Promoting activities of butylated hydroxyanisole and butylated hydroxytoluene on 2-stage urinary bladder carcinogenesis and inhibition of γ-glutamyl transpeptidase-positive foci development in the liver of rats

Abstract

Seven different forms of cytochrome P-450 have been purified from rat liver microsomes. The major 3-methylcholanthrene (MC) inducible cytochrome P-450 (form c) exhibits the greatest activity toward both benzo[a]pyrene (BP) (58 min^-1) and 7,12-dimethylbenz[a]anthracene (DMBA) (29 min^-1) and forms substantially high spin, high affinity complexes (K_d = 10 nM) with both hydrocarbons. Cytochrome P-450d, a minor MC-inducidble form, has far lower activity for metabolism of both polycyclic aromatic hydrocarbons (PAH), yet also forms high affinity complexs (K_d ˜ 100 nM) with both PAH, retaining the full high spin state of the free cytochrome. Although two phenobarbital (PB)-induced forms (P-450's b and e) differ by only 13 amino acids, they exhibit significant differences in metabolism of PAH and in complex formation. Whereas P-450b is only active in metabolism of DMBA (9.8 min^-1 versus 1.9 min^-1 for BP), P-450e has low activity for both substrates (3.3 and 1.2 min^-l). Nevertheless, P-450e forms a high affinity complex (K_d ˜ 100 nM) with both PAH that enhances the proportion of the high spin state (from 30% to 70%). Failure to displace n-octylamine (NOA) suggests binding that is removed from the heme. P-450b remains low spin in the presence of PAH and NOA is again not displaced. In addition, the two forms can be distinguished by their regioselectivities for both PAH. P-450' a, h, and pregnenolone-l6α-carbonitrile (PCN) exhibit little activity toward BP or DMBA, but P-450 PCN does form a low spin complex with BP (not DMBA). Regioselectivity in metabolism of DMBA by PB-induced microsomes does not agree with that of the major constituent forms. Only the minor, less active purified forms (e and a) mediate substantial 12-hydroxylation and 3,4-epoxidation of DMBA. Thus, additional factors in microsomal reactlons must contribute to these differences.