Cytochrome P-450-dependent bioactivation of 1,1-dichloroethylene to a reactive epoxide in human lung and liver microsomes.

We investigated the cytochrome P-450-dependent metabolism of 1, 1-dichloroethylene (DCE) by human lung and liver microsomes and compared the results from analogous experiments in mice. Metabolites were identified by HPLC analysis of their glutathione conjugates and/or hydrolyzed products and were detected by using [14C]DCE. The role of human CYP2E1 in the metabolic reactions was examined by comparing p-nitrophenol hydroxylase activities with levels of metabolites formed and by using the CYP2E1-selective inhibitor diallyl sulfone. The major products formed in microsomal incubations containing NADPH were the DCE-epoxide-derived glutathione conjugates 2-(S-glutathionyl)acetyl glutathione and 2-S-glutathionyl acetate. Lower levels of the acetal of 2,2-dichloroacetaldehyde were also detected. In lung samples from eight patients, the amounts of epoxide-derived conjugates formed ranged from 15.6 +/- 4.23 to 34.9 +/- 12.75 pmol/mg protein/min. The levels in murine lung were higher at 40.0 +/- 3.8 pmol/mg protein/min. In liver samples from five patients, conjugate levels ranged from 46.5 +/- 8.3 to 240.0 +/- 10. 5 pmol/mg protein/min, whereas levels in murine liver were 83.0 +/- 6.2 pmol/mg protein/min. Conjugate levels formed in human liver correlated with the relative levels of p-nitrophenol hydroxylase activity present, but this relationship was equivocal in human lung. Diallyl sulfone inhibited the formation of the glutathione conjugates (20-65%) in liver samples from all four patients, whereas only one of five human lung samples exhibited this inhibition (27%). These results demonstrated that the DCE-epoxide is a major metabolite formed by human microsomes and is mediated by CYP2E1 in liver and in some individuals in lung.

Immunochemical assay for recognition of 2-S-glutathionyl acetate, a glutathione conjugate derived from 1,1-dichloroethylene-epoxide.

Cytotoxicities induced by 1,1-dichloroethylene (DCE) are ascribed to cytochrome P450-dependent metabolism to an epoxide. Conjugation of the DCE-epoxide with glutathione (GSH) results in the formation of the conjugates 2-S-glutathionyl acetate (GTA) and 2-(S-glutathionyl) acetyl glutathione (GAG); GAG undergoes hydrolysis to form GTA, and thus GTA is a major metabolite of DCE metabolism. Our objective is to develop an antiserum against the chemically synthesized GTA, and for immunization, we have used a hapten that consists of GTA conjugated to bovine serum albumin (BSA) as the carrier protein and glutaraldehyde (GLUT) as a chemical cross-linker. The antisera were raised in rabbits and were characterized by using the following synthesized structural analogs: GTA, glycine-GLUT-BSA (GLY-GLUT-BSA), GTA-GLUT-ovalbumin (GTA-GLUT-OVB), GTA-1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-BSA (GTA-EDC-BSA), TRIS-GLUT-BSA, glutathione-GLUT-BSA (GSH-GLUT-BSA). The enzyme-linked immunosorbent assay (ELISA) and slot immunoblotting were used to characterize the specificity of the antisera. Noncompetitive ELISA experiments showed that the reaction of the antiserum with the antigen was concentration-dependent. In the competitive ELISA, GTA-GLUT-BSA inhibited binding efficiently; in contrast, the unconjugated GTA did not inhibit binding to the antigen. Competitive studies with the other analogs indicated low or minimal reactivities with the antibodies, which were blocked by incubation with GLY-GLUT-BSA. However, there was residual reactivity with the antigen that was not competitively inhibited by either the GTA-EDC-BSA or the GSH-GLUT-BSA conjugates. Slot-blotting experiments confirmed the findings of the ELISA studies and revealed high specificity of the antiserum to detect the hapten. These results demonstrated the successful development of polyclonal antibodies to detect GTA and hence DCE-epoxide.

Differential formation of 1,1-dichloroethylene-metabolites in the lungs of adult and weanling male and female mice: correlation with severities of bronchiolar cytotoxicity.

