Biomarkers and mechanisms of toxicity
Course summary
1) Introduction
- Overview of toxicity mechanisms
(with special respect to environmental contaminants)
- Concept of biomarkers - overview
2) Details on selected important toxicity mechanisms
- Membrane toxicity, enzyme inhibitions, Oxidative stress,
Genotoxicity, Detoxification, Nuclear Receptors (AhR, ER, AR ....),
Neurotoxins
3) Biomarkers
- In vitro and in vivo biomarkers / assays
- Applications in environmental studies
Toxicity - concept
Escher, B. I., Behra, R., Eggen, R. I. L., Fent, K. (1997), "Molecular mechanisms in ecotoxicology:
an interplay between environmental chemistry and biology", Chimia, 51, 915-921.
19621962
 Patuxent Wildlife Refuge, MA, USA http://www2.ucsc.edu/scpbrg/
Bitman et al. Science 1970, 168(3931): 594
Biochemistry
bird carbonate dehydratase
In vivo: shell thickening
In situ: bioaccumulation
-> bird population decline
Introduction
- Toxicokinetics
- Toxicodynamics
- Toxicity = effects
- Toxicity testing
Cause ­ effect paradigm:
nothing new....
`What is there which is not a poison?
All things are poison and nothing
without poison.
Solely the dose determines that
a thing is not a poison.
Paracelsus (1493 - 1541)
Toxicokinetics
- Processes involved in the fate of toxicant after entering
the organism:
: adsorbtion / membrane transport
: transport in body fluids
: distribution in body (fat / specific organs)
: transformation (liver / kidney ...)
& elimination (urine / bile / sweat)
Toxicokinetics
Toxicokinetics
- membrane -
Toxicokinetics
- membrane transport -
Toxicodynamics
Characterization of specifity & affinity:
homeostatic constants / coefficents (Ki; Kd):
Xen + Biol -> XenBiol (v1)
XenBiol -> Xen + Biol (v2)
K ~ v1 / v2
~ often expressed as concentrations (e.g. IC50)
As lower is ICx as stronger is the binding to specific
receptor and related toxic effect
Toxicodynamics
one compound - more targets
Targets (=receptors in toxicodynamics)
ANY BIOMOLECULE
Toxicity ?
Exposure & toxicity
- acute / chronic (exposure)
Effect & toxicity
- lethal (acute)
: mortality ­ definitive endpoint
: high concentrations
: easy to determine (single endpoint ­ death)
- nonlethal (chronic)
: organisms do not die - "less dangerous" (?)
(endocrine disruption, reproduction toxicity, immunotoxicity, cancerogenesis)
: difficult to determine (multiple endpoints)
: more specific ­ low concentrations / longer exposures
: reflected by specific biochemical changes (biomarkers)
Kidd, K.A. et al. 2007. Collapse of a fish population
following exposure to a synthetic estrogen. Proceedings
of the National Academy of Sciences 104(21):8897-8901
Controls +Ethinylestradiol
5 ng/L (!)
7 years
HO
OH
Chronic toxicity
Chronic toxicity is difficult to study and predict
­ time and cost consuming experiments
­ limited number of species (laboratory vs. natural species)
­ effect = combination of chemical exposure and life style, habits ...
­ metabolites or derivatives (not parent compounds) are often the active substances
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
OH
Cl
How to study (chronic) toxicity ?
In vitro studies (biochemical mechanisms)
+ easy to perform, short-term - ecotoxicological relevancy
+ highly controlled conditions - mostly with vertebrate cells
+ lower amounts of chemicals needed
(new cmpnds screening)
In vivo biotest testing
+ unique whole organisms - only few (ecologically
+ controlled conditions nonrelevant) organisms used
+ better ecological interpretation - mostly ACUTE assays
- chronic: long exposures
Field and in situ observations, epidemiological studies
Understanding mechanisms ...
... explains the effects
MECHANISMS
of chronic toxicity of POPs
Various chronic effects have uniform biochemical basis
­ principle studies with mechanistically based in vitro techniques
­ estimation of in vitro effects of individual compounds
understanding the mechanisms, prediction of hazard
­ application for risk assessment or monitoring
derivation of relative potencies ("toxic equivalents") -> RA
in vitro biomarkers - direct characterization of complex samples
HORMONE
Biochemical
effects
TOXIN
In vivo
effectsRECEPTOR
Estrogen receptor activation
1) female reproduction
disorders
2) male feminisation
3) tumor promotion
4) immunomodulations
5) developmental toxicity
SINGLE mechanism -> SEVERAL effects
=> understanding to mechanisms
may predict effects
Toxicity assessment
1) Biological target (molecule, cell, organism, population)
2) Chemical definition
3) Exposure of biological system to chemical
- variable concentrations
- defined or variable duration (time)
- conditions (T, pH, life stage ....)
