of en for two generations, some reduction in body weight

and microscopic changes of the kidneys and liver were observed. Liver

lesions were more prevalent among female than male rats. An 8 h

exposure to 4,000 ppm ethylenediamine was uniformly fatal to rats;

death was caused by kidney injury and lung damage. Thirty days of

inhalation exposure to 484 ppm of ethylenediamine killed all exposed

rats. Postmortem examination showed injury to the kidneys, liver and

lungs and hair loss. No deaths and a lesser degree of injury were seen

at 132 ppm and no injury was noted at 125 ppm. Undiluted liquid

ethylenediamine painted on the shaved skin of rabbits caused severe

skin irritation, blistering, and cell death. In rabbits, contact of the eye

with solutions containing more than 5 percent ethylenediamine

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produced serious corneal damage and partial corneal opacity.

Neutralized solutions of ethylenediamine were far less damaging to the

eyes of rabbits than alkaline ones.20

Effect on humans

Ethylenediamine is a strong irritant of the eyes and respiratory

tract and a skin and respiratory sensitizer.21 It is tolerable at a

concentration of 100 ppm for a duration of a few seconds; irritation of

the nose and respiratory tract, and asthmatic symptoms appear at a

concentration of 200 ppm; at 400 ppm, intolerable nasal irritation

occurs after an exposure of 5 to 10 seconds.22 Skin contact with the

liquid will produce irritation and, if contact is prolonged, burns and

blisters.23 Skin sensitization is accompanied by the development of a

chronic rash that disappears after exposure ends.24 Skin sensitization

is more likely when the skin is damaged.25 Workmen exposed to

vapours of ethylenediamine occasionally have reported seeing haloes

around lights and having blurred vision, effects that are likely to be

caused by corneal epithelial swelling or damage.

Signs and symptoms of exposure

Exposure to the vapors of ethylenediamine causes immediate

eye irritation, shortness of breath, respiratory irritation, and

asthmatic symptoms such as wheezing, faintness, anxiety and

dizziness.16 Contact of the eye with liquid ethylenediamine causes

acute pain and visual disturbances and may cause severe, permanent

corneal damage.20 Liquid spilled on the skin will cause redness,

burning, and, if exposure is severe, blistering and loss of the skin.

15

Acute exposure can cause skin sensitization and chronic asthma in

sensitive individuals. Ingestion of ethylenediamine causes severe

irritation and burns of the mouth and esophagus and nausea and

vomiting.26

TRACE ELEMENTS

Small quantities of mineral elements occurring in both plant

and animal tissues are called as trace elements. A trace element is

considered as essential for both man and animal life, if it meets the

listed conditions: i) it is present in all healthy tissues, ii) its

concentration from one species to the next is fairly constant, iii)

depending on the species studied, the amount of each element has to

be maintained within its required limit if the functional and structural

integrity of the tissues is to be safeguarded, and the growth, health

and fertility to remain unimpaired, iv) its withdrawal induces

reproducibly the same physiological and/or structural abnormalities

and v) its addition to the diet either prevents or reverses, the

abnormalities.27 Several trace elements are known to fulfill these

criteria of which the most well known are iron, zinc, manganese,

selenium, chromium, copper, cobalt, nickel, molybdenum and iodine.

Many of them act as catalysts in many enzymatic functions called as

metalloenzymes.28

The function of each element could be further divided into: (i)

biological action required to sustain optimum health, ii)

pharmacological action where supplements are used in treating

specific deficiency conditions, and iii) toxicological action where a dose

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exceeds the biochemical need.29 The significance of transition metals

in chemistry, however, is immence30, e.g. organometallic and bio-

inorganic chemistry, organic catalysis, etc. and hence there is a need

to have reliable and precise theoretical methods for their calculation.

COBALT

The occurrence of cobalt in the earth crust is about 25 ppm.

The dietary intake of cobalt per day is 40-50 µg. Concentration of

cobalt in human blood is 1.043 ppm. Cobalt is a central component of

vitamin B12. Minot and Murphy31 discovered that pernicious anemia

can be treated by feeding the patients with large amounts of liver

which contains vitamin B12. Since then cobalamin started gaining

importance. A normal person requires less than 10 µg of cobalamin

per day. Mammals require small amounts of cobalt which is the basis

of vitamin B12.

Cobalt-60 is an artificially produced radioactive tracer and

cancer treatment agent in radio therapy. Cobalt-60 is also used for

sterilization of medical supplies and radiation treatment of food for

sterilization (cold pasteurization). Cobalt compounds have been used

for centuries to impart a rich blue color to glass, glazes and ceramics.

Cobalt-nickel alloys are corrosion and wear resistant. Cobalt,

chromium and molybdenum alloys are used for prosthetic parts such

as hip and knee replacements.32 The alloys of aluminum, nickel and

cobalt, known as alnico, and of samarium and cobalt are used in

17

permanent magnets which can be used for recording media. Lithium

cobalt compounds are used in several chemical reactions as catalysts.

Absorption, transport, storage and excretion

The stomach secretes a glycoprotein called intrinsic factor,

which binds cobalamin in the intestinal lumen. This complex is

subsequently bound by a specific receptor in the lining of the ileum.

The complex of the cobalamin and glycoprotein is then dissociated by

a releasing factor and actively transported across the ileac membrane

into the blood stream. Cobalt in the form of cyanocobalamin is most

efficiently stored in kidneys and liver when administered orally in

physiological doses and ionic cobalt is excreted largely in the faeces.

Parenterally administered cobalt is excreted primarily through urine.

Role in biological systems

Cobalt is essential for the production of red blood cells. It acts

as coenzyme in several biochemical processes. It speeds up ATP

turnover. It is present as corrin coenzyme in glutamate mutase,

dialdehydase, Met synthase and arginase and in non-corrin form in

dipeptidase.

The cobalt in cobalamin can exhibit +1, +2 or +3 oxidation state.

It is in +3 oxidation state in hydroxocobalamin and OH-occupies the

sixth coordination site. This form, called B12a (Co3+), is reduced to a

divalent state, called B12r (Co2+) by a flavoprotein reductase. The B12r

(Co2+) form is further reduced by a second flavoprotein reductase to

B12s (Co+). NADH acts as a reductant in these reactions.

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The B12s form acts as the substrate for the final enzymatic

reaction that yields the active coenzyme derivatives of cyanocobalamin

and aquacobalamin. In these cobalamins when the base-sugar

phosphate moieties are removed by hydrolysis, they are called

cobinamides. Cobalamin enzymes catalyse three types of reactions: (i)

intramolecular rearrangements (ii) methylations, as in the synthesis of

L-methionine (Met) and (iii) reduction of ribo- and deoxyribo-

nucleotides.

Prokaryotic organisms such as Enterobacteria, E. Coli and

Salmonella have two proteins that catalyze the synthesis of Met from

homocysteine.33 One (Met synthase) contains cobalamin as a co-factor

and the other is cobalamin independent.

