diethyl-maleate and 1-2-dibromo-3-chloropropane

The metabolism of 1,2-dibromo-3-chloropropane (DBCP), measured as the formation of water soluble metabolites and metabolites covalently bound to macromolecules, was studied in isolated rat liver, kidney, and testicular cells, in subcellular fractions, and with purified rat and human glutathione S-transferases (GSTs). The rate of formation of water soluble metabolites in the cells were in the order liver > kidney > testis. The rate of covalent macromolecular binding of reactive DBCP metabolites in the different cell types was of the same relative order. Pretreatment of the cells with the GSH depleting agent diethyl maleate (DEM) markedly decreased the rate of covalent binding in all cell types. Both the overall metabolism and the formation of DBCP metabolites that covalently bound to macromolecules, were substantially higher in rat testicular cells compared to hamster testicular cells. Rat liver cytosol and microsomes, and various purified rat and human GSTs extensively metabolized DBCP to water soluble metabolites in the presence of GSH. When compared to isolated cells, substantially lower rates of binding per mg protein could be observed in subcellular fractions. Binding of DBCP was detected in the microsomal and cytosolic fractions in the absence of NADPH, though in microsomes fortified with a NADPH-regenerating system, the generation of reactive DBCP metabolites was approximately doubled. Studies with purified rat GST isozymes showed that the relative overall GSH conjugation activity with DBCP was in the following order: GST form 3-3 > 2-2 approximately 12-12 > 1-1 > 4-4 approximately 8-8 approximately 7-7. Furthermore, human GST forms also readily metabolized DBCP with activities of GST A1-2 > A2-2 approximately A1-1 > M1a-1a > M3-3 approximately P1-1.

Topics: Animals; Biotransformation; Cell Fractionation; Cell Separation; Cricetinae; Cytosol; Glutathione; Glutathione Transferase; Humans; Kidney; Kinetics; Liver; Male; Maleates; Microsomes; Propane; Rats; Rats, Wistar; Solubility; Testis

1,2-Dibromo-3-chloropropane (DBCP) induced DNA damage, measured by an automated alkaline elution method, in suspensions of rat liver parenchymal cells at low concentrations (1-10 microM). At much higher concentrations (0.5-2.5 mM), DBCP was metabolized to products that were mutagenic to Salmonella typhimurium TA100 co-incubated with the liver cells. At these higher concentrations a marked depletion of cellular glutathione was seen and at 2.5 mM DBCP was cytotoxic. Perdeuterated DBCP (D5-DBCP) caused less DNA damage in the liver cells than DBCP, most likely because of decrease in cytochrome P-450 dependent metabolism. A more pronounced decrease in mutagenicity occurred with D5-DBCP compared to DBCP, whereas the two compounds were equally cytotoxic. Preincubation of the liver cells with diethylmaleate or buthionine sulfoximine, to lower cellular levels of glutathione, decreased DBCP induced DNA damage. The decrease in DNA damage was proportional to the decrease in cellular glutathione levels. In contrast, diethylmaleate enhanced DBCP-induced bacterial mutagenicity and cellular cytotoxicity. The cytotoxic effect could be partly blocked by addition of ascorbate. From the data presented we suggest that: (i) cytochrome P-450 dependent oxidation as well as glutathione conjugation are involved in DBCP induced DNA damage, (ii) cytochrome P-450 dependent oxidation leads to formation of products mutagenic to bacteria and (iii) the cytotoxicity induced by DBCP in the liver cells in vitro is caused by oxidative damage following glutathione depletion and/or direct membrane damage.