The bronchiolar Clara cell cytotoxicant, 1,1-dichloroethylene (DCE), is selectively metabolized by CYP2E1 to metabolites including 2,2-dichloroacetaldehyde and DCE-epoxide. We have performed comparative studies in the lungs of adult and weanling male and female mice to determine their relative capacities to metabolize DCE. Levels of activities of p-nitrophenol hydroxylase, N-nitrosodimethylamine demethylase and NADPH-cytochrome P450 reductase were all significantly higher in adult female mice than in either adult male or weanling mice of both sexes. The quantities of 2,2-dichloroacetaldehyde (identified as its hydrolysis product, acetal) and the DCE-epoxide (identified as the GSH conjugates, 2-(S-glutathionyl) acetyl glutathione [B] and 2-S-glutathionyl acetate [C]) formed were significantly higher in lung microsomes from adult female mice than in those from either adult male or weanling mice of both sexes. Also, the metabolite levels formed in weanling mice were significantly higher than in adult male mice. The amounts of DCE-metabolites produced correlated with the relative severities of DCE-induced bronchiolar damage. The severities of bronchiolar injury were in the rank order adult female > weanling male and female > adult male mice, and coincided with the rank order of DCE-epoxide formation in these experimental groups of mice. In comparison with adult male and weanling male and female mice, adult female mice expressed highest levels of activities of CYP2E1-selective and reductase enzymes, formed most of the DCE-epoxide and were most susceptible to DCE-induced pneumotoxicity. These findings demonstrated sex-related differences in expression of activating enzymes and DCE metabolism in lung, and only in the adult female vs. female weanling mice were there age-related effects in regard to formation of both DCE-metabolites and cytotoxicity.

CYP2E1-dependent bioactivation of 1,1-dichloroethylene in murine lung: formation of reactive intermediates and glutathione conjugates.

We investigated the cytochrome P450-dependent metabolism of 1,1-dichloroethylene (DCE) in murine lung microsomal incubations. The metabolites were identified as their glutathione conjugates or hydrolyzed products, analyzed by HPLC and quantified with [14C]DCE. We determined the relative quantities of DICE metabolites formed in lung microsomal incubations and compared them to those produced in liver. Furthermore, we used antibody inhibition experiments to investigate the CYP2E1-dependent metabolism of DCE in lung. Our results demonstrated that reactive intermediates were generated from DCE in the lung microsomal incubations. The DCE epoxide (12.6 +/- 1.4 pmol/mg protein/min) was the major metabolite formed and was identified as two glutathione conjugates, 2-(S-glutathionyl) acetyl glutathione and 2-S-glutathionyl acetate. Lower levels of the acetal of 2,2-dichloroacetaldehyde (3.6 +/- 0.25 pmol/mg protein/min) were detected. The ratio of acetal to DCE epoxide was higher in lung (0.30 +/- 0.04) than in liver (0.12 +/- 0.02). Preincubation of microsomes with a CYP2E1-inhibitory monoclonal antibody resulted in a maximum inhibition of 50% in the formation of both the acetal and the glutathione conjugates derived from the DCE epoxide. These data demonstrated that lung CYP2E1 metabolizes DCE to reactive intermediates of which the DCE epoxide is both the major metabolite formed and an efficient scavenger of glutathione, implicating it as an important toxic species mediating DCE-induced lung cytotoxicity.

Protection from 1,1-dichloroethylene-induced Clara cell injury by diallyl sulfone, a derivative of garlic.

Bronchiolar Clara cell damage ensues after treatment of mice with 1,1-dichloroethylene (DCE). The cytotoxicity is mediated by CYP2E1, a cytochrome P450 isozyme that is highly localized in the Clara cells. Bioactivation of DCE produces the primary metabolites 2,2-dichloroacetaldehyde, which hydrolyzes to the acetal, and DCE epoxide, which reacts with glutathione to form the conjugates 2-(S-glutathionyl) acetyl glutathione [B] and 2-S-glutathionyl acetate [C]. In this study, we investigated the potential of diallyl sulfone (DASO2) to inhibit CYP2E1, to suppress the bioactivation of DCE to reactive intermediates and to abrogate DCE-induced Clara cell cytotoxicity. Our results showed that treatment of mice with DASO2 (100 mg/kg p.o.) produced decreases in CYP2E1-dependent p-nitrophenol hydroxylation that were apparent at 1 h. Enzyme activity plummeted to about 20% of the control by 2 h and remained at this low level from 3 to 8 h. Recovery of activity was evident at 16 h and returned to the control level by 24 h. Immunoreactivity of the CYP2E1 protein was decreased in immunoblots of lung microsomes from DASO2-treated mice. Treatment with DASO2 did not cause any structural alterations in lung tissue; in contrast, treatment with DCE (75 mg/kg) produced Clara cell damage. This lesion was not manifested in mice treated with DASO2 in conjunction with DCE. The lack of cytotoxicity observed in vivo correlated with a reduction of about 45% in the levels of both the acetal and the DCE epoxide-derived conjugates [B] and [C] in vitro. These results demonstrated that DASO2 significantly inhibited the CYP2E1 enzyme, decreased the production of DCE metabolites and protected Clara cells from DCE-induced cytotoxicity.