Agonistic and antagonistic action

No metal ion antagonism involving cobalt has been reported and

no ion other than cobalt has been found in nature complexed with the

corrin ring. The effects of cobalt and manganese on some enzymes in

the rat liver were studied.34 Manganese invariably inhibited the

activity of alkaline phosphatase, glucose-6-phosphatase, acid

phosphatase, cholinesterase and lipase, whereas cobalt affected only

the activities of acid phosphatase, glucose-6-phosphatase and lipase.

After combined treatment with both cobalt and manganese only the

activity of acid phosphatase decreased, while the activities of the other

enzymes remained within the normal range indicating an antagonistic

behavior of these essential trace elements.

19

Antidote effect

The aqueous solution of vitamin B12 was administered in doses

of 20, 40 and 60 mg/kg body weight on potassium cyanide

poisoning.35 The poison and antidote effects were monitored by

bioelectrical tests of the brain and heart. The advantage of vitaminB12

is its direct interaction with cyanide.

Deficiency

Deficiency of cobalt in human body leads to pernicious anemia.

The major metabolic defects are failure of propionate metabolism,

reactions involved in methyl group transfer and deoxyribose

reduction. It also causes injury to peripheral nervous system,

demyelination of nervous sheath and neuronal damage in the cerebral

cortex and cerebral ischemia. An insufficient level of cobalt in the diet

gives rise to wasting disease, known as bush sickness. Trace amounts

of vitamin B12 are essential for the synthesis of hemoglobin.

Toxicity

The threshold limit value for occupational exposure is 0.05

mg/m3. The LD50 for cobalt is 10-20 mg/kg. Cobalt has toxic action

on steroid producing cells in the adrenal gland in the testis. Cobalt

has higher cardio toxicity than general toxicity.36 After nickel and

chromium, cobalt is a major cause of contact dermatitis.37 Higher

levels of cobalt exposure show mutagenic and carcinogenic effects.38

The toxic effects of cobalt are cardiomyopathy, dyspnea, tachycardia,

20

abdominal pain, edema, hypothyroidism and hyperplasia. The

antidotes for cobalt poisoning are EDTA, L-histidine and L-cystiene.

NICKEL

Nickel was first suggested to be an essential element in 1936.

Rich sources are chocolates, nuts, dried beans, peas and grains.

Nickel belongs to 10th group and 4th period of d-block in the periodic

table. Its atomic number is 28 and atomic weight is 58.693 g/mol.

Nickel is silvery white, hard, malleable and ductile metal. It is a good

conductor of heat and electricity and is ferromagnetic. It is used in the

smaller coins, for plating iron, brass etc. and chemical apparatus and

in certain alloys such as German silver. The most common oxidation

state +2 through 0, +1, +3 and +4 are observed in Ni complexes. The

average nickel content in soil is around 20 ppm. Tea leaves are a rich

source of nickel which is 7.6 mg/kg of dried leaves. Chocolates, nuts,

dried pears and grains are also its rich sources.

Biochemistry of nickel

Nickel can activate or inhibit a number of enzymes that usually

contain other elements. The production or action of some hormones

(Prolactin, adrenaline, noradrenalin, aldosterone) responds to changes

in nickel concentration. Within cells, nickel alters membrane

properties and influences oxidation/reduction systems.39

Nickel is present in enzymes like urease. It is a binuclear Ni(II)-

containing metalloenzyme which accounts for 6% of the soluble

cellular proteins40-42 and catalyses the hydrolysis of urea to yield

21

ammonia and carbonate. It is present43 in archaebacteria or

methanogens, carbon monoxide dehydrogenase, methyl S-coenzyme-

M reductase and also in the membrane bound (Ni-Fe) hydrogenase,

which permits respiratory based energy production for the bacteria in

the mucosa.44, 45 Many but not all hydrogenases contain nickel in

addition to iron-sulfur clusters. Nickel centers are common in these

hydrogenases whose function is to oxidize rather than evolve

hydrogen. The nickel center appears to undergo changes in oxidation

state, and evidence has been presented that the nickel center might be

the active site of these enzymes.

The capacity to oxidize molecular hydrogen is a central

metabolic feature of a wide variety of microorganisms. H2 oxidation is

coupled with the reduction of electron acceptors such as oxygen,

nitrate, sulphate, carbon dioxide and fumarate. Hydrogenases are the

enzymes responsible for hydrogen metabolism in microorganisms.

Three types of metal containing hydrogenases have been defined: Iron

only or Fe hydrogenases46, Ni-Fe hydrogenases47 and Ni-Fe-Se

hydrogenases.48 The active site in Fe hydrogenase is a cluster of iron

atoms.

A Nickel-tetrapyrrole coenzyme, Co-F-430, is present in the

methyl CoM reductase and in methanogenic bacteria. The tetrapyrrole

is intermediate in structure between porphyrin and corrin. Changes in

redox states as well as in nickel coordination have recently been

observed. There is also a nickel-containing carbon monoxide

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dehydrogenase. Studies on chicks and rats suggest that nickel is

essential for proper liver function.

Ni2+ is a component of the enzyme, urease, present in a wide

range of plant species. In 1990 its function as redox metal in

biological systems was proved.49 Electron paramagnetic resonance

spectra have established the presence of both Ni2+ and Ni3+ in nickel

enzymes of aerobic and anaerobic bacteria. In these enzymes, the

coordination environment consists of nitrogen, oxygen and/or sulfur

ligands. For example, methanogenic bacteria possess an enzyme

called methyl-CoM reductase that contains a prosthetic group called

F430, which is a nickel porphynoid of unique structure. No other

transition metal can substitute for nickel in factor F430.

Subsequently, another nickel porphynoid natural product called

tunichlorin was identified.50 Tunichlorin has a reductive role in

tunicates similar to methyl-CoM reductase in bacteria.

Tunicates accumulate nickel selectively and contain fixed ratios

of nickel and cobalt.51 Many plant species also contain a relatively

constant nickel/cobalt ratio. The findings that nickel and cobalt form

porphynoid natural products and apparently occur in a rather

constant ratio in some living organisms suggest that these two

elements have interrelated biological functions. This suggestion is

supported by some findings.52

There is no nickel requirement or allowance set for humans.

However, because it is essential for several animal species, it seems

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reasonable to accept that it is required by humans. Many monogastric

animals have a dietary nickel requirement of less than 200 µg/g

diet.53 The dietary intakes of nickel in humans range between 60 and

260 µg/day.54 Antagonists to nickel are iron and copper.

Absorption and excretion

Less than 10% of ingested nickel is normally absorbed by

gastrointestinal tract. It is absorbed via the iron transport system.

Unabsorbed nickel is excreted through faeces. The plasma nickel is

excreted through urine which is complexed with histidine and aspartic

acid.

Antagonists

Iron and copper are antagonists to nickel.

Deficiency

The symptoms due to deficiency of nickel were studied55 on

chick, cow, goat, pig, rat and sheep. The signs of nickel deprivation

include depressed growth, reproductive performance and plasma

glucose. It also affects distribution and proper functioning of other

nutrients including calcium, iron, zinc and vitamin B12. Deficiency of

nickel also causes cellular level structures became disorganized and

membrane properties changed, abnormal bone growth, poor

absorption of ferric iron.