Topics: Animals; Biotransformation; Buthionine Sulfoximine; Cell Survival; DNA; DNA Damage; Dose-Response Relationship, Drug; Glutathione; In Vitro Techniques; Liver; Maleates; Methionine Sulfoximine; Mutagenicity Tests; Mutation; Propane; Rats; Salmonella typhimurium; Time Factors

The ability of 1,2-dibromo-3-chloropropane (DBCP), several methylated analogs of DBCP and perdeuterated DBCP (DBCP-D5) to cause DNA damage in isolated testicular cells from rats was measured by the alkaline elution technique. Of the methylated analogs studied, only the C3-methyl analog was capable of causing significant DNA damage at concentrations of 0-50 microM. In both time- (0-60 min) and concentration- (0-10 microM) dependent experiments, the testicular cell DNA damage caused by the perdeuterated analog of DBCP closely mimicked the damage resulting from DBCP itself. The lack of an isotope effect between DBCP-D5 and DBCP strongly suggests that metabolism via a cytochrome P-450-dependent pathway is not involved in the DNA-damaging effects of DBCP in rat testicular cells. In contrast, preincubation for 1 hr with diethylmaleate (DEM) inhibited DBCP-induced (10 microM) DNA damage in a concentration-dependent manner (0-500 microM DEM). The decrease in testicular DNA damage was proportional to the decrease in cellular nonprotein sulfhydryl levels. Similarly, it was shown that 1,2-dibromoethane (EDB), a structurally related halogenated alkane, produced DNA damage in isolated testicular cells in both a time- (0-60 min) and concentration- (0-600 microM) dependent fashion. The DNA damage produced by EDB (600 microM) was also inhibited by pretreatment of testicular cells with DEM (1 mM). The testicular genotoxicity induced by EDB is thought to involve its initial conjugation to glutathione and the subsequent formation of a reactive episulfonium ion. The data presented indicate that similar events may be occurring in DBCP-induced DNA damage in rat testicular cells.

Topics: Animals; Biotransformation; DNA; DNA Damage; Glutathione; Male; Maleates; Methylation; Oxidation-Reduction; Propane; Rats; Rats, Inbred Strains; Testis

Subcutaneous administration of the nematocide, 1,2-dibromo-3-chloropropane (DBCP), to adult, male, Fischer 344 rats transiently depleted hepatic and caput (head) epididymal nonprotein sulfhydryl (NPS) contents. NPS concentrations in the testis and kidney were not lowered by DBCP. Liver, kidney and testis all exhibited increases in tissue NPS concentrations 48 hr after treatment; the effects were most prominent in the outer medullary section of the kidney 24 hr after treatment with 80 mg/kg of DBCP. The glutathione-depleting agent diethyl maleate transiently lowered hepatic, renal and caput epididymal NPS concentrations in a dose- and time-dependent manner. Renal and caput epididymal NPS contents were increased relative to control 24 hr after diethyl maleate treatment. Single s.c. injections of DBCP produced dose-dependent lesions in the kidney, testis, caput epididymis and liver. Diethyl maleate treatment 90 min before DBCP treatment enhanced the nephrotoxic potency of DBCP as indicated by greater elevations of blood urea nitrogen and serum creatinine concentrations and by more severe renal tubular necrosis in diethyl maleate-pretreated animals than in vehicle controls, as determined 48 hr after DBCP exposure. Seminiferous tubular degeneration, as determined 48 hr post-DBCP treatment, was greater in rats pretreated with 600 mg/kg of diethyl maleate than in nonpretreated controls. When examined 16 days after DBCP treatment, however, the severity of testicular atrophy was virtually the same in rats pretreated with a lower dose of diethyl maleate (400 mg/kg) as in nonpretreated rats. These results indicate that DBCP is a depletor of hepatic and caput epididymal NPS in the acutely toxic dose range. Inasmuch as NPS concentrations were not lowered in two of the major target organs, kidney and testis, acute DBCP injury would not appear to be dependent on local glutathione depletion. However, the greater susceptibility of kidney and testis to DBCP injury after diethyl maleate pretreatment suggests an important role for NPS, particularly those in the liver, in modulating DBCP toxicities.

Topics: Animals; Antinematodal Agents; Dose-Response Relationship, Drug; Hydrocarbons, Halogenated; Kidney; Liver; Male; Maleates; Propane; Rats; Rats, Inbred F344; Sulfhydryl Compounds; Testis