Renee J. Krause

Biochemistry of 1,3-butadiene metabolism and its relevance to 1,3-butadiene-induced carcinogenicity

Recently, the roles of specific P450 isoforms, myeloperoxidase (MPO), GSH-S-transferase and epoxide hydrolase in the metabolism of 1,3-butadiene, and its major oxidative metabolite, butadiene monoxide (BM), were investigated. The results provided evidence for P450s 2A6 and 2E1 being major catalysts of 1,3-butadiene oxidation in human liver microsomes. cDNA-expressed human P450s 2E1, 2A6, and 2C9 catalyzed BM oxidation to meso- and (+/-)-diepoxybutane (DEB), but the rates of BM oxidation in mouse, rat, or human liver microsomes were much lower than the rates of 1,3-butadiene oxidation in these tissues. Human MPO catalyzed 1,3-butadiene oxidation to BM, but MPO incubations with BM did not yield DEB. Rates of BM formation in mouse and human liver microsomes were similar and were nearly 3.4-fold higher than that obtained with rat liver microsomes. However, rat liver epoxide hydrolase activity was nearly 2-fold higher than that of mouse liver microsomes. Rat and mouse liver GSH-S-transferases exhibited similar BM conjugation kinetics, but rats excreted more BM-mercapturic acids compared to mice given low equimolar doses of BM. BM reacted with guanosine and adenosine to yield N7-, N2-, and N1-guanosinyl and N6-adenosinyl adducts, respectively. These results may contribute to a better understanding of the biochemical basis of 1,3-butadiene-induced carcinogenicity.

Reduction of l-methionine selenoxide to seleno-l-methionine by endogenous thiols, ascorbic acid, or methimazole

Seleno-L-methionine (SeMet) can be oxidized to L-methionine selenoxide (MetSeO) by flavin-containing monooxygenase 3 (FMO3) and rat liver microsomes in the presence of NADPH. MetSeO can be reduced by GSH to yield SeMet and GSSG. In the present study, the potential reduction of MetSeO to SeMet by other cellular components and antioxidants was investigated. Besides GSH, other thiols (L-cysteine, or N-acetyl-L-cysteine) and antioxidants (ascorbic acid and methimazole) also reduced MetSeO to SeMet. This reduction is unique to MetSeO since methionine sulfoxide was not reduced to methionine under similar conditions. The MetSeO reduction by thiols was instaneous and much faster than the reduction by ascorbic acid or methimazole. However, only one molar equivalent of ascorbic acid or methimazole was needed to complete the reduction, as opposed to two molar equivalents of thiols. Whereas the disulfides produced by the reactions of MetSeO with thiols are chemically stable, methimazole disulfide readily decomposed at pH 7.4, 37 degrees C to yield methimazole, methimazole-sulfenic acid, methimazole sulfinic acid, methimazole S-sulfonate, 1-methylimidazole (MI) and sulfite anion. Collectively, the results demonstrate reduction of MetSeO to SeMet by multiple endogenous thiols, ascorbic acid, and methimazole. Thus, oxidation of SeMet to MetSeO may result in depletion of endogenous thiols and antioxidant molecules. Furthermore, the novel reduction of MetSeO by methimazole provides clear evidence that methimazole should not be used as an alternative FMO substrate when studying FMO-mediated oxidation of SeMet.

Renal cellular transport, metabolism, and cytotoxicity of S-(6-purinyl)glutathione, a prodrug of 6-mercaptopurine, and analogues

The disposition of S-(6-purinyl)glutathione (6-PG) and its metabolites, including the antitumor agent 6-mercaptopurine (6-MP), was characterized in freshly isolated renal cortical cells from male F344 rats to assess the ability of the kidney to convert 6-PG to 6-MP. The intracellular transport and accumulation of 6-PG and 6-MP, the metabolism of 6-PG to 6-MP, and the potential cytotoxicity of 6-MP, 6-thioxanthine (6-ThXan), and 6-thioguanine (6-ThGua) were determined. 6-PG and 6-MP were accumulated by renal cortical cells by time- and concentration-dependent processes, reaching maximal levels of 14.2 and 1.52 nmol/10(6) cells, respectively, with 1 mM concentrations of each compound. Treatment with acivicin, an inhibitor of 6-PG metabolism by gamma-glutamyltransferase, increased accumulation of 6-PG, and treatment with alpha-keto-gamma-methiolbutyrate, a keto acid cosubstrate that stimulates activity of the cysteine conjugate beta-lyase (beta-lyase), which generates 6-MP, decreased accumulation of 6-PG. Incubation of renal cells with 10 mM 6-PG generated 6-MP at a rate of 2.4 nmol/min per 10(6) cells, demonstrating that the beta-lyase pathway forms the desired product from the prodrug within the intact renal cell. Preincubation of cells with acivicin or aminooxyacetic acid, an inhibitor of the beta-lyase, decreased the net formation of 6-MP, demonstrating further the function of the beta-lyase. 6-MP, 6-ThXan, and 6-ThGua exhibited approximately equivalent cytotoxicity (45-55% release of lactate dehydrogenase with 1 mM at 2 hr) in isolated renal cells. Based on the known antitumor potency of these agents, this suggests that cytotoxicity and antitumor activity occur by distinct mechanisms. The high amount of accumulation of 6-PG and its subsequent metabolism to 6-MP, as compared with the relatively low amount of accumulation of 6-MP, in renal cells suggest that 6-PG can function as a prodrug and is a more effective delivery vehicle for 6-MP to renal cells than 6-MP itself. Administration of 6-PG may be an effective means of treating renal tumors or suppressing renal transplant rejection.

