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. Author manuscript; available in PMC: 2020 Dec 1.
Published in final edited form as: Xenobiotica. 2019 Apr 16;49(12):1414–1422. doi: 10.1080/00498254.2019.1566582

Concentration Dependence of Human and Mouse Aryl Hydrocarbon Receptor Responsiveness to Polychlorinated Biphenyl Exposures: Implications for Aroclor Mixtures

Hongxue Shi 1, Josiah E Hardesty 2, Jian Jan 1, Kimberly Z Head 3, K Cameron Falkner 3, Matthew C Cave 1,2,3, Russell A Prough 2
PMCID: PMC6764862  NIHMSID: NIHMS1030277  PMID: 30991879

Abstract

1. Aryl hydrocarbon receptor (AhR) ligands, including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated biphenyls (PCBs), are endocrine disrupting chemicals associated with nonalcoholic fatty liver disease. This study documents the species-specific differences between mouse (high affinity mAhR) and human AhR (hAhR) activation by PCB congeners and Aroclor mixtures.

2. AhR activation by TCDD or PCBs 77, 81, 114, 114, 126, and 169 was measured using luciferase reporter constructs transfected into either Hepa1c1c7 mouse or HepG2 human liver cell lines. The EC50 values were lower in Hepa1c1c7 cells than HepG2 cells for all compounds tested except PCB 81. The results for TCDD and PCB 126 were validated in primary human and mouse hepatocytes by measuring CYP1A1 gene transcript levels.

3. Because humans are exposed to PCB mixtures, several mixtures (Aroclors 1254; 1260; and 1260 + 0.1% PCB126 each at 10 μg/ml) were then tested. Neither Aroclor 1254 nor Aroclor 1260 increased luciferase activity by the transfected AhR reporter construct. The Aroclor 1260 + 0.1% PCB 126 mixture induced mAhR-mediated transactivation, but not hAhR activation in cell lines.

4. In summary, significant concentration-dependent differences exist between human and mouse AhR activation by PCBs. Relative effect potencies differed, in some cases, from published toxic equivalency factors.

Keywords: PCB, dioxin, TCDD, toxic equivalency factors, relative effect potency

Introduction

Polychlorinated biphenyls (PCBs) and dioxins are persistent organic pollutants (POPs) which have been associated with endocrine and metabolic disruption (Heindel et al., 2017). PCBs have been classified into dioxin-like (DL) and non-dioxin-like (NDL) congener classes. DL PCBS, in particular, may irreversibly bind to hepatic cytochrome P450s and bioaccumulate in liver (Diliberto et al., 1997). Cohort studies demonstrate associations between environmental PCB exposures and suspected nonalcoholic fatty liver disease (NAFLD) (Cave et al., 2010, Serdar et al., 2014, Kumar et al., 2014, Yorita Christensen et al., 2013, Kim et al., 2011, Rantakokko et al., 2015, Clair et al., 2018). NAFLD has historically been considered the hepatic manifestation of obesity and metabolic syndrome. However, endocrine and metabolism disrupting chemicals (EDCs/MDCs) targeting liver have recently been proposed to impact NAFLD pathogenesis (Heindel et al., 2017). Animal models of dioxin or DL PCB exposures confirm a potential role for these chemicals in NAFLD (Wahlang et al., 2014b, Hennig et al., 2005, Wahlang et al., 2017, Angrish et al., 2012, Gadupudi et al., 2016). However, more data are required to understand fully the mode of action of DL PCBs in NAFLD.

DL PCBs have non-ortho (co-planar) or mono-ortho (non-planar) substitution and are ligands for the aryl hydrocarbon receptor (AhR). The AhR is a transcription factor that belongs to the Per-Arnt-Sim family (Murray et al., 2014). Unliganded AhR resides in the cytoplasm of cells as part of a multi-protein complex and upon ligand binding, translocates to the nucleus, forming a heterodimer with AHR nuclear translocator protein (ARNT). The AhR-ARNT heterodimer binds to the dioxin response element (DRE) on the 5’-flanking regions of most dioxin-responsive genes, and regulates expression of target genes, prototypically the Cyp1 family. However, AhR activation also appears to have a mechanistic role in NAFLD (Lee et al., 2010). Many dioxin and PCB toxicology studies have been performed in rats, while mice have been more typically used in studies of obesity-associated diseases, including NAFLD; especially C57BL/6 mice. Obtaining a better understanding of the potential differences in the responses of murine AhR (mAhR) vs. human AhR (hAhR) to DL PCBs is critical to create the most relevant murine models of DL PCB-associated NAFLD using a reverse translational approach. The objective of this manuscript is to address that knowledge gap.

