Among sensitizers tested in the previous

Among 22 sensitizers tested in the previous study, six test chemicals were pre- or pro-haptens (Nepal et al., 2018a). Among 6 chemicals, hydroquinone and 2-aminophenol, strong sensitizers, were falsely classified as non-sensitizers due to the lack of metabolic activation (Nepal et al., 2018a). Literatures have shown that peroxidase and tyrosinase would be required to convert hydroquinone and 2-aminophenol to 1,4-benzoquinone and 2-aminophenoxazin-3-one, respectively (Kettle and Winterbourn, 1992; Washington et al., 2015). Both enzymes would be present in liver cells and assisted to convert those chemicals to their reactive forms (Amanzada et al., 2011; Brown et al., 2001). Although the presence of these enzymes in liver microsomes would be minimal, our present results showed that the incorporation of induced microsomes clearly classified those chemicals as sensitizers, indicating that metabolism by other enzymes to sensitizing metabolite(s) would be possible (Table 2). Moreover, the percent suppression of β-galactosidase was higher with pooled S-9 factions than with pooled microsomes, suggesting that the enzymes present in cytosolic fraction in S-9 would preferably activate 2-aminophenol (Fig. 2). Among pre- or pro-haptens studied in our previous study, four of them, such as isoeugenol, eugenol, benzocaine, and cinnamaldehyde, were correctly predicted as sensitizers even without any assistance of liver microsomes. The results indicated the likelihood of autoxidation of pre-haptens to active haptens. Furthermore, among several pre- and pro-haptens studied in the present study, most of the chemicals were identified as true positives regardless either with or without the addition of microsomes. However, some chemicals, such as hydroquinone, 2-aminophenol, 3-aminophenol, p-phenylenediamine, aniline, and benzo(a)pyrene studied in the current study, were only able to suppress β-galactosidase activity with the assistance of liver microsomes. The results suggested that the LacZ gene expression system in E. coli cultures that we developed would be performed in both with and without metabolic activation system as in the Ames test (Flamand et al., 2001). Interestingly, test chemicals, such as propyl gallate and lauryl gallate gave a reduced response in the presence of microsomes. Conversely, a high percent suppression was achieved in no microsome-added group (Table 2). The results indicated the toxic behavior of parent compounds, but not by their metabolites. These results were also consistent with the results from Booth et al. (1959) and Nakagawa et al. (1995), where metabolic products, such as gallic Lck Inhibitor and 4-O-methyl gallic acid, were measured. Because the toxicity of parent compounds, propyl gallate and lauryl gallate, was much higher than their metabolites, this might be a possible reason why lesser or no suppression of β-galactosidase enzyme was observed by propyl gallate or lauryl gallate when incubated with microsomes (Table 2; Nakagawa et al., 1995). Among LLNA-categorized sensitizers tested, limonene, a weak sensitizer in LLNA studies, was detected as false negative in both with and without addition of liver microsomes (Table 2). For the metabolism of limonene, CYP2C9 enzyme was known to be required (Miyazawa et al., 2002). Although further studies are needed, the failure to suppress β-galactosidase by limonene would indicate that the metabolite(s) produced would not have a sensitizing potential. In a pharmacokinetics of d-limonene in a rat model, two major metabolites, dihydroperillic acid and perillic acid, were identified from plasma. Interestingly, perillic acid has been studied as a protective agent against many disorders, such as radiation-induced oxidative stress, cytokine profile, DNA damage, and intestinal toxicity, indicating the non-toxic effects of limonene metabolites (Pratheeshkumar et al., 2010). Among 11 non-sensitizers that were adopted from our previous study, 3 non-sensitizers, such as salicylic acid, methyl salicylate, and 4-hydroxybenzoic acid, showed higher suppressive effects on β-galactosidase in the microsome study (Table 2). In addition, only 2 chemicals, such as salicylic acid and methyl salicylate, showed false positive results in the S-9 study (Supplementary Table 1). In previous study, salicylic acid was considered as the only false positive, which was also consistent with the present results (Table 2). In fact, several conflicting results of salicylic acid were existed in literatures. Salicylic acid, which was considered as a non-sensitizer in several studies (Gerberick et al., 2007), was also categorized as a weak sensitizer and weak skin irritant by some researchers (Arif, 2015; Lee et al., 2014). Moreover, salicylic acid could be hydroxylated to 2,3-dihydrobenzoic acid and 2,5-dihydroxybenzoic acid, and further decarboxylated to 1,2-dihydroxybenzene (catechol) (Stumpf, 2016; Ohmoto et al., 1991). Of interest, catechol was itself considered as a skin sensitizer (Hirose et al., 1999), although the production was not quantitated in the present study. Moreover, from centuries, salicylic acid was used as a skin peeling agent (Arif, 2015). Because of this effect, salicylic acid might showed higher percent suppression of β-galactosidase activity. Similarly, methyl salicylate is a methylated product of salicylic acid. Although the percent suppression of β-galactosidase activity was within the cut-off range of 17.3% in microsome-unused group, with the addition of microsomes, the suppression of β-galactosidase activity was increased to be positive. The results indicated the conversion of methyl salicylate to salicylic acid when acted upon by microsomal enzymes. This hypothesis is further supported by the reports that have stated of easier conversion of methyl salicylate to salicylic acid by blood and liver esterase and could be the reason of methyl salicylate poisoning (Dasgupta and Wahed, 2014). This further supported the skin sensitizing or irritating behavior of salicylates. 4-Hydroxybenzoic acid also exhibited the similar phenomenon of β-galactosidase suppression similar to methyl salicylate. 4-Hydroxybenzoic acid (p-salicylic acid) which showed no suppressive effect when incubated in the absence of liver microsome (Table 2). However, after the incubation with liver microsomes, the suppression was increased to be falsely positive. This result might indicate the conversion of 4-hydroxybenzoic acid to hydroquinone from the assistance of liver microsome. Moreover, from the same study by Stumpf (2016), 4-hydroxybenzoic acid could be converted to hydroquinone following hydroxylation and decarboxylation. These results might provide a possible evidence that some non-sensitizers might be converted into metabolites that are more potent to suppress β-galactosidase following metabolism by microsomes. Meanwhile, 4-hydroxybenzoic acid, a false positive in microsome-incorporated group, was obtained to be true negative with the S-9 fractions-incorporated group. This result suggested that, with the existence of lesser enzymes in S-9 fractions, the conversion of 4-hydroxybenzoic acid to hydroquinone would be rather limited.