Time-dependent decreases of atrial natriuretic peptide release from the isolated rat atrium: evidence for a readily releasable pool.

In vitro, atrial distension causes a rapid increase in atrial natriuretic peptide (ANP) release. This stretch-induced release, however, declines to baseline levels within minutes without significant depletion of the total hormone stores. It has been observed that the basal rate of ANP release from isolated atria also declines over time despite evidence that the tissue retains its viability. We examined this time-dependency of ANP release from isolated rat atria and some parameters that may explain the diminishing release. Mean ANP secretion was 60 pg/min for both spontaneously beating and electrically paced preparations. Although ANP secretion steadily declined over time, there was no time-dependent effect on the amplitude of intracellularly recorded action potentials. The total ANP content in atria obtained after dissection was 133 +/- 28.9 micrograms/g (n = 3) which was not significantly different from atria that were perfused for 3 h (137 +/- 21.2 micrograms/g; n = 3). Only the 28 amino acid circulating form of ANP was released. The ANP mRNA appeared to be partially degraded in atria after 30 min equilibration or after perfusion for 3 h. These results demonstrated that ANP release from isolated atrial preparations declines steadily despite the maintenance of normal electrophysiological activity. This decline was not due to significant depletion of the ANP stores suggesting that a readily releasable pool of ANP exists and represents only a small fraction of the total hormone stores. Finally, degradation of ANP mRNA implies a reduction of de novo synthesis in our preparation which suggests that the observed depletion of the releasable pool was related to a decline in newly synthesized ANP.

Reaction of glutathione with the electrophilic metabolites of 1,1-dichloroethylene.

1,1-Dichloroethylene (DCE) requires cytochrome P450-catalyzed bioactivation to electrophilic metabolites (1,1-dichloroethylene oxide, 2-chloroacetyl chloride and 2,2-dichloroacetaldehyde) to exert its cytotoxic effects. In this investigation, we examined the reactions of these metabolites with glutathione by spectroscopic and chromatographic techniques. In view of the extreme reactivity of 2-chloroacetyl chloride, primary reactions are likely to include alkylation of cytochrome P450, conjugation with GSH to give S-(2-chloroacetyl)-glutathione, or hydrolysis to give 2-chloroacetic acid. Our results showed conjugation of GSH with 1,1-dichloroethylene oxide, through formation of the mono- and di-glutathione adducts, 2-S-glutathionyl acetate and 2-(S-glutathionyl) acetyl glutathione, respectively. The observed equilibrium constant between the hydrate of 2,2-dichloroacetaldehyde and S-(2,2-dichloro-1-hydroxy)ethylglutathione was estimated from 1H-NMR experiments to be 14 +/- 2 M-1. Thus, 2,2-dichloroacetaldehyde is unlikely to make a significant contribution to GSH depletion as GSH concentrations above normal physiological levels would be necessary to form significant amounts of S-(2,2-dichloro-1-hydroxy)ethylglutathione. We also compared the formation of the glutathione conjugates in rat and mouse liver microsomes using 14C-DCE. The results demonstrated a species difference; the total metabolite production was 6-fold higher in microsomes from mice, compared with samples from rat. Production of DCE metabolites in hepatic microsomes from acetone-pretreated mice was 3-fold higher than those from untreated mice suggesting a role for P450 2E1 in DCE bioactivation. These results indicate that the epoxide is the major metabolite of DCE that is responsible for GSH depletion, suggesting that it may be involved in the hepatotoxicity evoked by DCE. Furthermore, this metabolite is formed to a greater extent in mouse than in rat liver microsomes and this difference may underlie the enhanced susceptibility found in the former species.