PCBs are polyhalogenated aromatic hydrocarbons and consist of up to 10 chlorine atoms attached to a biphenyl ring. PCBs were manufactured as mixtures (US tradename Aroclor) and sold by the percentage of chlorine in the mix. For example, Aroclor 1254 is 54% chlorine by mass, while Aroclor 1260 is 60% chlorinated. Of the 209 theoretical PCB congeners, approximately 130 were present in commercial PCB mixtures. PCB mixtures were used in a variety of industrial applications, such as insulating agents for electrical transformers. Approximately 1.3 million tons were produced worldwide before PCBs were banned (Ockenden et al., 2003). The primary routes of human PCB exposure are ingestion of contaminated food (Schecter et al., 2003) or breast milk, and inhalation of contaminated air (Ampleman et al., 2015). PCB metabolism varies considerably with low molecular weight PCBs being metabolized at a higher rate than the more heavily chlorinated PCBs. This leads to bioaccumulation patterns in adipose that greatly favor retention of the more highly chlorinated congeners being similar to those present in Aroclor 1260. However, there is an important exception. Aroclor 1260 is not believed to contain significant amounts of DL PCBs (Wahlang et al., 2014a, Wahlang et al., 2014b, BattelleMemorialInstitute, 2012).

To estimate the human risk assessment of AhR activation by mixtures of xenobiotic compounds, the World Health Organization (WHO) has established toxic equivalency factors (TEFs) (Van den Berg et al., 1998) which are used to determine the total dioxin toxic equivalency (TEQ). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is considered to be the prototypical ligand for AhR. Accordingly, the potent TCDD has been assigned a TEF of 1. The TEFs of other chemicals are calculated based on relative effect potency (REP) values compared to TCDD. PCB 126, a non-ortho substituted PCB, is the single greatest contributor to the overall PCB toxic equivalency. PCB 126 has been assigned a TEF of 0.1. Other DL PCBs are believed to be much less potent. For example, the TEFs of all mono-ortho substituted PCBs are 0.00003 (Van den Berg et al., 2006).

However, species- and strain-specific differences in xenobiotic-induced AhR activation are known to exist. Thus, if differences exist between the affinity for TCDD or the PCBs relative to TCDD, then there will be differences in the exposures required to activate the AhR across species and strain. Studies have compared the potency of TCDD and DL PCB congeners in inducing CYP mRNA levels in human and rat immortalized cell lines and primary hepatocytes (Zeiger et al., 2001, Silkworth et al., 2005, Schrenk et al., 1995, Peters et al., 2004). These data support the conclusion that the sensitivity of human AhR is lower than that of rat in response to TCDD or some DL PCB congener exposures. The difference between the primary amino acid sequences in the AhR ligand-binding pocket (Ramadoss and Perdew, 2004), and the differential ability to recruit coactivators by AhR (Flaveny et al., 2008) were thought to be responsible for the different AhR sensitivity observed across these species. The difference between the affinities of the human and the high affinity C57B/6 strain AhRb1 receptor (Thomas et al., 2002) is due to a single amino acid residue (valine 381 in humans and alanine 375 in the C57B/6 mouse) (Ramadoss and Perdew, 2004), resulting in decreased affinity of the human receptor for 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin. Mouse C57BL/6 strain is a species predominantly used in PCB-related fatty liver studies (Wahlang et al., 2014b, Gadupudi et al., 2016, Hennig et al., 2005, Wahlang et al., 2013, Wahlang et al., 2017, Wahlang et al., 2016, Larsson et al., 2015, Peters et al., 2006, Strapacova et al., 2018, van Ede et al., 2016). Given these factors, we hypothesize that selected DL PCB congeners, and Aroclor mixtures with DL PCBs will more potently activate the murine AhR compared to the human AhR. Understanding these species differences is important for translational studies investigating PCBs in NAFLD and other metabolic disorders in humans.

