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 03 – 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) •… natural mutagenesis 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) Most common patterns: • G - the most frequent target (highly nucleophilic character) • T=T at the same strand • G=G crosslinks 1212569_21823227.jpg logo_mu_cerne.gif Example: Complex system of SOS repair proteins induced in E. coli by DNA damage (induction and/or elevated levels of SOS-repair also used as a „biomarker of genotoxicity“) 1212569_21823227.jpg logo_mu_cerne.gif How is the information stored in DNA decoded and used 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 à Mutation fixed in 50% of cells after the first replication 1212569_21823227.jpg logo_mu_cerne.gif INSERTION DELETION à shifts in reading frame 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: oxidation (deamination) à CG to à TA 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 Bioactivation of aflatoxin à genotoxicity Výsledek obrázku pro aflatoxin activation Výsledek obrázku pro aflatoxin producer AFLATOXIN sources Výsledek obrázku pro aflatoxin source Výsledek obrázku pro aflatoxin source 1212569_21823227.jpg logo_mu_cerne.gif •INTERCALATORS 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 intercalator 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 àMutations can be: àInherited (inheritable) or somatic àImpacts of mutations àLethal àNon-lethal •Impacts of point mutations à(a) silent … silent à(b) missense àChanges in protein structure and then function – various effects - both adverse (disease incl. cancer; lower fitness) or beneficial (evolution) à(c) nonsense and (d) frameshift àUsually lethal • Wrap-up: Mutations and genotoxicity Somatic mutations that occur earlier in development are generally present in a larger fraction of body cells. 1212569_21823227.jpg logo_mu_cerne.gif Single nucleotide polymorphism SNP - YouTube Also further discussed at „biomarkers“ of susceptibility 1212569_21823227.jpg logo_mu_cerne.gif Mutations (alleles) and evolution http://www.anselm.edu/homepage/jpitocch/genbi101/13_03bPesticideResist-L%20copy.jpg