Triphenyltin acetate-mediated in vitro inactivation of rat liver cytochrome P-450

The in vitro effects of the organotin (OT) compound triphenyltin acetate (TPTA) on cytochrome P-450 content and functions were investigated in liver microsomes from untreated, phenobarbital (PB)- or beta-naphthoflavone- (betaNAF) pretreated rats. At a concentration of 0.5 mM, TPTA caused a marked loss in the spectrally detectable content of cytochrome P-450 up to 27% of its original value, along with an increase in the inactive form cytochrome P-420. Both effects were most pronounced in betaNAF-treated microsomes, which showed a shift in the hemoprotein absorption maximum from 448 nm to 451 nm, but in all cases TPTA failed to affect either cytochrome b5 or total heme content, or to increase the production of malondialdehyde. These results suggest that lipid peroxidation of microsomal membranes or damage to the heme moiety should be excluded as contributing factors in the hemoprotein loss. TPTA also produced a concentration-related functional inactivation of cytochrome P-450 that was most pronounced in betaNAF-exposed microsomal preparations, as denoted by a striking reduction in the ethoxyresorufin O-deethylase (EROD) activity (IC50 = 0.088 mM). In contrast, the activities of cytochrome P-450-independent microsomal enzymes such as NADPH cytochrome c reductase and indophenyl acetate esterase (IPA-EST) were not markedly affected even by 0.5 mM TPTA (-30%). As assessed by Lineweaver-Burk plots, the mechanism of inhibition appeared to be noncompetitive for IPA-EST and of mixed type (competitive-noncompetitive) for EROD. Among sulfhydryl-containing compounds, dithiothreitol was considerably more effective than albumin and reduced glutathione in preventing cytochrome P-450 inactivation and even was able to partially reverse the hemoprotein damage when added after TPTA; glycerol, which is known to protect the hydrophobic environment of cytochrome P-450, was as effective as albumin. This study indicates that TPTA behaves as an almost specific and powerful in vitro inhibitor of cytochrome P-450-dependent monooxygenases, apparently through the interaction with critical sulfhydryl groups of the hemoprotein.