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Four decades after its discovery, the aryl hydrocarbon receptor (AHR), a ligand-inducible transcription factor (TF) activated by the persistent environmental contaminant 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), remains an enigmatic molecule with a controversial endogenous role. Here, we have assembled a global map of the AHR gene regulatory network in female C57BL/6 mice orally gavaged with 30 µg/kg of TCDD from a combination of previously published gene expression and genome-wide TF-binding data sets. Using Kohonen self-organizing maps and subspace clustering, we show that genes co-regulated by common upstream TFs in the AHR network exhibit a pattern of co-expression. Directly bound, indirectly bound, and non-genomic AHR target genes exhibit distinct expression patterns, with the directly bound targets associated with highest median expression. Interestingly, among the directly bound AHR target genes, the expression level increases with the number of AHR-binding sites in the proximal promoter regions. Finally, we show that co-regulated genes in the AHR network activate distinct groups of downstream biological processes. Although the specific findings described here are restricted to hepatic effects under short-term TCDD exposure, this work describes a generalizable approach to the reconstruction and analysis of transcriptional regulatory cascades underlying cellular stress response, revealing network hierarchy and the nature of information flow from the initial signaling events to phenotypic outcomes. Such reconstructed networks can form the basis of a new generation of quantitative adverse outcome pathways.

Phospholipase A2 (PLA2) has been postulated to play a role in the regulation of cytokine expression. Therefore, the objective of the present study was to investigate the effects of PLA2 inhibitors p-bromophenacyl bromide (BPB) and arachidonyl trifluoromethyl ketone (AACOCF3) on interleukin-2 (IL-2) expression in murine primary splenocytes. Pretreatment of the splenocytes with both BPB and AACOCF3 suppressed phorbol 12-myristate 13-acetate plus ionomycin-induced IL-2 secretion in a concentration-dependent manner. Inhibition > 90% of IL-2 secretion was observed at 1 microM BPB and 10 microM AACOCF3 compared to the respective vehicle control. Likewise, IL-2 steady-state mRNA expression was inhibited by both PLA2 inhibitors in a concentration-dependent fashion with > 90% inhibition at 1 microM BPB and 20 microM AACOCF3. Taken together, these data demonstrated that PLA2 inhibitors BPB and AACOCF3 are robust inhibitors of IL-2 expression at both the mRNA and protein levels in murine splenocytes. Moreover, these findings suggest that drugs and chemicals which inhibit PLA2 may have marked effects on T-cell function.

Previous studies have demonstrated that repeated (14 day) administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) enhances the suppression of humoral immunity in DBA/2 (Ah-low responder) mice relative to the effect seen with identical cumulative doses after a single treatment (cumulative doses of 4.2, 14.0, and 42 mg/kg). In the present studies, we have explored this phenomenon further by determining the status of several specific parameters, which might account for the increase in antibody suppression in the DBA/2 strain following repeated TCDD exposures. Included in these studies was the induction of hepatic and splenic microsomal 7-ethoxyresorufin-o-deethylase (EROD; P4501A1) activity and biodistribution of the administered TCDD into various target organs and tissues. Changes in lymphocyte subpopulations within the spleen were also assessed by flow cytometry following both single and repeated dosing. All studies made use of direct comparisons between DBA/2 (Ah-low responder) and B6C3F1 (Ah-high responder) female mice. Results of these studies demonstrate that the enhanced suppression of humoral immunity in DBA/2 mice following repeated exposure to TCDD is not directly associated with increases in liver microsomal EROD activity and does not appear to be correlated with changes in the pattern of biodistribution or amount of TCDD within the liver or spleen of these animals. In contrast, the most significant changes that occurred following repeated dosing in either strain were observed in the levels of microsomal EROD activity and immune cell ratios within the spleen. This effect was characterized as an increase in microsomal EROD activity, and a corresponding reduction in the numbers of a non-B/non-T cell population in the spleen.

The goal of these studies was to characterize the role played by antigen challenge in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced immunosuppression. The effects of single exposure to TCDD (4.2, 14, and 42 microg/kg) in B6C3F1 mice on the reverse plaque assay (RPA; no sensitization) and the sheep red blood cell (SRBC) antibody response were compared. While the RPA was suppressed in a dose-dependent fashion with significance at the two highest doses, a much more dramatic effect was noted with the primary anti-SRBC response: a suppression was noted at the lowest dose, which was comparable to that observed with the highest dose in the RPA. Subsequent studies compared the RPA in B6C3F1 (Ah-high responder) and DBA/2 (Ah-low responder) mice after both single and repeated exposure to identical cumulative doses of TCDD (4.2, 14, and 42 microg/kg). The repeated exposures consisted of 14 consecutive daily treatments of 0.3, 1.0, and 3.0 microg/kg. The results indicated only a slight difference in the effects of TCDD in the two strains after single exposure, and even less difference after repeated exposure. Moreover, administering TCDD on different days relative to the SRBC challenge indicated a suppression in both strains when given 1, 2, or 3 days before antigen challenge, on the day of antigen challenge, or 1 or 2 days after antigen challenge. The only day of administration where the suppression was attenuated was 3 days after antigen challenge. These results confirm an important relationship between antigen challenge and TCDD exposure on immunosuppression.

2-Acetylaminofluorene (AAF) inhibited in a dose dependent manner mouse spleen cell blastogenesis in response to phorbol 12-myristate 13-acetate (PMA)/Ionomycin (Io) activation, the T-cell lectin, concanavalin A (Con A), and following stimulation by alloantigens as measured by the mixed lymphocyte response (MLR). AAF also markedly suppressed the T-cell dependent antibody forming cell (AFC) response to sRBC. AAF was most inhibitory on both the sRBC IgM AFC response and Con A stimulated proliferation when added during the first 24 h following initiation of culture. Direct addition of high concentrations of AAF (100 microM) to spleen cell cultures at 48 h following Con A stimulation produced a very modest inhibition (< 20%) of T-cell proliferation as compared to 90% when added at the time cultures were initiated. Similarly, AAF (75 and 100 microM) produced a greater than 80% inhibition of the in vitro AFC response when spleen cells were sensitized with antigen in presence of AAF. In contrast, no inhibition of the IgM AFC response was produced when AAF (75 microM) was added to spleen cell cultures 48 or 72 h after antigen sensitization. Con A-triggered cell-cycle progression was attenuated at the G1 stage by the addition of AAF (50 and 100 microM) with no inhibition of S to G2/M phase transition. These results suggest that the mechanism of AAF-mediated immune suppression is through a blockade of cell cycle progression from G1 to S phase.