Biomarkers lBiomarkers - 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. l l lToxicology – present status: l - identification of markers of long-term risks : human (health, toxicology and carcinogenesis) : ecotoxicology – early markers of toxic effects Biomarkers - summary lBiomarker: l change which occurs as response to "stressors" (xenobiotics, disease, temperature...) which extend the adaptive response beyond the normal range lIn vivo biomarkers: l changes measured in stressed animals ("classical biomarkers") l lIn vitro biomarkers in vitro testing to characterize potencies of xenobiotic to induce specific biological activity (genotoxicity, estrogenicity, dioxin-like activity, tumor promotion ...) l = biological potencies (markers) of potential hazards Biomarkers & Exposure l l l l l lh: homeostatic conditions lc: reversible stage lr: irreversible effects of pollutants - lBiomarkers: - temporal change – B5, B2; short period: B4 l - continuous increase – B3 l - repeated occurrence (B5) – irreversible change 1850 Biomarker intensity Health effect intensity Stressor / time Stressor / time Biomarkers at different levels of biological organisation C:\Documents and Settings\Ludek Blaha\Dokumenty\katedra\vyuka\Biomarkery-Mechanismy\_SCAN_Gen&Apll_Toxicology\1842.jpg Biomarkers - classification lCategorization US National Academy of Sciences l - Biomarkers of exposure l - Biomarkers of response or effect l - Biomarkers of susceptibility l lContinuum exists among biomarkers l example: adducts of toxicant with DNA ? biomarker of exposure / ? response l l lSpecific (selective) in vivo biomarkers -Biomarkers selectively reflecting specific types (mechanisms) of toxicity -E.g. inhibition of AcCholE : exposure = organophosphates; effect = neurotoxicity l+ provides specific information l- multiple biomarkers must be measured in parallel l lNon-specific (non-selective) in vivo biomarkers -Biomarkers of general stress -E.g. induction of Heat Shock Proteins (hsp) l+ general information about stress l- sensitive to many "stressors" (temperature, salinity ...) l lIn vivo biomarkers - sampling l -Non-destructive (non-invasive) : blood / haemolymph collection & analyses : skin, feather, hair … : life of the organism not affected - -Destructive (invasive) l : whole animal -> multiple biomarker evaluation EXAMPLE - Paracetamol l l l l l l l (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 C:\Documents and Settings\Ludek Blaha\Dokumenty\katedra\vyuka\Biomarkery-Mechanismy\_SCAN_Gen&Apll_Toxicology\1843.jpg Human biomarkers – example 1857 Human biomarkers – example 1847 194 Further examples Toxicity biomarkers What kind of biomarkers to measure ? lDo we know possible exposure (toxicant) ? l : specific biomarkers ? estrogenic effects in effluents ? dioxin-like effects, mutagenicity in urban areas ? neurotoxicity (AcChE) in rural areas l lDo we expect complex exposures/contamination ? l - integrated approach needed l - nonspecific biomarkers (hsp) … Multiple biomarker evaluation 1851 Biomarkers of susceptibility Toxicokinetics & Biomarkers of susceptibility 1876 Biomarkers of susceptibility 1875 Biomarkers of susceptibility lMetabolism and genotype l - genetic polymorphism in detoxification enzymes l - variability in specific isoenzymes l - susceptibility to „activate“ toxicants: example: N-acetylation of arylamines – NAT2 l - familial cancers l - susceptibility to genotoxins l - susceptibility to drugs (including anticancer drugs) Example: genetic polymorphism SNPs - single nucleotide polymorphism lSNP -> affects protein functions l lMany genotypes (from many individuals) must be sequenced to identify SNPs l(Some) SNPs identified for some (few) genes l Example: cyclophosphamide toxicity Genetic polymorphism Example: genetic polymorphism Alleles known to be involved in polymorphism Biomarkers of EXPOSURE Biomarkers of Exposure lBiomarkers of … internal / effective dose depending on toxicokinetics l - 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) l l l - effective dose - the chemical interacted with the biological target = ADDUCTS TOXICANT ADDUCTS with BIOMOLECULES l1) Selective adducts (chemical-specific) l - DNA aducts: styrene-oxide-O6-guanine; N7-guanyl-aflatoxin B1; - hemoglobin-pesticides l l - extraction and chemical determination (HPLC, GC) l l2) Non-selective adducts 1880 l2) Non-selective aducts – binding with DNA (proteins) but no further information on the structure of aduct (causative agent) l - Analysis: - 32P-postlabelling assay - DNA-strand breaks – - comet assay l - identification of oxy-DNA 8-hydroxy-2´-deoxyguanosine – TOXICANT ADDUCTS with BIOMOLECULES 1846 32P-postlabelling assay TLC result A - 2-5 = various adducts B - controls Comet assay 1 1 1 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 1886 Occup. exposure (Low / Intermed. / High) Occupational Non-exposed (NS) vs. Exposed (S)