Liver plays a pivotal role

Liver plays a pivotal role in metabolism of xenobiotic compounds, such as environmental contaminants and drugs. Some of these compounds generate hepatic damage and impair its function. It was well-known that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) could induce pathological changes in mouse and rat liver (Fernandez-Salguero et al., 1996; Gupta et al., 1973). In addition, TCDD upregulates genes encoding enzymes for metabolizing xenobiotics, including cytochrome P450 (CYP) 1A (Zeiger et al., 2001), CYP1B (Xu et al., 2005), and anterior gradient protein 2 homolog (AGR2) (Ambolet-Camoit et al., 2010) in cell lines. Liver is also a significant organ for amino 5-Carboxymethylester-UTP transport in the body. Amino acid availability and transport play essential roles in hepatocellular growth, development or regeneration after trauma, necrosis, or other damage (Mailliard et al., 1995). A human hepatoma cell line, HepG2, has been universally applied to detect the toxic effects of hepato-toxins such as apoptosis, necrosis and toxicogenomic effect (Marinković et al., 2010; Fernandez-Salguero et al., 1996; Gupta et al., 1973). Genes associated with amino acid metabolism (e.g. amino acid transporter solute carrier gene families) were found to be regulated by TCDD in HepG2 cells (Frueh et al., 2001; Puga et al., 2000). Jennen et al. (2011) have also shown that amino acid metabolism was widely impacted in HepG2 exposed to TCDD by a cross transcriptomics and metabonomics study. Thus, the transport and metabolic pathway of amino acid are responsive to dioxin exposure in HepG2 cells. However, the related responsive genes are still not fully understood. B0AT1 is a neutral amino acid transporter encoded by SLC6A19 gene, which plays an important role in transporting neutral amino acids (NAA) and Na+, involving in protein digestion and absorption, and mineral absorption. It has been reported that mutations in SLC6A19 result in several diseases, such as hartnup disorder (Bröer, 2009), iminoglycinuria (Vanslambrouck et al., 2010) and hyperglycinuria (Bröer et al., 2008). B0AT1 is highly expressed in kidney and intestine, compared to lower expression in other tissues such as pancreas, stomach, liver, duodenum and ileocecum (Kleta et al., 2004). The tissue-specific expression of B0AT1 might be due to transcriptional or epigenetic regulation of SLC6A19 gene (Tümer et al., 2013). However, whether SLC6A19 is one of the responsive genes in dioxin-induced alterations in amino acid transportation in HepG2 cells is still not known. In this study, we elaborated three issues as follow: (1) the effects of dioxin and dioxin-like compounds on SLC6A19 expression; (2) whether these effects were mediated by AhR; (3) whether TCDD affected the expression of B0AT1. Given the function of B0AT1 in amino acid transportation and metabolism, the data of the present study will provide new insight in understanding the molecular basis of the interfering effects of dioxin on the metabolic processes in hepatocytes.
Materials and methods
Results
Discussion In this study, we reported that SLC6A19 expression was upregulated by dioxin and dioxin-like chemical compounds in HepG2 cells. These compounds are well-known AhR agonists, and the action mechanism for their effects likely involves AhR. The activation of AhR upregulates expression of various genes, most of which belong to well-characterized metabolic enzymes for xenobiotics (Nebert et al., 2000). Besides, these metabolizing enzyme systems are usually followed by activation of detoxic function in liver, and the liver also plays critical roles in nutrient metabolism, including glucose, lipids and amino acids (Dentin et al., 2005; Häussinger et al., 1991). NAAs have an irreplaceable role in preserving normal physiological processes, growth and/or development of liver. B0AT1 transports all the NAAs and eight essential amino acids, preferentially large aliphatic NAAs, including leucine, isoleucine, valine, methionine, phenylalanine, tryptophan, threonine and histidine (Bröer, 2009). It was reported that dysfunction of B0AT1 caused several diseases, resulting in the inability to absorb tryptophan in intestine or reabsorb neutral amino acids in kidney (Seow et al., 2004) and tryptophan deficiency (Verrey, 2007; Seol et al., 2008). Moreover, cellular absorption of amino acids may promote the ability of cells to survive and proliferate, particularly for tumor cells and cancer cells, which requires enhanced amino acid transportation (Fuchs and Bode, 2005; Ganapathy et al., 2009; McGivan and Bungard, 2007; Medina, 2001). Currently there is controversy regarding the effect of dioxins on cell proliferation, which has been elaborated by several reviews (Schwarz et al., 2000; Mandal, 2005; Bock and Köhle, 2005). In this study, we have identified regulatory effects of TCDD and certain dioxin-like compounds on SLC6A19 gene expression which encodes the NAA transporter B0AT1. Moreover, the upregulation of SLC6A19 may result in increased expression of B0AT1 in the cells. However, whether the upregulation of SLC6A19 and B0AT1 lead to enhancement of NAA transportation and subsequently affect cell proliferation still needs further investigation. B0AT1 requires accessory subunits to modify their expression and activity at the cell surface, such as collectrin in the kidney, and angiotensin-converting enzyme 2 (ACE2) in the small intestine (Fairweather et al., 2015). There have been reports elaborating expression and roles of ACE2 and collectrin in B0AT1 functioning in liver, such as in liver fibrosis and immunity (Österreicher et al., 2009; Lambert et al., 2010; Fairweather et al., 2015). However, the effects of dioxin on these accessory proteins of B0AT1 remain to be answered. In our recent transcriptome dataset, ACE2 was slightly inhibited and collectrin remained no change in TCDD-treated (10−8 M, 24 h) HepG2 cells compared to solvent control (Data not shown). Thus, sophisticated study of dioxin effect on the function of B0AT1 and its molecular chaperones is needed for further explore the toxicological significance of the upregulation of B0AT1 by dioxin in HepG2 cells. Dioxin may affect amino acid transport and metabolism via multiple gene alterations related to the processes. Although some genes belonging to the same or different transporter families from that of SLC6A19 were not altered based on our transcriptome dataset, such as SLC6A14, SLC3 or 36 family genes, we did find other transporters being changed upon TCDD treatment, such as SLC7A5, which could be upregulated by about 2-fold in TCDD-treated HepG2 cells (Data not shown).