Relationship between hepatic DNA damage and methylene chloride-induced hepatocarcinogenicity in B6C3F1 mice.

Methylene chloride (MC) induced DNA damage in freshly isolated hepatocytes from mice and rats, which was detectable as single-strand (ss) breaks by alkaline elution. The lowest in vitro concentration of MC needed to induce DNA damage in mouse hepatocytes (0.4 mM) was much lower than for rat hepatocytes (30 mM), and is close to the calculated steady-state concentration of MC in the mouse liver (1.6 mM) at a carcinogenic dose (4000 p.p.m. by inhalation). DNA ss breaks were also detectable in hepatocyte DNA from mice which had inhaled 4000 p.p.m. MC for 6 h, but not in hepatocyte DNA from rats similarly exposed. In studies with hepatocytes cultured overnight in the presence of buthionine sulfoximine to deplete glutathione (GSH), subsequent exposure to MC resulted in less DNA damage in the GSH-depleted cells. This shows that conjugation of MC with GSH is important in its activation of DNA-damaging species in the liver. The GSH pathway of MC metabolism produces two potential DNA-damaging species, formaldehyde and S-chloromethylglutathione (GSCH2Cl). Formaldehyde is known to cause DNA ss breaks in cells. However, the lowest concentration of formaldehyde required to induce a significant amount of DNA ss breaks in mouse hepatocytes (0.25 mM) is unlikely to be formed following in vitro or in vivo metabolism of MC at concentrations that induce similar amounts of DNA damage. That formaldehyde does not play a role in this DNA damage has been confirmed in experiments with CHO cells exposed to MC and an exogenous activation system from mouse liver (S9 fraction). Formaldehyde was responsible for the DNA- protein cross-linking effect of MC, but did not cause the DNA damage leading to ss breaks. These DNA ss breaks are likely to be caused by GSCH2Cl. The results suggest a genotoxic mechanism for MC carcinogenicity in the mouse liver, and support the proposal that the observed species differences in liver carcinogenicity result from differences in the amount of MC metabolism via the GSH pathway in the target organ.

[1]  J. Allen,et al.  Further evidence that dichloromethane does not induce chromosome damage , 1990, Journal of applied toxicology : JAT.

[2]  J. Ashby,et al.  Evaluation of dichloromethane as an inducer of DNA synthesis in the B6C3F1 mouse liver. , 1989, Carcinogenesis.

[3]  F. A. Smith,et al.  Physiologically based pharmacokinetics and the risk assessment process for methylene chloride. , 1987, Toxicology and applied pharmacology.

[4]  M. W. Anders,et al.  Metabolism of dihalomethanes to carbon monoxide--III. Studies on the mechanism of the reaction. , 1978, Biochemical pharmacology.

[5]  F. Guengerich,et al.  Covalent binding of 1,2-dihaloalkanes to DNA and stability of the major DNA adduct, S-[2-(N7-guanyl)ethyl]glutathione. , 1986, Cancer research.

[6]  A. Hsie,et al.  Mutagenicity and cytotoxicity of haloethanes as studied in the CHO/HGPRT system. , 1981, Mutation research.

[7]  R. Grafström.  In vitro studies of aldehyde effects related to human respiratory carcinogenesis. , 1990, Mutation research.

[8]  R. Hilf,et al.  A fluorometric method for determination of oxidized and reduced glutathione in tissues. , 1976, Analytical biochemistry.

[9]  R. Maronpot,et al.  Effect of varying exposure regimens on methylene chloride-induced lung and liver tumors in female B6C3F1 mice. , 1993, Carcinogenesis.

[10]  H J Clewell,et al.  Metabolism of inhaled dihalomethanes in vivo: differentiation of kinetic constants for two independent pathways. , 1986, Toxicology and applied pharmacology.

[11]  T. Green.  A Biological Data Base for Methylene Chloride Risk Assessment , 1989 .

[12]  B. Ames,et al.  Revised methods for the Salmonella mutagenicity test. , 1983, Mutation research.

[13]  H. Ottenwälder,et al.  DNA binding assay of methylene chloride in rats and mice , 2004, Archives of Toxicology.

[14]  N. Ozawa,et al.  Evidence for formation of an S-[2-(N7-guanyl)ethyl]glutathione adduct in glutathione-mediated binding of the carcinogen 1,2-dibromoethane to DNA. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[15]  C. Wolf,et al.  Cytochrome P-450 mediated genetic activity and cytotoxicity of seven halogenated aliphatic hydrocarbons in Saccharomyces cerevisiae. , 1980, Mutation research.

[16]  W. Ross,et al.  Relationship between DNA damage and survival in formaldehyde-treated mouse cells. , 1980, Mutation research.

[17]  J. Kanno,et al.  Effect of methylene chloride inhalation on replicative DNA synthesis in the lungs of female B6C3F1 mice. , 1993, Environmental health perspectives.

