DDE and PCB 153 independently induce aryl hydrocarbon receptor (AhR) expression in peripheral blood mononuclear cells

Abstract

Recent studies have demonstrated that compounds inducing pro-inflammatory cytokines enhance AhR expression. The aim of this study was 2-fold: (1) to determine if two pro-inflammatory compounds, dichlorodiphenyldichloroethylene (DDE) and 2,2′,4,4′,5,5′-hexa-chlorobiphenyl (PCB 153), independently affect AhR gene expression in peripheral blood mononuclear cells (PBMC); and (2) if affected, to determine whether the mechanism involved was due to AhR activation or to a pro-inflammatory effect of the chemicals. PBMC isolated from healthy individuals were incubated in the presence of DDE (10 µg/ml) and PCB 153 (20 ng/ml) over time and AhR and CYP1A1 expression was assessed with a real-time PCR technique. The results indicated there was over-expression of the AhR mRNA in PBMC when the cells were treated with DDE and PCB 153. No changes in expression levels of CYP1A1 mRNA were found. Importantly, when the cells were exposed to DDE and PCB 153 in the presence of an antagonist of tumor necrosis factor (TNF)-α, the over-expression of AhR was abolished; as expected, the expression of CYP1A1 was unaffected. In conclusion, these studies demonstrated for the first time an increment of AhR expression “in vitro” in PBMC treated with two pro-inflammatory environmental pollutants, DDE and PCB153. Moreover, the over-expression of AhR was dependent of TNFα induced by DDE and PCB 153 and was independent of AhR activation.

• Aryl hydrocarbon receptor
• AhR
• CYP1A1
• DDE
• PCB 153
• TNFα

Introduction

The aryl hydrocarbon receptor (AhR) belongs to the basic helix-loop-helix (bHLH)/PAS (Per/Arnt/Sim) family of transcription factors (Hahn, 2002; Tian, 2009). In its basal or resting state, AhR remains predominantly cytoplasmic as part of a protein complex with the molecular chaperone heat shock protein 90 (HSP90), p23, and aryl hydrocarbon receptor interacting protein (XAP2; Perdew, 1988). Various cellular stresses activate AhR and lead to a conformational change that results in the exposure of the nuclear localization sequence (NLS) and AhR nuclear translocation. AhR then dissociates from the protein complex and binds to the AhR nuclear translocator (ARNT; McGuire et al., 1994). The AhR/ARNT heterodimer binds to promoter regions of target genes that contain the AhR DNA binding consensus sequence. Thus, the AhR is classified as a ligand-activated transcription factor that mediates expression of a suite of pleiotropic responses, including biotransformation enzymes (Nebert et al., 2004; Nebert & Dalton, 2006). AhR is also a key mediator of toxic effects induced by a great number of organic pollutants (Gu et al., 2000; van den Berg et al., 1998). Activation of the AhR has been shown to cause a range of adverse effects in vertebrates, including hepatotoxicity, immune suppression, reproductive and endocrine impairment, teratogenicity, and carcinogenicity, among many (Kawajiri & Fujii-Kuriyama, 2007).

The AhR is mainly expressed in liver cells, but it is also present in blood cells (Prigent et al., 2014; Schote et al., 2007; Siest et al., 2008). Also, an induction of the cytochromes P450 1A1 (CYP1A1) and 1B1 (CYP1B1) mRNA expression as AhR activation markers has been observed in blood cells (Prigent et al., 2014). Moreover, increasing experimental evidence supports an important role for AhR in the regulation of immune responses. Early studies on AhR deficient mice reported altered lymphocyte numbers in the spleen, increased incidence of infection (Fernandez-Salguero et al., 1995), and resistance to dioxin induced immunosuppression (Vorderstrasse et al., 2001), suggesting that blood cell-mediated immune responses are among the most sensitive targets of dioxin toxicity. Other studies have suggested that AhR ligation in blood cells does not lead to immunosuppression but, instead, enhances the secretion of various mediators implicated in immune pathology (Zhu et al., 2014).

In actuality, there is substantial evidence for mutual cross-talk between inflammatory mediators and AhR (Drozdzik et al., 2014; Ke et al., 2001; Kobayashi et al., 2008; Tian 2009; Umannová et al., 2007; Vogel et al., 2014). Recent studies have been demonstrated that compounds inducing pro-inflammatory cytokines also enhance AhR expression (Champion et al., 2013; Drozdzik et al., 2014; Kobayashi et al., 2008; Umannová et al., 2007; Vogel et al., 2014; Vondrácek et al., 2011). Thus, the aim of this study was 2-fold: (1) to determine if dichlorodiphenyldichloroethylene (DDE) and 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB 153; two pro-inflammatory compounds (Alegría-Torres et al., 2009; Cardenas-Gonzalez et al., 2013; Kwon et al., 2002)) independently affect AhR gene expression in peripheral blood mono-nuclear cells (PBMC); and (2) if affected, to determine whether the mechanism involved was due to AhR activation or to a pro-inflammatory effect induced by the chemicals.

