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

The importance of understanding mechanisms
DDT story


1962
© Patuxent Wildlife Refuge, MA, USA
http://www2.ucsc.edu/scpbrg/

Možné negativní důsledky chemického znečištění pro organismy poprvé přinesla do širšího povědomí
kniha S.S. 1962 - spojila zřejmý úbytek populací ptáků („charismatické druhy“) s používáním DDT -
pesticid proti komárům (a přenášeným chorobám) - i přes celosvětový zákaz (tzv. Stockholmská úmluva
OSN - obdoba Kjotského protokolu) je jeho výroba velmi levná: užití dodnes v Africe, Indii … od
40.let považován za zcela neškodný a zázračný („netoxický“) ….

Bitman et al. Science 1970, 168(3931): 594
Biochemistry
bird carbonate dehydratase
In vivo: shell thining
In situ: bioaccumulation
-> bird population decline

Možný mechanismus byl navržen až po řadě let
- inhibice ptačí karbonátdehydratázy v žláze tvořící skořápku vajíčka (BTW - enzym, který v našich
krvinkách zajišťuje přeměnu CO2 na HCO3z tkání
- in vivo -> in situ
Proč tento obrázek? - 1) až za 30 let od uvedení na trh se poznalo co vlastně DDT dělá …, 2) ukázka
jak se malá změna na biochemické úrovni promítne až na úroveň ekosystémů …

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.

Mechanisms vs. biomarkers ?
-Chemical enters organism (& may be metabolized/detoxified, transported, released)
-Chemical reacts with target (e.g. DNA) and changes a specific nucleotide (e.g. G à de-oxo-G)
-Elevated de-oxo-G in blood
-Toxicokinetics
-
-
-Toxicodynamics + toxicity mechanism
-
-(Selective) biochemical marker = information about exposure and/or effect)

Biomarkers
lChanges in biological systems
l … induced by various stressors, stimuli etc.,
l … with a sufficently long half-life (so they can be measured),
l … which allow location where in the biological system change occur and to quantify the change.
l
lExamples:
lproducts of metabolism/toxicity reaction (small MW molecules)
lchanges in enzymatic activities
lnew presence (or absence) of certain proteins
lstructural (histopathological) changes
l
lApplications in medicine: Hippocrates – urine colour ~ health status
lBiomarkers in present - identification (and prediction) of long-term risks
: humans – carcinogenesis
: ecotoxicology – early markers of toxic effects
l
l
l

Introduction to general toxicology
l- Toxicokinetics
l- Toxicodynamics
l
l- Toxicity = effects
l
l- Toxicity testing
l
l

Cause – effect paradigm:
nothing new….
‘What is there which is not a poison?
lAll things are poison and nothing without poison.
l
lSolely the dose determines that a thing is not a poison.
l
2
Paracelsus (1493 - 1541)

Toxicokinetics
l- Processes involved in the fate of toxicant after entering the organism:
l
l : adsorbtion / membrane transport
l : transport in body fluids
: distribution in body (fat / specific organs)
l : transformation (liver / kidney ...)
l & elimination (urine / bile / sweat)
l

E3
Toxicokinetics


Toxicokinetics
- membrane -
proteins

Toxicokinetics
- membrane transport -


Toxicodynamics
lCharacterization of specifity & affinity:
l homeostatic constants / coefficents (Ki; Kd):
l Xen + Biol -> XenBiol (v1)
l XenBiol -> Xen + Biol (v2)
l
l K ~ v1 / v2
~ often expressed as concentrations (e.g. IC50)
l
lAs 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 ?
lExposure & toxicity
l - acute / chronic (exposure)
l
lEffect & toxicity
l - lethal (acute)
l : mortality – definitive endpoint
: high concentrations
l : easy to determine (single endpoint – death)
l
l - nonlethal (chronic)
: organisms do not die - "less dangerous" (?)
(endocrine disruption, reproduction toxicity, immunotoxicity, cancerogenesis)
l : difficult to determine (multiple endpoints)
l : more specific – low concentrations / longer exposures
: reflected by specific biochemical changes (biomarkers)

Chronic toxicity
lChronic 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
–

MECHANISMS
of chronic toxicity of POPs
lVarious chronic effects have uniform biochemical basis
–
–principle studies with mechanistically based in vitro techniques
–
–
–
–
–
–estimation of in vitro effects of individual compounds
lunderstanding the mechanisms, prediction of hazard
–
–application for risk assessment or monitoring
lderivation of relative potencies ("toxic equivalents") -> RA
lin vitro biomarkers - direct characterization of complex samples
HORMONE
Biochemical
effects
TOXIN
In vivo
effects
RECEPTOR

Estrogen receptor activation
1)female reproduction disorders
2)
2)male feminisation
3)
3)tumor promotion
4)
4)immunomodulations
5)
5)developmental toxicity
SINGLE mechanism -> SEVERAL effects
=> understanding to mechanisms
may predict effects

Understanding mechanisms ...
... explains the effects at higher levels
Organism
Population & beyond

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

Toxicity assessment
l1) Biological target (molecule, cell, organism, population)
l2) Chemical definition
l3) Exposure of biological system to chemical
l - variable concentrations
l - defined or variable duration (time)
l - conditions (T, pH, life stage ....)
l4) Effect assessment
l - changes in relationship to concentrations
l5) Dose-response evaluation & estimation of toxicity value
(! concentration): LDx, ICx, ECx, LOEC/LOEL, MIC ...
l

How to study (chronic) toxicity ?
lIn 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)
–
lIn vivo biotest testing
–+ unique whole organisms - only few (ecologically
–+ controlled conditions   nonrelevant) organisms used
–+ better ecological interpretation - mostly ACUTE assays
– - chronic: long exposures
l
lField and in situ observations, epidemiological studies

Effect assessment - procedure
auto0 AcuteToxScheme_RM HaF-adult arenic~4 Daphnia Dania pict
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)