BITC significantly enhanced the gene expression of

BITC significantly enhanced the gene expression of the Nrf2-dependent genes, such as NQO1 and HO-1 (Fig. 4), as well as the ALDH genes (Fig. 2). Nrf2 is a key transcriptional factor which activates the expression of the genes that contain ARE in their promoter. Nrf2 translocates to the nucleus where it binds to the ARE sequence, associated with its target genes and proteins. We confirmed that BITC increased both the protein expression and its nuclear translocation of Nrf2 (Fig. 5A and B). Furthermore, the pretreatment of the Nrf2 siRNA decreased both the basal and BITC-enhanced total ALDH activities, suggesting that the BITC-enhanced ALDH activity is regulated by the Nrf2-dependent pathway. However, the Nrf2-dependency was varied between the basal and BITC-induced expressions of ALDH1A1, ALDH2 and ALDH3A1; the ALDH1A1 gene required Nrf2 for its basal, but not the BITC-induced expression; ALDH2 only required Nrf2 for the BITC-induced expression and; ALDH3A1 required Nrf2 for both its basal and BITC-induced expressions. These results suggested that the Nrf2-dependent pathway is, at least partly, involved in the BITC-induced gene expression of ALDHs. In addition to the Nrf2-dependent pathway, other signing pathways are also postulated. The inhibition of the STAT3 signaling pathway or its activator EZH2 genetically or pharmacologically diminished the level of the ALDH(+) cells and clonogenicity (Shao et al., 2014). AMP-activated protein kinase regulates the ALDH1A1 and ALDH2 expressions (Choi et al., 2011) (Kiefer et al., 2012). The gene expression of ALDH3s was shown to be mediated by an aryl hydrocarbon receptor- (Sládek, 2003) and NF-κB-dependent pathway (Weiner, 2007). The retinoic Mupirocin response element is responsible for down-regulation of ALDH1A1 through interaction of the retinoic acid receptor and CCAAT/enhancer binding protein β (Elizondo et al., 2000). Even though other signaling pathways might be involved in the BITC-enhanced gene expression of ALDH1A1 and ALDH2, Nrf2 is one of the key transcriptional factors for the BITC-enhanced ALDH activity. The cytotoxic effect of acetaldehyde has also been reported in various cellular models which are derived from the liver (Kurose et al., 1997), gastrointestinal tract (Rao, 1998), gonads (Mapoles et al., 1994), immune system (Sisson and Tuma, 1994) and brain (Yan et al., 2016). We also confirmed that acetaldehyde dose-dependently induced a cytotoxicity in mouse hepatoma Hepa1c1c7 cells (Fig. 7A). Tanaka et al. found that acetaldehyde triggered oxidative stress via mitochondrial superoxide production in the esophageal keratinocytes and that ALDH2-deficient cells were highly susceptible to the ethanol- or acetaldehyde-mediated toxicity (Tanaka et al., 2016). ALDH2−/− mice are more susceptible to the ethanol and acetaldehyde-induced toxicity than the wild type mice (Yu et al., 2009). Because the ALDH status is responsible for the sensitivity of the acetaldehyde-induced toxicity, we examined the effect of BITC on the acetaldehyde-induced cytotoxicity. BITC (1 μM) actually impaired the acetaldehyde cytotoxicity (Fig. 7A), even though BITC at supraphysiological concentrations is cytotoxic (data not shown). However, BITC did not impair the acetaldehyde-induced cytotoxicity in the Nrf2-knockdown cells as well as in the ALDH3A1-knockdown cells. These findings suggested that the inhibitory effect of BITC on the acetaldehyde-induced cytotoxicity is controlled by the Nrf2-dependent pathway and, at least in part, ALDH3A1 activity. The present data are consistent with a previous study showing that Nrf2−/− mice underwent a significant reduction in their ability to detoxify acetaldehyde (Lamlé et al., 2008). Anni et al. suggested another plausible mechanism for impairment of the acetaldehyde-induced toxicity which involves cysteine and glutathione (Anni et al., 2003). BITC not only induced the gene expression of ALDHs, but also the phase 2 genes including HO-1 (Fig. 4B), GST (Nakamura et al., 2000) and GCLC (Miyoshi et al., 2008). All of the HO-1, GST and GCLC genes have been reported to play an effective role in protection against oxidative stress (Ghattas et al., 2002) (Zou et al., 2014) (Franklin et al., 2002). Therefore, the BITC-induced phase 2 enzymes cannot be ruled out in the beneficial effect on amelioration of the acetaldehyde-induced cytotoxicity.