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Ecotoxicology - Biology 5868 Levels of Biological Organization Toxic Effects - Molecular & Cell October 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 Organization A. 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; two categories 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 different levels are not uniform 1 - Data v. Information (information helps make decisions) IV. Subjects for toxicity tests/biomarker assessment 1. natives 2. 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 assurance 4. 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 2 - 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 mixedfunction 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 major systems: 1. Immune system 2. Stress response system 3 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, immune function, inflammation NOTE - 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 Biomarkers acetylcholinesterase (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 enzymes e.g. photolyase, endonuclease, exonuclease, ligases some question of induction stress proteins; i.e. hsps (heat shock proteins) metallothioneins immunological biomarkers competitive receptor binding (e.g. AhR, ER, AR) *anti-clotting proteins (e.g. response to coumarin-based rodenticides) hemoglobin adducts (e.g. carboxyhemoglobin) *vitellogen induction (%) 4 DNA strand breakage *micronuclei formation *chromosomal sequence mutations - must be inducible - must respond to specific stressor VIII. Methods Enzymes: conventional assays Proteins: 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 ÷ effect contaminant ÷ 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 5 - microarray: identify patterns; method for identifying mechanism e.g. apoptosis, cell cycle, DNA repair, CYPA1 or diagnostic (predictive) array Protein level: - caveat for genomics approach 1. degradation - messages transcribed but not translated however, 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) effects 3) mechanism (for toxicants with unknown pathways) 6 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 system Endocrine Disruptor Cancer GeneChip (Affymatrix); e.g. rat U34 chip 7 High Throughput Assays - goal of much assay development - both assay and analysis - need: pattern recognition software QSAR or QSAM-based NOTE - high throughput means automation Caveats, continued - more caveats for systems biology approach 2. 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/chromosome C 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 8 C C C C C C C - metals bind to phosphate groups or bases - Cu binds between bases; competes with normal H-bonding - Hg forms strong crosslinks between strands of DNA Adducts - activated forms of PAHs - covalently bound assays: 32P-post-labeling - sensitive; 1 adduct/1010 bases detect using GC/MS for specific adducts ELISAs fingerprinting Thymine-Thymine dimers - UV damage 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) Mutation analysis Repair enzymes Apoptosis Tumors (see below) Enzyme Analysis C 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 -brain essentially irreversible (recovery ~4 mo) biopsy - sacrifice -blood plasma AChE & BuChE more rapidly metabolized reversible no 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 in exposed animal than in naive 9 - two major classes: 3-methylcholanthrene-incucible (CYP1A) phenobarbital-inducible (CYP2B) - assessment methods: enzyme assays Western blots ELISAs mRNA 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 slices C hepatocytes C cultured cells - primary - cell line C transfection - stable - transient - insert genes of interest - look for receptor binding - activation of gene 10 - use as indicator of toxin Metabolites of contaminants C rapid metabolism (e.g. PAHs) = not find contaminant in tissues C 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 mammals C indicators of exposure and effects (potential and actual) - macrophage counts - T-lymphocyte functions - specific antibodies Blood Chemistry C indicators of general animal health - complete blood counts (RBCs, leucocytes) hemoglobin hematocrit mean cell volume mean corpuscular hemoglobin platelets blood chemistry (e.g. alkaline phosphates, (-glutamyltransferase, alanine aminotransferase) C problem - lack of specificity Terata C mutation v. developmental 11 C C e.g. frog malformations (UV-B), fish-eating bird bill malformation (DDT/DDE) problem - other causes possible; e.g. parasites, regeneration Imposex C “imposed sex” C gastropods; generally development of male genitalia in females; prevent egg laying C 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 Histopathology C classical techniques C best used on most sensitive tissues; e.g. gills liver gonads kidney lungs C caution to observe primary or secondary effects C challenge to quantify effects C main limitations are field validation in species in question C cost an issue Tumors/Carcinogenesis C 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 thinning C 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. 12