Material and Methods

Reagents

TCDD, PCBs 77, 81, 114, 126, 169; and Aroclor 1254 (lot 124–191) and Arochlor 1260 (lot 021–020–1A) were purchased from AccuStandard (New Haven, CN). The individual chlorinated compounds were found to be 99.5% pure (AccuStandard, New Haven, CN). Lipofectamine; Opti-MEM and Wamouth’s medium; fetal bovine serum (FBS); Hank’s Balanced Salt Solution (HBSS, 10×); probes for human CYP1a1 (Hs01054797_g1) and GAPDH (Hs02786624_g1) as well as mouse Cyp1a1 (Mm00487218_m1) and GAPDH (Mm99999915_g1) for Taqman Gene Expression Assays were ordered from Thermo Fisher Scientific (Waltham, MA). Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from VWR (Radnor, PA). Antibiotics; 0.25% trypsin with EDTA; Type 1 collagen; and ITSTM + Premix Universal Culture Supplement were obtained from Corning Inc. (Corning, NY). Cell culture Lysis 5× buffer and the Luciferase Assay System were obtained from Promega (Madison, WI). Chlorophenol red-β-D-galactopyranoside and Collagenase D were from obtained from Sigma-Aldrich, (St. Louis, MO). RNA STAT-60 was purchased from Amsbio (Austin, TX). The QuantiTect® Reverse Transcription Kit was obtained from QIAGEN (Qiagen, Valencia, CA). iTag Universal Probes Supermix was purchased form Bio-Rad (Hercules, CA). 1,2-benz[a]anthracene (BA); dimethyl sulfoxide (DMSO); ethanol; 2-propanol; chloroform; and all other’s reagents were obtained from Sigma Aldrich (St. Louis, MO).

Liver Cell Line Culture, Transfection, and Treatment

Human HepG2 and mouse Hepa1c1c7 cells were purchased from the American Type Culture Collection (Manassas, MD), and were cultured in DMEM completed medium supplement with 10% FBS and 1% antibiotics, and incubated in 5% CO2 and 95% humidity at 37°C. HepG2 cells and Hepa1c1c7 cells were sub-cultured every 3–4 days. The HepG2 and Hepa1c1c7 cells were plated into 24-well plates, and transfected at 40%–50% confluence. DMEM medium was replaced by Opti-MEM medium 30 min prior to transfection. Cells were transfected with 150 ng of a pGl3-promoter-based reporter plasmid pXRE-SV40-Luc that has been described previously (Wahlang et al., 2014a) and 150 ng of pCMV-β (Stratagene, CA) using lipofectamine (Thermo-Fisher) according to the manufacturer’s instructions. Following an overnight recovery period, cells were treated for 24 hours with either: TCDD (at concentrations ranging from 0.1 pM to 1 μM); PCB congeners (at concentrations ranging from 1 nM to 10 μM); Aroclors 1254, 1260, or 1260+0.1% PCB 126 (10 μM each); or 1,2-benz[a]anthracene (BA, 10 μM). PCB 126 was spiked into the Aroclor 1260 in order to create a PCB mixture that is more representative of human bioaccumulation (Wahlang et al., 2014a). A dose of 0.1% of PCB 126 was chosen based on the serum PCB levels measured in the National Health and Nutrition Examination Survey (NHANES) 2003–2004. DMSO was used as a solvent control, and the final concentration was less than 0.2%. Cells were harvested using1× cell culture lysis buffer. Chlorophenol red β-D-galactopyranoside was used to determine β-galactosidase activity to normalize transfection efficiency. Following incubation, β-galactosidase enzyme activity was determined spectrophotometrically at 595 nm using the Bio-Tek Synergy HT multi-mode micro plate reader (Bio-Tek USA, Winooski, VT). Luciferase reporter assay was performed with a Promega Luciferase Assay System on an Orion L micro plate luminometer (Berthold Detection Systems, Pforzheim, Germany).

Primary Mouse and Human Hepatocyte Culture and Treatment

Livers from 10-week-old male C57BL/6 mice were used to prepare hepatocytes by digestion with collagenase D. Hepatocytes were resuspended in cold Waymouth’s medium, and cultured for 12 hours in type 1 collagen pre-coated 12-well plates (Aparicio-Vergara et al., 2017). Primary human hepatocytes were obtained from BioreclamationIVT (Baltimore, MD). Primary human hepatocytes were plated according to the supplier’s instructions in 12-well plates and cultured overnight. TCDD (0.01 nM – 1 μM) and the single PCBs (1 nM −10 μM) were administered to the primary hepatocytes at various concentrations for 24 hours. Cells were lysed and mRNA was isolated using STAT-60. RNA purity and quantity were assessed with a Nanodrop spectrometer (ND-1000, Thermo Scientific, Wilmington, DE) using ND-1000 V3.8.1 software. cDNA was synthesized using a Qiagen QuantiTect Reverse Transcription Kits. PCR reaction was performed using Promega iTag Universal Probes Supermix, with Cyp1a1/CYP1a1 [mouse Cyp1a1 (Mm00487218_m1) and human CYP1A1 (Hs01054797_g1)] and GAPDH [murine (Mm99999915_g1) and human (Hs02786624_g1)] GAPDH Taqman primers probe sets with a Bio-Rad CFX384TM Real-Time System (Hercules, CA). CYP1a1/1A1 expression level was determined using 2−ΔΔCt methods.

