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 toxicology – 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 assessment for characterization of xenobiotic potencies to induce specific biological activity (genotoxicity, estrogenicity, dioxin-like activity, tumor promotion ...) Biomarkers at different levels of biological organisation Biomarkers - classification Categorization according to US Nat. Academy of Science - Biomarkers of exposure - Biomarkers of response or effect - Biomarkers of susceptibility Continuum exists adducts with DNA ? response / ? exposure Biomarkers & sampling invasive / non-invasive Biomarkers - Paracetamol • paracetamol • parent compound measurement - exposure • activation to reactive metabolite (N-ac-p-benzoquinone, NAPQI) by CYPs; reaction with GSH / measurement – levels of CYPs; levels of GSH – susceptibility) • GSH-NAPQI conjugate – exposure, susceptibility • NAPQI-protein adducts -> toxicity: exposure, effective dose • adaptations: GSH depletion, inhibition of protein synthesis – biomarkers of response • 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 Specific (selective) in vivo biomarkers - Biomarkers selectively reflecting specific types (mechanisms) of toxicity - E.g. inhibition of AcCholE : exposure = organophosphates; effect = neurotoxicity + specific information - multiple biomarkers must be measured 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 ...) In vivo biomarkers - Non-destructive : blood / haemolymph collection & analyses : skin, feather, hair ... contamination - Destructive : whole animal -> multiple biomarker evaluation 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 varying exposure / contamination ? - integrated approach - non-specific biomarkers (hsp) as predictors of stress level Multiple biomarker evaluation Biomarkers & Exposure h: homeostatic conditions c: reversible stage r: irreversible effects of pollutants Biomarkers: - transitory – B5, B2; short period: B4 - continuous increase – B3 - repeated appearance (B5) – irreversible change Biomarkers of Exposure Biomarkers of - internal dose (short / long term) – Cd in urine, DDE in fat tissues - should be easy to sample (urine, breath) - effective dose - the chemical interacted with the target = ADDUCTS Biomarkers of Exposure - ADDUCTS Selective aducts (chemical-specific) - DNA aducts: styrene-oxide-O6-guanine; N7-guanyl-aflatoxin B1; hemoglobin-pesticides - chemical determination (HPLC/GC) Aselective aducts – binding with DNA (proteins) but no info on structure of aduct - 32P-postlabelling assay - identification of oxy-DNA (8-hydroxy-2´-deoxyguanosine) - DNA-strand breaks – alkaline unwinding assay or comet assay) Genetic damage in fish exposed to BaP PAH-DNA adducts Biomarkers of susceptibility Metabolism - variability in specific enzymes - susceptibility to modify toxicants: N-acetylation of arylamines – NAT2 - null genotypes for conjugation enzymes (GSTM1) Genotype - familial cancers & susceptibility to genotoxins Biomarkers of susceptibility Biomarkers of susceptibility In vivo biomarkers of effects / response Do we know the agent ? Do we expect the effect ? : specific biomarkers / non-specific changes Behaviour and Clinical biomarkers Pathology Clinical chemistry Enzymatic changes Protein synthesis Oxidative stress markers + Human: Excretory products in urine Tumor genes and tumor markers cancer genes ras, myc, a-fetoprotein (AFP) suppressor genes p53, Rb Behaviour and clinical biomarkers Parameters evaluated - body weight - food consumption - fitness & welness Interpretation : ? biomarkers ? effects already demonstrated in vivo - biomarkers of existing serious stress / intoxication Behaviour and clinical biomarkers Pathology (-) Destructive methods, Time consuming, Professional requirements (+) High relevance – organ/tissue changes - microscopy of internal organs : non-specific changes in internal organs : specific changes in liver (dioxin-like POPs, cyanobacterial toxins) : intersex / imposex formation (xenoestrogenicity) - immunohistochemistry & microscopy : determination of specific changes : Fluorescein (FITC)- labeled antibodies (Ab) applications - determination of vitellogenin in male organs (anti-Vtg Ab) - autoimmunity (anti-nuclear Ab, ANA, in exposed organisms) - chromosomal abnormalities & micronuclei evaluation : karyotype biomarkers : non-destructive (blood samples; plant tissues) Pathology - Liver damage by microcystins Pathology – Intersex microscopy Immunohistochemical determination of Vtg in male fish Immunohistochemistry of ANA in autoimmune serum Chromosomal aberations Micronuclei determinations Clinical chemistry Non-destructive Often specific interpretation - determination of enzymatic activities in blood - response to tissue/organ damage - muscle damage: creatine kinase in serum : isozymes - tissue specific (brain, muscle, heart); - heart attack – isozymes of lactate dehydrogenase (LDH) - liver damage – AST (....), ALT (....) in blood : cyanotoxins, dioxin-like POPs Example – changes in rat serum enzymes after CCL4 exposure Enzymatic changes Inhibitions of AcChE (organo-phosphates) d-Aminolevulinic Acid Dehydratase (ALAD) (lead - Pb) Proteinphosphatases (microcystins) Inductions of detoxication & oxidative stress enzymes (hepatopancreas / liver / blood) MFO [CYP classes - EROD / MROD / BROD] Phase II enzymes (GSTs) Glutathion metabolism enzymes (GPx, GRs) (+) Rapid enzymatic assays, specific responses (-) Some ~ EXPOSURE biomarkers AcChE inhibition assay Model Substrate (butyryl-thio-choline, acetyl-thio-choline) - cleaved by AcChE -> formation of free –SH groups - SH: thiol reactive probes: Ellman´s reagent (DTNB) - DTNB-S-choline: yellow colour (spectrophotometry A420) AcChE inhibition mechanism & effects in birds AcChE inhibition mechanism & effects in birds PPase inhibition assay Model substrates cleaved by PPase ^32P-labelled protein -> free ^32P radioactivity 6,8-difluoro-4-methylumbelliferyl phosphate -> fluorescence MFO (CYP) activities MFO (CYP) activities Potencies to induce CYPs (AhR) TEFs for selected PCDDs TEFs for PCBs Phase II conjugation enzymes - GSTs GST activity - example GSH-related oxidative stress enzymes PROTEIN SYNTHESIS Heat Shock Proteins (hsp) Heat Shock Proteins (hsp) HSP determination - example Induction of SOD in plants - protein electrophoresis + immunoblotting Vitellogenin Vitellogenin in fish - ELISA Vitelin-like proteins in mussels Oxidative stress markers Oxidative stress markers Malonyldialdehyde (MDA) GSH & MDA - modulation / - example