[18]  A. Castonguay,et al.  Modulation by glutathione of DNA strand breaks induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and its aldehyde metabolites in rat hepatocytes. , 1992, Carcinogenesis.

[19]  W. Jongen,et al.  Mutagenic effect of dichloromethane on Salmonella typhimurium. , 1978, Mutation research.

[20]  J. Ashby,et al.  Lack of uds activity in the livers of mice and rats exposed to dichloromethane , 1987, Environmental and molecular mutagenesis.

[21]  R. Graves,et al.  The role of formaldehyde and S-chloromethylglutathione in the bacterial mutagenicity of methylene chloride. , 1994, Mutation research.

[22]  C. Bolognesi,et al.  Improved microfluorometric DNA determination in biological material using 33258 Hoechst. , 1979, Analytical biochemistry.

[23]  M. Casanova,et al.  Dichloromethane (methylene chloride): metabolism to formaldehyde and formation of DNA-protein cross-links in B6C3F1 mice and Syrian golden hamsters. , 1992, Toxicology and applied pharmacology.

[24]  H. Heck,et al.  Effects of formaldehyde exposure on the extractability of DNA from proteins in the rat nasal mucosa. , 1983, Toxicology and applied pharmacology.

[25]  C. Harris,et al.  Mutagenicity of formaldehyde in Chinese hamster lung fibroblasts: synergy with ionizing radiation and N-nitroso-N-methylurea. , 1993, Chemico-biological interactions.

[26]  A. J. Gandolfi,et al.  Characterization of the hepatic DNA damage caused by 1,2-dibromoethane using the alkaline elution technique. , 1981, Carcinogenesis.

[27]  B. Elliott,et al.  Inactivity of methylene chloride in the mouse bone marrow micronucleus assay. , 1987, Mutagenesis.

[28]  B. Anderstam,et al.  Chemical reactivity and mutagenicity of some dihalomethanes. , 1983, Chemico-biological interactions.

[29]  C. Harris,et al.  DNA repair in human bronchial epithelial cells. , 1982, Carcinogenesis.

[30]  T. Green.  The metabolic activation of dichloromethane and chlorofluoromethane in a bacterial mutation assay using Salmonella typhimurium. , 1983, Mutation research.

[31]  E. Zeiger,et al.  The role of glutathione in the bacterial mutagenicity of vapour phase dichloromethane , 1992, Environmental and molecular mutagenesis.

[32]  B. Ketterer,et al.  Theta, a new class of glutathione transferases purified from rat and man. , 1991, The Biochemical journal.

[33]  R. Maronpot,et al.  Ras proto-oncogene activation in liver and lung tumors from B6C3F1 mice exposed chronically to methylene chloride. , 1993, Carcinogenesis.

[34]  A. Zhitkovich,et al.  A simple, sensitive assay to detect DNA-protein crosslinks in intact cells and in vivo. , 1992, Carcinogenesis.

[35]  W. Humphreys,et al.  Expression of mammalian glutathione S-transferase 5-5 in Salmonella typhimurium TA1535 leads to base-pair mutations upon exposure to dihalomethanes. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[36]  W. Jongen,et al.  Mutagenicity testing of dichloromethane in short-term mammalian tests systems. , 1981, Mutation research.

[37]  R H Reitz,et al.  In vitro metabolism of methylene chloride in human and animal tissues: use in physiologically based pharmacokinetic models. , 1989, Toxicology and applied pharmacology.

[38]  A. Thilagar,et al.  Induction of chromosome damage by methylene chloride in CHO cells. , 1983, Mutation research.

[39]  R. Maronpot,et al.  Inhalation exposure to a hepatocarcinogenic concentration of methylene chloride does not induce sustained replicative DNA synthesis in hepatocytes of female B6C3F1 mice. , 1993, Carcinogenesis.

[40]  F. Guengerich,et al.  Selectivity of rat and human glutathione S-transferases in activation of ethylene dibromide by glutathione conjugation and DNA binding and induction of unscheduled DNA synthesis in human hepatocytes. , 1990, Cancer research.

[41]  W. Caughey,et al.  METABOLISM OF DIHALOMETHANES TO CARBON MONOXIDE , 1974 .

[42]  T. Green,et al.  Macromolecular interactions of inhaled methylene chloride in rats and mice. , 1988, Toxicology and applied pharmacology.

[43]  R. Maronpot,et al.  Methylene chloride-induced tumorigenesis. , 1993, Carcinogenesis.

[44]  M. W. Anders,et al.  Metabolism of dihalomethanes to formaldehyde and inorganic halide. I. In vitro studies. , 1976, Drug metabolism and disposition: the biological fate of chemicals.

[45]  L. L. Smith,et al.  Methylene chloride--an inhalation study to investigate pathological and biochemical events occurring in the lungs of mice over an exposure period of 90 days. , 1992, Fundamental and applied toxicology : official journal of the Society of Toxicology.

[46]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.