Role of isosafrole as complexing agent and inducer of P-450LM4 in rabbit liver microsomes

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