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Ecotoxicology - Biology 5868Levels of Biological OrganizationToxic Effects - Molecular & CellOctober 1-8, 2004 Patrick K. Schoff, Ph.D.

Concepts:I. Levels of Biological Organization

molecular, cell tissue, organ, individual, population, community, ecosystem,landscape, global

II. Toxicity Testing: Assays for toxicological effects at each Level of Biological OrganizationA. What questions are we asking?

- will change at each LOB- human toxicology - effect of toxicant on individual- ecotoxicology - effect of toxicant on population

B. What is the scope of the answers?- in ecotox, usually we are looking for answers at the upper levels of biol org (e.g.

population or above)

III. Biomonitoring: use of biological system to evaluate current status of ecosystem; twocategories of biomonitoring programs: 1. Exposure (most current biomonitoring)2. Effects (evaluate toxicological status without having to establish mechanism)

- qualitative assessment

NOTE - biomonitoring can be performed at any level of Biological Organization; e.g.< bioaccumulation/biotransformation/biodegradation< biochemical monitoring< physiological and behavioral< population parameters< community parameters< ecosystem effects

NOTE - techniques of monitoring at differentlevels are not uniform

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- Data v. Information (information helps make decisions)

IV. Subjects for toxicity tests/biomarker assessment1. natives2. exotics or non-native models (e.g. Xenopus, fathead minnow, white rat)

Advantages and Disadvantages to both:Natives:

- represent population and ecological community under surveillance- no control over genetic background or variation within group- (usually) little known about toxicological background of native species

Exotics:- database (i.e. biological information) generally larger- some control of source (genetics)- realism/relevance lacking

V. Concept of biomonitoring (or biomarker) efficiency:E = Ui/Bi

where E is efficacy of biomonitoring methodology, Ui is the concentration at which undesirable effects upon the population or ecosystem in

system i occur, Bi is the concentration at which the biomonitoring methods can predict the undesirable

effect in system i

NOTE - efficient system can predict effects of given toxicants

VI. Biomarkers - NOTE - Biomarkers at all levels (including e.g. molecular, tissue, behavioral,

community) - Newman and Ungers’ criteria:

1. measurable before adverse effect at higher level of biol org (chicken before the egg?)2. rapid, inexpensive, easy (possibly the most important - in a pragmatic sense)3. quality control/quality assurance4. specific to single toxicant or class of toxicants (hardly likely)5. concentration-effect relationship should exist (linear over exposure range - best)6. applicable to broad range of sentinel species (do they exist?)7. linkage of biomarker changes with some toxicant-related decrease in individual fitness

(desirable, not necessary)8. system should be familiar; incorporate qualities of organism that influence biomarker

(asking a lot, especially if using native species)

- molecular, physiological (and some behavioral) tests used as indicators of toxicant exposure;possibly eventually used as predictors of effects

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- to date, biomarkers have not proven to be predictive of effects at population, community,or ecosystem levels

- however, useful as measures of exposure, e.g. provide clinical evidence of causative agents indicators of exposure; e.g.

certain enzyme systems only inhibited by a few classes of chemicals; - induction of certain detoxification mechanisms such as specific mixed-

function oxidases used as indicator of exposure- even if agent is below detectable levels; presence of certain enzymes in blood

plasma used as indication of lesions or other damage to specific organs; - good monitoring tool

- Biomarker types- exposure - quantifying only biologically-active toxins- efffects - integrate effects of multiple stressors

- elucidate mechanisms

- Biomarker tests (general categories)- Tier I - screening -Tier II - mechanism

Major problem at molecular level: signal:noise ratio- NOTE - most molecular biomarkers are induced enzymes or protein processing

molecules- the titers of these molecules will naturally fluctuate

- tox signal will be on top of existing signal- NOTE that some of the signal will be existing tox signal (for “normal” tox)

This is a major problem in measuring response at the molecular level of two majorsystems:1. Immune system2. Stress response system

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Immunological biomarkers- both cellular and molecular response- stimulation of immune system provides evidence of both exposure and impact- cellular response - macrophages (either enhance or inhibit activity)

rates of phagocytosis = assay- molecular response - antibodies

- rate of antibody production- antibodies in response to specific challenges

General Stress Response system: adrenocorticotropic hormone (ACTH)- induced by stress

corticosteroids (e.g. cortisol) (effects on intermediary metabolism, immunefunction, inflammationNOTE - handling causes stress

how to distinguish between: - chronic stress v. induced stress (e.g. contaminant) v. acute

stress (e.g. handling)- e.g. case of fish - netted, look at blood levels of corticosteroids- e.g. Galapagos marine iguanas

heat shock proteins (chaperones or chaperonins)- hsp 90, 70, 58 - 60, 20 - 30 kDa; ubiquitin (related to hsp) = 7 kDa; - also, glucose-regulated stress proteins ~100 - 75 kDa