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Toxicity

Excess intake of nickel and accumulation of nickel results in

interference with normal protein-metal binding and catalysis, as well

as regeneration of reactive oxygen species.56-58

Toxicity59 has occurred in workers exposed to nickel dust or

nickel carbonyl fumes in refining. Increased risk of nasal and lung

cancers was linked to occupational nickel exposure. Environmental

sources of lower levels of nickel include tobacco/cigarette smoking,

dental or orthopedic implants, stainless steel kitchen utensils and in

expensive jewelry. Repeated exposure may lead to asthma and contact

dermatitis, symptoms of which may worsen if the diet is high in

nickel. Excessive nickel in tissue is pro-oxidant (damaging

chromosomes and other cell components) and alters hormone and

enzyme activities. These effects can change glucose tolerance, blood

pressure, response to stress, growth rate, bone development and

resistance to infections. Under some conditions, large amounts of

nickel may precipitate magnesium deficiency or cause of accumulation

of iron or zinc.

COPPER

Copper belongs to 11th group and 4th period of d-block in the

periodic table. Its atomic number is 29 and atomic weight is

63.546 g mol-1. Copper in its pure state has a pinkish or peachy color.

It is a ductile metal. It has second highest thermal and electrical

conductivity, after silver. Copper is an essential trace nutrient to all

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higher plant and animal life. In animals including humans, it is found

primarily in the blood stream, as a co-factor in various enzymes and

in copper-based pigments. However, excess amounts of copper can be

poisonous and even fatal to organisms.

Copper is germicidal, via the oligodynamic effect. For example,

brass door knobs disinfect themselves of many bacteria within a

period of eight hours. Antimicrobial properties of copper are effective

against other pathogens; in colder temperature, longer time is

required to kill bacteria.

Biochemistry of copper

Copper is an essential element for life on earth. It is one of the

transition elements found at the active site of proteins. Human body

requires 2-3 mg/day copper (30 µg/kg body-weight according to

WHO60) through diet for healthy growth. Cereals and vegetables are

sources of copper. Animal liver and various shell fish contain on

average more than 20 mg/kg copper. Meats were found to have an

average content of about 2.5 mg/kg copper. In nature, a wide variety

of copper proteins are essential constituents of aerobic organisms,

including hemocyanins and enzymes that activate O2 promoting

oxygen atom incorporation into biological substrates.

Absorption, transport and excretion

Normal diet contains 2-5 mg of copper, one third of which is

absorbed by the body in the stomach and duodenum. Absorption of

copper is dependent largely on its chemical form. Complexes of some

26

amino acids have a higher rate of absorption than that of copper

sulphate. But its absorption is inhibited by phytates, ferrous sulphide,

zinc, molybdenum and cadmium, if they are present in the diet.

The absorbed copper is transported mainly by the plasma of

which 93% is bound to ceruloplasmin and the remaining is bound to

amino acids and albumin. Ceruloplasmin is synthesized in the liver

and contains six or seven copper atoms per molecule. This

incorporates copper into cytochrome oxidase and other enzymes. The

amino acids include histidine, threonine and glutamine which form

binary and ternary complexes of copper. The therapeutic action of

copper has been attributed to the solubility of metallic copper in

human sweat and diffusion of copper-amino acid complexes into the

human body.61 Albumin is the principal carrier in the portal blood

between the intestine and liver. The fraction of copper that is bound to

albumin is accessible to cells and serves as sole source of metal for

their metabolic needs.

The highest concentrations of copper are found in liver (6.6

µg/g) and brain (5.4 µg/g) while the whole body average for most

vertebrates is 1.5-2.5 µg/g of fat free tissue. The total body copper of

man has been estimated at 80 mg and plasma copper ranges from 80-

150 µg/100 ml, the levels being slightly higher in women.

A large production of absorbed copper is excreted along with

bile and faces, so that a negligible amount appears in urine. 1% of the

intake is excreted in urine and a negligible quantity in sweat.

27

Role in biological systems

Copper is largely rejected from cells, but outside the cell it is

essential for the metabolism of many hormones and connective

tissues. The copper-containing enzymes and proteins constitute an

important class of biologically active compounds. The biological

functions include electron transfer, dioxygen transportation,

oxygenation, oxidation, reduction and disproportionation.62, 63

Though copper is present in about 12 enzymes, there are four

copper containing enzymes that play key roles in the clinical

biochemistry of copper. (1) Its role in iron absorption and transport,

(2) The monoamine oxidase enzyme may account for the role of copper

in pigmentation and control of neurotransmitters and neuropeptides,

(3) Lysyl oxidase is essential for maintaining the integrity of connective

tissue, a function that explains disturbances in lungs, bones, and the

cardiovascular systems in patients with copper deficiency and

(4) Cytochrome C oxidase and super oxide dismutase play a central

role in the terminal steps of oxidative metabolism and they defend

against the superoxide radical intoxication.

Copper compounds are well known for their special color, good

stability and natural abundance of the metal. It is relatively

environmental-friendly making it attractive for technical

applications.64 Organometallic copper complexes are colorful

compounds that may be used in photo-induced and ground state

redox property.65 Cu(I), with d10 electronic configuration, adopts

28

preferentially the tetrahedral coordination geometry. For Cu(II) (d9

electronic configuration), penta- or hexa- coordination is found. Thus,

upon oxidation of Cu(I) to Cu(II), a large structural change occurs

which often is accomplished by solvent coordination. Hence, copper’s

main role in vivo would be in redox reactions and it plays a major role

in many of the electron transfer processes, but does not appear to

function in any non-redox metalloproteins. The copper enzymes

particularly involved in electron transfer reactions are the blue

proteins (azurines) where the copper is in the +2 oxidation state.

The accumulation of copper66 by hepatocytes is initiated by the

binding of copper in either a Cu(His)2 complex or a Cu(His)(Alb)

ternary complex, followed by transfer of the metal alone across the cell

membrane.

Correlated with the enzymatic activity, the copper proteins

exhibit unique spectroscopic properties and accordingly, the proteins

are divided into mainly three types. Type I copper proteins (also called

Blue copper proteins) are known to have one copper ion at the active

site. This ion shows some remarkable spectroscopic features: an

intense absorption around 600 nm, with an extinction coefficient of

about 3000 M-1cm-1. Another characteristic feature is the extremely

small hyperfine splitting in the EPR spectrum. Type II copper proteins

have no distinct unique properties. The spectroscopic data of these

proteins are comparable to those of normal copper compounds. Type

III proteins contain antiferromagnetically coupled copper dimers.

29

These proteins are diamagnetic and therefore are EPR silent. In some

proteins, all three types of copper sites are present. Such proteins are

proposed to classify as Type IV. The melanin pigment of the skin is

also a copper containing protein. The copper pigment, plastocyanin is

found to be ubiquitous in green plants and probably participates in

the electron transport chain during photosynthesis.

Copper is an important element in oxidation catalysts for

laboratory and industrial use. Interest in the complexes stems from

the diverse occurrence of copper proteins which function as highly

efficient bio-oxidation catalysts. Copper-dioxygen adducts are

suggested as key reaction intermediates in these enzymatic reactions.

