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Biomarkers

David Barberbarberd@mail.vetmed.ufl.edu

392-2243 x.5540

Challenge with ecotoxicology

• Identify causative agent(s) in a complexmixture of agents when inputs of thecausative agent may be sporadic

Biomarker

• What is a biomarker? Quantifiable biochemical, histological or

physiological measures that relate in a dose-or time-dependant manner the degree ofdysfunction produced by contaminants (Mayeret al., 1992; in: Biomarkers, edited by Huggettet al., SETAC Press)

– Also called bioindicator

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Types of Biomarkers• Biomarker of Exposure

– Presence of xenobiotic substance or a metabolitewithin an organism

• Biomarker of Effect– Measurable biochemical, physiologic, behavioral

changes in an organism that are recognized disease or health impairment

• Biomarker of Susceptibility– Endpoints that are indicative of altered biochemical or

physiologic state that predispose individual tochemical or infectious agents.

From Metcalf and Orloff, 2004

• Idea is to identify effects before theybecome a serious problem

They can act as integrative measures atthe suborganismal level to indicateadverse conditions preceding population-level effects

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BiologicalResponse

Exposure/Stress LevelHigh Low

Molecular

Biochemical

CellularTissue

SystemicOrganism

PopulationEcosystem

Biomarkers Can be Measured at DifferentLevels of Biological Organization:

Paradigm for ecological assessment

Temporal ResponseMin/hours Days Weeks/Months Years

BiologicalGenerations

Ecological Relevance

Mechanistic Relevance

Criteria for useful biomarkers• Accuracy• Reproducibility• Sensitivity• Specificity• Plausibility

– How good is the link with outcome• Temporal characteristics• Ease of sampling• Throughput

Potential sources for biomarkerevaluation

• Field collection of samples– Air, water, soil, tissues

• Caged animals• Archived samples• Lab exposures

– Really for development and validation

• Each source has pros and cons. Choicebased on availability and use of data.

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Biomarkers of Exposure

• Usually the exogenous chemical, itsmetabolites, or product of interactionbetween chemical and a target molecule.– Usually measured in easily obtained samples– May not identify source of exposure

Examples of exposurebiomarkers

• Short-lived chemical– Identify metabolites such as p-nitrophenol

from methyl parathion, or TCP fromchlorpyrifos in urine

– Biliary metabolites of PAHs

• Long-lived chemical– Identify PCBs, dioxin, OCPs directly in blood

or tissue

Adducts

• DNA• Protein• Indicative of reaction of active form of

compound with biological material• Usually requires mass spec for specific

adducts• Can also use 32P post-labeling

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From Winter et al., 2004, Mutation Res, v. 552

Biomarker of Effect

• A measurable biochemical or physiologicalchange in a biological system that iscorrelated with the development ofadverse effects– Usually early in the process, so is predictive of

effect

Cholinesterase Inhibition• The organophosphate and carbamate

insecticides are generally potent inhibitors ofAChE, some also inhibit BChE and CBxE. OPsare irreversible, carbamates are reversible

• AChE is found in brain and RBC, others arefound in plasma and tissue

• Inhibition of RBC AChE or plasma BChE is goodmarker of exposure, inhibition of brain AChE iswell correlated with toxicity (though really needat least 50% inhibition to cause observable signsof toxicity).

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• Blood is good because it doesn’t require killingthe animal, can also do serial sampling

• Activity usually remains depressed for days toweeks after OP exposure. This is good,because chemical itself is rapidly hydrolyzed inbody and in environment.

• Activity varies with species, temperature, age(length), sex; so care must be used withinterpretation. Good to have concurrent controlsfrom non-contaminated site.

From Whitehead et al.,2005, Ecotoxicology 14.

Metabolic Enzyme Induction• Some P450s are inducible by exposure to

xenobiotics• CYP1A1 is strongly induced by compounds that

bind to the arylhydrocarbon receptor (AhR)• CYP1A1 is major catalyst of ethoxyresorufin-O-

dealkylation (EROD)• Assay is easy, but requires samples of liver to

perform. Samples must be handled carefully tomaintain activity.

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• Western blotting for P450s is more isoformspecific and not as sensitive to handling.Many species cross-react, but must usecare to validate.