VII. Examples of Molecular Biomarkersacetylcholinesterase (AChE) inhibition

blood plasma (non-destructive)brain (destructive)

MFOs (~150 varieties; possibly >400; possibly >700)including:EROD (ethoxyresorufin O-deethylase)AHH (aryl hydrocarbon hydroxylase)

DNA repair enzymese.g. photolyase, endonuclease, exonuclease, ligasessome question of induction

stress proteins; i.e. hsps (heat shock proteins)metallothioneinsimmunological biomarkerscompetitive receptor binding (e.g. AhR, ER, AR)*anti-clotting proteins (e.g. response to coumarin-based rodenticides)hemoglobin adducts (e.g. carboxyhemoglobin)*vitellogen induction (%)

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DNA strand breakage*micronuclei formation*chromosomal sequence mutations

- must be inducible - must respond to specific stressor

VIII. MethodsEnzymes: conventional assaysProteins: Western blots, etc.

VIII. “-omics” Approaches (Systems Biology)gene expression is altered either directly or indirectly by toxic exposure

- more sensitive (most sensitive of LBO)

Principle: DNA ÷ protein ÷ effectcontaminant ÷ induction of gene ÷ protein (e.g. P450, hsp) ÷ effect/detox

DNA level: - find message for tox response gene- use PCR-based method to quantify gene response- patterns of gene expression = fingerprints = biomarker of exposure

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- microarray: identify patterns; method for identifying mechanisme.g. apoptosis, cell cycle, DNA repair, CYPA1or diagnostic (predictive) array

Protein level:- caveat for genomics approach

1. degradation - messages transcribed but not translatedhowever, proteins will have biological activity (NOTE -considerable degradation of proteins, also)

- protein approach: identify tox-proteins- e.g. 2-D gels

- multiple proteins induced by toxicant: “suite” of tox-response proteins- analysis of 1) exposure

2) effects3) mechanism (for toxicants with unknown pathways)

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e.g. 2-D gels of control v. treated cells/tissues- show response in certain proteins- appearance/disappearance; intensity reveals pathways

Protein/peptide Microarray- antibodies or affinity matrix for selected proteins on matrix- protein preparation from control v. exposed cells/tissues/organism

- examples of commercial genomics-based technologies:ToxBlot (AstraZeneca) chips; e.g.

Immune systemEndocrine DisruptorCancer

GeneChip (Affymatrix); e.g. rat U34 chip

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High Throughput Assays - goal of much assay development- both assay and analysis- need: pattern recognition software

QSAR or QSAM-basedNOTE - high throughput means automation

Caveats, continued - more caveats for systems biology approach2. is gene/protein pivotal in pathway for toxicity?3. is pathway reversible?4. does gene/protein expression lead to altered cell or tissue function?5. is the pathway extensible to other species? (cross-species extrapolation)

- need “bridging” pathways

Cell, molecular-based assays = save animals- of importance to society (groups), government (funding), scientists (ethics, $)

IX. Thresholds- define thresholds, hormesis- no threshold contaminants, e.g. ionizing radiation- threshold shifts according to effect being measured; e.g. because of degradation of

messages & proteins, effects threshold for contaminant would be different

X. Cellular and Tissue Effects / Toxicity Tests

DNA/chromosomeC Genotoxicity - damage by a physical or chemical agent to genetic material

e.g.: - oxidative damage - free radicals produced breaks in one or both sides of molecule- oxyradicals also oxidize bases- xenobiotics and metabolites may form adducts - covalent bond to base

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- metals bind to phosphate groups or bases- Cu binds between bases; competes with normal H-bonding- Hg forms strong crosslinks between strands of DNA

C Adducts- activated forms of PAHs - covalently bound

assays: 32P-post-labeling - sensitive; 1 adduct/1010 basesdetect using GC/MS for specific adductsELISAsfingerprinting

C Thymine-Thymine dimers - UV damageC DNA/chromosome techniques

- strand breakage - alkaline unwinding assay- fragmentation - Comet assay- chromosome analysis - SCE - sister chromatid exchange (NOTE - SCE not damage, but

occurs at mutational “hotspots”either conventional labeling or flow cytrometry

- micronuclei (abnormal mitosis)- karyotyping (e.g. aneuploidy)

