Gene expression changes induced by skin sensitizers in the KeratinoSens™ cell line: Discriminating Nrf2-dependent and Nrf2-independent events

Abstract

The KeratinoSens™ assay is an in vitro screen for the skin sensitization potential of chemicals. It is based on a luciferase reporter gene under the control of the antioxidant response element of the aldoketoreductase gene AKR1C2. The transferability, reproducibility, and predictivity of the KeratinoSens™ assay have been investigated in detail and it is currently under assessment at the European Center for Validation of Alternatives to animal testing (ECVAM). Here we investigate the sensitizer-induced gene expression in the KeratinoSens™ cell line at the mRNA level and discriminate Nrf2-dependent and Nrf2-independent events by using siRNA to better characterize this test system at the molecular level. The results show that (i) the sensitizer-induced luciferase signal in KeratinoSens™ cells is completely dependent on Nrf2. The same holds true for the luciferase induction observed for the false positive chemical Tween80, indicating that the false positive result is not due to recruitment of an alternative transcription factor. (ii) Luciferase induction parallels the induction of endogenous Nrf2-dependent genes, indicating that the luciferase signal is representative for the sensitizer-induced Nrf2-response. (iii) The induction by sensitizers of additional genetic markers related to heat shock proteins and cellular stress could be reproduced in the KeratinoSens™ cell line and they were shown to be Nrf2-independent. These results confirm that the KeratinoSens™ cell line is a rapid and adequate screening tool to assess the sensitizer-induced Nrf2-response in keratinocytes.

Keywords: 2,4-dinitrochlorobenzene; ARE; DMSO; DNCB; FCS; Gene expression; Keap1; Kelch-like ECH-associated protein 1; KeratinoSens™; Keratinocytes; LLNA; Nrf2; SDS; Skin sensitization; Small interfering RNA; antioxidant response element; dimethylsulfoxide; fetal calf serum; local lymph node assay; nuclear factor-erythroid 2-related factor 2; qRT-PCR; quantitative real time polymerase chain reaction; sodium dodecyl sulfate.