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Reduced population of GulfSturgeon in Suwannee River
• What factors contribute to populationlevels?
• How would you determine which one isbeing affected in this case?
• If you believe it is an anthropogenicchemical that is impacting the population,How do chemicals get into water supplies?
• What happens to chemicals during thisprocess?
Populations
# of individuals in population
Reproduction
Emigration
Mortality
Immigration
•How long an effect takes to manifest inpopulation levels often depends onlongevity and rate of reproduction
Population structure
Age Class
0 2 4 6 8 10
% o
f all in
div
idu
als
in a
ge
cla
ss
0
5
10
15
20
25
30
Chemical Effects on Populations
Concentration of Chemical "X"
0 10 20 30 40 50 60
Bir
th a
nd
De
ath
Ra
tes
0
20
40
60
80
100
120
Birth Rate
Death Rate
Chemical Inputs into WaterSources
From “Fundamentals of Ecotoxicology”, Newman and Unger, eds.
Suwannee River Watershed
What’s happening to chemicals asthey enter bodies of water?
• Dilution• Microbial and photodegradation
– Depends on chemical• Binding to particulates and organic matter
– Leads to sedimentation– Often related to hydrophobicity
• Bioaccumulation– May be none in the water, but very high in food items– Can lead to large differences in effect across species
due to diet
What are the challenges foridentifying impacts in aquatic
toxicology?• Systems are complex
– Biological and chemical complexity• Contaminant concentrations are often low• Inputs are often sporadic• In many aquatic systems, contaminants
dissipate quickly due to flow• This ain’t CSI…
– Analytical methods are incredibly sensitive,but you need to know what you are looking for
How can we narrow down thesearch?
•Look for changes in the organism that areindicative of exposure to specific chemicalsor classes of chemicals•Biomarkers or bioindicator
Quantifiable biochemical, histological orphysiological measures that relate in a dose-ortime-dependant manner the degree of dysfunctionproduced by contaminants (Mayer et al., 1992; in:Biomarkers, edited by Huggett et al., SETACPress)
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
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
• Long-lived chemical– Identify PCBs, dioxin, OCPs directly in blood
or tissue
Elimination of Chlorpyrifos incatfish
Barron et al., 1991, TAAP 108:474
Examples of exposurebiomarkers
• Short-lived chemical– Identify metabolites such as p-nitrophenol
from methyl parathion, or TCP fromchlorpyrifos in urine
– Biliary metabolites of PAHs
Adducts
• When it is difficult to measure measurematerial directly, it is possible to look atreaction products of material
• Really only works for compounds that arereactive or have reactive metabolites– DNA– Protein– Indicative of reaction of active form of
compound with biological material
BAP activation and adduct formation
From Winter et al., 2004, Mutation Res, v. 552
Limitations of exposure biomarkers
• Provide information about absorbed dose,but don’t tell you anything about whetheror not the exposure caused an effect
• Detection can be misleading due tosensitivity of modern analyticalinstrumentation
Concentration analogies• One-Part-Per-Billion
one 4-inch hamburger in a chain of hamburgers circling the earth at theequator 2.5 timesone silver dollar in a roll of silver dollars stretching from Detroit to Salt LakeCityone kernel of corn in a 45-foot high, 16-foot diameter siloone sheet in a roll of toilet paper stretching from New York to Londonone second of time in 32 years
• One-Part-Per-Trillionone square foot of floor tile on a kitchen floor the size of Indianaone drop of detergent in enough dishwater to fill a string of railroad tankcars ten miles longone square inch in 250 square milesone mile on a 2-month journey at the speed of light
• One Part Per Quadrillionone postage stamp on a letter the size of California and Oregonone human hair out of all the hair on all the heads of all the people in theworldone mile on a journey of 170 light years
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
Cholinergic neurotransmission
Cholinesterase Inhibition
•Organophosphateand carbamateinsecticides bind toAChE and inhibit theenzyme•Allows ACh tobuildup, leading tooverstimulation ofreceptors withensuing SLUDsymptoms
Cholinesterase Inhibition
• The organophosphate and carbamateinsecticides are generally potent inhibitors ofAChE, some also inhibit BChE and CBxE. OPsare irreversible, carbamates are reversible
• 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).
• 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.
Reference site Exposed site
Metabolic Enzyme Induction• Some P450s are
inducible by exposureto xenobiotics
• CYP1A1 is stronglyinduced bycompounds that bindto thearylhydrocarbonreceptor (AhR) suchas PAHs and dioxin
• How do you measureCYP1A?
• 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.
• Western blotting for P450s is more isoformspecific and not as sensitive to handling. Manyspecies cross-react, but must use care tovalidate.
• Many compounds can induce CYP1A1• Induction lasts for some time• There are non-lethal methods of analyzing P450
activity, but most are not useful in ecologicalspecies
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
Source: Sepúlveda, Gallagher, Gross. (2004). Ecotoxicol. 13: 291-301.
2
4
6
8
10
12
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
Endocrine Disruption
• Many chemicals have the ability to alter functionof the endocrine system
• May lead to reproductive or developmentalproblems
• A major group is chemicals that are considered“estrogenic” or interact with the estrogenreceptor
• How can you determine if an organism has beenexposed to an estrogenic substance?
Oocyte development andVitellogenein
• major component of eggyolk.
• synthesized in the liver inresponse to estradiol andthen secreted into bloodfor transport todeveloping follicles.
• Normally absent or verylow 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.
• Protein levels remain high in blood for weeksfollowing 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
Oxidative Stress
Consequences of Oxidative Stress
• Lipid peroxidation– Leads to loss of membrane fluidity which can alter activity of
membrane proteins and membrane permeability– Formation of reactive aldehyde products, 4-HNE, acrolein– Oxidized fatty acids can have biological activity- isoprostanes
• Protein oxidation– Oxidation of residues in proteins, especially thiols, can lead to
loss of activity and changes in redox status that modulate genetranscription
• DNA oxidation– Oxidation of DNA bases leads to strand breaks and mutations
Antioxidant enzymes• Superoxide Dismutase (SOD)
– Converts superoxide to hydrogen peroxide– Two forms Cu,Zn-SOD (cytosolic), MnSOD (mitochondrial)
• Catalase– Converts hydrogen peroxide to water and oxygen– Not functional on long chain peroxides, e.g. lipid hydroperoxides
• Glutathione Peroxidase– Selenoprotein in mitochondria and cytosol– Converts peroxides to alcohol and water at expense of GSH– Acts on hydrogen peroxide and organic hydroperoxides– Some GSTs will also act similarly on LOOH
ROH + H20
ROOH GSH
GSSG NADPH
NADP
Markers of Oxidative Stress
• GSH/GSSG ratio• TBARS
– Measures MDA, product of lipid peroxidation– Isoprostanes are more specific (F2)
• Enzymes– Catalase– SOD– GSH peroxidase
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
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.
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.