Copper or copper containing enzymes may be involved in

neurotransmission in the locus ceruleus. The copper containing

amine oxidases, capable of oxdatively deaminating primary amines,

constitute a family of enzymes, derived from plant, animal and fungal

sources. These proteins contain two Cu(II) ions per molecule and a

single organic cofactor, pyrroloquinoline quinine (PQQ) capable of

reacting with phenyl hydrazine. Electron spin-echo spectroscopic

studies reveal that two different populations of imidazole are ligated to

Cu(II). In ascorbate oxidase one of the copper ions is in a distorted

tetrahedral coordination with two histidines, one methionine and one

cysteine.

Copper-cytrochrome C provides further evidence of the power of

the protein to act as a macrocyclic ligand. The EPR and electronic

30

spectral studies carried out over the pH range 4.0-11.0 indicated that

the central metal ion is six coordinate. Most copper-porphyrins do not

usually complex with two axial Lewis base ligands. At the extremes of

pH the molecule dimerises, but it probably, retains one axial ligand.

During catalysis Cu(II) dissociates from the enzyme tyrosinase and is

reduced to Cu(I).

Copper is required to fix calcium in the bones and to build and

repair all connective tissues. Imbalances can contribute to

osteoporosis and bone spurs.67 Copper is needed in the final steps of

the Krebs energy cycle called the electron transport system. Grave’s

disease (hyper thyroidism) usually is due to stress and copper

imbalance.67 Copper is closely related to estrogen metabolism and is

required for women’s fertility and to maintain pregnancy.67 Copper

stimulates production of the neurotransmitters, epinephrine,

norepinephrine and dopamine. It is also required for monoamine

oxidase, an enzyme related to serotonin production.67

Agonistic and antagonistic action

Copper interferes with iron metabolism in various ways.

Deficiencies, as well as excess of copper have deleterious effects on

iron metabolism. Deficiency leads to anemia while an excess causes

hemolytic anemia and methemoglobinaemia. It competes with iron

mainly at four metabolic sites viz., (1) gastrointestinal absorption,

(2) mobilization from cells, (3) utilization for heme synthesis and

(4) reutilization in the reticuloendothelial cells.

31

Copper exhibits strong physiological interactions with other

elements. As a result of similarities in the chemistry of copper and

zinc, they share some common ligands and zinc and cadmium may

antagonize the absorption of copper by competing for the same

binding sites. Copper is known to reverse the zinc toxicity. The

intestinal absorption of copper was blocked until the zinc was

eliminated.68 The toxic effects of molybdenum and cadmium are also

reduced by copper.

Deficiency

Deficiency of copper leads to Menkes Disease69 genetic disorder

arises from the widespread defect in intracellular copper transport.

Copper deficiency is characterized by anemia, eutropenia,

oateopenia70 impairs the growth of low birth weight babies71 or infants

treated for conditions associated with malnutrition72, causes neonatal

ataxia, acromotrichia (lack of pigmentation), connective tissue defects

(including bone disorders and cardiovascular failure), defect in

phospholipid synthesis and spontaneous bone fractures, increases

free protoporphyrin in erythrocytes and decreases reproductive

capacity and milk production. Deficiency of copper in plants has been

associated with a reduction in chlorophyll content. Human copper

deficiency.73 results in neutropenia, which has been suggested to

result from an arrest of cell maturation.

32

Toxicity

High accumulation of copper in the liver, kidney and brain

causes Wilson’s disease74 (hepatolenticular degeneration) arises due to

genetic disorder in copper metabolism. Ultimately the accumulation of

copper leads to nervous disorders (mild tremors) and pathological

lesions especially in the liver. In extreme cases the light brown circles

referred to as Kayser-Fleischer rings surrounding the iris are seen,

which are caused by deposition of copper salts in the Cornea.75 The

alterations in its cellular homeostasis are connected to serious

neurodegenerative diseases, like Alzheimer’s disease76 (due to

increased concentration of copper), familial amyotrophic lateral

Scelerosis77, 78 and prion diseases79 even though there is no evidence

of teratogenic, mutagenic or carcinogenic action. In addition, there is

increased excretion of amino acids and dicarboxylic amino acid

peptides. The condition is believed to be a result of impaired synthesis

of ceruloplasmin and is a hereditary defect transmitted by a single

autosomal recessive gene. There is an increase in absorption of copper

from the gastrointestinal tract with a reduced faecal excretion and an

increased quantity of copper in the urine. The deposition of copper in

the brain produces tremor, rigidity, dysphasia and the signs of

Parkinsonism along with some abnormal mental symptoms. The renal

deposition of copper can interfere with enzyme systems responsible for

the transport of material across epithelial membranes and results in

an increased excretion of amino acids, calcium, phosphate, glucose

and bicarbonate. In the liver, cellular necrosis followed by fibrosis

33

leading to cirrhosis can occur. The treatment of Wilson’s disease

involves the administration of various chelating agents like BAL, D-

penicillamine, trien etc. which are capable of mobilizing the excess

copper.

SOLVENTS

1, 4-DIOXAN

1, 4-Dioxan (Dox) (C4H8O2) (Fig. 1.6), often called dioxin

because the other isomers of dioxan are rare. Dioxan is a clear,

colorless, flammable heterocyclic flammable heterocyclic organic

liquid at room temperature and pressure with a mild, pleasant, ether-

like faint pleasant odour80. This colorless liquid is mainly used as a

stabilizer for the solvent trichloroethane. It is an occasionally used

solvent for a variety of practical applications as well as in the

laboratory. Dioxan is soluble in water and most organic solvents.80 It

is byproduct of the ethoxylation process in the production of materials

used in cosmetics, notably sodium myreth sulfate and sodium laureth

sulfate.81

Fig. 1.6: 1, 4-Dioxan

34

Dox is used as a solvent in a wide range of organic products like

lacquers, paints, varnishes, paint and varnish removers, wetting and

dispersing agent in textile products, dye baths and stain and printing

compositions, cleaning and detergent preparations, cements,

cosmetics, deodorants, fumigants, emulsions, and polishing

compositions. It is also used as a stabilizer for chlorinated

solvents. 82Dox is used as a solvent in chemical synthesis, as a fluid

for scintillation counting, and as a dehydrating agent in the

preparation of tissue sections for histology.83, 84

Reactivity

Conditions contributing to instability are heat, sunlight or

flame. Incompatibilities are contact of dioxan with oxidizing agents. It

reacts violently with hydrogen in the presence of Raney nickel (above

2100 C/4100 F) with decaborane, which is impact-sensitive; with

triethynylaluminum, which is sensitive to heating or drying; and with

sulfur trioxide. There is a potentially explosive reaction with nitric acid

in the presence of perchloric acid. Hazardous decomposition products

are toxic vapors (such as carbon monoxide) may be released in a fire

involving dioxan when dioxin undergoes thermal oxidative degradation

and special precautions are dioxan is hydroscopic and will produce

peroxides in the presence of moisture. Dioxan-containing peroxides

should not be distilled to dryness because of the potential explosion of

non-volatile peroxides.