Statistical Analysis

Concentration-response analyses were performed on SigmaPlot 11.0 software (Systat Software Inc., San Jose, CA) using a Four Parameter Logistic Curve. EC50 values are presented as the mean ± standard deviation. Other statistical analyses performed using Graphpad Prism software (GraphPad Software Inc., La Jolla, CA). Data in the figures are presented as mean ± SEM. Statistical evaluation of the data were performed using one-way analysis of variance (ANOVA) followed by the Dunnett’s post hoc test to compare all groups with the control sample for multiple groups. For all statistical comparisons, p-values ≤ 0.05 was considered statistically significant. For the PCB congeners, relative effect potencies were determined by dividing the EC50 for that congener by that of TCDD.

Results

Concentration-response curves for CYP1 gene family expression and the luciferase reporter activated by TCDD

Primary human and mouse hepatocytes were prepared and after plating, they were treated with varying concentrations of TCDD (0.1 pM to 1 μM). mRNA levels were measured by quantitative PCR as described in Methods. TCDD had an EC50 value for induction of the AhR-dependent target gene of 0.45 ± 0.34 nM in primary human hepatocytes, and 0.04 ± 0.02 nM in primary mouse hepatocytes (Fig. 1 A&B), documenting that the murine receptor displays a higher affinity for TCDD than does the human receptor in primary cells. Subsequently, HepG2 and Hepa1c1c7 cells were transfected with the AhR-dependent (pXRE-SV40-Luc) reporter plasmid, then treated with TCDD to generate a concentration-response plot. TCDD potently induced the AhR luciferase reporter activity in both the human and murine immortalized cell lines (Fig. 1 C&D). Based on the concentration-response data, TCDD had an EC50 value of 0.4 ± 0.1 nM in HepG2 cells, whereas the EC50 value in Hepa1c1c7 cells was 0.03 ± 0.01 nM. These results confirm that TCDD is a more potent activator of mAhR than hAhR (approximately 13.3-fold). TCDD-induced luciferase activity was greater in HepG2 cells (maximum response 83-fold) compared to Hepa1c1c7 cells (maximum response 2.2-fold). A higher basal level of expression was observed in Hepa1c1c7 cells. These results suggest that primary liver cells and immortalized hepatoma cells display similar concentration responses to AhR ligands and can be used to obtain ligand affinity data for AhR, as reported by others (Harris et al., 1993, Strapacova et al., 2018, Peters et al., 2006, Jones and Anderson, 1999).

Figure 1. Concentration-response curves for the luciferase reporter activation and CYP1A1/Cyp1a1 gene family expression activated by TCDD.

Figure 1.

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AhR target gene CYP1A1/Cyp1a1 induction was measured at 24 h in primary human (A) and mouse (B) hepatocytes after the treatment with various concentration of TCDD (0.1 pM to 1 μM). AhR-dependent luciferase activity was measured at 24 h in HepG 2 cells (C) and Hepa1c1c7 cells (D) treated with the indicated concentration of TCDD (0.1 pM to 1 μM). Data are presented as mean ± SEM.

Concentration-response curves forCyp1a1/CYP1A1 gene family expression and the luciferase reporter activated by non-ortho PCB 126

Many of the PCB congeners in Aroclor 1254 and 1260 are co-planar PCBs and expected to be ligands for AhR. Therefore, we focused on these congeners to establish which were the best ligand activators for human and murine AhR. Using primary human and mouse hepatocytes, real-time PCR was used to measure CYP1A1 and Cyp1a1 mRNA levels following PCB 126 treatment. PCB 126 induced CYP1A1 mRNA levels with an EC50 value of 195 ± 35 nM in primary human hepatocytes, while the EC50 value for Cyp1a1 in primary mouse hepatocytes was 12.3 ± 7.9 nM (Fig. 2 A&B). Thus, PCB 126 induced transcription of the AhR target gene approximately 16-fold more potently in the primary mouse vs. human hepatocytes. However, the maximal increase in gene expression did not differ significantly by species in the primary hepatocytes (human CYP1A1 45.1-fold induction vs. murine Cyp1a1 53.3-fold, respectively).