Cellular and molecular basis for species, sex and tissue differences in 1,3-butadiene metabolism

Species differences in 1,3-butadiene (BD) bioactivation and detoxication have been implicated in the greater sensitivity of mice to the carcinogenic effects of BD compared to rats, but the molecular basis for species differences in BD metabolism is not well understood. Previous and recent work conducted in this laboratory has examined the relative rates of BD oxidation to epoxybutene (EB) in male and female B6C3F1 mouse tissues, characterized the major cytochrome P450 enzymes involved in BD bioactivation in these tissues, and determined the potential utility of the freshly isolated hepatocyte model to investigate species differences in metabolism of BD and related compounds. Collectively, the results suggest a role for P450s 2E1, 2A5, and 4B1 in sex and tissue differences in BD bioactivation in the mouse. When coordinated metabolism of EB was investigated in male B6C3F1 mouse and Sprague-Dawley rat hepatocytes, the hepatocytes from both species were found to catalyze EB oxidation to meso- and (+/-)-diepoxybutane (DEB), EB hydrolysis to 3-butene-1,2-diol (BDD), and EB conjugation to form GSH conjugates (GSEB). The metabolite area under the curve (AUC) exhibited dependence on the EB concentration used. However, the EB activation/detoxication ratios with the mouse hepatocytes were much higher than the ratios obtained with the rat hepatocytes. These results illustrate the potential utility of the hepatocyte model for estimating flux through competing metabolic pathways and predicting in-vivo metabolism of EB. Collectively, the results may allow a better understanding of the molecular and kinetic basis of species differences in BD metabolism and may lead to a more accurate assessment of human risk.

S-(1,2,2-Trichlorovinyl)-l-cysteine Sulfoxide, a Reactive Metabolite of S-(1,2,2-Trichlorovinyl)-l-cysteine Formed in Rat Liver and Kidney Microsomes, Is a Potent Nephrotoxicant

Previously, we have provided evidence that cytochromes P450 (P450s) and flavin-containing monooxygenases (FMOs) are involved in the oxidation of S-(1,2,2-trichlorovinyl)-l-cysteine (TCVC) in rabbit liver microsomes to yield the reactive metabolite TCVC sulfoxide (TCVCS). Because TCVC is a known nephrotoxic metabolite of tetrachloroethylene, the nephrotoxic potential of TCVCS in rats and TCVCS formation in rat liver and kidney microsomes were investigated. At 5 mM TCVC, rat liver microsomes formed TCVCS at a rate nearly 5 times higher than the rate measured with rat kidney microsomes, whereas at 1 mM TCVC only the liver activity was detectable. TCVCS formation in liver and kidney microsomes was dependent upon the presence of NADPH and was inhibited by the addition of methimazole or 1-benzylimidazole, but not superoxide dismutase, catalase, KCN, or deferoxamine, consistent with the involvement of both FMOs and P450s. Rats given TCVCS at 230 μmol/kg i.p. exhibited acute tubular necrosis at 2 and 24 h after treatment, and they had elevated blood urea nitrogen levels at 24 h, whereas TCVC was a much less potent nephrotoxicant than TCVCS. Furthermore, pretreatment with aminooxyacetic acid enhanced TCVC toxicity. In addition, reduced nonprotein thiol concentrations in the kidney were decreased by nearly 50% 2 h after TCVCS treatment compared with saline-treated rats, whereas the equimolar dose of TCVC had no effect on kidney nonprotein thiol status. No significant lesions or changes in nonprotein thiol status were observed in liver with either TCVC or TCVCS. Collectively, the results suggest that TCVCS may play a role in TCVC-induced nephrotoxicity.

Reactive Metabolites of 1,3-Butadiene: DNA and Hemoglobin Adduct Formation and Potential Roles in Carcinogenicity

1,3-Butadiene (BD), a petrochemical widely used in the manufacture of synthetic rubber and plastics, has recently been added to the list of chemicals Known To Be Human Carcinogens based upon epidemiological and mechanistic data indicating a causal relationship between occupational exposure to BD and excess mortality from lymphatic and/or hematopoietic cancers (U.S. Department of Health and Human Services, 2000). Long-term BD inhalation studies in mice and rats have also been associated with genotoxicity and carcinogenicity, with mice being much more sensitive to BD-induced carcinogenicity than rats (Melnick et al, 1990; Owen et al, 1987). Because BD toxicities are believed to be mediated by reactions of BD metabolites with nucleophilic sites on macromolecules, studies in our laboratory have focused on the characterization of potential metabolic pathways of BD bioactivation, in terms of the enzymes involved, the metabolites formed, and the kinetics of the reactions. Bioactivation reactions of several primary and secondary BD metabolites were also examined. In addition, the reactions of butadiene monoxide (BMO), a primary metabolite of BD, with macromolecules were characterized.