35

Effects of Human Exposure

Dioxan is absorbed by all routes of administration.85 In

humans, the major metabolite of dioxan is β-hydroxyethoxyacetic acid

(HEAA) and the kidney is the major route of excretion.86 The enzyme

responsible for HEAA formation has not been studied, but data from

Young et al.87 indicate saturation does not occur up to an inhalation

exposure of 50 ppm for 6 hours. Under these conditions the half-life

for dioxan elimination is 59 min (plasma) and 48 min (urine).

Although physiologically based pharmacokinetic (PBPK) modeling

suggests HEAA is the ultimate toxicant in rodents exposed to dioxan

by ingestion, the same modeling procedure does not permit such a

distinction for humans exposed by inhalation.88

Several anecdotal reports have appeared in which adverse

health effects due to chronic dioxan exposure are described. Barber

described dioxane exposed factory workers89, some of whom exhibited

signs of liver changes, increased urinary protein and increased white

blood cell counts, and some of whom died from apparent acute

exposures. Although the kidney and liver lesions were considered

manifestations of acute exposure, the author suggested a chronic

component that was manifested by increased white blood cells. A case

was reported in which a worker, who died following exposure by

inhalation and direct skin contact to high (unspecified) dioxan levels,

exhibited lesions in the liver, kidneys, brain and respiratory system.

However, the effects could not be easily separated from the effects due

to high intake of alcohol.90

36

In a German study91,92 74 workers exposed to dioxan in a

dioxan-manufacturing plant (average potential exposure duration - 25

years) underwent evaluation for adverse health effects. Air

measurements indicated dioxan levels varied from 0.01 to 13 ppm.

Clinical evaluations were applied to 24 current and 23 previous

workers. Evidence of increased (i.e., abnormal) aspartate

transaminase (also known as serum glutamate-oxalacetic

transaminase or SGOT), alanine transaminase (serum glutamate

pyruvate transaminase or SGPT), alkaline phosphatase and gamma

glutamyltransferase activities (liver function) was noted in these

workers, but not in those who had retired. The indicators of liver

dysfunction, however, could not be separated from alcohol

consumption or exposure to ethylene chlorohydrin and/or

dichloroethane.

A follow-up mortality study93 was conducted on 165 chemical

plant manufacturing and processing workers who were exposed to

dioxan levels ranging from less than 25 to greater than 75 ppm

between 1954 and 1975. Total deaths due to all causes, including

cancer, did not differ from the statewide control group, but the data

were not reanalyzed after removing the deaths due to malignant

neoplasms. The study is limited by the small number of deaths and by

the small sample number. The study did not assess hematologic or

clinical parameters that could indicate adverse health effects in the

absence of mortality.

37

Yaqoob and Bell94 reviewed human studies on the relationship

between exposure to hydrocarbon solvents - including dioxan - and

renal failure, in particular rare glomerulonephritis. The results of their

analysis suggest that such solvents may play a role in renal failure,

but dioxan was not specifically discussed. Of interest to the

discussion on chronic exposure to dioxan is the suggestion that the

mechanism of the disease process involves local autoimmunity with

decreased circulating white blood cells.

Effects of Animal Exposure

In rats, the major metabolite of dioxan is HEAA, which is

excreted through the kidneys.95 Exposure to dioxan by ingestion

results in saturation of metabolism above 100 mg/kg given in single

dose. Saturation of metabolism was also observed as low as 10 mg/kg

if dioxan was administered in multiple doses. Dioxan itself is not

cleared through the kidney. A decrease in metabolic clearance with

increasing dose has been interpreted as the saturation of metabolism

at the higher doses.96

For Sprague-Dawley rats, the metabolic fate of inhaled dioxan

(head only exposure) was based on one air concentration (50 ppm). At

this level, nearly all the dioxan was metabolized to HEAA since HEAA

represented 99 percent of the total dioxan + HEAA measured. The

plasma half-life for dioxan under these conditions was 1.1 hr. The

absorption of dioxan through the inhalation pathway could not be

exactly determined, because of a high inhalation rate (0.24

liters/min), calculated on the basis of complete absorption96,97.

38

Although the high inhalation rate could be dioxan related, another

explanation may be the stress incurred when the jugular veins were

cannulated as part of the experiment. Extensive absorption by

inhalation is also inferred from the high tissue/air partition

coefficients.98

Although the PBPK modeling suggests that in rat the parent

dioxan is a better dose surrogate than HEAA for exposure by

ingestion, the inhalation modeling did not use more than one

inhalation dose. No studies were located on the biological or

biochemical properties of HEAA or the properties of the enzyme(s) that

are responsible for the transformation of dioxan into HEAA.

Rats were exposed by inhalation to dioxan (111 ppm; 7 h/day, 5

days/week) for 2 years.98 Increased mortality and decreased body

weight gains, compared to unexposed control rats, were not observed.

Among the male rats, decreased blood urea nitrogen (kidney function),

decreased alkaline phosphatase (cholestatic liver function), increased

red blood cells, and decreased white blood cells were observed.

In another inhalation study, rats were exposed to dioxan at

levels of 0.15, 1.3, and 5.7 ppm.99 Frequency was not specified, but

the duration is given as 90 successive days. At the end of the 3-

month exposure, increased SGOT activity at the two highest doses

and increased SGPT activity at all doses were measured in the sera of

the exposed rats. Rats exposed to the highest dose also exhibited

increased urinary protein and chloride levels, each of which returned

39

to control levels during an unspecified recovery period. Pilipyuk et al.

99also report changes in the minimum time (ms) required for an

electric stimulus to result in excitation of extensor and flexor muscles.

Although they considered the changes to be a reflection of adverse

effects due to exposure to dioxan, Torkelson et al.98 not considered the

hematologic and clinical changes of toxicologic importance. In

particular, toxic manifestations are usually associated with increased

blood urea nitrogen and alkaline phosphatase levels, whereas these

levels decreased in the Torkelson et al.98 investigation.

Kociba100 exposed rats (Sherman) to dioxan by ingestion of

drinking water for up to 2 years. The drinking water levels were 0,

0.01, 0.1, and 1.0 percent, which were converted to daily intake

according to measured rates of water consumption during exposure.

Exposure to the highest level resulted in decreased body weight gain

and increased deaths but exposure related hematologic changes did

not occur. Histopathologic examination revealed evidence of

regeneration of hepatic and kidney tissues in rats exposed to 1.0 or

0.1 percent, but not in rats exposed to 0.01 percent dioxan. On the

assumption of total absorption of dioxan from the gastrointestinal

tract, the exposure levels in female and male rats are as follows:

0.01%-18 ppm/F, 9.3 ppm/M; 0.1% -144 ppm/F, 91 ppm/M.

The teratogenic potential of dioxan was studied in

rats.101 Dioxan was administered by gavage at doses of 0, 0.25, 0.5,

and 1.0 mL/kg-day, on gestation days 6-15, and observations

continued through day 21. Dams exposed to the highest dose

40

exhibited non-significant weight loss and a significant decrease in food

consumption during the first 16 days. During the remaining 5 days,

food consumption increased, but the weight gain reduction in the

presence of dioxan continued. At the 1.0 mL/kg-day dose, mean fetal

weight and ossified sternebrae were also reduced. The inability to

separate the developmental toxicity from maternal or embryo toxicity

renders these data inconclusive as to the developmental toxicity of

dioxan. If toxicity to the dam and/or embryo exists, the NOAEL for

dioxan (based on density = 1.03 gm/mL) is 517 mg/kg-day.

