1
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.
Toxicology ­ present status:
- identification of markers of long-term risks
: human (health, toxicology and carcinogenesis)
: ecotoxicology ­ early markers of toxic effects
Biomarkers - summary
Biomarker:
change which occurs as response to "stressors"
(xenobiotics, disease, temperature...) which extend
the adaptive response beyond the normal range
In vivo biomarkers:
changes measured in stressed animals
("classical biomarkers")
In vitro biomarkers
in vitro testing to characterize potencies of xenobiotic to induce specific biological activity
(genotoxicity, estrogenicity, dioxin-like activity, tumor promotion ...)
= biological potencies (markers) of potential hazards
Biomarkers
& Exposure
h: homeostatic conditions
c: reversible stage
r: irreversible effects of pollutants
Biomarkers:
- temporal change
­ B5, B2; short period: B4
- continuous increase ­ B3
- repeated occurrence (B5)
­ irreversible change
Biomarker
intensity
Health effect intensity
Stressor / time
Stressor / time
Biomarkers
at different
levels
of biological
organisation
Biomarkers - classification
Categorization US National Academy of Sciences
- Biomarkers of exposure
- Biomarkers of response or effect
- Biomarkers of susceptibility
Continuum exists among biomarkers
example: adducts of toxicant with DNA
? biomarker of exposure / ? response
Specific (selective) in vivo biomarkers
- Biomarkers selectively reflecting specific types
(mechanisms) of toxicity
- E.g. inhibition of AcCholE :
exposure = organophosphates; effect = neurotoxicity
+ provides specific information
- multiple biomarkers must be measured in parallel
Non-specific (non-selective) in vivo biomarkers
- Biomarkers of general stress
- E.g. induction of Heat Shock Proteins (hsp)
+ general information about stress
- sensitive to many "stressors" (temperature, salinity ...)
2
In vivo biomarkers - sampling
- Non-destructive (non-invasive)
: blood / haemolymph collection & analyses
: skin, feather, hair ...
: life of the organism not affected
- Destructive (invasive)
: whole animal -> multiple biomarker evaluation
EXAMPLE
- Paracetamol
(1) paracetamol
(2) parent compound measurement - biomarker of exposure
(3) activation to reactive metabolite (N-ac-p-benzoquinone, NAPQI) by CYPs; reaction with
GSH / measurement ­ levels of CYPs; levels of GSH ­ susceptibility)
(4) GSH-NAPQI conjugate ­ exposure, susceptibility
(5) NAPQI-protein adducts -> toxicity: exposure, effective dose
(6) adaptations: GSH depletion, inhibition of protein synthesis ­ biomarkers of response
(7) protein alkylation -> degeneration of hepatocytes: necrosis -> increase concentrations of
bile acids, bilirubin in plasma; start of inflamation in degraded tissue ­ response / effect
Human biomarkers ­ example Human biomarkers ­ example
Further
examples
Toxicity
biomarkers
What kind of biomarkers to measure ?
Do we know possible exposure (toxicant) ?
: specific biomarkers
? estrogenic effects in effluents
? dioxin-like effects, mutagenicity in urban areas
? neurotoxicity (AcChE) in rural areas
Do we expect complex exposures/contamination ?
- integrated approach needed
- nonspecific biomarkers (hsp) ...
3
Multiple biomarker evaluation
Biomarkers of susceptibility
Toxicokinetics
& Biomarkers
of susceptibility
Biomarkers of susceptibility
Biomarkers of susceptibility
Metabolism and genotype
- genetic polymorphism in detoxification enzymes
- variability in specific isoenzymes
- susceptibility to ,,activate" toxicants:
example: N-acetylation of arylamines ­ NAT2
- familial cancers
- susceptibility to genotoxins
- susceptibility to drugs (including anticancer drugs)
Example: genetic polymorphism
SNPs - single nucleotide polymorphism
SNP -> affects protein
functions
Many genotypes (from
many individuals)
must be sequenced
to identify SNPs
(Some) SNPs identified
for some (few) genes
4
Example: cyclophosphamide toxicity
Genetic
polymorphism
Example: genetic polymorphism
Alleles
known
to be involved
in polymorphism
Biomarkers of EXPOSURE
Biomarkers of Exposure
Biomarkers of ... internal / effective dose
depending on toxicokinetics
- internal dose (short / long term)
­ Cd in urine, DDE in fat tissues
- should be easy to sample (urine, breath)
- instrumental analytical methods (analyses of toxicant)
- effective dose
- the chemical interacted with the biological target
= ADDUCTS
TOXICANT ADDUCTS with
BIOMOLECULES
1) Selective adducts
(chemical-specific)
- DNA aducts:
styrene-oxide-O6-guanine;
N7-guanyl-aflatoxin B1;
- hemoglobin-pesticides
- extraction
and chemical determination (HPLC, GC)
2) Non-selective adducts
5
2) Non-selective aducts
­ binding with DNA (proteins) but no further information
on the structure of aduct (causative agent)
- Analysis:
- 32P-postlabelling assay
- DNA-strand breaks
- comet assay
- identification of oxy-DNA
8-hydroxy-2´-deoxyguanosine
TOXICANT ADDUCTS with
BIOMOLECULES
32P-postlabelling assay
TLC result
A - 2-5 = various adducts
B - controls
Comet assay Example results - Comet assay vs. radiation
8-hydroxy-2´-deoxyguanosine analysis
Oxidative damage to DNA
- many causes
- 8-OH-dG is the most common DNA marker
Analysis:
- HPLC
- immunochemistry (ELISA)
PAH-DNA adducts
Occup. exposure
(Low / Intermed. / High)
Occupational
Non-exposed (NS)
vs.
Exposed (S)