• Many compounds can induce CYP1A1• Induction lasts for some time• There are non-lethal methods of analyzing

P450 activity, but most are not useful inecological species

Proteins: CYP450sEROD = Ethoxyresorufin-O-deethylase Activity

Effects of Paper Mill Effluents onLargemouth Bass Reproduction

Cedar Creek

Palatka

Etonia Creek

Rice Creek

Welaka

Dunn’sCreek

10 Km0

N

Mainstream Reference

Tributary ReferenceSt. Johns River

Location of Paper Mill Plant(direction of River flow is North)

Tributary Effluent-exposed

Tributary Effluent-exposed

Field Sites in North-East Florida

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Source: Sepúlveda, Gallagher, Gross. (2004). Ecotoxicol. 13: 291-301.

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4

6

8

10

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Tributary

Reference

Mainstream

Reference

Tributary

Exposed

Mainstream

Exposed

pmol

/reso

rufin

/mg/

min

FemalesMales *

10 10

7

6

10

10 6

5

*

Hepatic EROD activity as a measureof exposure to paper mill effluents

Proteins: CYP450s

From Pretti et al., 2001, CBC Part C v. 130

Vitellogenin• Phospholipoglycoprotein that is major

component of egg yolk.• Vitellogenin is synthesized in the liver in

response to estradiol and then secreted intoblood for transport to developing follicles.

• Normally absent or very low in male and juvenileegg laying animals.

• Induction of Vtg is fairly sensitive marker ofeffect for estrogenic compounds in males andjuveniles. Decreased Vtg in females has beencorrelated with poor reproductive success.

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• Protein levels remain high in blood forweeks following exposure.

• Western blots, ELISA, and mRNA assays• Not clear how well Vtg induction correlates

with adverse effects.

From Diniz et al., 2005, STE, v. 349

∂ -ALAD• Aminolevulinic acid dehydratase• Makes porphobilinogen in the heme synthesis

pathway• Inhibited by lead exposure• Can be measured relatively simply in RBCs• Use concurrent references or reactivate with

zinc to determine inhibition• Fairly specific to lead, though other things can

interfere (need examples)

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From Martinez-Lopez et al., 2004, Arch. Env. Cont.Toxicol., v. 47

Oxidative Stress

• Imbalance of oxidants and antioxidants• Often caused by chemicals that produce

free radicals• Results in membrane, protein, and DNA

oxidation

Markers of Oxidative Stress

• GSH/GSSG ratio• TBARS

– Measures MDA, product of lipid peroxidation– Isoprostanes are more specific (F2)

• Enzymes– Catalase– SOD– GSH peroxidase

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DNA damage• Chromosomal aberrations

– SCE• Mutations

– HPRT– Direct sequencing

• Oxidative damage– 8-oxodG– Strand breaks

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Metallothionein

• Small, thiol rich protein that strongly bindsmany divalent metals including cadmium,zinc, cobalt, copper and mercury

• Induced in response to these metals, somay serve as a potential biomarker

• Fairly complicated to assay MTs• May be faster and cheaper to just look at

metals themselves

Heat Shock Proteins (HSP)• Proteins that are thought to serve primarily as molecular

chaperones, promoting correct folding of proteins• Classified according to MW (e.g, HSP25, 70, 90), but

can be constitutive and inducible forms that are similar insize (HSP70)

• Are induced by improperly folded proteins which activateHSF and increase HSP expression

• Can be induced by temperature, infection, ischemia,xenobiotic toxicity (metals, oxidative stress)

• For many species, there are good Ab available that areuseful for Westerns and immunohistochemistry

• Not specific to a particular chemical or class ofchemicals, but more indicative of cellular stress

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Toxicogenomics• The sequencing of many genomes and

development of arrays and bioinformatics hasproduced a potentially powerful tool

• Ability to analyze thousands of responsessimultaneously may allow identification of“fingerprint”

• Some success at separating classes of toxicants• Problems include timing (genes change rapidly),

cost, most work not clearly related to adverseeffects at higher levels of organization.

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Biomarkers of susceptibility

• Indicator of ability to respond to a toxicchemical challenge– May be inherent (genetic) or acquired (due to

exposure, diet, etc).

Examples of Biomarkers ofSusceptibility

• Deficient DNA repair– Xeroderma Pigmentosum (XP)– Ataxia telangiectasia (AT)– BRCA

• Deficient metabolism– P450 variants

From Mohrenweiser, 2002. In Biomarkers of EnvironmentallyAssociated Disease

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• Induced susceptibility– P450 inhibition

• Grapefruit juice• Drugs

– GST inhibition– MDR inhibition

• Diet is a major factor in this area for humans.Works both ways: increased and decreasedsusceptibility.

Concluding Thought

• To really use biomarkers, must haveexposure and effect. Without both, it isvery difficult to establish causality as mostbiomarkers of effect are not specific.