It is well known that

It is well-known that TCDD is the most potent ligand of AhR and it regulates gene expression, such as CYP1A1, via activation of AhR (Mimura and Fujii-Kuriyama, 2003). Besides Tomblin et al. (2016) have recently shown that TCDD via AhR regulated L-type amino BMS-509744 transporter 1 expression in MCF-7 and MDA-MB-231 breast cancer cells. Thus, we hypothesized that TCDD increased the expression of SLC6A19 through the classic molecular mechanism medicated by AhR (Landers and Bunce, 1991; Denison et al., 2011). We tested the role of AhR in dioxin-induced effect by knocking down AhR expression and inhibiting the activation of AhR in HepG2 cells. Our data demonstrated that AhR took part in the dioxin-mediated upregulation of SLC6A19. In addition, three other AhR agonists also have been found to increase the expression of SLC6A19 via AhR. However, details of how AhR related to the increase in SLC6A19 gene expression are still to be identified. It was generally believed that activated AhR complex bind to specific DNA sequences, DREs, located in the upstream of a variety of genes, including CYP1A1, AGR2, Tgfb1, 2, and 3, Tgfbi, IL1rap (Berger, 2002; Kimura, 2013). Transcriptionally active regions are usually marked by histone modifications, among which histone 3 lysine 4 tri-methylation (H3K4me3) is one of the most commonly used histone markers (Berger, 2002). Since ChIP-seq data of H3K4mes shows likely transcriptionally active sites for transcription factor binding (Kimura, 2013), we analyzed the transcriptionally active region of human SLC6A19 in UCSC Genome Browser by adding H3K4me3 ChIP-seq data Hub. Peaks of H3K4me3 showed that there were 11 putative DRE sequences in a cluster of DREs around 2800 bps and 5100 bps downstream of the first intron (Fig. S3), which overlaps with the H3K4me3 peaks (http://www.pubfacts.com/detail/23739122/Histone-modifications-for-human -epigenome-analysis). The DREs located region could be potential location for AhR exerting its regulation on SLC6A19 transcription. SLC6A19 expression in the intestine could be regulated at three different levels involving promoter methylation, histone modification, and opposing transcription factors (Tümer et al., 2013). Thus epigenetic and posttranscriptional mechanisms should be considered and investigated for dioxin-induced SLC6A19 upregulation in HepG2 cells. Although the selective AhR agonists all increased the expression of SLC6A19, we also observed that there were differences among their effects (Table 1). TCDD, PBDDs and PBDFs have different regulation potential on induction of cytochrome P450 mRNA and “CALUX” assay (Birnbaum et al., 2003; Guruge et al., 2009; Behnisch et al., 2001). It is likely that they have different toxic equivalency factors (TEFs). TEFs reflect relative potency of AhR activation compared with TCDD. In general, it is believed that the toxicity of dioxins and dioxins-like compound is consistent with their TEFs. The highest relative potency values were observed for 1,2,3,7,8-PeCDD (TEF = 1) at 0.69 compared to TCDD using CALUX analysis in mouse hepatoma H1L6.1c3 (Samara et al., 2009). Besides, 1,2,3,7,8-PeBDD had weaker binding affinities with AhR compared with TCDD (Mason et al., 1987). Our results showed the similarly results. TCDD and 1,2,3,7,8-PeCDD were similarly potent inducers for SLC6A19 expression in HepG2 cells (Table 1). And the induction of SLC6A19 by TCDD was more obvious than those of PeCDF (TEF = 0.3) and PCB126 (TEF = 0.1) (Fig. 1 B &Table 1). CH223191, the ligand-selective antagonist of AhR, was used to assess the abilities of four selected dioxins and dioxin-like PCB in SLC6A19 induction via AhR. In rat hepatoma cells, CH223191 could totally inhibit the AhR activation induced by TCDD (10−9 M) and of PCB126 (10−8 M) in CALUX assays (Zhao et al., 2010). Our present data showed similar potency of CH223191 antagonization to the effects of TCDD (TEF = 0.1, 10−8 M) and PCB126 (TEF = 0.1, 10−8 M) on SLC6A19 expression. Interestingly, CH223191 could only partially inhibited the induction of SLC6A19 by 2,3,4,7,8-PeCDF (TEF = 0.3, 10−8 M). The differential antagonization of CH223191 suggest potential differences in action mechanism of structurally different dioxin congeners for the gene regulation.