Materials and methods

Cell culture

Heparinized blood from six healthy volunteers (aged 20–30 years) was used to isolate PBMC by Ficoll-Hypaque density-gradient centrifugation (Sigma, St Louis, MO). After washing with cold phosphate-buffered saline (PBS) solution, the pooled PBMC were grown at 10⁶cell/ml in RPMI 1640 (Sigma) supplemented with 10% fetal bovine serum, L-glutamine 2 mM, 50 U penicillin/ml and 50 mg streptomycin/ml (Gibco Invitrogen Corp., Carlsbad, CA). Cells were incubated in 5% CO₂ at 37 °C for at least 24 h prior to dosing. The Ethics Committee of the School of Medicine, Universidad Autonoma de San Luis Potosi approved this study.

Tumor necrosis factor (TNF)-α, DDE and PCB 153 exposure

As TNFα is able to induce AhR expression (Drozdzik et al., 2014; Kobayashi et al., 2008), we treated PBMC with 10 ng of TNFα/ml (Sigma) as a positive control. After the incubation, fresh stock solutions of 100 mg/ml of DDE and PCB 153 (each Sigma) were made in isopropanol and stored at −40 °C. The cells were then treated (in triplicate) with aliquots of the stock solution to yield final concentrations of 10 µg DDE/ml or 20 ng PCB 153/ml for 2, 5, and 8 h. To avoid the effects of the vehicle (non-exposed cells, NE), the level of isopropanol in the medium did not exceed 0.5%. To demonstrate that any observed effect on AhR mRNA expression when PBMC were treated with DDE and PCB 153 was regulated by TNFα (induced by both chemical compounds), other sets of PBMC were treated with DDE (10 μg/ml, 2 h) and PCB 153 (20 ng/ml, 2 h) in the presence of anti-TNFα monoclonal antibody (100 ng/ml; MAb1; eBioscience, San Diego, CA) as a TNFα antagonist. Cell viabilities post-treatment were routinely assessed using a trypan blue exclusion assay (values were always >95%).

Total RNA isolation

After incubation and treatment, the cells were collected by centrifugation and washed with cold phosphate-buffered saline (PBS, pH 7.4), and then total RNA was isolated using TRIZOL (Invitrogen) reagent following manufacturer instructions. The final RNA concentration was determined by spectrophotometric analysis at 260 nm in a NanoDrop ND1000 system (Thermo Fisher Scientific, Waltham, MA). RNA quality was assessed by electrophoresis over a 1.2% agarose gel. All samples were stored at −80 °C until use.

cDNA synthesis and real-time quantitative RT-PCR

The oligonucleotide sequences of primers used to detect marker mRNA expression are shown in Table 1. Primers were obtained from Gibco Invitrogen. cDNA was synthesized with 1 μg total RNA using reverse transcriptase superscript II (Invitrogen) under the following conditions: 25 °C for 10 min, 35 °C for 90 min, 94 °C for 5 min, and 4 °C for 5 min. Then 1.0 µl of cDNA (100 ng/µl) was mixed with 1.0 µl of sense and antisense primers (20 pM), 1 X PCR buffer, 200 µM dNTPs, and 1.0 U DNA Taq polymerase. Real-time PCR was performed using LightCycler FastStart DNA MasterPlus SYBR Green I Kit and a LightCycler v1.5 system (Roche Applied Science, Mannheim, Baden-Württemberg, Germany). cDNA was amplified under the following conditions: initial cDNA concentration of 10 ng/µl in a reaction volume of 10 µl; denaturation step for 10 s at 95 °C, annealing step for 10 s at 60 °C, and extension step for 20 s at 72 °C for 40 cycles. All PCR reactions were performed in triplicate, with each run containing at least one negative and one positive control, and three independent experiments were carried out to verify reproducibility of the results. The transcript concentration of each gene (AhR or CYP1A1) relative to 18s rRNA expression was calculated using the comparative threshold cycle (Ct) method.

Table 1. Nucleotide mRNA sequences for target human genes used in qRT-PCR approaches.

