Non-coplanar polychlorinated biphenyls (PCBs) are direct agonists for the human pregnane-X receptor and constitutive androstane receptor, and activate target gene expression in a tissue-specific manner

doi:10.1016/j.taap.2012.05.016

Toxicology and Applied Pharmacology

Volume 263, Issue 1, 15 August 2012, Pages 7-13

https://doi.org/10.1016/j.taap.2012.05.016 Get rights and content

Abstract

The polychlorinated biphenyl group possesses high environmental persistence, leading to bioaccumulation and a number of adverse effects in mammals. Whilst coplanar PCBs elicit their toxic effects through agonism of the aryl hydrocarbon receptor; however, non-coplanar PCBs are not ligands for AhR, but may be ligands for members of the nuclear receptor family of proteins. To better understand the biological actions of non-coplanar PCBs, we have undertaken a systematic analysis of their ability to activate PXR and CAR-mediated effects.

Cells were exposed to a range of non-coplanar PCBs (99, 138, 153, 180 and 194), or the coplanar PCB77: Direct activation of PXR and CAR was measured using a mammalian receptor activation assay in human liver cells, with rifampicin and CITCO used as positive controls ligands for PXR and CAR, respectively; activation of target gene expression was examined using reporter gene plasmids for CYP3A4 and MDR1 transfected into liver, intestine and lung cell lines.

Several of the non-coplanar PCBs directly activated PXR and CAR, whilst the coplanar PCB77 did not. Non-coplanar PCBs were also able to activate PXR/CAR target gene expression in a substitution- and tissue-specific manner.

Non-coplanar PCBs act as direct activators for the nuclear receptors PXR and CAR, and are able to elicit transcriptional activation of target genes in a substitution- and tissue-dependent manner. Chronic activation of PXR/CAR is linked to adverse effects and must be included in any risk assessment of PCBs.

Highlights

► Several Non-coplanar PCBs are able to directly activate both PXR and CAR in vitro. ► PCB153 is the most potent direct activator of PXR and CAR nuclear receptors. ► Non-coplanar PCB activation of CYP3A4/MDR1 reporter genes is structure-dependent. ► Non-coplanar PCB activate CYP3A4/MDR1 reporter genes in a tissue-dependent. ► PCB153 is the most potent activator of PXR/CAR target gene in all tissues.

Introduction

Polychlorinated biphenyls (PCBs) were historically used in a large number of industrial applications, including coolants, plasticisers, lubricants and insulators (Safe, 1984, Safe, 2001). Their high chemical stability has resulted in environmental persistence, and coupled with their highly lipophilic nature raises the potential for bioaccumulation in higher mammals, including humans.

There are 209 potential PCB congeners, of which 36 congeners are considered to be environmentally threatening due to environmental prevalence, bioaccumulation in animal tissues and known toxic effects (McFarland and Clarke, 1989). These congeners can be divided into non-ortho PCBs that have a coplanar configuration, and ortho‐substituted PCBs with a non-coplanar configuration. PCB congeners with zero or one ortho-chlorine substituent (e.g. PCB77) exhibit toxic effects similar to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and these are mediated through the aryl hydrocarbon receptor (AhR); thus, these PCBs are referred to as dioxin-like PCBs. These adverse effects include hepatotoxicity, elevated blood lipids, immune suppression, reproductive and developmental toxicity and carcinogenicity (Safe, 1984, Safe, 2001). Due to these highly undesirable properties, PCB manufacture was banned in the late 1970s and their usage severely restricted. However, due to the highly persistent nature of these chemicals, it is estimated that there remains over 750,000 tonnes of PCBs in the biosphere, and hence they still represent an important potential toxic contaminant.

