Download 1 Ecotoxicology - Biology 5868 Levels of Biological Organization

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
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- 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
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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 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
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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, 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 (%)
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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
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- 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)
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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 system
Endocrine Disruptor
Cancer
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-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
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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
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
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- 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
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hepatocytes
C
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 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
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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
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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.
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