Gene	Sequence
AhR	FP: 5′-CGCCAGCTCAGAACAGGGGC-3′
	RP: 5′-GGTCCCCGGAACCCGGTGTA-3′
CYP1A1	FP: 5′-TGCCTGCCCCTACCCTGGAC-3′
	RP: 5′-GCGCTCTGATCCCCGCAGAC-3′
18s rRNA	FP: 5′-CGGCTACCACATCCAAGGAA-3′
	RP: 5′-GCTGGAATTACCGCGGCT-3′

Statistical analysis

The data from real-time (RT)-PCR were expressed as the average of six experiments (±SD). To evaluate differences over time (2, 5, and 8 h) at a fixed dose of 10 µg DDE/ml or 20 ng PCB 153/ml, a one-way analysis of variance (ANOVA) followed by a Tukey’s post-hoc test was applied; p values <0.05 were considered statistically significant. JMP IN 5.0.1.2 software (SAS Institute Inc., Cary, NC) was used for the statistical analysis.

Results

The effect of TNFα on AhR mRNA expression in PBMC in vitro was first assessed (Figure 1A). It was found that the cytokine induced a statistically significant over-expression of AhR in the PBMC compared with levels seen in the control (NE) cells. However, this increase was not statistically significant after 8 h of exposure to the TNFα. On the other hand, it was seen that TNFα did not affect expression of CYP1A1 mRNA. These outcomes suggested to us that AhR mRNA over-expression induced by TNFα was independent of AhR activation (Figure 1B).

Figure 1. Effect on relative (A) AhR and (B) CYP1A1 gene expression in PBMC exposed to TNFα (10 ng/ml) for different durations. Mean [±SD] of six independent experiments is shown. mRNA for selected genes were assessed using qRT-PCR and normalized against 18s rRNA. *p < 0.05 versus non-exposed cells (NE). NE = cells exposed to vehicle control. As no significant differences were found between cells exposed to vehicle control after 5 or 8 h of exposure, only data from the 2 h of exposure is shown.

In previous studies, it was demonstrated that 10 µg DDE/ml was able to induce pro-inflammatory cytokines in PBMC (Alegría-Torres et al., 2009; Cárdenas-González et al., 2013). Using this concentration to ascertain the effect of DDE on AhR mRNA expression (Figure 2A), it was seen that DDE induced a significant increase in AhR mRNA expression after 2 h of exposure compared to that seen in the NE cells. The increase in AhR mRNA expression was maintained up through 8 h of exposure. PCB 153, also a pro-inflammatory compound (Kwon et al., 2002) induces pro-inflammatory cytokines in PBMC in vitro (unpublished data). In the present study, 20 ng PCB 153/ml caused a significant increase in AhR mRNA expression after 2 h of exposure (Figure 3A). Similar to the DDE findings, the increase in AhR mRNA expression was maintained up through 8 h of exposure. These results in human PBMC clearly demonstrated that both pro-inflammatory DDE and PCB 153 enhanced AhR transcript expression.

Figure 2. Effect on relative AhR (A) and CYP1A1 (B) gene expression in PBMC exposed to DDE (10 µg/ml) for different durations. Mean [±SD] of six independent experiments is shown. mRNA for selected genes were assessed using qRT-PCR and normalized against 18s rRNA. *p < 0.05 versus non-exposed (NE) cells.

Figure 3. Effect on relative (A) AhR and (B) CYP1A1 gene expression in PBMC exposed to PCB 153 (20 ng/ml) for different durations. Mean [±SD] of six independent experiments is show. mRNA for selected genes were compared using qRT-PCR and normalized against 18s rRNA. *p < 0.05 versus non-exposed (NE) cells.

As CYP1A1 is a well-accepted marker that serves as an indicator of AhR activation in most cells, including PBMC (Komura et al., 2001; Sciullo et al., 2010; Siest et al., 2008), CYP1A1 expression levels in PBMC were monitored to evaluate AhR activation. The results indicate there was no change in expression levels of CYP1A1 mRNA in the PBMC treated with 10 µg DDE/ml (Figure 2B) or 20 ng PCB 153/ml (Figure 3B) for all treatment durations.

To demonstrate that the observed over-expression of AhR mRNA when PBMC were treated with DDE and PCB 153 was regulated by TNFα (a cytokine induced by both chemical compounds; see Alegría-Torres et al., 2009; Cardenas-Gonzalez et al., 2013; Kwon et al., 2002), PBMC were treated with DDE (10 μg/ml, 2 h) or PCB 153 (20 ng/ml, 2 h) in the presence of a TNFα antagonist (i.e. anti-TNFα MAb). When the cells were exposed to DDE in the presence of the MAb, the AhR over-expression was abrogated (Figure 4A); as expected, CYP1A1 expression was unaffected (Figure 4B). No significant change in expression of AhR and CYP1A1 were observed when PBMC were treated with the MAb alone (compared to expression levels of both mRNA in NE cells). Similar results to those obtained in PBMC treated with DDE were observed when PBMC were treated with PCB 153 (Figures 5A and B).