Figure 2. Concentration-response curves for the luciferase reporter and CYP1A1/Cyp1a1 gene family expression activated by non-ortho (co-planar) PCB 126.

Figure 2.

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AhR-dependent luciferase activity was measured at 24 h in HepG 2 cells (A) and Hepa1c1c7 cells (B) treated with the indicated concentration of PCB 126 (1 nM to 10 μM). AhR target gene CYP1a1 induction was measured at 24 h in primary human (C) and mouse (D) hepatocytes after the treatment with various concentration of PCB 126 (1 nM to 10 μM). Data are presented as mean ± SEM.

Because PCB 126 is thought to be the congener with the greatest contribution to the overall TEQ provided by PCB mixtures, the concentration dependence in both human HepG2 cells and mouse Hep1c1c7 cells was examined next. PCB 126 had an EC50 value of 250 ± 150 nM in HepG2 cells, while EC50 value in Hepa1c1c7 cells was 4.7 ± 3.2 nM (Fig. 2 C&D). Thus, PCB 126 activated the reporter construct approximately 53-fold more potently in the murine compared to the human cell line. PCB 126’s relative effect potency was similar in HepG2 (0.002) vs. Hepa1c1c7 (0.006) cells. PCB 126 induced luciferase activity to a significantly greater level in HepG2 cells (maximum response 31.6-fold) compared to Hepa1c1c7 cells (maximum response 5.9-fold). Except for level of induction, these luciferase reporter results were similar to the AhR-dependent induction of CYP1A1 message in primary hepatocytes, confirming the concordant results in receptor activation of both systems.

Concentration-response curves for the luciferase reporter activated by co-planar PCBs

We sought to characterize the ability of other co-planar PCBs in Arochlor 1260 to activate the human and murine AhR. The U.S. Environmental Protection Agency has defined 12 PCB congeners as having TEF values large enough to be significant contributors to AhR activation (BattelleMemorialInstitute, 2012). Like TCDD and PCB 126, PCB 77 more potently increased luciferase activity in Hepa1c1c7 cells compared to HepG2 cells (Fig. 3 A&B). The EC50 value for PCB 77 was 108 ± 39 nM in Hepa1c1c7 cells. It is unclear if a maximal response was obtained in HepG2 at the highest PCB 77 concentration tested (10 μM). However, the estimated EC50 was ≥ 4000 nM. Thus, the REP for PCB 77 was 0.0003 in the murine system and ≤ 0.00009 in the human system.

Figure 3. Concentration-response curves for the luciferase reporter activated by non-ortho (co-planar PCBs): PCB 77, PCB 81, PCB 114, and PCB 169.

Figure 3.

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AhR-dependent luciferase activity was measured at 24 h in HepG 2 cells (A) and Hepa1c1c7 cells (B) treated with the indicated concentration of the indicated PCBs (at concentrations ranging from 1 nM to 10 μM). Data are presented as mean ± SEM.

Unlike the other compounds tested, PCB 81 had an approximately 5-fold higher EC50 value in Hepa1c1c7 cells (32.0 ± 17.7 nM), compared to the EC50 value in HepG2 cells (6.8 ± 2.7 nM) (Fig. 3 A&B). Thus, this congener may be more potently in activating hAhR than mAhR. The REPs for this congener also differed by species: human=0.06 and mouse=0.0009. PCB 81-induced maximal luciferase activity was higher in HepG2 cells (34.6-fold induction) compared to Hepa1c1c7 cells (2.3-fold induction).

PCB 114 induced luciferase activity more potently in the mouse vs. human hepatoma cell line (Fig 3 A&B). Based on the concentration-response data, the EC50 value for PCB 114 was 796 ± 661 nM in Hepa1c1c7 cells. Like PCB 77, PCB 114 may or may not have induced a maximal response in HepG2 cells at the highest concentration tested (10 μM). However, the estimated EC50 was ≥ 5000 nM. PCB 114’s REP was 0.00004 in the murine system and ≤ 0.00007 in the human system.

PCB 169 was the least potent congener tested. PCB 169 increased luciferase activity in both model systems (Fig 3 A&B). The EC50 for PCB 169 was 25.7 ± 4.6 nM in Hepa1c1c7 cells. Like PCBs 77 and 114, PCB 169 was much less potent in HepG2 cells and may or may not have achieved maximal luciferase activity at the highest concentration tested. The estimated EC50 was ≥ 7000 nM. PCB 169’s REP was 0.001 in Hepa1c1c7 cells and ≤ 0.00005 in human cells. For TCDD and PCBs 77, 81, 114, 126, 169, the EC50 values determined by the luciferase reporter assays are summarized in Table 1, while the relative effect potencies determined by these values are provided in Table 2. The corresponding WHO PCB TEFs (Van den Berg et al., 2006) are also provided in Table 2 for comparison.

