Activation of microsomal glutathione S-transferase and inhibition of cytochrome P450 1A1 activity as a model system for detecting protein alkylation by thiourea-containing compounds in rat liver microsomes

The recent development of several promising new thiourea-containing drugs h as renewed interest in the thiourea functionality as a potential toxicophor e. Most adverse reactions of thiourea-containing compounds are attributed t o the thionocarbonyl moiety. Oxidation of these thionocarbonyl compounds by flavin-containing monooxygenases (FMO) and cytochrome P450 isoenzymes (P45 0)to reactive sulfenic, sulfinic, or sulfonic acids leads to alkylation of essential macromolecules. To more rationally design thiourea-containing dru gs, structure-toxicity relationships (STRs) must be derived. Since for the development of STRs a large number of thiourea-containing compounds must be investigated, it is important to develop rapid in vitro assays for alkylat ing potential. In this study, the utility of activation of microsomal gluta thione S-transferase (mGST) and inactivation of P450 1A1 as markers of the alkylating potential of metabolites of thiourea-containing compounds was in vestigated. It was found that metabolites of thiourea-containing compounds inactivate P450 1A1 in a time-dependent manner, as evidenced by a decrease in 7-ethoxyresorufin O-dealkylation (EROD) activity. An extent of inactivat ion of P450 1A1 by 100 mu M N-phenylthiourea (PTU)of 64% was found after 10 min. This inactivation was dependent on the presence of NADPH and the pres ence of the thionosulfur, since the carbonyl analogue of PTU was not found to inactivate P450 1A1, and was partially prevented by heat treatment of th e microsomes which is known to selectively inactivate FMO enzymes. Inactiva tion of P450 1A1 could be reversed by treatment with dithiothreitol, indica ting the formation of disulfide bonds. However, thiourea-containing compoun ds also inhibited the EROD activity of P450 1A1 in a competitive manner. Th is property complicates the usefulness of the EROD activity of P450 1A1 as a marker for the alkylating potential of thiourea-containing compounds. It was found that metabolites of thiourea-containing compounds could transient ly activate the mGST. A maximal level of activation by 100 mu M PTU of 162 +/- 16% was found after 10 min. Activation of mGST by 100 mu M PTU was depe ndent on the presence of NADPH and the presence of the thionosulfur, since the carbonyl analogue of PTU was not found to activate mGST. Activation was completely prevented by heat treatment of the microsomes, indicating invol vement of FMO in the bioactivation process. Finally, a series of structural ly diverse thiourea-containing compounds were tested for their ability to a ctivate mGST. It appeared that their potency in alkylating mGST was inverse ly related to their V-max/K-m valve for the FMO enzyme. From this study, it is concluded that, whereas activation of mGST in rat liver microsomes may be a useful system with which to investigate the relationship between struc ture and alkylating potential of thiourea-containing compounds in vitro, in activation of P450 1A1 is not.