Figure 4. Effect on relative (A) AhR and (B) CYP1A1 gene expression in PBMC exposed to DDE (10 µg/ml) and an antagonist of TNFα (100 ng anti-TNFα/ml) after 2 h of exposure. Mean [±SD] of three independent experiments is shown. mRNA for selected genes were compared using qRT-PCR and normalized against 18s rRNA. *p < 0.05 versus non-exposed cells (NE) and **p < 0.05 versus DDE-exposed cells.

Figure 5. Effect on relative (A) AhR and (B) CYP1A1 gene expression in PBMC exposed to PCB 153 (20 ng/ml) and an antagonist of TNFα (100 ng anti-TNFα/ml) after 2 h of exposure. Mean [±SD] of six independent experiments is shown. mRNA for selected genes were compared using qRT-PCR and normalized against 18s rRNA. *p < 0.05 versus non-exposed cells (NE) and **p < 0.05 versus PCB 153-exposed cells.

Discussion

A wide range of chemicals can bind to the AhR and activate AhR-associated pathways, including environmental contaminants like 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and other compounds such as tryptophan derivatives, flavonoids, and biphenyls (Hu et al., 2007). Interestingly, recent studies also have indicated that AhR activation plays diverse roles in cell proliferation, adhesion, and migration, as well as inflammation (Champion et al., 2013; Esser et al., 2009; Stevens et al., 2009). However, nothing is currently known about the effect of non-dioxin-like AhR ligands (compounds unable to activate AhR) on the modulation of AhR expression and/or activation in PBMC.

The more important finding in our study was that DDE and PCB 153 up-regulated expression of AhR mRNA in PBMC. In this regard, it has been demonstrated that chemical compounds known to be pro-inflammatory are involved in AhR expression induction in several cellular types (Champion et al., 2013; Drozdzik et al., 2014; Kobayashi et al., 2008; Umannová et al., 2007; Vondrácek et al., 2011). For example, a recent study performed in Caco-2 cells demonstrated that PMA induces AhR transcript expression (Champion et al., 2013). Moreover, those investigators showed that this effect at least partially involved the nuclear factor-κB (NF-κB) pathway. Also, it was demonstrated that the activation of NF-κB promotes an inflammatory loop, via pro-inflammatory cytokines expression as interleukin (IL)-1β and TNFα, and finally induction of AhR expression (Champion et al., 2013). In this respect, in a recent study, Alegría-Torres et al. (2009) suggested that pro-inflammatory molecules (such as IL-1β and TNFα), generated when PBMC were treated with DDE in vitro, could be mediated by NF-κB activation (Alegría-Torres et al., 2009). Consistent with the suggestion made by Alegría-Torres et al. (2009), it has been demonstrated that DDT and its metabolites can induce NF-κB activation in several cellular types (Frigo et al., 2004; Kim et al., 2004). Similar findings were found for PCBs (Bezdecny et al., 2007; Glauert et al., 2008; Myhre et al., 2009; Wang et al., 2008). In line with the mechanism proposed by Champion et al. (2013), our data showed that the over-expression of AhR was dependent on the TNFα induced by DDE and PCB 153. In this context, a recent study performed by Drozdzik et al. (2014) found this pro-inflammatory cytokine was able to induce AhR gene expression in the human salivary cell line (HSY; we also note the same effect in PBMC). Moreover, a similar finding to that here (i.e. decreased AhR expression over time when PBMC were treated with TNFα) was seen in the Drozdzik et al. (2014) study. However, this finding is somewhat surprising, and it would be important in future studies to elucidate the mechanism for which the AhR expression decreased over time.

Our results are also in concordance with the data obtained by Kobayashi et al. (2008) that assessed AhR expression levels in isolated fibroblast-like synoviocytes (FLS) treated with various cytokines. That study found that TNFα up-regulated both AhR mRNA and protein. To confirm these findings, FLSs were treated with the humanized monoclonal anti-TNFα antibody, infliximab, which resulted in decreased AhR expression. Those findings indicated that AhR expression in FLSs was stimulated in the presence of TNFα, similar to our results here. Taking into account that data and our own, we hypothesize that TNFα induced by DDE and PCB 153 (Alegría-Torres et al. 2009; Cárdenas-González et al., 2013), likely via an NF-κB pathway (Bezdecny et al., 2007; Frigo et al., 2004; Glauert et al., 2008; Kim et al., 2004; Myhre et al., 2009; Wang et al., 2008), was able to induce AhR mRNA expression.

