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  • Prior to proceeding to the main

    2021-10-14

    Prior to proceeding to the main tests, essential conditions were optimized. At first, we tested whether the parameters that were successful in inducing LacZ gene for the production of β-galactosidase enzymes in E. coli culture system in our previous study would be sufficient to induce LacZ gene in the current microsome-incorporated system (Nepal et al., 2018a). To do so, parameters, such as concentration dependencies of IPTG and ONPG along with their pH requirements for the maximum induction of β-galactosidase enzymes were determined. Consistent with the previous study, IPTG at 30 μM in LB media, pH 7.0, and ONPG at 2 mg/ml in Z-buffer, pH 7.5, were noticed to be sufficient for the induction of LacZ gene to produce β-galactosidase enzyme (Fig. 1). The results indicated that the incorporation of microsomal fraction in E. coli cultures would not significantly affect the results. The addition of S-9 fractions also gave no interferences (data not shown). Thereafter, to investigate the applicability of test method incorporated with metabolic activation system in the previously developed system with E. coli cultures, several pre- and pro-haptens were preliminarily tested. At first, the effects of either uninduced S-9 fraction or microsome were compared with either induced S-9 fraction or microsome. The percent suppression of β-galactosidase by several pre- or pro-haptens following the activation with induced S-9 or microsomes were much higher than uninduced S-9 or microsomes, indicating some sensitizers would require metabolic activation (data not shown). Therefore, the induced fraction of either S-9 or microsomes were chosen for the subsequent experiments. Next, the percent suppression of β-galactosidase by pre- or pro-haptens in the presence of pooled liver S-9 fractions or microsomes induced by individual inducers was investigated. The induced fractions were originally prepared for the xenobiotic metabolism studies (Noh et al., 2015). Because the individual inducer-induced microsomes were thought to have high activities of only few xenobiotic-metabolizing enzymes, we decided to use pooled liver fractions to use pan CYP-induced metabolic activation system. In the test system, E. coli Erlotinib Hydrochloride with optimal cell densities (OD600 0.5 - 0.6) were added into LB broth medium, pH 7.0, containing 30 μM of IPTG and treated with various test chemicals at 0.6 mM. Either induced liver microsomes at 2 mg/ml, S-9 fractions at 2.5 mg/ml or potassium phosphate buffer, pH 7.4, as control was added into the incubation mixture along with 1 mM of NADPH. After a 6-h incubation, the percent suppression of β-galactosidase was determined colorimetrically by using 2 mg/ml of ONPG. As shown in Fig. 2, the percent suppression of β-galactosidase by test chemicals, such as p-hydroquinone, 2-aminophenol, and 3-aminophenol, were much higher with both pooled microsome- and S-9 fraction-added groups than with control groups. Likewise, when we compared the percent suppression of β-galactosidase between microsome-added and S-9-added groups, except 2-aminophenol, a higher percent suppression was observed with pooled microsome-added group. 2-Aminophenol showed higher percent suppression with S-9 fractions than microsomes, which signified that the enzymes responsible for metabolic activation of 2-aminophenol would reside in S-9 fractions than in microsomal fractions. Conversely, no differences were observed with chemicals, such as isoeugenol and geraniol, regardless of with or without the addition of liver fractions, indicating that these pre- or pro-haptens would be strong enough to cause sensitization by themselves. Likewise, the addition of either S-9 fractions or microsomes in E. coli cultures did not affect the results on non-sensitizers, lactic acid and 2-propyl alcohol. The results also indicated that the incorporation of metabolic activation system in E. coli cultures could be applicable to improve predictive capacity for identifying skin sensitizers requiring metabolism. Because of the better results with microsomes, we primarily chose pooled induced microsome rather than S-9 fractions for the subsequent experiments. However, it would be apparent that several additional metabolic enzymes along with specific CYP enzymes would exist in S-9 fractions. Therefore, it was worth to explore the effects of S-9 fractions on chemicals that would only be responsive to certain enzymes besides CYP enzymes. Therefore, pooled induced S-9 fractions were also tested in the present study.