C Mutation analysisC Repair enzymesC ApoptosisC Tumors (see below)

Enzyme AnalysisC Cholinesterase activity

- OPs and carbamates- diagnostic - finding $50% inhibition of brain AChE (plus residual OP or carbamate) =

cause of death (birds)fish - more variable; 40 - 80% inhibition

-brainessentially irreversible (recovery ~4 mo)biopsy - sacrifice

-blood plasmaAChE & BuChEmore rapidly metabolizedreversibleno biopsy

C Cytochromes P450- e.g. various oxidations, etc.- inducible, therefore, can use them as exposure diagnostic

e.g. if phenobarbital class induced, treatment with sedative less effective inexposed animal than in naive

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- two major classes: 3-methylcholanthrene-incucible (CYP1A)phenobarbital-inducible (CYP2B)

- assessment methods:enzyme assaysWestern blotsELISAsmRNA

C Hemoglobin synthesis- ALAD (*-aminolevulinic acid dehydratase): sensitive to Pb - reversible by zinc

C Porhyria- heme sythesis = 12 step enzymatic process; vulnerable to metals, etc- many chlorinated organics affect hemoglobin synthesis; hexachlorobenzene, PCBs- result in accumulation of highly carboxylated porphyrins- liver, plasma, urine, feces- HPLC determination of particular porphyrin- NOTE - contaminant has effect on necessary pathway - not just homeostatic response

Heat Stress Proteins (detect by Westerns, etc.; see above)Genomics, Proteomics (see above)

Vitellogenin C yolk protein found in many species (e.g. fish)

- gene found in males and females- induced by estrogens- diagnostic for estrogen exposure in males and immature females- sensitive assay

Tissue Slices, cell preps, cell culture (e.g. vitellogenin in liver)C liver slicesC hepatocytesC cultured cells

- primary - cell line

C transfection- stable- transient - insert genes of interest - look for receptor binding - activation of gene

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- use as indicator of toxin

Metabolites of contaminantsC rapid metabolism (e.g. PAHs) = not find contaminant in tissuesC metabolites found in eg:

- blood plasma- bile - relatively high concentrations, “clean” not many lipids

Oxidative Damage (also, see above)C PAHs, halogenated aromatic hydrocarbons, heavy metals, selenium, industrial solvents

- and metabolites- cause adaptive responses in antioxidant systems

C modifications of cellular macromolecules- ultimately, cell and tissue damage

C changes in antioxidant system: both biomarker and protective; e.g.- glutathione (GSH, GSSG reductase)- catalase- SOD- peroxidases

C problem - lack of specificity

Immune Competence (also, see above)C phylogenetically - well established

- earthworms (components) to mammalsC indicators of exposure and effects (potential and actual)

- macrophage counts- T-lymphocyte functions- specific antibodies

Blood ChemistryC indicators of general animal health

- complete blood counts (RBCs, leucocytes)hemoglobinhematocritmean cell volumemean corpuscular hemoglobinplateletsblood chemistry (e.g. alkaline phosphates, (-glutamyltransferase, alanine

aminotransferase)C problem - lack of specificity

TerataC mutation v. developmental

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C e.g. frog malformations (UV-B), fish-eating bird bill malformation (DDT/DDE)C problem - other causes possible; e.g. parasites, regeneration

ImposexC “imposed sex”C gastropods; generally development of male genitalia in females; prevent egg layingC most often associated with organo-tins (e.g. tributyl-, triphenyl-, methacrylate-)

- v. sensitive; dogwhelk - 0.1 ppb (µg/L) produces penis in &- 0.1 µg/L inhibits development, shell growth in oysters

Inflammation

Necrosis

HistopathologyC classical techniquesC best used on most sensitive tissues; e.g.

gillslivergonadskidneylungs

C caution to observe primary or secondary effectsC challenge to quantify effectsC main limitations are field validation in species in questionC cost an issue

Tumors/CarcinogenesisC most widely occurring aberration

- esp. in fish- esp. in bottom-feeding fish; e.g.

flatfish (marine)bullheads (freshwater)

- exception: walleye and sauger in Great Lakes region; also salmonids- large #s sampled; tumors often visible from outside

C however, impossible to link to specific carcinogen- also, neoplasms from viruses, parasites

Eggshell thinningC DDE affects estrogen-dependent Ca2+-transport activity in shell gland

- NOTE - shell partially used during development; assay must be done early in incubation

Knockouts/Knockins, etc.