Reductive activation of 1,1-dichloro-1-fluoroethane (HCFC-141b) by phenobarbital- and pyridine-induced rat liver microsomal cytochrome P450

Abstract

1. During anaerobic reductive incubation of liver microsomes, from either the pyridine- or phenobarbital-treated rat, with 1,1-dichloro-1-fluoroethane (HCFC-141b) in the presence of a NADPH-regenerating system, a time- and dose-dependent formation of reactive metabolites was detected as indicated by a depletion of added exogenous glutathione. 2. A statistically significant, dose-dependent loss of both cytochrome P450 and microsomal haem was also observed under these experimental conditions. Furthermore, a statistically significant decrease of p-nitrophenol hydroxylase and pentoxyresorufin O-depentylase activity was measured in microsomes from the pyridine- and phenobarbital-induced rat, respectively indicating that both P4502E1 and P4502B undergo substrate-dependent inactivation. 3. Both reactive metabolite formation and P450 inactivation were almost completely inhibited by previous bubbling of the incubation mixture with carbon monoxide, indicating that interaction of the substrate with a free and reduced P450 haem iron is required for substrate bioactivation and enzyme loss. 4. The presence in the incubation mixture of the spin-trap N-t-butyl-alpha-phenylnitrone (PBN) and the carbene trap 2,3-dimethyl-2-butene (DMB) largely prevented both glutathione depletion and P450 loss. This suggests that free radical and carbene intermediates formed by the metabolic activation of the substrate are involved in the inactivation of P450 and the loss of its prosthetic haem group.

Bioactivation to free radicals and cytotoxicity of 1,1-dichloro-1-fluoroethane (HCFC-141b)

“…These results indicate overall that CYP2E1 and, probably, also CYP2B and CYP3A are both involved in the activation of HCFC-141b. Finally, the concentration-dependent depletion of glutathione found in the present study, con® rms a direct interaction of HCFC-141b reactive metabolites with intracellular glutathione, as suggested previously (Tolando et al 1996). However, glutathione depletion was found to be associated with but not responsible for LDH release.…”

“…Indeed, under strictly anaerobic conditions, HCFC-141b was reductively metabolized by pyridine-induced rat liver microsomes to free radicals, as indicated by the formation of typical ESR spectra in the presence of the spin-trapping agent PBN. It is conceivable, therefore, that free radical intermediates of HCFC-141b may also be involved in the modi® cation of microsomal membrane lipids and contribute, together with the haem loss, to the inactivation of microsomal P450 observed in Tolando et al (1996).…”

“…In addition, biotransformation of HCFC-141b to 2,2-dichloro-2-ethanol in vitro was enhanced when rats were treated with pyridine, an inducer of P4502E1 (Loizou and Anders 1993). Moreover, previous work has shown that during anaerobic incubation with HCFC-141b of liver microsomes from PYR-and PB-treated rats, the compound is activated to reactive metabolites (Tolando et al 1996). Under these conditions, HCFC-141b caused molecular and functional damage of P450, as indicated by the concentration-dependent loss of total P450 content and isoform-speci® c activities observed in both PYR and PB microsomes.…”

“…Analysis by NMR spectroscopy of urine of human subjects exposed to 1000 ppm HCFC-141b showed resonance spectra attributable to 2,2-dichloro-2-uoroethyl glucuronide (major metabolite) and dichloro¯uoroacetic acid (minor metabolite) (Tong et al 1998). Previous work in the authors' laboratory has reported that the anaerobic incubation of PYR-or phenobarbital (PB)-induced rat liver microsomes with HCFC-141b in the presence of NADPH, results in a dosedependent loss of P450 activity and content and, to a lower extent, in the inactivation of its prosthetic haem group (Tolando et al 1996). These eå ects were accompanied by a time-and dose-dependent depletion of exogenous glutathione and were attributed to the formation of HCFC-141b reactive metabolites by rat liver microsomal P450.…”

“…HCFC-141b has a low ozone-depleting potential of 0.10± 0.12 and a low global warming potential of 0.12 relative to the corresponding CFC-11 references of 1.0 (ECETOC 1994). HCFC-141b is oxidatively metabolized by P450 both in vitro (Loizou and Anders 1993) and in vivo (Loizou andAnders 1993, Tong et al 1998) to 2,2-dichloro-2-¯uoroethanol and it was reductively activated to reactive metabolites by rat liver microsomes resulting in P450 loss (Tolando et al 1996).…”