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 05 – Mechanisms - DNA OPVK_MU_stred_2 1212569_21823227.jpg logo_mu_cerne.gif DNA -principal molecule for life -structure and function carefully checked -changes rapidly repaired -irreversible changes à cell death (physiologically by apoptosis) • •Mutagenesis à MUTATIONS • à variability and evolution • or à damage to DNA (structure or coding) •… naturally billions of nucleotides/day à most are repaired •… stress-induced à toxicity http://upload.wikimedia.org/wikipedia/commons/thumb/e/e4/DNA_chemical_structure.svg/800px-DNA_chemi cal_structure.svg.png 1212569_21823227.jpg logo_mu_cerne.gif DNA damage and its effects Cellular changes à Health and evolutionary consequences 1212569_21823227.jpg logo_mu_cerne.gif •Damage of DNA is carefully controlled • constitutively expressed repair systems • •Sudden changes in DNA • à induction of additional repair enzymes (e.g."SOS-repair“ in bacteria - biomarker of DNA damage) DNA repair 1212569_21823227.jpg logo_mu_cerne.gif Various types of molecular changes in DNA ... and corresponding repair systems Note! •Not all nucleotides are affected in the same rate (mutations occur only at specific sites due to physicochemical properties) • • G is commonly affected • T=T at the same strand • G=G crosslinks 1212569_21823227.jpg logo_mu_cerne.gif Complex system of SOS repair proteins induced in E. coli by DNA damage 1212569_21823227.jpg logo_mu_cerne.gif •POINT mutationts • Base exchanges • Deletions / Insertions • à Impacts of point mutations (a) silent, (b) missense, (c) nonsense, (d) frameshift • •CHROMOSOMAL mutations • à large scale impact • • TYPES of mutations 1212569_21823227.jpg logo_mu_cerne.gif BASE – EXCHANGE 1212569_21823227.jpg logo_mu_cerne.gif INSERTION DELETION Reading frame shift 1212569_21823227.jpg logo_mu_cerne.gif http://academic.pgcc.edu/%7Ekroberts/Lecture/Chapter%207/07-21_PointMutations_L.jpg Impacts of point mutations à (a) silent, (b) missense, (c) nonsense, (d) frameshift 1212569_21823227.jpg logo_mu_cerne.gif Large – chromosomal mutations 1212569_21823227.jpg logo_mu_cerne.gif •PHYSICAL FACTORS • •Ionizating radiation • - direct interactions with NA • - interactions with water à formation of OH* (and other oxygen radical species – ROS) •à Various impacts on bases and strands • •UV radiation • - interaction with aromatic cycles (bases) à base dimerization (T=T) • What are the agents inducing mutations? MUTAGENS 1212569_21823227.jpg logo_mu_cerne.gif Ionizing radiation effects on DNA 1212569_21823227.jpg logo_mu_cerne.gif •CHEMICALS • •1) Small electrophilic molecules (attracted by nucleophilic/basic sites … e.g. in DNA) •2) Other reactive molecules * alkylating and arylating agents – covalent adducts * specifically intercalating agents •3) Base analogs • inserted during replication instead of nucleotides • •Some compounds may require “activation” by metabolism pro-mutagen (pro-carcinogen) à mutagen (carcinogen) What are the agents inducing mutations? MUTAGENS 1212569_21823227.jpg logo_mu_cerne.gif •HNO2, HSO3- Hydroxylamine (HO-NH2), Methoxyamine (CH3-O-NH2) • •Example: deamination leading to GC à AT shift • Small molecules à deamination of bases 1212569_21823227.jpg logo_mu_cerne.gif •Covalent binding to NA (alkylation of bases, crosslinks in dsDNA) •Alkylsulphates, Nitro-urea, N-nitroso-alkyles, cis-platinum cisplatin cyclophosphamide ALKYLating compounds Nitrourea 1212569_21823227.jpg logo_mu_cerne.gif •Covalent binding, aromatic „adducts“ with bases (see also discussion at biomarkers) • •Mycotoxins (Aflatoxins) – requires activation • •PAHs (benzo[a]pyrene) – requires activation •PAH derivatives - 2-AA, 2-AF (grill products) - NQO – model mutagen in experiments • •... many others • ARYLating compounds http://www.uoguelph.ca/%7Edjosephy/lab/images/mutagens.gif 1212569_21823227.jpg logo_mu_cerne.gif Bioactivation of benzo[a]pyrene à genotoxicity BaP is oxidized to epoxides and OH-derivatives during detoxification (CYP450) à increased reactivity (including binding to bases ... primarily G or A) (Similar bioactivation e.g. at aflatoxin) 1212569_21823227.jpg logo_mu_cerne.gif •Compounds with characteristic structures “fitting” into DNA à both noncovalent and covalent intercalation Intercalating agents http://what-when-how.com/wp-content/uploads/2011/05/tmp32C166_thumb1.jpg Example 1 – ETHIDIUMBROMIDE - experimental dye – visualization of DNA - intercalation à sharing of electrones with bases à high fluorescence 1212569_21823227.jpg logo_mu_cerne.gif C:\Documents and Settings\Ludek Blaha\Plocha\E3.GIF Intercalating agents Other examples -Anticancer drug - doxorubicin - - - - - - Psoriasis treatment – psoralen - - - - - - -Experimental research compnds (e.g. acriflavine) C:\Documents and Settings\Ludek Blaha\Dokumenty\katedra\vyuka\Biomarkery-Mechanismy\_molekularni biologie\598-599.jpg 1212569_21823227.jpg logo_mu_cerne.gif •Structure similarity with natural bases à Incorporation into DNA during replication à Base exchange mutations • •Example •5-Br-Uracil (anticancer drug) –AT à GC shift Base analogs 1212569_21823227.jpg logo_mu_cerne.gif Mutations (alleles) and evolution http://www.anselm.edu/homepage/jpitocch/genbi101/13_03bPesticideResist-L%20copy.jpg