K L Kunze

Selective inactivation of cytochrome P-450 isozymes by suicide substrates

Abstract The autocatalytic destruction of cytochrome P-450 by the following six substrates has been investigated in vivo and in vitro with microsomal and purified, reconstituted rat liver enzymes: 2-isopropyl-4-pentenamide (AIA), 1-ethinylcyclopentanol, 17α-propadienyl-19-nortestosterone, fluroxene, 5,6-dichloro-1,2,3-benzothiadiazole (DCBT), and 1-aminobenzotriazole (ABT). Administration of the first three substrates to rats pretreated with either phenobarbital (Pb) or 3-methylcholanthrene (3-MC), or their incubation with hepatic microsomes from such rats, produced a larger decrease in cytochrome P-450 levels in the membranes from Pb- than 3-MC-treated rats. Comparable losses, however, were observed in microsomes from rats pretreated with both Pb and 3-MC when the last three agents were used. Similar experiments were carried out using the major cytochrome P-450 isozymes purified from liver microsomes of Pb- or 3-MC-treated rats. The Pb isozyme was inactivated during catalytic turnover of all six substrates while only three substrates (DCBT, ABT, and fluroxene) were found to inactivate the 3-MC isozyme. Oxygen consumption studies with purified enzymes have shown that AIA is not a measurable substrate for the 3-MC isozyme, a fact which explains its failure to inactivate this isozyme. Similar studies with the Pb isozyme establish that one enzyme molecule is inactivated for approximately every 230–320 AIA molecules processed by the enzyme.

Differential inhibition of hepatic ferrochelatase by the isomers of N-ethylprotoporphyrin IX

Abstract The four isomers of N-ethylprotoporphyrin IX have been synthesized. The two isomers with the N-ethyl group on pyrrole rings A or B inhibit rat liver ferrochelatase as effectively as the corresponding N-methyl analogues, whereas those with the N-ethyl moiety on rings C or D are 30–100 times less effective. The ability of N-alkyl porphyrins to inhibit ferrochelatase thus depends not only on the size of the N-alkyl group but also on its precise location on the porphyrin face.

Inactivation of hepatic cytochrome P-450 by allenic substrates

Abstract Hepatic microsomal cytochrome P-450 from phenobarbital-pretreated rats is destroyed by 17-α-propadienyl-19-nortestosterone, 1-propadienylcyclohexanol, and 1,1-dimethylallene. Substantial activity is also exhibited by 1-propadienylcyclohexanol against the enzymes in microsomes from 3-methylcholanthrene pretreated rats. The destructive process requires NADPH, is not inhibited by glutathione, and is paralleled by equimolar loss of hepatic heme but not by in vivo accumulation of identifiable “green” pigments. Attempted metabolism of allenes can thus result in destruction of cytochrome P-450.

Shift of the acetylenic hydrogen during chemical and enzymatic oxidation of the biphenylacetylene triple bond

Abstract Oxidation of 1-[13C]biphenylacetylene by either meta-chloroperbenzoic acid or by liver microsomes from phenobarbital-pretreated rats yields (after esterification) methyl 2-biphenylacetate in which the biphenyl group is still attached to the labeled carbon. This demonstrates that it is the terminal hydrogen and not the biphenyl group which is involved in the 1,2-shift previously shown to accompany this oxidative transformation. The implications of a 1,2-hydrogen shift for acetylene group metabolism in other substrates are discussed.

Inhibition of hepatic ferrochelatase by the four isomers of N-methylprotoporphyrin IX.

Summary The four isomers of N-methylprotoporphyrin IX, obtained by chemical synthesis and purified by high pressure liquid chromatography, have been shown to be equally potent as inhibitors of the ferrochelatase enzymes from rat liver and from cultured chick embryo liver cells.

Exogenous heme restores in vivo functional capacity of hepatic cytochrome P-450 destroyed by allylisopropylacetamide

Abstract Administration of allylisopropylacetamide (AIA) produces a dose-related destruction of the heme moiety of the phenobarbital-induced subspecies of hepatic cytochrome P-450. This results in delayed plasma disappearance of the inactivating agent as determined after injection of [14C]AIA. In phenobarbital-pretreated rats, infusion of heme reversed this AIA-mediated impairment of the plasma disappearance of [14C]AIA. In the absence of phenobarbital pretreatment, cytochrome P-450 destruction by AIA was minimal and heme infusion failed to enhance plasma disappearance of [14C]AIA. Since exogenously administered heme is incorporated into hepatic cytochrome P-450 in vivo , these observations suggest that the infused heme restored the functional capacity of the phenobarbital-induced mixed function oxidase system by substituting for the prosthetic heme moiety destroyed by AIA. Heme infusion is a potentially useful therapeutic modality for enhancing drug biotransformation after intoxication with compounds that inactivate cytochrome P-450.

INACTIVATION OF CYTOCHROME P-450 BY SUICIDE SUBSTRATES. PARTIAL STRUCTURE OF THE RESULTING PROSTHETIC-HEME ADDUCTS

The following substrates alkylate the prosthetic-heme of cytochrome P-450 during catalytic interaction with the enzyme: 2-isopropyl-4-pentenamide, methyl 2-isopropyl-4-pentenoate, norethisterone, 1-ethynylcyclohexanol, 1-ethynylcyclopentanol, and 3-phenoxy-1-propyne. Mass spectrometric studies suggest that an oxygen atom is incorporated into each substrate prior to or during the alkylation process.