Differential haemin-mediated restoration of cytochrome P-450 N-demethylases after inactivation by allylisopropylacetamide

Abstract

Administration of allylisopropylacetamide (AIA) to phenobarbital-pretreated rats results in the destruction of several phenobarbital-inducible cytochrome P-450 isoenzymes and a correspondingly marked loss of benzphetamine N-demethylase and ethylmorphine N-demethylase activities. Accordingly, the ion-exchange h.p.l.c. or DEAE-cellulose-chromatographic profile of solubilized microsomal preparations from such rats revealed a marked decrease in the cytochrome P-450 content of several eluted fractions compared with that of microsomes from corresponding non-AIA-treated controls. Incubation of liver homogenates from such rats with haemin restores not only cytochrome P-450 content from 35 to 62% of original values, but also benzphetamine N-demethylase and ethylmorphine N-demethylase activities, from 23 to 67%, and from 12 to 36% of original values respectively. Moreover, the chromatographic profiles of microsomes prepared from such homogenates indicated increases of cytochrome P-450 content only in some fractions. Reconstitution of mixed-function oxidase activity of cytochrome P-450 by addition of NADPH: cytochrome P-450 reductase to these fractions indicated that incubation with haemin restored benzphetamine N-demethylase activity predominantly, but ethylmorphine N-demethylase activity only minimally. After injection of [14C]AIA, a significant amount of radiolabel was found covalently bound to protein in chromatographic fraction III, and this binding was unaffected by incubation with haemin. Furthermore, the extent of this binding is apparently equimolar to the amount of cytochrome P-450 refractory to haemin reconstitution in that particular fraction. Whether such refractoriness reflects structural inactivation of the apo-cytochrome remains to be determined. Nevertheless, the evidence presented very strongly argues for AIA-mediated inactivation of multiple phenobarbital-induced isoenzymes, only a few of which are structurally and functionally reparable by haemin.

Formation of glutathionyl-spironolactone disulfide by rat liver cytochromes P450 or hog liver flavin-containing monooxygenases: a functional probe of two-electron oxidations of the thiosteroid?

“…Liver microsomes were prepared as described before (18), from livers that were perfused in situ with ice-cold 0.15 M KCl solution, removed, and homogenized. Liver microsomes were prepared as described before (18), from livers that were perfused in situ with ice-cold 0.15 M KCl solution, removed, and homogenized.…”

Incorporation of haemoglobin haem into the rat hepatic haemoproteins tryptophan pyrrolase and cytochrome P-450

“…Such increases might have been even greater had benzphetamine N-demethylation also been monitored at the time these studies were conducted. Recent findings indicate that exogenous haem restores benzphetamine N-demethylase activity to a much greater extent than that of ethylmorphine N-demethylase (Bonkowsky et al, 1984;Bornheim et al, 1985). Table 2.…”

Amplification of cci4toxicity by chlordecone: Destruction of rat hepatic microsomal cytochrome p‐450 subpopulation

“…These isozymes are regulated not only by hormonal and dietary factors (Levin et al, 1975;Wascman et al, 1985), but also by exposure to xenobiotics that can induce or suppress specific cyt P-450 isozymes. The pioneering studies by Kotake and Funae (1980) and the more recent reports (Bansal et al, 1985;Bornheim et al, 1985;Holm and Kupfer, 1985;Iverson and Franklin, 1985;Bornheim and Correia, 1986) have demonstrated that ion-exchange HPLC may be employed not only for separation and purification of multiple cyt P-450 isozymes, but also for screening the inductive or suppressive effects of xenobiotics on particular isozyme species.…”