PowerPoint Presentation: Genotoxicity BY: - NEERAJ KUMAR Animal Biotechnology Lab Kurukshetra University Kurukshetra PowerPoint Presentation: Introduction Causes How to measure genotoxicity Biomarkers of Exposure Biomarkers of Effects Biomarkers of Susceptibilty Application of genotoxicity Conclusion References Contents PowerPoint Presentation: Toxic effect of any Physical , Chemical , and Biological agent on genetic material(DNA/ RNA) Genotoxic agents Genotoxicity results in mutations which may results in tumor or cancer Introduction PowerPoint Presentation: Physical Agents Radiations like X-rays Chemical Agents Hydrocarbons, Aromatic amines, Benzene etc. Biological Agents Viruses like retrovirues Causes PowerPoint Presentation: How To Measure Genotoxicity PowerPoint Presentation: Biomarker of Exposure Biomarker of Effect Biomar ker of Susceptibility(Gene polymorphism) Cytogenetic Biomarkers Biochemical Biomarkers Biomarkers PowerPoint Presentation: Toxic agents or their metabolites that can be measured in body or after excretion. These agents or their metabolites bind to biomolecules to form Adducts. e.g DNA Adducts, Protein Adducts Presence of these Adducts can be measured and used as a biomarkers of exposure Biomarker of Exposure PowerPoint Presentation: Chemical Species bound to DNA Detected By: TLC or HPLC Mass Spectrometry(MS) Radioimmunoassay(RIA) ELISA DNA Adducts PowerPoint Presentation: Chemical species bound to proteins Haemoglobin Adducts Albumin Adducts Immunological detection of intact proteins Hydrolysis of Adducts and analysis of chemical species GC-MS Protein Adducts PowerPoint Presentation: Effects of toxic agents that can be detected by various assays Cytogenetic Biomarkers Chromosomal Aberrations Micronucleus(MN) Sister chromatid Exchange(SCE ) Biochemical Biomarkers Comet Assay/Single Gel electrophoresis Oxidative Stress Assays Ames Test Biomarkers of effects Chromosomal Aberrations(CAs): Chromatid Type Chromosomal Aberrations(CAs) Chromatid Break Chromatid Gap Cytogenetic Biomarkers Chromosomal Type: Chromosomal Breaks Breaks in both chromatids Chromosomal Gaps Gaps in both chromatids Chromosomal Type Others Aberrations: Others Aberrations Acentric Dicentric Micronucleus: Small nucleus like structure formed from chromosome fragments or whole chromosome that lag behind during anaphase and fail to integrated into daughter nuclei Genotoxic agents induce Micronucleus formation Micronucleus PowerPoint Presentation: CBMN ANALYSIS BLOOD COLLECTION BLOOD LYMPHOCYTES CULTURE HARVESTING OF LYMPHOCYTES HYPOTONIC TREATMENT AT 37⁰C SLIDE PREPERATION FOR CBMNs SLIDE ANALYSIS INCUBATION AT 5% C0 2 & 37⁰C WASHING RESULT Sister chromatid Exchange(SCE): Reciprocal exchange of DNA between two sister chromatids. Sister chromatid Exchange(SCE) Genotoxic agents induce SCEs. PowerPoint Presentation: Sister chromatid Exchange Analysis Incubation at 5% C0 2 & 37⁰C Microscopic Slide Analysis Slide Preparation & Staining for SCE Washing of Cell Pellets Blood Collection Lymphocyte Culture SCE Statistical Analysis PowerPoint Presentation: Comet Assay/Single Gel electrophoresis Detect DNA damage i.e Single Strand breaks(SSBs) Damaged DNA migrate more toward anode forming comet apperance Length of tail is proportional to extent of DNA damage Whole blood or harvested lymphocytes Biochemical Biomarkers PowerPoint Presentation: Comet present in Exposed Sample Electrophoresis under Alkaline condition (pH >13) Placed in lysis solution for cell lysis Slide analyzed under Fluorescent Microscope Blood Collection Slide preparation Slide Staining with EtBr Comet Analysis Single Cell Gel Electrophoresis (comet Assay) Oxidative Stress Markers : Oxidative stress is oxidative modification of biological components like nucleic acids, proteins, lipids by reactive