Table 1.

Relative Potency Values (REPs)

Chemical Typea HepG2 Hepa1c1c7 WHO-TEFc Hepa1c1c7/HepG2
TCDD 1 1 1 1
PCB126 n 0.002 0.006 0.1 3
PCB77 n ≤ 0.00009 0.0003 0.0001 ≥ 3.3
PCB81 n 0.06 0.0009 0.0003 0.015
PCB114 m ≤ 0.00007 0.00004 0.00003 ≥ 0.5
PCB169 n ≤ 0.00005 0.001 0.03 ≥ 20
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a

n means non-ortho (co-planar); m means mono-ortho.

Effects of concentration of individual PCBs or PCB mixtures in the AhR-mediated transcription assay system

The purpose of these experiments was to determine if exposures to PCB mixtures at environmentally relevant levels that resemble human bioaccumulation patterns differentially activated human vs. murine AhR. Although Aroclor 1260 contains high molecular weight PCBs that bio-accumulate in human adipose tissue, the use of Aroclor 1260 mixtures for research in animal models has been criticized because it is relatively low in the lower molecular weight DL PCB congers relative to the levels of PCBs in human adipose. Thus, for future translational studies, it is important to determine the impact of PCB mixtures potentially containing more dioxin-like PCBs on AhR activation. Therefore, Aroclor 1254 (10 μg/ml final), Aroclor 1260 (10 μg/ml final), or a mixture of Aroclor 1260 (10 μg/mL final) plus 0.1 % PCB 126 (31 nM final) were added to the primary hepatocytes or immortalized cell lines to compare their effects on AhR activation. The concentration of Aroclor 1260 used has previously been shown not to cause cellular toxicity (Wahlang et al., 2014a). DMSO vehicle solvent was used as the negative control. Positive controls included PCB 126 (10 μM) and 1,2-benz[a]anthracene (BA, 10 μM). As anticipated, vehicle control did not induce CYP1A1 mRNA levels in the primary hepatocytes (Fig. 4A–B) or increase luciferase activity in the immortalized liver cell line reporter constructs (Fig. 4C–D); but the positive controls did (Fig. 4A–D). In HepG2 cells, luciferase activity was increased 67-fold by BA and 42-fold by PCB 126 (10 μM) (Fig. 4C). In Hepa1c1c7 cells, luciferase activity was increased 5-fold by BA and 3.7-fold by PCB 126 (10 μM) (Fig. 4D). Likewise, the fold induction of CYP1a1 mRNA was higher in the primary human hepatocytes than Cyp1a1 was in the primary mouse hepatocytes (Fig. 4A–B). Neither Aroclor 1254 nor Aroclor 1260 (10 μg/mL) activated the AhR in the human or rodent systems (Fig. 4A–D). However, the Aroclor 1260 plus PCB126 mixture induced Cyp1a1 mRNA in primary mouse hepatocytes (Fig. 4B), and increased luciferase activity in the Hepa1c1c1c7 cell line (3.0-fold) (Fig. 4D). No effect was seen in either of the two human systems. In summary, neither Aroclor 1254 nor Aroclor 1260 (10 μg/ml) activated the mAhR or the hAhR. The concentrations of the DL PCBs in Aroclor 1254 and 1260 were apparently too low to activate the mAhR or hAhR receptors at this dose. In contrast, Aroclor 1260 plus 0.1 % PCB 126 (10 μg/ml) activated mAhR but not the hAhR. The level of mAhR activation was slightly lower than PCB 126 (31 nM) treatment alone, indicating that the other PCB congeners in Aroclor 1260 may effect mAhR activation by PCB 126 (Bannister et al., 1987).

Figure 4. Effects of PCB mixtures on CYP1A1/Cyp1a1 expression and luciferase reporter activity.

Figure 4.

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AhR-dependent induction of CYP1A1/Cyp1a1 in either human primary human (A) and mouse (B) hepatocytes and AhR-dependent luciferase activity in HepG2 cells (C) or mouse primary hepatocytes (D) were measured after treatment for 24 h with either Aroclor 1254 (10 μg/ml), Aroclor 1260 (10 μg/ml), PCB 126 (31 nM), or a mixture of Aroclor 1260 (10 μg/ml) plus 0.1 % PCB 126 (10 ng/ml ≈ 31 nM), and as a positive control 1,2-benz[a]anthracene (BA, 10 μM). Data are presented as mean ± SEM. **p<0.01, and ***p<0.001 VS vehicle.

