Hypoxia perturbs aryl hydrocarbon receptor signaling and CYP1A1 expression induced by PCB 126 in human skin and liver-derived cell lines
Introduction
Polychlorinated biphenyls (PCBs) are a group of synthetic organic chemicals consisting of 209 congeners that accumulate and persist in the environment (Safe, 1993). PCBs that are more highly chlorinated have much slower rates of metabolism, and therefore quickly accumulate in the human body. Human blood serum levels of PCBs in a population in Alabama, USA range from 0.003 μM to 6.5 μM (Hansen et al., 2003). Exposure to PCBs is known to cause a broad range of adverse health effects and due to their persistence and ubiquitous distribution, PCBs pose a serious hazard to human and animal health (Crinnion, 2011, Ludewig et al., 2008, Silberhorn et al., 1990).
3,3′,4,4′,5-Pentachlorobiphenyl (PCB 126) is a coplanar and the most potent dioxin-like PCB. As such, PCB 126 can bind to and activate the aryl hydrocarbon receptor (AhR), an immediate early cellular response to xenobiotics and mediator of many of the toxic effects of PCB exposure. Upon ligand binding, the AhR translocates to the nucleus and forms a heterodimer with the aryl hydrocarbon receptor nuclear translocator (ARNT). This complex binds to and activates the promoters of genes containing xenobiotic response elements (XREs). Binding to these regulatory sequences induces transcription and expression of AhR target genes such as the cytochrome P450 (CYP) 1 family, including CYP1A1 and CYP1B1 (Beischlag et al., 2008).
Similar to the AhR, hypoxia-inducible factor-1α (HIF-1α) heterodimerization with ARNT is a prerequisite for transcriptional activation of hypoxia-inducible genes (Kewley et al., 2004). HIF-1α is an important sensor of oxygen environments and regulates responses to reduced oxygen availability (hypoxia). In the context of sufficient oxygen concentrations (normoxia), HIF-1α is continuously hydroxylated by oxygen-dependent prolyl hydroxylase domain-containing enzymes (PHDs) which target HIF-1α for subsequent ubiquitination and degradation. Under limiting oxygen concentrations, HIF-1α is stabilized allowing it to translocate to the nucleus where it binds to ARNT. The HIF-1α:ARNT heterodimer binds to hypoxia response element (HRE) sequences in hypoxia-inducible target genes, and subsequently induces gene transcription (Majmundar et al., 2010).
Since ARNT is critical for both the AhR and hypoxia pathways, it seems likely that a crosstalk exists that can affect the regulation of gene expression. This may be important in the context of organs such as the liver, which contains zones of various oxygen tensions, and is responsible for a large proportion of PCB metabolism (Oinonen and Lindros, 1998). Interestingly, regional induction of CYP1A1 has been observed in rat livers. Rats exposed to low doses of PCB 126 induced CYP1A1 mainly in the perivenous zone where oxygen is scarce (Chubb et al., 2004). Numerous studies have analyzed the effect of hypoxia on AhR target genes in different cell lines after exposure to exogenous ligands (Kim and Sheen, 2000, Nie et al., 2001). However, these studies have focused on using 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as an AhR ligand and used chemical compounds such as cobalt chloride (CoCl2) to stabilize HIF-1α and induce a hypoxic response. Only one study in topminnow hepatocarcinoma cells showed that hypoxia could inhibit a PCB 126 induced AhR response (Fleming et al., 2009). Overall, the crosstalk between HIF-1α and the AhR using PCBs as AhR ligands and genuine hypoxia has not been extensively studied in human cell lines.
In an effort to understand PCB-induced AhR function and target gene expression in different oxygen environments, we exposed human hepatocellular carcinoma and human keratinocyte cell lines to PCB 126 in normal oxygen (21% O2) or hypoxia (1% O2). Our results show that hypoxia significantly interfered with AhR signaling and could inhibit AhR target gene expression and function.
Section snippets
Chemicals and PCB treatment
3,3′,4,4′,5-Pentachlorobiphenyl (PCB 126), 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB 153), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were provided by Dr. H. J. Lehmler of the Iowa Superfund Research Program at The University of Iowa. 6,2′,4′-Trimethoxyflavone (TMF) was purchased from Sigma (St. Louis, MO). All stock solutions were made in dimethyl sulfoxide (DMSO) and the appropriate amount of stock solution was added to the cell culture medium. PCBs were used at concentrations ranging from 2
Hypoxia inhibits PCB 126 induced expression of CYP1A1
Hepatocellular carcinoma (HepG2) and keratinocyte (HaCaT) cell lines were used to assess the effect of hypoxia on AhR-dependent gene expression. Cell lines were treated with either 3 μM TCDD (positive control) or varying doses of the dioxin-like coplanar PCB 126 under normoxic or hypoxic conditions. The accumulation of CYP1A1 mRNA was then determined by qRT-PCR. TCDD and PCB 126 significantly induced expression of CYP1A1 mRNA in both HepG2 cells (Figs. 1B, C) and HaCaT cells (Figs. 1E, F). The
Discussion
Both AhR and HIF-1α are members of the class I bHLH/PAS (basic helix-loop-helix/PER-ARNT-SIM) protein family and form obligate heterodimers with their common binding partner ARNT, a member of the class II bHLH/PAS protein family, prior to actively transcribing their respective target genes (Kewley et al., 2004). Given the importance of ARNT in both the xenobiotic responsive AhR and hypoxia responsive HIF-1α pathways, it may be likely that ARNT is a central regulator of these pathways and can
Conclusions
Due to the accumulation and long half-lives of PCBs, these organic contaminants pose a significant health risk to both humans and wildlife in the environment. As certain organs can experience varying concentrations of oxygen under normal physiologic conditions, it is critical to study the influence of oxygen concentrations on PCB-induced physiological processes in the human body.
The results in this study show that hypoxia interfered with AhR binding to genomic target gene sequences and that AhR
Conflict of interest statement
The authors declare that no conflicts of interest exist.
The following are the supplementary data related to this article.
Acknowledgments
This work was supported by National Institutes of Health (NIH) grant P42 ES013661-5099 (FED), P42 ES004940-5083 (BWF), and P50 ES006694. SUV received salary support from SRP Training Core P42 ES013661-5110. SUV further received a Superfund Research Program K.C. Donnelly Externship Administrative Supplement. PLS received salary support from NIH grant 2T32 ES007091-31. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The
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