Whilst the majority of risk assessment interest has focussed on the coplanar dioxin-like PCBs, non-coplanar PCBs are also highly environmentally prevalent, with PCB138, 153 and 180 being widely present in the environment and thus must also be included in any hazard/risk assessment (Safe, 1994). Non-coplanar PCBs (e.g. PCB153) possess ortho‐chlorine substituents on the biphenyl ring that twists the structure away from a single plane. This significantly reduces the affinity for AhR, and indeed these compounds are likely to act as inhibitors of AhR-mediated activation (Suh et al., 2003). Non-coplanar PCBs are more likely to act as ligands for members of the nuclear receptor family of transcription factors (Wu et al., 2009). This activity may be important considering that not all of the adverse effects associated with PCB exposure are mediated via AhR. For example, the hydroxylated metabolites of PCBs are weak oestrogen receptor agonists, as well as inhibitors of oestrogen sulphotransferase; as such, PCBs may have an endocrine disrupting effect, which may underlie their reproductive toxicity (Kester et al., 2000). Thus it is important to assess the hazard of non-coplanar PCBs to humans to inform a full risk assessment.

It has been suggested that non-coplanar PCBs may act as ligands for the constitutive andorstane receptor (CAR) and/or the pregnane‐X receptor (PXR) (Jacobs et al., 2005, Schuetz et al., 1998), and may thus activate CAR/PXR target genes expression. Due to this activity, this group of PCBs is often referred to as ‘Phenobarbital-like PCBs’.

There is thus a need to examine the non-AhR‐mediated effects of PCBs, and specifically the non-coplanar phenobarbital-like PBCs, for comprehensive risk assessments to be undertaken. As such, we have examined the ability of the non-coplanar PCBs PCB99, 138, 153, 180 and 194 to directly activate CAR and PXR, as well as their ability to activate target gene expression in liver, lung and intestinal cells in vitro.

Section snippets

Reagents

Fugene-6 transfection reagent was purchased from Roche Diagnostics, Lewes, UK. Unless otherwise stated all other chemicals were of molecular biology grade and obtained from Sigma-Aldrich (Poole, UK). The Huh7 human hepatocellular carcinoma cell line (Nakabayashi et al., 1982) was a kind gift from Dr Steve Hood (GSK, Ware, UK). Human intestinal (Caco2) and lung (A549) cell lines were purchased from ECACC (Porton Down, UK). All cells were routinely cultured in minimal essential medium with

Results

A range of PCB congeners were selected for study that encompassed the coplanar PCB77, and the non-coplanar PCB99, 138, 153, 180 and 194 (Fig. 1). Initially, we confirmed that only the coplanar PCB77 was able to activate AhR-mediated gene expression. Human liver, intestine and lung cell lines were exposed to each of the PCBs (10 μM) for 48 h and CYP1A1 transcript levels measured by Q-PCR. PCB77 elicited a significant increase in CYP1A1 transcript levels in all three cell lines; Caco2 cells were

Discussion

Previous data has provided indirect evidence for the interaction of non-coplanar PCBs with the nuclear receptors PCR and CAR. Using a reporter gene system, Tabb et al. (2004) were able to demonstrate that highly chlorinated, non-coplanar, PCBs were able to impact on PXR‐mediated transcription. Interestingly, they demonstrated that these highly chlorinated PCBs were able to activate reporter gene expression of target genes via mouse and rat PXR, but inhibited human PXR-mediated transcriptional

Conflict of interest

None.

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Citation Excerpt :
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Non-coplanar polychlorinated biphenyls (PCBs) are direct agonists for the human pregnane-X receptor and constitutive androstane receptor, and activate target gene expression in a tissue-specific manner

Abstract

Highlights

Introduction

Section snippets

Reagents

Results

Discussion

Conflict of interest

Pharmacol. Res.

Cell

Chemosphere

J. Lipid Res.

J. Biol. Chem.

Environ. Int.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Toxicol. Appl. Pharmacol.

Environ. Int.

Biochim. Biophys. Acta

Anal. Biochem.

Environ. Int.

Toxicol. Lett.

Toxicol. Appl. Pharmacol.

Trends Endocrinol. Metab.

Toxicol. Lett.

Sci. Total Environ.

Transcriptional regulation of PXR: identification of a PPRE within the proximal promoter responsible for fibrate-mediated transcriptional activation of PXR

Drug Metab. Dispos.