4) Effect assessment
- changes in relationship to concentrations
5) Dose-response evaluation & estimation of toxicity value
(! concentration): LDx, ICx, ECx, LOEC/LOEL, MIC ...
Effect assessment - procedure
Cu addition
Effect concentrations
expressed in total/dissolved Cu
Extrapolation =
PNECs or EQCs expressed in
total / dissolved Cu
Effect assessment - results
50
100
LC50 [concentration]
in mg/L or % effluent
Threshold:
No Observed Effect
Concentration (NOEC)
Mechanisms of toxicity - overview
- What is the "toxicity mechanism"
- interaction of xenobiotic with biological molecule
- induction of specific biochemical events
- in vivo effect
- Biochemical events induce in vivo effects
(mechanisms)
- Changes of in vivo biochemistry reflect the
exposure and possible effects (biomarkers)
Factors affecting the toxicity
Xenobiotic
- physico-chemical characteristics
- solubility / lipophilicity
- reactivity and redox-characteristics
- known structural features related to toxicity
(organophosphates)
- structurally related molecules act similar way
- bioavailability & distribution (toxicokinetics)
Biological targets (receptors)
- availability (species- / tissue- / stage- specific effects)
- natural variability (individual susceptibility)
Concentration of both Xenobiotic and Receptor
Mechanisms of toxicity - specificity
- Tissue-specific mechanisms (& efffects)
- hepatotoxicity; neurotoxicity; nefrotoxicity; haematotoxicity
- toxicity to reproduction organs;
- embryotoxicity, teratogenicity, immunotoxicity
- Species-specific mechanisms
- photosynthetic toxicity vs. teratogenicity
- endocrine disruption ­ invertebrates vs. vertebrates
- Developmental stage-specific mechanisms
- embryotoxicity: toxicity to cell differenciation processes
BIOMARKERS
Biomarkers - markers in biological systems with a sufficently
long half-life which allow location where in the biological
system change occur and to quantify the change.
Applications in medicine:
Hippocrates ­ urine colour ~ health status
Toxicology ­ present status:
- identification of markers of long-term risks
: humans ­ carcinogenesis
: ecotoxicology ­ early markers of toxic effects
Cellular toxicity mechanisms - overview
Membrane nonspecific toxicity (narcosis)
Inhibition of enzymatic activities
Toxicity to signal transduction
Oxidative stress ­ redox toxicity
Toxicity to membrane gradients
Ligand competition ­ receptor mediated toxicity
Mitotic poisons & microtubule toxicity
DNA toxicity (genotoxicity)
Defence processes as toxicity mechanisms and biomarkers
- detoxification and stress protein induction
NARCOSIS / nonspecific toxicity
- All organic compounds are narcotic in particular ("high")
concentrations
- Compounds are considered to affect membranes;
nonspecific disruption of fluidity and protein function
- Related to lipophilicity (logP, Kow): tendency of
compounds to accumulate in body lipids (incl.
membranes)
Narcotic toxicity to fish: log (1/LC50) = 0.907 . log Kow - 4.94
- The toxic effects occur at the same "molar volume" of all
narcotic compounds (volume of distribution principle)
Volume of distribution principle
Enzyme inhibition - toxicity mechanism
- Millions of enzymes (vs. millions of compounds)
: body fluids, membranes, cytoplasm, organels
- Compound - an enzyme inhibitor ?
- Enzymology: interaction of xenobiotics with enzymes
- Competitive vs. non-competitive:
active site vs. side domains
- Specific affinity ­ inhibition (effective) concentration
- What enzymes are known to be selectively affected ?
- Nonspecific inhibitions (!)
Compound affects high osmomolarity or pH ...
Enzyme inhibition - toxicity mechanism
Enzyme inhibition - toxicity mechanism
Enzyme inhibition - examples
Acetylcholinesterase (organophosphate pesticides)
Microsomal Ca2+-ATPase (DDE)
Inhibition of hemes ­ respiratory chains (cyanides)
d-Aminolevulinic Acid Dehydratase (ALAD) inhibition
(lead - Pb)
Inhibition of proteinphosphatases (microcystins)
Non-competitive inhibition ­ changes in terciary structure
(metals: toxicity to S-S bonds)
Acetylcholinesterase inhibition
by organophosphate pesticides
Inhibition of Ca2+-ATPase by DDE
Ca2+:
general regulatory molecule
contractility of muscles
calcium metabolism in bird eggs
stored in ER
(endo-/sarcoplasmatic reticulum)
concentrations regulated by
Ca2+-ATPase
Inhibition of hemes by cyanide
oxidations in respiratory chains; Hemoglobin
ALAD inhibition by lead (Pb)
PPase inhibitions by microcystins
NH
OCH3
H3C CH3
Y
O
NH X
COOH
R1
NH
O
H3C
N
O
H2C
R2
HN
O
HOOC
O
H
H
H
H
H
H
H3C
H
Microcystins ­ produced in eutrophied waters by
cyanobacteria; kg ­ tons / reservoir