Identification of pre- and pro-haptens with a β-galactosidase-expressing E. coli culture system for skin...

Although several in vitro approaches were successful in separating chemicals as skin sensitizers and non-sensitizers, none of the available methods completely mimics the absolute in vivo scenario of skin sensitization. One of the major challenges with currently available systems would be the limited or no metabolic capacity to activate pre- or pro-haptens to reactive metabolites in the system. In the present study, E. coli cells with β-galactosidase-expressing LacZ gene were combined with either induced rat liver S-9 fractions or microsomal fractions to detect pre- or pro-haptens to cause skin sensitization. Following optimization of some experimental conditions, we examined 20 sensitizers classified as pre- or pro-haptens and 11 non-sensitizers in these E. coli cultures by incubating bacterial cells and test chemicals with and without S-9 or microsomal proteins. After a 6-h incubation in the presence of IPTG, cells were lyzed to determine the suppression of β-galactosidase enzyme. A cut-off of 17.3% was applied to determine the percent suppression of β-galactosidase activity by test chemicals to classify skin sensitizers and non-sensitizers. Among chemicals tested, 19 pre- or pro-haptens were categorized as true positives and 8 non-sensitizers were categorized as true negatives. Thereby, the overall sensitivity, specificity and accuracy achieved with microsome-incorporated and S-9 fraction-incorporated group were 95.0%, 72.7% and 87.1% and 80.0%, 81.8% and 80.6%, respectively. The results suggested that the present bacterial system incorporated with the microsomal activation system could be considered as a useful alternative method to classify not only direct-acting sensitizers but also pre- or pro-haptens requiring metabolic activation in vitro.