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Rabbit P450 2E1 was stably expressed in Chinese hamster ovary cells after cotransfection with pRC/CMV-2E1 and pFR400 which expresses murine dihydrofolate reductase with a single arginine to leucine substitution at position 22. This mutation permits amplification of expression with increasing methotrexate concentrations in CHO-K1 cells that are not dihydrofolate reductase deficient. After amplification with 1 μM methotrexate, a representative clone expressed about 15 pmol of P450 2E1/mg microsomal protein. Cells from a single 35-mm plate catalyzed the formation of 1.02 nmol 6-hydroxychlorzoxazone/10 6 cells/h or about 127 pmol/mg total cell protein/min. The enzyme was rapidly labeled when pulsed with [ 35 S]-methionine. Initial pulse-chase experiments indicate that the expressed protein has a half-life of 4.8 h.

The ethanol-induced rabbit liver microsomal cytochrome P-450, P-450LM3a, has been shown previously to efficiently catalyze the demethylation of N-nitrosodimethylamine (NDMA) with a Km of 2.9 mM. Since the predominant Km in hepatic microsomes from ethanol-treated rabbits is 0.07 mM, the role of P-450LM3a in the activation of this carcinogen has been uncertain. In the present study, antibodies to P-450LM3a were shown to almost completely inhibit NDMA demethylation by the purified P-450 in a reconstituted system as well as the low-Km activity of liver microsomes from control or ethanol-treated rabbits. In contrast, the antibody did not inhibit the high-Km NDMA demethylase activity in the microsomes. These results indicate that P-450LM3a is the major P-450 responsible for the low-Km NDMA demethylase activity. In addition, evidence is provided for the existence of a cytochrome immunochemically similar to P-450LM3a in liver microsomes from rats, mice, and guinea pigs that effectively catalyzes the demethylation of NDMA.

SUMMARY: Isosafrole serves as an excellent inducing agent in rabbits for Wq, the same isozyme as that which is induced by 3-methylcholanthrene and 5,6-benzoflavone. Since the isosafrole adduct formed with isozyme 4 is unstable, the uncomplexed cytochrome may be purified in very good yield (over 30%, based on total P-450) from liver microsomes of animals given this agent. Isosafrole forms adducts with both isozyme 2 (the phenobarbital-inducible form of P-450) and isozyme 4 in the reconstituted system containing NADPHcytochrome P-450 reductase, phosphatidylcholine, and NADPH under aerobic conditions, but with the latter cytochrome the reaction has a lower Km and maximal rate. The addition of synergists to insecticides has a sparing action, so that lower doses of the insecticide then have the same effect (11. Many synergists were found to exhibit substrate binding spectra upon addition to hepatic microsomes (21, and it was proposed that the methylenedioxyphenyl synergists may act by affecting the integrity of the electron transfer chains involved in mixed function oxidation reactions (3). Philpot and Hodgson (4) reported that an ethyl isocyanide-like difference spectrum with absorption maxima at 455 and 428 nm was produced by the addition of piperonyl butoxide and a reduced pyridine nucleotide to mouse liver microsomes, and Franklin (5) demonstrated that various methylenedioxyphenyl compounds (piperonyl butoxide, methylenedioxybenzene, safrole, and isosafrole) are metabolized by rat liver microsomes in the presence of oxygen and NADPH (but not NADH) to give products which exhibit an isocyanide-like absorption spectrum with the reduced cytochrome P-450. Safrole and isosafrole cause the induction of a unique hepatic microsomal hemoprotein in the rat (6) which is present as a stable complex (7); this form of cytochrome P-450 has been purified to electrophoretic homogeneity as the isosafrole metabolite complex (8,9). The dissociation of the complex by added substrates to generate free cytochrome P-450 has been described (10,111, and a linear relationship has been observed between the amount of complex decomposed and the ability of the

This laboratory has recently reported the isolation of an ethanol-inducible form of rabbit liver microsomal cytochrome P-450, designated isozyme 3a. In view of the reports of others that the hepatotoxicity of acetaminophen is increased in ethanol-treated animals and the human alcoholic, we have determined the activity of the six available P-450 isozymes in the activation of the drug to give an intermediate which forms a conjugate with reduced glutathione. Isozymes 3a, 4, and 6, all of which are present in significant amounts in the liver microsomes from rabbits chronically administered ethanol, exhibited the highest activities in the reconstituted enzyme system, whereas isozymes 3b and 3c were 10- to 20-fold less effective, and phenobarbital-inducible isozyme 2 was essentially inactive, even in the presence of cytochrome b5. The results obtained thus indicate that induction by ethanol of P-450 isozyme 3a (or a homologous enzyme in other species) may contribute to the toxicity of acetaminophen but that other cytochromes also play a significant role.

This laboratory has previously reported the occurrence in rabbit liver microsomes of a non-inducible form of cytochrome P-450, designated P-450 lm 3b because of its electrophoretic mobility relative to that of phenobarbital-inducible P-450 lm 2 and 5,6-benzoflavone-inducible P-450 lm 4 . In the present study, P-450 lm 3b was purified to electrophoretic homogeneity and a specific content of over 19 nmol per mg of protein by chromatographic procedures carried out in the presence of detergents. The isolated cytochrome has a minimal molecular weight of 52,000 and exhibits absorption maxima at 418, 537, and 571 nm in the oxidized state, 412 and 547 nm in the reduced state, and 451 and 555 nm as the CO complex. In a reconstituted system containing NADPH-cytochrome P-450 reductase and phosphatidylcholine, P-450 lm 3b has relatively high activity in the hydroxylation of testosterone in the 6β and 16α positions as well as significant activity toward a number of other substrates tested. The NADPH oxidase activity of P-450 lm 3b is less than half that of P-450 lm 2 and lm 4 .