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Triphenyltin acetate-mediated in vitro inactivation of rat liver cytochrome P-450
1Department of Animal Pathology, University of Turin, Italy. cnebbia@veter.unito.it
Abstract:
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.
Insights
Triphenyltin acetate (TPTA) significantly reduces cytochrome P-450 content and function in rat liver microsomes, particularly in beta-naphthoflavone-pretreated samples. This organotin compound acts as a potent inhibitor, likely via sulfhydryl group interaction.
Area of Science:
- Biochemistry
- Toxicology
- Enzymology
Background:
- Cytochrome P-450 enzymes are crucial for xenobiotic metabolism.
- Organotin compounds are environmental pollutants with known toxic effects.
- Understanding TPTA's interaction with P-450 is vital for assessing its toxicological profile.
Purpose of the Study:
- To investigate the in vitro effects of triphenyltin acetate (TPTA) on rat liver cytochrome P-450 (CYP450).
- To determine the impact of TPTA on CYP450 content, spectral properties, and enzymatic functions.
- To elucidate the mechanism of TPTA-induced CYP450 inhibition.
Main Methods:
- Incubation of rat liver microsomes with varying concentrations of TPTA.
- Spectrophotometric analysis of CYP450 content and P450/P420 conversion.
- Assay of ethoxyresorufin O-deethylase (EROD) activity as a measure of CYP450 function.
- Measurement of CYP450-independent enzyme activities (NADPH cytochrome c reductase, IPA-EST).
- Lineweaver-Burk kinetic analysis to determine inhibition mechanisms.
- Assessment of protective effects of sulfhydryl-containing compounds and glycerol.
Main Results:
- TPTA significantly decreased spectrally detectable CYP450 content and increased inactive CYP420, most notably in beta-naphthoflavone-pretreated microsomes.
- TPTA caused a concentration-dependent functional inactivation of CYP450, with a significant reduction in EROD activity (IC50 = 0.088 mM).
- No significant effects were observed on cytochrome b5, total heme content, or malondialdehyde production, excluding lipid peroxidation and heme damage.
- TPTA did not markedly affect CYP450-independent enzymes like NADPH cytochrome c reductase and IPA-EST.
- Dithiothreitol effectively prevented and partially reversed CYP450 inactivation, suggesting inhibition via critical sulfhydryl groups.
Conclusions:
- TPTA is a potent in vitro inhibitor of CYP450-dependent monooxygenases.
- The inhibition mechanism appears to involve interaction with critical sulfhydryl groups of the hemoprotein.
- TPTA's effects are most pronounced in microsomes pretreated with inducers like beta-naphthoflavone.
- These findings highlight TPTA's specific toxicity towards the CYP450 system.
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