Discussion

One objective of this study was to test the hypothesis that selected dioxin-like PCB congeners and Aroclor mixtures more potently activate mAhR compared to hAhR. The positive control, TCDD, had 13-fold higher EC50 values in HepG2 than Hepa1c1c7. In addition, the EC50 values were similar to prior studies (Peters et al., 2006, Larsson et al., 2015, Brennan et al., 2015). The EC50-based potencies for the PCB congeners in HepG2 cells in rank order were: PCB 81 > PCB 126 > PCB 77 > PCB 114> PCB 169 (Table 1). These results were validated for PCB 126 using primary human hepatocytes (Figure 1A). However, the maximal fold induction of CYP1A1 and Cyp1a1 mRNA levels were similar in the primary human and mouse hepatocytes. Thus, the differences in maximal inducibility of the luciferase reporter are most likely a cell line-dependent effect, possibly a result of rapid decrease in AhR protein levels after translocation in Hepa1c1c7 cells (Suzuki and Nohara, 2007).

Notably, the maximal fold induction of luciferase activity was always higher in HepG2 and Hepa1c1c7 for TCDD and all PCB congeners tested. The EC50-based REPs in HepG2 cells were: PCB 81 = 0.06; PCB 126 = 0.002; PCB 77 ≤ 0.00009; PCB 114 ≤ 0.00007; PCB 169 ≤ 0.00005. These human PCB potencies and REPs were consistent with a previously published study (Zeiger et al., 2001).

The PCB congener EC50 values were lower (range 6.3 to 194-fold, Table 1) in Hepa1c1c7 cells than HepG2 cells except for PCB 81. This suggests that the congeners were, in general, more potent in mice than humans. The rank order of the PCB EC50-based potencies in Hepa1c1c7 cells was PCB 126 > PCB 169 > PCB 81 > PCB 77 > PCB 114 (Table 1). The REPs in the murine cell line were: PCB 126 = 0.006; PCB 169 = 0.001; PCB 81 = 0.0009; PCB 77 = 0.0003; PCB 114 = 0.00004 (Table 2). These data demonstrate that, with the exception of PCB 81, PCBs have 3- to12- fold higher (Table 2) REPs in mouse than human. However, previously published REPs were generally higher in the rat, indicating that PCBs have even higher relative effect potencies in rat than mouse (Zeiger et al., 2001). These species differences should be taken into consideration in translational studies of PCB or dioxin-induced endocrine disruption and fatty liver disease.

The WHO 2005 TEF values (provided in Table 2) are based on a combination of rodent and human REPs. The species most commonly used in PCB studies is the rat, so the WHO 2005 TEF values likely reflect this (Harris et al., 1993). The toxicity of a mixture of dioxins or dioxin-like chemicals can be defined by the TEQ. The TEQ can be calculated by the sum of the concentrations of individual chemicals (Ci) multiplied by their TEFs. The WHO 2005 TEF for PCB126 is 0.1, which is 50-fold higher than the human REP determined in the present study. While the 2005 WHO TEFs for PCBs 77 and 114 were similar to the human REPs calculated by this study, they deviated for PCB 81 (200-fold lower) and PCB 169 (>300 fold higher). Some Authors have proposed revising the 2005 WHO TEFs by more heavily weighting human-based REPs to improve human risk assessment (van Ede et al., 2016). The data provided by the present study may be used to support that effort.

Humans are exposed to PCB mixtures rather than individual congeners. These mixtures include both DL and NDL PCBs. Previously, we have shown that an environmentally dose of Aroclor 1260 (20 mg/kg) was a ‘second hit’ leading to the progression of fatty liver disease in animal models (Wahlang et al., 2017, Wahlang et al., 2016, Wahlang et al., 2014b). However, dioxin-like PCBs are not major constituents of Aroclor 1260. Aroclor 1260 at a dose of 200, but not 20 mg/kg was associated with increased expression of the AhR reporter gene, Cyp1a2 (Wahlang et al., 2014b). In recent in vivo studies, we spiked supplemental PCB 126 (0.1%) into the Aroclor 1260 which was added at 10 μg/ml for Arochlor 1260 and 32 nM for PCB126 (Shi et al., 2018). Altered hepatokines and Pnpla3 expression contributing to altered fat metabolism and NAFLD was noted.

