O I.2 Metabolic activation and detoxification of carcinogens in relation to DNA adduct levels and cancer susceptibility in humans

Sol: Metabolism of mutagens and carcinogens

SS

SESSION I: Metabolism of mutagens and carcinogens ORALS

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in human carcinogen-target tissues are being examined as biomarkers of individual cancer susceptibility and of carcinogen exposure. Chemistry of DNA-carclnogen ad duds: Formation and biological effeds

F. Peter Guengerich, William W. Johnson, Mi-Sook Kim, Michael Milller, Sophie Langouet. Vanderbilt Uniu, Nashville. TN. USA Understanding the chemistry of reactions of electrophiles with DNA is an important goal in the field of mutagenesis. Several cases are considered. Aflatoxin BI> (AFBJl exo-8,9-epoxide is a very unstable P450 reaction product, with tl/2 = I s in H20 at 25· C (k = 0.6 S·I). However, at high concentrations of DNA (>0.4 mg mI- J), >97% of the epoxide is conjugated with DNA to form the guanyl_N7 adduct. Kinetic analysis of the binding data yielded an apparent Kd = 1.4 mM and leal (conjugation) = 35 S-I. The conjugation could be observed directly by stopped-flow fluorescence methods, with K m = 5.8 mM and kal. = 42 S-I. DNA also catalyzes the hydrolysis of the epoxide, with k - 0.2 s·J. The reaction of AFB, epoxide with DNA is characterized by intercalation between base pairs, acidcatalyzed hydrolysis by a peripheral proton field, and consequently a very facile SN2 reaction with the guanyl N7 atom. Glutathione (GSH) transferases catalyze the conjugation of ethylene dibromide with GSH to form the half-mustard S-(2-bromoethyl)GSH, which reacts with DNA to form the N 7_, N 2_, and 06_guanyl adducts, with at least the N 7 adduct involving an SN2 mechanism. A set of oligonucleotides with G substituted by each of these adducts at a single site has been synthesized and examined with four different polymerases. All three adducts can block polymerases and miscode. 2-Chlorooxlrane, the oxidation product of vinyl chloride, reacts with DNA to form adducts in the order N 7.(2.oxoelhyl)G :» 1,N6~-A > 5,6,7.9tetrahydrO;7-hydroxy-9-oxoimidazo[I,2-aJimidazole(HOethanoG) > N2,3~_ G > 3,N ~-C > 1,N2~-G. 1,N2~-G and HOethanoG have each been inserted into oligonucleotides and shown to be blocking and miscoding. Both are mutagenic in Escherichia coliIM13 phage, with 1,N2~-G showing more mutations. (Support by USPHS R35 CA44353, P30 ESOO267) Keyword(s): Chemistry; aflatoxin; ethylene dibromide

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Metabolic activation and detoxilicatlon of carcinogens In relation to DNA adduct levels and cancer susceptlblllty In humans

Fred F. Kadlubar. Division ofMolecular Epidemiology, National Centerfor Toxicological Research, Jefferson. Arkansas 72079, USA The metabolic activation and detoxification pathways associated with the carcinogenic aromatic amines (AAs), polycyclic aromatic hydrocarbons (PAHs), and nitrosamines (NAs) provide an excellent model of metabolic polymorphisms that appear to modulate human carcinogenesis. The wide variations in the biotransformation of these agents, whether due to genetic or environmental factors, can be a significant factor in individual cancer susceptibility in humans. Our current studies are concerned with human cancers of the urinary bladder, prostate, breast, ovary, colon & rectum, pancreas, lung, and esophagus. and the role of enzymatic systems that are known to play a major role in the biotransformation of carcinogenic AAs, PAHs, and NAs. These include the: cytochromes P450 (CYPIA2, CYPIBI, CYPIAI, CYP2C9, CYP2E I, and CYP2A6); acetyltransferases (NATI and NATI); sulfotransferases (SULTlAI); glucuronosyl transferases (UGTls); and glutathione Stransferases (GSTAI, GSTMI, and GSTPI). The influence of metabolic polymorphisms in these enzymatic systems on the levels of DNA adduets

Keyword(s): activation; detoxification; DNA adducts

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Cytosol oflluman erythrocytes increases the mutagenIcity of ethylene oxide

Franz Oesch, Jan Hengstler. Institute of Toxicology, University of Mainz. 0.55131 Mainz. Germany Ethylene oxide is a water-soluble gas, widely used in industrial processes and in sterilization of heat-sensitive materials. Large interindividual differences in sensitivity of humans to toxic and genotoxic effects of ethylene oxide have been reported by us and others. In this study we examined mechanisms of extra- and intracellular modification of ethylene oxide mutagenicity and genotoxity. Addition of human erythrocyte homogenate or intact human erythrocytes to the preincubation mixture significantly increased ethylene oxide mutagenicity in the Ames Test using Salmonella typhimurium TA 1535 (I h preincubation; assay without S-9 Mix). Due to interindividual differences the increase in ethylene oxide mutagenicity by human erythrocytes ranged between 2- and 4-fold. Addition of theophylline to the preincubation mixture in a dose range between 0.OO~.5 mglml dose dependently inhibited the increase in ethylene oxide mutagenicity caused by human erythrocytes. To examine whether theophylline might influence the susceptibility of humans in vivo 20 individuals were stimulated to drink 1.5 I of a standard preparation of black tea within I h, whereas controls drank 1.5 I of water during the same time period. Erythrocyte homogenate prepared from tea drinkers (2 h after the beginning of tea drinking) caused a 28% significantly (p < 0.01) smaller increase in ethylene oxide mutagenicity compared to controls. The factor in human erythrocytes, which increases the mutagenicity of ethylene oxide, which by ultracentrifugation has been shown to be located predominantly in the cytosol of erythrocytes and is relatively heat-insensitive (1 h at 6' C), still has to be identified. Keyword(s): Ethylene oxide