It is known that CYP1A1 expression is regulated by AhR activation (Whitlock, 1999). During activation, the AhR translocates to the nucleus and forms a heterodimer with ARNT (Denison et al., 1986; Elferink et al., 1990); this, in turn, functions as a transcription factor, binding to dioxin response elements (DRE) and inducing expression of various genes encoding drug-metabolizing enzymes, including CYP1A1 (Hamada et al., 2006). Thus, induction of cyto-chrome P4501A1 (CYP1A1) is a useful marker of AhR-mediated signal transduction and has been used as an endpoint to assess dioxin/dioxin-like compound toxicity in animal models and cell culture system as well as molecular/genetic studies of mechanisms of AhR-mediated gene transcription (Whitlock, 1993, 1999). In the current study, when CYP1A1 expression levels in the PBMC were assessed to evaluate AhR activation, no change in expression levels of CYP1A1 were observed in PBMC treated with any of the test regimens. These results indicated that over-expression of AhR mRNA induced by TNFα, DDE, and PCB 153 was AhR activation-independent. However, it is still necessary to determine whether the AhR over-expression found in PBMC in this study was associated with a transcriptionally-active form of AhR. In this regard, a recent study showed that resting human blood T-cells (like those used in our study) were unable to over-express mRNA CYP1A1 when treated with AhR agonists; this suggested to us an inactive form of AhR. However, active T-cells are able to induce over-expression of mRNA CYP1A1 when treated with TCDD and benzo[a]pyrene, suggesting an active form of AhR (Prigent et al., 2014).

Finally, Umannová et al. (2007) investigated possible interactions of TNFα with ligands of the AhR and known liver carcinogens like TCDD and coplanar 3,3′,4,4′,5-pentachloro-biphenyl (PCB 126) in contact-inhibited rat liver WB-F344 cells. In that study, TNFα had no significant effect on proliferation/apoptosis ratio in the WB-F344 cells. However, it significantly potentiated the proliferative effects of low pM doses of both TCDD and PCB 126, leading to an increase in cell numbers as well as an increased percentage of cells entering cell cycle S-phase. Those results suggested to us that TNFα could significantly amplify effects of AhR ligands on de-regulation of cell proliferation control. However, Umannová et al. (2007) did not elucidate the molecular mechanism by which TNFα amplified effects of AhR ligands. We hypothesize that the mechanism by which TNFα amplified the effects of AhR ligands was by inducing an over-expression of the AhR. In this context, Hollingshead et al. (2008) showed that TCDD treatment in combination with IL-1β or phorbol 12-myristate 13-acetate (PMA) resulted in a marked synergistic induction of IL-6 levels over that seen without AhR activation in MCF-7 cells. Since TCDD induces IL-6 expression through the AhR pathway, those authors suggested this synergistic effect could be partly explained by an inflammation-induced increase in AhR expression.

It is important to remember that humans are usually exposed to complex chemical mixtures rather than to individual compounds (Domínguez-Cortinas et al., 2013; Perez-Maldonado et al., 2004, 2013; Orta-Garcia et al., 2014; Trejo-Acevedo et al., 2012). Moreover, it was noted that populations are exposed to pro-inflammatory compounds (as DDE and PCB153) and compounds such as PAHs, PCBs, and TCCD (AhR agonists) simultaneously (Costilla-Salazar et al., 2011; Martínez-Salinas et al., 2011; Perez-Maldonado et al., 2013; Trejo-Acevedo et al., 2012). Thus, it is important to assess potential toxicological interactions between components of a chemical mixture in exposed populations. As we noted above, pro-inflammatory molecules can significantly amplify the effects of AhR ligands (Hollingshead et al., 2008; Umannová et al., 2007).

Conclusion

This study was the first to demonstrate an increase in AhR mRNA expression in vitro in PBMC treated with either of two pro-inflammatory environmental pollutants, i.e. DDE and PCB153. Moreover, the over-expression of AhR mRNA was seen to be dependent of the TNFα that was induced by the DDE and PCB 153 and was independent of AhR activation. However, it is still important that any future studies evaluate the expression of AhR at the protein level and the functional status (and regulation) of the product AhR.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

This work was supported by grant from the Consejo Nacional de Ciencia y Tecnología, Mexico, SSA/IMSS/ISSSTE-CONACYT-2013-01-202013.