1, 2-PROPANEDIOL

l, 2–Propanediol (C3H8O2) is a small, hydroxyl substituted

hydrocarbon. Its trade names are propylene glycol (PG),

monopropylene glycol, 1, 2-dihydroxy propane, trimethyl glycol.

Commercially it is manufactured by direct hydrolysis of propylene

oxide by water.102

Fig. 1.7: Propylene glycol

PG is a widely used compound with diverse applications. It is

one of the most commonly used humectants-substances that have a

high affinity for water and have a stabilizing action on the water

content of a material. It is used to maintain moisture within a narrow

range in certain food products, such as coconut and marsh mallows,

41

as well as in tobacco.103 It has been extensively employed in the

pharmaceutical industry as a solvent for drugs, as a stabilizer for

vitamins and in pastes for medicinal purposes. It is also used to

absorb extra water and maintain moisture in certain cosmetics, and is

a solvent for food colors and flavors. It is used in antifreeze and

deicing solutions. It is used as a solvent in the paint and plastics

industries, and to make polyester compounds. It is used as a

substitute for ethylene glycol, monoalkyl ethers in all purpose

cleaners, coatings, inks, nail polish, lacquers, latex paints, and

adhesives. It is also used to create artificial smoke or fog used in fire-

fighting training and in theatrical productions.104 PG is used as a

chemical intermediate in the production of unsaturated polyester

resins, liquid detergents, deicing fluids, antifreeze/engine coolant,

paints and coatings. It is one of the chemicals ‘generally recognized as

safe’ (GRAS) by the U. S. Food and Drug Administration.

It is released into the environment from industrial disposal and

from consumer products containing this chemical. It does not

bioaccumulate in organisms and rapidly biodegrades in the soil and in

the water.105 This process is oxygen-demanding and can deplete

dissolved oxygen levels in water.106 It is exposed by dermal contact or

ingestion of PG containing products. Inhalation from such products

may also occur. Oral exposure occurs through its use in food, tobacco

products and in prescription and over-the-counter medicines.107 In

most mammals, part of the absorbed PG is eliminated unchanged by

42

the kidney, while other portion is excreted as a glucuronic acid

conjugate.108 PG and ethanol are substrates that compete for alcohol

dehydrogenase in the initial step of metabolism. Less oxygen is

required by PG for oxidation, accounting for the decreased oxygen

consumption than dextrose for production of energy.

PG is oxidized by alcohol dehydrogenase to D and L forms of

lactaldehyde, then to lactate by aldehyde dehydrogenase. The lactate

is further metabolized to pyruvate, carbon dioxide and water. Lactate

also contributes to glucose formation through glyconeogenic

pathway.109 Lactate, via phosphoenol pyruvate can be detoxified into

glucose and stored as glycogen.110 In alternate route of metabolism the

conversion of lactaldehyde to methyl glyoxal by alcohol dehydrogenase

and then to lactate by glyoxalase and reduced glutathione is observed

(Fig. 1.8).

The other possible metabolic pathway in which PG can be

phosphorylated, converted to acetol phosphate, lactaldehyde phosph-

ate, lactyl phosphate and lactic acid111 is given in (Fig.1.9). Excess

production of lactic acid resulting from very large exposures can

produce a metabolic anionic gap and acidosis.112 Serum levels of PG

greater than 180 mg/L can result in toxicity.113

43

Fig. 1.8: Propylene glycol metabolism in mammals

44

Fig. 1.9: Phosphorylated Propylene glycol metabolism in mammals

45

SIGNIFICANCE OF PROPOSED STUDY

Co(II), Ni(II) and Cu(II) are the essential metal ions and any

variation in their biological concentrations leads to metabolic

disorders. With this in view the author has investigated the

protonation equilibria of L-Asp and ethylenenediamine and their

binary and ternary complexes with the metals in Dox- and PG-water

mixtures of varying compositions.

Dox and PG are selected in these studies to maintain the

dielectric properties of the medium in comparable levels to those of

the physiological fluids since the polarity at the active site cavities

should generally be applicable when it is possible to compare ligand

binding to the metal ion in protein and mixed solvent environments.

These solutions are expected to mimic the physiological

conditions where the concept of the equivalent solution dielectric

constant114 for protein cavities is applicable. The studies carried out

on these systems under the present experimental conditions are

useful to understand (i) the role played by the active site cavities in

biological molecules, (ii) the type of complex formed by the metal ion

and (iii) the bonding behavior of the protein residues with the metal

ion. The species refined and their relative concentrations under the

present experimental conditions represent the possible forms of metal

ions in the biological fluids.

Speciation analysis has become important in human biology,

nutrition, toxicology and in clinical practice. However, concentrations

46

of essential, endogenous elements are frequently controlled

homeostatically, and instances of pathology arising from altered

speciation of these elements are relatively rare. When a major ligand is

deficient it is the total concentration, rather than its distribution

among species, is of most diagnostic significance. On the other hand,

speciation profoundly influences both the toxicity and bioavailability

of an element.

Further, computer augmented modeling studies were carried

out to arrive at the best fit chemical models and to check their

validity. The results of these investigations are presented in the

subsequent chapters.

47

References

1. Templeton D. M., Ariese, F., Cornelis, R., Danielsson, L. G.,

Muntau, H., Van Leeuwen, H. and Lobinski, L., Pure Appl.

Chem. 72 (2000)1453.

2. Templeton, D. M., Analysis 26 (1998) 68.

3. Templeton, D. M., Fresenius J. Anal. Chem. 363 (1999) 505.

4. Andrea, M. O., Springer-Verlag (1984) 359.

5. Lehninger, A. L., Nelson, D. L. and Cox, M. M., Principles of

Biochemistry, 3rd Ed., Worth Publishing, NY (2000).

6. Krebs, H. A. and Henseleit, K. Hoppe-Seyler’s Z. Physiol.

Chem. 210 (1932) 33.

7. Plimmer, R. H. A., Monographs on biochemistry 18 (2010)

112.

8. Balch Phyllis, A., and James, F., “Amino Acids.” Prescription

for Nutritional Healing. (Ed.) A. C. Tecklenberg. Penguin

Putnam Inc. NY 3 (2000) 42.

9. Koyuncuoglu, H., Psychopharmacol. 54 (1977) 187.

10. Paoletti, P., Pure Appl. Chem. 56 (1984) 491.

11. Martell, A. E. and Smith, R. M., Critical Stability Constants

Vol. 2. Plenum Press, NY (1975).

12. Hiromichi, F., Kenichi, M., Kita, Y., Ozora, K. and Yuruke,

O., Org. Lett. 9 (2007) 1687.

13. Arash, L., Habibi, M. H., Harrington, R. W., Morteza, M. and

William, C., J. Fluorine Chem.127 (2006) 769.

48

14. Karsten, E., Erhard, H., Roland, R. and Hartmut, H.,

"Amines, Aliphatic" in Ullmann's Encyclopedia of Industrial

Chemistry, Wiley-VCH Verlag, Weinheim (2005).

