ABSTRACT
The catalytic activity of cytochrome P450 enzymes is known to be affected by presence of organic solvents in in vitro assays. However, these effects tend to be variable and depend on the substrate and CYP450 isoform in question. In the present study, we have investigated effect of ten water miscible organic solvents (methanol, ethanol, propanol, isopropanol, acetone, acetonitrile, dimethylsulphoxide, dimethylformamide, dioxane and PEG400) on water soluble substrates of CYP450, metoprolol and imipramine, at 0, 0.1, 0.25, 0.5, 0.75 and 1% v/v concentration in rat liver microsomes. Organic solvents studied had a concentration dependent inhibitory effect on the metoprolol and imipramine metabolism activity. Metoprolol metabolism was found to be more susceptible to the organic solvents, almost all the ten solvents had more or less inhibitory effect compared to imipramine metabolism. Except acetone, PEG400 and dimethylsulphoxide, all solvents had ~50% inhibition of total metoprolol metabolism activity, while in case of imipramine metabolism activity, only n-propanol, isopropanol and PEG400 had ~50% inhibition at 1% v/v. Interestingly, methanol, dimethylsulphoxide and acetonitrile had negligible effect on the imipramine metabolism (less than 10% inhibition at 1% v/v) while, total metoprolol metabolism activity was substantially inhibited by these solvents (MeOH 52%, DMSO 29% and ACN 47% at 1% v/v). In both cases, dioxane was found to be the most inhibitory solvent (~90% inhibition at 1% v/v).
ABSTRACT
Organic solvents used for solubilization of the substrates/NCEs are known to affect the activity of cytochrome P450 enzymes. Further, this effect varies with the solvents used, the substrates and CYP450 isoforms in question. In the present study, we have investigated the effect of ten commonly used water miscible organic solvents (methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, acetone, dimethyl sulphoxide, N,N-dimethyl formamide, dioxane and polyethylene glycol 400) on p-nitrophenol hydroxylase activity at 0, 0.1, 0.25, 0.5, 0.75 and 1% v/v concentration in rat liver microsomes. All the solvents studied showed concentration dependent inhibition of the p-nitrophenol hydroxylase activity except acetonitrile which showed activation of the activity at concentration range studied. Out of ten solvents studied, dioxane was found to be the most inhibitory solvent (inhibition >90% at 0.25% v/v concentration). Overall, solvents like dimethyl sulphoxide, dimethyl formamide and dioxane appeared to be unsuitable for characterizing p-nitrophenol hydroxylase (CYP2E1-mediated) reactions due to a high degree of inhibition. On the other hand, methanol and acetonitrile at concentrations <0.5% v/v appeared to be appropriate solvents for substrate solubilization while evaluating CYP2E1-mediated catalysis. The results of this study imply that caution should be exercised while choosing solvents for dissolution of substrate during enzyme studies in liver microsomes.
