1212569_21823227.jpg logo_mu_cerne.gif 1212570_28446780.jpg logo_mu_cerne.gif Luděk Bláha, PřF MU, RECETOX www.recetox.cz BIOMARKERS AND TOXICITY MECHANISMS 11 – BIOMARKERS of EXPOSURE and SUSCEPTIBILITY OPVK_MU_stred_2 1212569_21823227.jpg logo_mu_cerne.gif Biomarkers of Exposure •Biomarkers of internal and effective dose depends on toxicokinetics • Biomarkers of internal dose (short / long term) – examples: Cd in urine, DDE in fat tissues – - should be easy to sample (urine, breath) – - instrumental analytical methods (analyses of toxicant) • • • Biomarkers of effective dose - the chemical interacted with the biological target à analyses of ADDUCTS • Two types of adducts: selective and non-selective 1212569_21823227.jpg logo_mu_cerne.gif SELECTIVE ADDUCTS OF TOXICANTS with BIOMOLECULES •SELECTIVE = CHEMICAL-SPECIFIC • •Adducts with DNA styrene-oxide-O6-guanine N7-guanyl-aflatoxin B1 • •Hemoglobin-pesticides adducts • • •Methods of analyses: • - analytical chemistry • - extraction from biological sample • - chemical determination by HPLC or GC (or ELISA) • 1212569_21823227.jpg logo_mu_cerne.gif 1880 1212569_21823227.jpg logo_mu_cerne.gif PAH-DNA adducts PAH (polycyclic aromatic hydrocarbons) * often high variability * may have difficult interpretation 1886 Occup. exposure (Low / Intermed. / High) Occupational Non-exposed (NS) vs. Exposed (S) 1212569_21823227.jpg logo_mu_cerne.gif •– binding of toxicant to macromolecule (DNA, proteins) with no further information on the structure of actual adduct (i.e. causative agent is not clear but the „exposure“ clearly happened) • •Typical nonselective biomarker of exposure methods - for DNA damage: • - 32P-postlabelling assay • - oxidized DNA: 8-hydroxy-2´-deoxyguanosine – Non-selective adducts 1212569_21823227.jpg logo_mu_cerne.gif 1846 32P-postlabelling assay principle •Digestion of NA •Enzymatic labelling with 32P (kinase) •TLC (or HPLC) analyses of the product (Not an absolute method = comparison with controls always needed) TLC result (thin layer chromatography) A - 2-5 = various adducts B - controls 1212569_21823227.jpg logo_mu_cerne.gif 8-hydroxy-2´-deoxyguanosine - Analysis Oxidative damage to DNA - many causes à 8-OH-dG is the most common marker of DNA oxidation Analysis: analytical chemistry methods - HPLC - immunochemistry-based methods ELISA 1212569_21823227.jpg logo_mu_cerne.gif Biomarkers of susceptibility 1212569_21823227.jpg logo_mu_cerne.gif Importance of susceptibility biomarkers 1875 1212569_21823227.jpg logo_mu_cerne.gif Toxicokinetics determines susceptibility of an individual at various levels à Biomarkers of susceptibility Will the individual be sensitive? Will patient respond to a drug? Esp. METABOLISM is important and used as a BM of susceptibility (mostly known for prototypical enzymes, like CYPs, for prototypical chemicals such as DRUGS…) 1876 1212569_21823227.jpg logo_mu_cerne.gif Biomarkers of susceptibility •Susceptibility depends on genotype >> metabolism • - genetic polymorphism in detoxification enzymes • - variability in specific isoenzymes • àExample: susceptibility to „activate“ toxicants: example: N-acetylation of arylamines (NAT2 gene) àsusceptibility to genotoxins à àfamily cancers àsusceptibility to drugs (including anticancer drugs) 1212569_21823227.jpg logo_mu_cerne.gif Example: genetic polymorphism SNPs - single nucleotide polymorphism •SNPs à affects protein functions • à in specific cases (see example) some SNPs identified • •To identify SNP as a biomarker • Many genotypes (from many individuals) must be sequenced and compared with phenotype (e.g. responsiveness to certain drug) • •à application: PERSONALIZED MEDICINE • 1212569_21823227.jpg logo_mu_cerne.gif Cyclophosphamide (anticancer drug) and its toxicity Genetic polymorphism 1212569_21823227.jpg logo_mu_cerne.gif Example: genetic polymorphism Alleles known to be involved in polymorphism http://www.deenabio.com/wp-content/uploads/2011/04/SNPQuickReference.jpg 1212569_21823227.jpg logo_mu_cerne.gif Personalized medicine http://www.bayerpharma.com/html/images/upload/Oncology/personalisierte_medizin_en.jpg SNP diagnostics 1) DNA isolation 2) Multiplication of specific gene (eg. CYP) 3) SNP identification ... Molecular biology methods such as * NA sequencing * Probe pairing ... number of variants • http://upload.wikimedia.org/wikipedia/en/0/01/SNP-invader-1.jpg