15. Kotti, S. R. S. S., Timmons, C. and Li, G., J. Chem. Biol. Drug

Design 67 (2006) 101.

16. Schenectady, Material safety data sheet No. 325, Genium

Publishing Corporation, NY (1993).

17. Sax, N. I. and Lewis, R. J., Dangerous properties of industrial

materials. 7th ed., Van Nostrand Reinhold Company, NY

(1989).

18. Documentation of the threshold limit values and biological

exposure indices. 6th ed. Cincinnati, OH., American

Conference of Governmental Industrial Hygienists (ACGIH)

(1991).

19. Ethylenediamine. Cincinnati, OH: U. S. Department of Health

and Human Services (DHHS), Public Health Service, Centers

for Disease Control (PHSCDC), National Institute for

Occupational Safety and Health (NIOSH), Division of

standards Development and Technology Transfer (DSDTT),

Technical Information Branch (TIB), Registry of toxic effects of

chemical substances (1991).

20. Grant, W. M., Toxicology of the eye. 3rd ed.

Springfield, IL: Charles C. Thomas Publisher, NY (1986) 919.

49

21. Sittig, M., Handbook of toxic and hazardous chemicals and

carcinogens. 3rd ed, Noyes Publications, Park Ridge, NJ

(1991).

22. Hathaway, G. J., Proctor, N. H., Hughes, J. P. and Fischman,

M. L. Proctor and Hughes' chemical hazards of the workplace.

3rd ed., Van Nostrand Reinhold, NY (1991).

23. Patnaik, P., “A comprehensive guide to the hazardous

properties of chemical substances”. Van Nostrand Reinhold.

NY (1992).

24. Clayton, G. and Clayton, F. “Patty's industrial hygiene and

toxicology. 3rd rev. ed”. John Wiley & Sons. NY (1981-1982).

25. Gosselin, R. E., Smith, R. P. and Hodge, H. C., “Clinical

toxicology of commercial products. 5th ed”. Baltimore, M. D.,

Williams & Wilkins (1984).

26. Hazardous substances data bank: Ethylenediamine, National

Library of Medicine, Bethesda, M. D., (1992).

27. Cotzias, G. C., Trace substances., Environ. Health. Proc. Univ.

Mo. Ann. Conf. 5 (1967).

28. Underwood, E. J., Trace elements in Human and Animal

Nutrition, Academic Press, 4th Ed., NY (1977)230.

29. Venchikov, A. I., Trace elements in human and animal

nutrition, Academic press, 3rd Ed., NY (1974).

30. Berthon, G., Handbook of Metal-ligand Interactions in

biological Fluids, Marcel Dekker, NY (1995).

50

31. Minot, G. R. and Murphy, W. P., J. Am. Med. Assoc. 87

(1926) 470.

32. Matthew, J., “Super alloys”, ASM International (2002).

33. Matthews, R. G. and Drummond, J. T., Chem. Rev. 90 (1990)

1275.

34. Rama, S. V. S., Prakash, R. and Agarwal, V. P., Ash. Hig.

Rada. Toksikol. 36 (1985) 365.

35. Bovykin, B. A., Eksp. Klin. Farmakol. 57 (1994) 57.

36. Chekunova, M. P., Frolova, A. D. and Minkina, N. A., Gig.

Sanit. 7 (1986) 29.

37. Basketter, D. A., Angelini., G., Ingber, A., Kern, P. S and

Menne, T., “Nickel, Chromium and Cobalt in consumer

products” (2003).

38. “Report on Carcinogens”, 11th Ed. Cobalt sulphate., Nat. Toxi.

Prog. (2008).

39. Balazs, R., Jorgenson, O. S. and Hack, N., Neurosci. 27

(1988) 437.

40. Hu, L. T. and Mobley, H. L., Immun. 58 (1990) 992.

41. Mulrooney, S. B. and Hausinger, R. P., FEMS Microbiol. Rev.

27 (2003) 239.

42. Dunn, B. E., Champbell, G. D., Perz – perz, G. I. and Blaser,

M., J. Biol. Chem. 265 (1990) 9494.

43. Hansinger, R. P., Biochemistry of Nickel, Plenum, NY (1993).

44. Maier, R. J., Fu, C., Gilbert, J., Moshiri, F., Olson, J. and

Plaut, A. G., FEMS Microbiol. Lett. 141 (1996) 71.

51

45. Olson, J. W. and Maier, R. J., Science 298 (2002) 1788.

46. Adams, M. W. W., Biochim. Biophys. Acta 1020 (1990) 115.

47. Cammack, R., Nature 373 (1995) 556.

48. Kolodziej, A. F., Prog. Inorg. Chem. Wiley, NY 41 (1994) 493.

49. Poellot, R. A., Shuler, T. R., Uthus, E. O. and Nielsen, F. H.,

Proc. Natl. Acad. Sci. USA 44 (1990) 80.

50. Cammack, R., Adv. Inorg. Chem. 32 (1998) 297.

51. Bible, K. C., Buytendorp, M., Zierath, P. D. and Rinehart, K.

L., Proc. Natl. Acad. Sci. USA 85 (1998) 582.

52. Anke, M., Groppedl, B., Krause, U. and Langer, M., Nickel in

Trace Elements in Man and Animals. Plenum, NY (1988) 467.

53. Wever, R. and Kren, B. E., Vanadium in biological Systems.

Kluwer, Dordrecht, Netherlands (1990) 81.

54. Nielsen, F. H., Zimmerman, T. J., Shuler, T. R., Brossart, B.

and Uthus, E. O., J. Tr. Elem. Exp. Med. 2 (1989) 21.

55. Nielsen, F. H., “Nickel in Trace Elements”, in Human and

Animal Nutrition.” W. Mertz (Ed), Academic Press, San Diego,

California 1 (1987) 245.

56. Misra, M., Olinski, R., Dizdaroglu, M. and Kasprzak. K. S.,

Chem. Res. Toxicol. 6 (1993) 33.

57. Chakrabarti, S. K., Bai, C. and Subramanian, K. S., Toxicol.

Appl. Pharmacol. 170 (2001) 153.

58. Kawanishi, S., Inoue, S., Oikawa, S., Yamashita, N.,

Toyokuni, S., Kawanishi, M. and Nishino, K., Free Radical

Biol. Med. 31 (2001) 108.

52

59. Nielsen, F. H., Shils. M. E., Olson, J. A., Shike, M., Ross, A.

C. and Williams, W., Other Trace elements. Inc., Modern

Nutrition in Health and Disease. 9th Ed Baltimore, MD (1999)

283.

60. “Trace Elements in Human Nutrition”. Tech. Report Series.

No.532. WHO, Geneva (1973).

61. Walker, W. R, Reeves, R. R., Brosnan, M. and Coleman, G.

D., Bioinorg. Chem. 7 (1977) 271.

62. Holm, R. H., Kennepohl, P. and Solomon, E. I., Chem. Rev.

(1996) 96.