oxygen species. Reactive oxygen species include free radicles and peroxides. Binding of Reactive oxygen species leads to Damage. Reactive oxygen species are formed during metabolism of genotoxic agents. Oxidative Stress Markers Oxidative Stress Markers : Oxidative Stress Markers Biomarkers Availability Frequently used assays Lipid peroxidation Malondialdehyde (MDA) Plasma, Urine, Serum, Saliva Spectrophotometrically Protein Oxidantion Protein carbonyls Plasma, Serum ELISA DNA Oxidation 8-hydroxy-2- deoxyguanosine Plasma, Urine, Serum HPLC-MS GC-MS PowerPoint Presentation: Biomarkers Availability Frequently used assays Enzymatic Antioxidants Catalase Glutathion Peroxidase Superoxide Dismutase(SOD) Plasma, Serum Spectrophotometrically Non-Enzymatic Antioxidants Glutathione Carotenoides Ascorbic Acid Plasma, Serum Plasma, Serum Plasma, Serum Spectrophotometrically, HPLC GC-MS, HPLC-MS Spectrophotometrically, Ames Test: Ames Test PowerPoint Presentation: Indicator of natural charactersticks of individuals that make them more susceptible to effect of an exposure to a chemical. Susceptibility may be due to genotype of individuals or different factors like Age, diet etc. Biomarkers of Susceptibilty PowerPoint Presentation: Wiencke et.al studied effect of trans-stillbene oxide on Sister chromatid Exchange(SCE). Detoxified by conjugation to glutathione Conjugators have GST M I (+ve) gentoype Non-Conjugators have GST M I (-ve) gentoype PowerPoint Presentation: trans-stillbene oxide + Normal SCEs GST M I (+ve) gentoype Individuals trans-stillbene oxide + Increased SCEs GST M I (-ve) gentoype Individuals GST M I (-ve) gentoype Individuals are more susceptible to trans- stillbene oxide than GST M I (+ve) gentoype PowerPoint Presentation: Assessment of genotoxicity in occupational exposed individuals Testing of new pharmaceutical product for its safety Testing of various products used by humans like Cosmetics, Food products. Applications of Genotoxicity PowerPoint Presentation: Genotoxicity is ability of any agent to damage genetic material. Many assays are used to measure genotoxic potential of genotoxic agents. Researech is needed to discover new biomarkers and techniques for assessment of genotoxicity. Conclusion PowerPoint Presentation: Richard J. Albertini a, Diana Anderson b, George R. Douglas c, Lars Hagmar d,Kari Hemminki e, Franco Merlo f, A.T. Natarajan g, Hannu Norppa h,David E.G. Shuker i, Raymond Tice j, Michael D. Waters k, Antero Aiti, IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. Mutation Research 2000 ; 436 : 111–172 . Yuji Naito 1), Masaichi-Chang-il Lee 2), Yoji Kato 3), Ryoji Nagai 4), Yoshikazu Yonei, Oxidative Stress Markers. Anti aging medicine 2010; 115: 56-65 . A. L. Brooks, Biomarkers of exposure and dose: state of the art. Radiation Protection Dosimetry 2001; 9: 739–46 . R. R. Tice, E. Agurell,2 D. Anderson, Burlinson, Hartmann, H.Kobayashi, Y. Miyamae,E. Rojas,J.-C. Ryu, and Y. F. Sasaki, Single Cell Gel/Comet Assay: Guidelines for In Vitro and In Vivo Genetic Toxicology Testing. Environmental and Molecular Mutagenesis 2000; 35: 206-221 . References PowerPoint Presentation: Madle S, Korte A, Ball R. Experience with mutagenicity testing if new drugs :view point of a regulatory agency. Mutat Res 1987; 182:187-92 Wassom J. Origins of genetic toxicology and the environmental Mutagen Society. Environ Mol Mutagenesis 1992; 14:1-6. Tennant R, Margolin B, Shelby, M, Zeiger E, Haseman J, Spalding J, et al. Prediction of chemical carcinogenicity in rodents from in vivo genotoxicity assays. Science 1987 ; 236:933-41. H.J. Evans, Molecular mechanisms in the induction of chromosome aberrations, in: D. Scott, B.A. Bridges, F.H. Sobels (Eds.), Progress in Genetic Toxicology, Elsevier North Holland Biomedical Press, 1977; 57–74 .