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    • Liver is the house for a

    2022-08-18

    Liver is the house for a variety of xenobiotic-metabolizing U-73122 that produce reactive oxygen species as well as reactive metabolites (Johansson et al., 2009; Noh et al., 2017; Puntarulo and Cederbaum, 1998). When the enzymes act upon pre- or pro-haptens, the chemicals would be activated to reactive metabolites to cause skin sensitization (Johansson et al., 2009). In this regard, upon biotransformation of pre- or pro-haptens by hepatic enzymes, the reactive metabolites would act upon β-galactosidase expression system in E. coli, and then the production of the enzyme would be suppressed. Alternatively, they could also directly affect the activity of expressed β-galactosidase. By either way, the β-galactosidase activity reduced by test chemicals would be a toxic marker in the skin sensitization test, as we proposed in the previous report (Nepal et al., 2018a). Although there are very limited literatures for CYP-mediated activation of topically applied chemicals (Natsch et al., 2013), it is highly evident that the metabolic activation might be necessary for identifying those pre- or pro-haptens from in vitro alternative tests where metabolic activation capacity is very limited. Skin S-9 or microsomal fractions would be the best choice for the activation of sensitizers in the test. However, it is very complex to extract S-9 fractions or microsomes from skin for the test development, and the metabolic capacity of skin microsomes would not be high enough for activating sensitizers during the short period of chemical exposure time in any alternative tests available. In this regard, even in the well-known Ames test, Aroclor 1254-induced rat liver S-9 fractions are adopted for the maximum induction of xenobiotic-metabolizing enzymes including CYPs. Therefore, for a testing system that is readily performed for the identification of toxic signs following activation, more general metabolic activation system would be preferable than skin microsomes in the test. Because the purpose of proposed test here would be to better determine the sensitizing potential of certain chemicals that require metabolic activation, liver metabolic system was selected in the present study. In our recent study, we developed a method to screen the skin sensitization potential of chemicals by measuring percent suppression of β-galactosidase activity by test chemicals in E. coli cells containing β-galactosidase-expressing LacZ gene in the presence of IPTG (Nepal et al., 2018a). It was thought that, by IPTG induction, the rapid expression of β-galactosidase in E. coli cells would be vulnerable to toxic insults by test chemicals, so that reactive sensitizers would suppress directly or indirectly on the expression of β-galactosidase as the sensitizer interacts with carrier proteins. From the total numbers of 33 chemicals with 22 sensitizers (weak to extreme) and 11 non-sensitizers, the accuracy of 87.7% was achieved (Nepal et al., 2018a). Nonetheless, two strong pre- and pro-haptens, hydroquinone and 2-aminophenol, were falsely classified as non-sensitizers in the study (Nepal et al., 2018a). In the present study, we investigated the applicability of E. coli cells with β-galactosidase-expressing system combined with either induced rat liver S-9 fractions or microsomal fractions to identify pre- and pro-haptens, based on their skin sensitizing and non-sensitizing potential from LLNA.
    Materials and methods
    Results In our previous study, we investigated suppressive effects of 22 skin sensitizers and 11 non-sensitizers on the β-galactosidase activity (Nepal et al., 2018a). Chemicals were predicted as skin sensitizers or non-sensitizers based on percent suppression of β-galactosidase by test chemicals in which a cut-off percent of 17.3 was applied. As mentioned, two LLNA categorized sensitizers, p-hydroquinone and 2-aminophenol, which were known as pre- or pro-haptens, were determined to be false negatives. To accurately predict the sensitizing potential of certain chemicals, a system that could detect not only direct acting sensitizers but also pre- or pro-haptens must exist. Therefore, a system incorporated with either S-9 fractions- or microsome-mediated metabolic activation system was designed U-73122 in the present study. The percent suppression of β-galactosidase by 31 chemicals were studied in the current study, where 20 chemicals of either pre- or pro-haptens were selected from literatures as listed in Table 1. Eleven non-sensitizers were the same as we studied previously for testing whether the results would be the same as in the direct incubation system when metabolic activation systems are supplemented (Nepal et al., 2018a).