63. Mukherjee, R. N., Comprehensive Coordination Chemistry-II

From biology to nanotechnology, Mc Cleary, J. A. and Meyer,

J. (Eds) Elsevier 5 (2003).

64. Halcrow, M. A., J. Chem. Soc. Dalton Trans (2003) 4375.

65. Li, T., Huang, J. W., Ma, L., Zhang, Y. Q. and Ji, L. N., Trans.

Met. Chem. 28 (2003) 288.

66. Van den Berg, G. J. and McArdle, H. J., Biochim. Biophys.

Acta 276 (1994) 1194.

67. Nolan, K., J. Orthomol. Psych. 12 (2008) 270.

68. Hoffmann, H. N., Phyliky, R. L. and Fleming, C. R.,

Gastroenterology 94 (1986) 508.

69. Waggoner, D. J., Bartnikas, T. B. and Gitlin, J. D., Neurobiol.

Disease 6 (1999) 221.

70. Cardana, A., Baertl, J. M. and Graham, G. G., Pediatrics 34

(1964) 324.

53

71. Soo, T. L., Simmer, K., Carlson, L. and McDonald, L., Arch.

Dis. Child. 63 (1988) 79.

72. Castillo-Duran, C. and Uavy, R., Am. J. Clin. Nutr. 47 (1988)

710.

73. Patrice, M. and Percival, S. S., J. Nutr. Immunol. 3 (1994) 5.

74. Vulpe, C., Levinson, B., Whitney, S., Packman, S. and

Gitschier, J., Nat. Genet. 3 (1993) 7.

75. Sterlieb, I., Med. Radiogr. Photogr.42 (1966) 14.

76. Barnham, K. J., Masters, C. L. and Bush, A. I., Nat. Rev.

Drug. Discovery 3 (2004) 205.

77. Valentine, J. S. and Hart, P. J., Proc. Natl. Acad. Sci. USA.

100 (2003) 3617.

78. Bruijn, L. I., Miller, T. M. and Cleveland, D. W., Annu. Rev.

Neurosci. 27 (2004) 723.

79. Brown, D. R. and Kozlowski, H., J. Chem. Soc. Dalton Trans

(2004) 1907.

80. Budavari, S., The Merck index, 12th Ed., Whitehouse Station,

NJ, Merck & Co. (1996) 559.

81. Black R. E., Hurley F. J. and Havery D. C. J. AOAC

International 84 (2001) 666.

82. Lewis, R. J. Jr, Hawley’s Condensed Chemical Dictionary,

12th Ed., New York, Van Nostrand Reinhold (1993) 426.

83. Grant, R., and Grant, C., Grant and Hackh’s Chemical

Dictionary. R. Grant and C. Grant, eds. 5th ed. New York:

McGraw-Hill Book Co. (1987) 189.

54

84. HSDB. Hazardous Substances Data Bank. National Library

of Medicine, Bethesda, MD (TOMESÒ CD-ROM version).

Denver, CO: Micromedex, Inc. (1995) (edition expires

7/31/95).

85. CARB. 1999. Air toxics emissions data collected in the Air

Toxics Hot Spots Program CEIDARS Database as of January

29 (1999).

86. Young, J. D., Braun, W. H., Gehring, P. J., Horvath, B. S.

and Daniel, R. L., Toxicol. Appl. Pharmacol. 38 (1976) 643.

87. Young, J. D., Braun, W. H., Rampy, L. W., Chenoweth, M. B.

and Blau, G. E., J. Toxicol. Environ. Health 3 (1977) 507.

88. Reitz, R. H., McCroskey, P. S., Park, C. N., Andersen, M. E.

and Gargas, M. L., Toxicol. Appl. Pharmacol. 105 (1990) 37.

89. Barber, H., Haemorrhagic nephritis and necrosis of the liver

from dioxane poisoning. Guy’s Hospital Report. 84 (1934)

267.

90. Johnstone, R. T., AMA Arch. Ind. Health 20 (1959) 445.

91. Thiess, A. M., Tress, E. and Fleig, I., Arbeitsmed. Sozialmed.

Praventivmed. 11 (1976) 35.

92. NIOSH. Criteria for a Recommended Standard. Occupational

Exposure to Dioxane. National Institute for Occupational

Safety and Health, Centers for Disease Control, Public Health

Service, Department of Health Education and Welfare.

Publication No. (1977) 77.

55

93. Buffler, P. A., Wood, S. M., Suarez, M. S. and Kilian, D. J., J.

Occup. Med. 20 (1978) 255.

94. Yaqoob, M. and Bell,G. M. Renal Failure., 16 (1994) 425.

95. Braun, W. H. and Young, J. D., Toxicol. Appl. Pharmacol. 39

(1977) 33.

96. Young, J. D., Braun, W. H. and Gehring, P. J. Health. 4

(1978) 709.

97. U.S. EPA (U.S. Environmental Protection Agency).

Recommendations for and Documentation of Biological

Values for Use in Risk Assessment. Chapter 4, United States

Environmental Protection Agency (1988).

98. Torkelson, T. R., Leong, B. K. J., Kociba, R. J., Richter, W.

A., and Gehring, P. J. Toxicol. Appl. Pharmacol. 30 (1974)

287.

99. Pilipyuk, Z. I., Gorban, G. M., Solomin, G. I. and

Gorshunova, A. I. Space Biology and Aerospace Medicine 11

(1977) 70.

100. Kociba, R. J. Chronic toxicity study of dioxan in the drinking

water of Sherman rats. Midland, MI, Dow Chemical Company

(1974).

101. Giavini, W., Vismara, C. and Broccia, M. L., Toxicol. Lett. 26

(1985) 85.

102. Propylene Glycols, Chemical Economics Handbook (2000).

103. Van Nostrand Reinhold. Van Nostrand’s Scientific

Encyclopedia, NY 1 (1989) 1479.

56

104. ToxFAQs for Ethylene Glycol and Propylene Glycol.

Agency For Toxic Substances and Disease Registry.

105. Klecka, G. M., Carpenter, C. L. and Landenberger, B. D.,

Ecotoxicol. Environ. Saf., 25 (1993) 280.

106. Sills, R. D. and Blakeslee, P. A., The environmental impact of

deicers in airport stormwater runoff. (Chelsea, Ed) Lewis

Publishers, Inc. (1992).

107. HSDB. Propylene glycol. National Library of Medicine

(2002).

108. LaKind, J. S., McKenna, E. A., Hubner, R. P. and Tardiff, R.

G., Crit. Rev. Toxicol. 29 (1999) 331.

109. Christopher, M. M., Eckfeldt, J. H. and Eaton, J. W., Lab.

Invest. 62 (1990) 114.

110. Wittman, J. S. and Bawin, R. R., Life Sci. 15 (1974) 515.

111. Ruddick, J. A., Toxicol. Appl. Pharmacol. 21 (1972) 102.

112. Morshed, K. M., L’Helgoualch, A., Nagpaul, J. P., Amma, M.

K. and Desjeux, J. F., Biochem. Med. Metab. Biol. 46

(1991)145.

113. Arbour, R. and Esparis, B., Chest 118 (2000) 545.

114. Sigel, H., Martin, R. B., Tribolet, R., Haring, U. K. and Malini

Balakrishnan, R., Eur. J. Biochem. 152 (1985) 187.