The present manuscript investigated potential species differences in the ability of this mixture, as well as Aroclor 1254 and Aroclor 1260, to activate the AhR. At the dose given, Aroclor 1260 did not activate mAhR (Figure 4B), consistent with a previous animal study (Wahlang et al., 2014b). The other PCBs in Aroclor 1260 did not appear to interfere with the ability of the ‘spiked-in’ PCB 126 to activate mAhR. Aroclor 1254 also did not activate either hAhR or mAhR at the dose administered. However, the amounts of dioxin-like PCBs in Aroclor 1254 varies considerably by lot (Burgin et al., 2001). We used lot 124–191 rather than lots like lot 6024, which contains approximately 10-fold higher DL PCBs. Thus, induction may have been observed with Aroclor 1254, had we used the other lot. We propose that Aroclor 1260 + 0.1% PCB 126 administered at a 20 mg/kg dosage could be an appropriate PCB exposure for use in vivo mouse studies of endocrine and metabolic disruption (Shi et al., 2018). However, it is important to remember that species differences exist. Perhaps the use of ‘humanized mice’ expressing the hAhR would be suitable to further dissect the impact of complex PCB mixtures in human health and disease.

This study has several limitations which could impact future translational studies of PCBs and dioxins in obesity, fatty liver, and metabolic syndrome. In mice, different AhR ligands may activate different subsets of genes leading to the concept of selective modulators of the AhR (Nault et al., 2013). It is unclear whether the human AhR behaves in a similar manner. The possibility that dioxin-like PCBs modulate other AhR-dependent metabolic processes differently than they modulate CYP1A1 is not excluded by this study. As the human AhR has higher affinity for either dietary indole metabolites or tryptophan metabolites than the mouse AhR (Flaveny et al., 2009), in the future it may be important to determine whether PCBs act more like the dietary and endogenous ligands or more like dioxin. The toxicity of DL PCBs may also be mediated, in part, by interactions with other receptors or cell signaling process. For example, PCBs potently inhibited signaling of the EGFR (Hardesty et al., 2018, Hardesty et al., 2017). Moreover, PCB 126 had the highest potency for EGFR inhibition (up to IC50 = log −16.07 ± 0.31 M) of all congeners tested (Hardesty et al., 2018). It is important to consider the impact of DL PCBs acting through other receptors apart from the AhR in human health and disease.

In summary, these results demonstrate that for most of the PCB congeners tested, both the potencies and REPs were higher for mouse than human AhR. PCB 81 was an exception. In some cases, the human REPs deviated significantly from the 2005 World Health Organization toxic equivalency factors. Neither Aroclor 1254 nor Aroclor 1260 activated the human or mouse AhR at the concentration tested. However, Aroclor 1260 spiked with a low concentration of supplemental PCB 126 was able to activate mAhR, but not hAhr. Therefore, it is important to consider potential species differences in AhR signaling when modeling PCB-related liver and metabolic diseases in mice and to ascertain how such models can be translated to PCB-induced toxicity in humans.

Supplementary Material

S1

Acknowledgments:

The authors would like to acknowledge the University of Louisville Alcohol Research Center (ULARC), the Hepatobiology and Toxicology COBRE for use of core facilities, and Dr. David W. Hein and his lab members for providing primary human hepatocytes.

Funding information: This work was supported in part by the National Institute of Environmental Health Sciences [1R01ES021375, F31ES028982, R35ES028373, P42ES023716], the National Institute of General Medical Sciences [P20GM113226], and the National Institute on Alcohol Abuse and Alcoholism [P50AA024337].

Abbreviations:

AhR

aryl hydrocarbon receptor

ARNT

AhR nuclear translocator

BA

1,2-benz[a]anthracene

CYPs

cytochromes P450

DL PCB

dioxin-like PCB

DMSO

dimethyl sulfoxide

DRE

dioxin response element

EC50

half maximal effective concentration

EDC

endocrine disrupting chemical

hAhR

human aryl hydrocarbon receptor

mAhR

murine aryl hydrocarbon receptor

MDC

metabolism disrupting chemical

NAFLD

nonalcoholic fatty liver disease

NDL PCB

non-dioxin-like PCB

NHANES

National Health and Nutrition Examination Survey

PCB

polychlorinated biphenyls

POPs

persistent organic pollutants

REP

relative effect potency

TCDD

2,3,7,8-tetrachlorodibenzo-p-dioxin

TEF

toxic equivalency factor

TEQ

toxic equivalency

WHO

World Health Organization

Footnotes

Conflicts of Interest: The authors declare no actual or potential conflicts of interest regarding this work.

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