To ensure the induction of CYP enzymes by
To ensure the induction of CYP enzymes by several inducers, specific monooxygenase activities of variously induced, uninduced and pooled liver microsomes were compared (Fig. 3). Although 3-MC, phenobarbital, acetone, and dexamethasone could induce CYP1A-, CYP2B-, CYP2E1-, and CYP3A-selective enzyme activities by 15-, 6-, 5-, and 3.4-fold, respectively, the inductions of other enzymes in individual microsomes were lesser. Meanwhile, the fold inductions of all tested CYP enzymes were evenly high in pooled liver microsomes. With pooled liver microsomes, induction folds of 10, 4, 5, and 2.2 were achieved for CYP1A-, 2B-, 2E1- and 3A-selective marker enzymes, respectively, when compared to uninduced control microsomes (Fig. 3). Because of none or little evidences of metabolic pathways of test chemicals or the lack of information on CYP enzymes required to metabolize certain test chemicals, it would be reasonable to use pan CYP-induced microsomes. Therefore, use of pooled liver microsomes would be safer than using uninduced or single protein-enriched microsomes. Moreover, the induction folds of pooled microsomes were much higher than the uninduced microsomes. Therefore, pooled liver microsomes were chosen for the subsequent experiments. As presented in Fig. 4A, the optimum concentrations of liver microsomes were tested for the maximum suppression of β-galactosidase by pre- and pro-haptens after their activation to reactive haptens. E. coli sphingosine-1-phosphate with optimum cell densities were mixed with LB broth containing 30 μM of IPTG and various chemicals at 0.6 mM in the presence of various concentrations of microsomes. After mixing, NADPH at 1 mM was added into the tubes to start the reaction at 37 °C. After 6-h of incubation, the samples were lyzed to determine β-galactosidase activity. As noted in Fig. 4A, although the results with 1.5 mg/ml of liver microsome were acceptable, the results obtained with 2 mg/ml of liver microsome were better to separate sensitizers and non-sensitizers. Thus, 2 mg/ml was chosen as the optimal concentration of microsomes according to the maximum activation of pre- or pro-haptens to reactive metabolites. Likewise, the optimal incubation time required to achieve the maximum suppression of β-galactosidase activity was determined (Fig. 4B). E. coli cells with optimum cell densities were mixed with LB broth containing 30 μM of IPTG, various chemicals at 0.6 mM along with induced liver microsome at 2 mg/ml and NADPH at 1 mM concentration. The samples were incubated for given time points from 3 h to 24 h. Following respective incubation time, the cells were lyzed and β-galactosidase activity was determined. As shown in Fig. 4B, incubation for 6 h at 37 °C would be sufficient to achieve the maximum suppressive effect on β-galactosidase activity. Similarly, the pooled liver S-9 fractions needed for maximum suppression of β-galactosidase by several test chemicals were also determined in the present study (Supplementary Fig. 2). The results indicated that the pooled liver S-9 fraction at 2.5 mg/ml was the best in terms of the suppression of β-galactosidase. It was also observed that, with the increase in the concentration of S-9 fractions, the expression of β-galactosidase by IPTG was lowered, when compared with the microsome-supplemented studies (data not shown). Subsequently, effects of 4 common solvents on β-galactosidase activity were determined with 6 testing chemicals including 4 pre- or pro-haptens and 2 non-sensitizers in microsome-supplemented system. It was assumed that the vehicles used to dissolve test chemicals would affect CYP enzymes, which would affect the metabolism of test chemicals, and ultimately would affect the suppressive effects on β-galactosidase activity. The solvent affecting enzyme’s potency to activate test chemicals might cause less suppression of β-galactosidase by test chemicals. E. coli cells with optimum cell densities were mixed with LB broth containing 30 μM of IPTG, and treated with various chemicals at 0.6 mM that were dissolved in either DMSO, acetonitrile, acetone or methanol. The final concentration of each vehicle was 0.4%. Pooled liver microsomes at 2 mg/ml and NADPH at 1 mM was added into the samples and incubated at 37 °C for 6 h. As shown in Fig. 5, only marginal differences were observed between the solvents tested except methanol. It seemed that acetonitrile and DMSO showed relative consistent results. Thus, acetonitrile was chosen as the primary solvent in the main study. If the chemicals were not dissolved in acetonitrile, DMSO was used as a second choice. The vehicle used to dissolve each test chemical was listed in Table 2. The same solvent system was chosen for the experiments with pooled liver S-9 fractions.