Universidade Federal de Santa Catarina

Centro de Ciências Biológicas

Departamento de Bioquímica

Laboratório de Biomarcadores de Contaminação Aquática e Imunoquimica - LABICAI

Maria Risoleta Freire Marques, PhD.

Florianopolis, SC, Brazil November, 2002

LABICAI Research projects:LABICAI Research projects:

Biomarkers

Defense Proteins

ECOTOXICOLOGY

COMPARATIVE IMMUNOLOGY

Aquatic Organisms

How to evaluate the impact of xenobiotics?

Why are some xenobiotics more toxic than others?

What determines the “resistance/susceptibility”of organisms?

What are the biochemical and immunological responses of organisms?

Xenobiotics x Environment x Organisms

Environmental monitoring surveys

Quantitative

Analysis

water

sediment

organisms

BIOMARKERS

“Biological alterations that express the exposure and/or the toxic effects of contaminants present in

the environment”(WALKER et al., 1996)

Biochemical and Molecular Techniques

No information about the effects causedto organisms and are expensive

Adams et al. (1989)

Biochemical, Physiological

and Immunological

Responses

1- early detection of contamination

2- risk for species and/or population

3- evaluation and degree of contamination

4- range and effect of xenobiotics (severity)

Some Biomarkers

P450 Isoforms (Phase I)MetallothioneinsConjugation enzymes (Phase II)Antioxidant enzymesStress proteins

CYTOCHROME P450

Catalyses both detoxification and bioactivation reactions (metabolism of xenobiotics and endogenous compounds)

(JOSEPHY, 1997; TERAMITSU et al., 2000)

Family of proteins (Monooxigenases) (45-55 kDa); Heme group; Anchored into the smooth ER.

SubfamilyCYP1A

PAHs

PCBs

Dioxins

EROD

ImmunodetectionRT-PCR

P450 enzymes different but overlapping specificities

“Corcoroca”

From the South of Caribbean to the South of Brazil (Rio Grande do Sul)

Coastal zones; surface waters (turbidity) (FIGUEIREDO, 1977)

“Cangoá”

From Panama to the North of Argentina

Bottom (muddy), Transparent waters.(MENEZES & FIGUEIREDO, 1980)

Paralonchurus brasiliensis (Steindachner, 1875) SCIAENIDAE

Orthopristis ruber (Cuvier, 1830) POMADASYIDAE

MOMAM Project

Southest coast of Brazil (Includes Rio de Janeiro)

Methods: “Immunoblot” (Western blotting) (KLOEPPER-SAMS et al.,1987)

“Dot-Blot” (MCDOUGAL et al., 1983).

Microsomal fraction

Transfer to nitrocellulose

membrane

Primary antibody: mouse Mo Ab 1-12-3 against scup CYP1A ; Secondary antibody: goat anti- mouse IgG horseradish peroxidase conjugate.

`chemiluminesence

detectionDensitometry

SCION Image

CYP1A Immunodetection

CYP1A “Immunoblotting” (Sep/99)

CYP1A “Dot-Blot” (Dec/00)

C (+) = Positive Control, Oreochomis niloticus treated with 20mg/Kg of methylcholantrene.

Cytochrome P450 1A (CYP1A) Aug/00 e Dec/00

Ago/00

Dez/00

PHAs

METALMETALLLOTHIONEINSOTHIONEINS

• Low-molecular weight, heat-stable, cysteine-rich proteins Low-molecular weight, heat-stable, cysteine-rich proteins that bind to metals.that bind to metals.

• Participate directly in metal detoxification processes as well Participate directly in metal detoxification processes as well as in the cellular homeostasis of essential metals, such as as in the cellular homeostasis of essential metals, such as intracelular Zn and Cu intracelular Zn and Cu (donate these metals to metalloproteins).(donate these metals to metalloproteins).

• Their synthesis is induced by a variety of metalic ions, such as Their synthesis is induced by a variety of metalic ions, such as Cd, Cu, Zn, Hg, Co, Ni, Bi and Ag protection against Cd, Cu, Zn, Hg, Co, Ni, Bi and Ag protection against metal toxicity.metal toxicity.

MetalMetalllothioneins (MT) - othioneins (MT) - primary structure features:primary structure features:

Class IClass I - Similar to horse MT (kidney); 38 MTs, including aminoacid sequences found in fish (Pleuronectes platessa), crab (Scylla serrata), lobster(Homarus americanus) and oyster (Crassostrea virginica);

Class II Class II - Proteins phylogenetically distant from those found in mammals, such as those described in yeast;

Class III Class III - Oligopeptides isolated from plants and microorganisms Euglena gracilis and Schizosaccharomyces pombe.

MT - Laboratory experiments

Brown mussel (Perna perna)

Cd exposure (300µg/L) - 7 and 15 days

Gill tissue and digestive gland excised

Sample preparation HPLC analysis (Molecular exclusion - DIOL 150)

0

10

20

30

40

50

60

4.9 6.4 6.9 7.3 7.6 8.4 9.4 9.7 10.7

Time (minutes)

Control

7 Days

15 Days

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

5

4,9 6,4 6,9 7,3 7,6 8,4 9,4 9,7 10,7

Tempo (minutos)

Cd

(p

pm

)

Controle

7 Dias

15 Dias

Identification and partial

characterization of Cd-binding proteins

in Perna perna

(digestive gland)

HPLC

ICP-MS

20

MK (kDa)

17 kDA

10

15

STRESS PROTEINS

First described

Several physical and chemical agents also induce their synthesis

Drosophila melanogaster: temperature, heat-shock proteins - Hsp

Found in different organisms Conserved proteins

hsp 90hsp 70hsp 60hsp 20-30Ubiquitin

Families

787570

kDa

20 ug/L 100 ug/L 70 ug/L 50 ug/L

Immunodetection of Hsp70 isoforms in Perna perna (Brown mussel)

Animals exposed to Cu under laboratory conditions

Gill tissue

Correlation with mRNA levels (Northern blotting)

In situ biomonitoring

LABICAI - UFSC

Florianópolis, SC, BRAZIL

Florianópolis

Island of Santa Catarina

Universidade Federal de Santa Catarina - UFSC

Praia Mole

Praia Brava

In situ monitoring using mollusks (mussels and oysters)

Praia do Sambaqui

Hercílio Luz Bridge

THANK YOU FOR YOUR ATTENTION!!