Genetic Regulation of Aryl Hydrocarbon Hydroxylase Induction: V. SPECIFIC CHANGES IN SPIN STATE OF CYTOCHROME P450 FROM GENETICALLY RESPONSIVE ANIMALS

Abstract

Abstract The genetically controlled expression of hepatic aryl hydrocarbon (benzo[a]pyrene) hydroxylase induction in inbred and hybrid mice by aromatic hydrocarbons in vivo is associated, in freshly prepared oxidized microsomes, with increases in high spin cytochrome P450 having a set of electron paramagnetic resonance signals at g = 8.0, 3.7, and 1.7. Phenobarbital causes similar rises (2- to 3-fold) in both high (g = 8.0, 3.7, and 1.7) and low spin (g = 2.4, 2.3, and 1.9) hepatic P450 from any of the inbred or hybrid mice examined. Acetone treatment of microsomes in vitro destroys the g = 8.0, 3.7, 1.7 signals without affecting the hydroxylase activity, whereas Triton X-100 or sodium dodecyl sulfate affects the enzyme activity more so than the high spin signals. Substrate binding produces interconversion between high and low spin P450. In vitro treatment of microsomes with sodium dodecyl sulfate or sodium deoxycholate or at 55° for 5 min causes the irreversible loss of high and low spin signals concomitant with a rise in the g = 5.95 signal. Induction of the hydroxylase activity is associated with the increased g = 8.0, 3.7, and 1.7 signal heights in the kidney from 3-methylcholanthrene-treated rabbit and in the liver or kidney from 3-methylcholanthrene-treated rat, but not in liver from aromatic hydrocarbon-treated rabbit. Hence, aryl hydrocarbon hydroxylase induction by polycyclic hydrocarbons and the increase in high-spin cytochrome P450 are not always necessarily related. These results indicate that aromatic hydrocarbon treatment of the genetically responsive animal, rather than treatment of any animal with aromatic hydrocarbons per se, causes an increase in a P450 species which has iron in a preferred high spin configuration. This preferred spin state may reflect a change near the P450 active site, such as the presence of new apoenzyme(s) or a different configuration. There is another high spin protein iron having a g = 5.95 signal; this most likely represents a denatured form of P450.