Transgenic Overexpression of Aryl Hydrocarbon Receptor Repressor (AhRR) and AhR-Mediated Induction of CYP1A1, Cytokines, and Acute Toxicity

Abstract

Background:The aryl hydrocarbon receptor repressor (AhRR) is known to repress aryl hydrocarbon receptor (AhR) signaling, but very little is known regarding the role of the AhRR in vivo.Objective:This study tested the role of AhRR in vivo in AhRR overexpressing mice on molecular and toxic end points mediated through a prototypical AhR ligand.Methods:We generated AhRR-transgenic mice (AhRR Tg) based on the genetic background of C57BL/6J wild type (wt) mice. We tested the effect of the prototypical AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the expression of cytochrome P450 (CYP)1A1 and cytokines in various tissues of mice. We next analyzed the infiltration of immune cells in adipose tissue of mice after treatment with TCDD using flow cytometry.Results:AhRR Tg mice express significantly higher levels of AhRR compared to wt mice. Activation of AhR by TCDD caused a significant increase of the inflammatory cytokines Interleukin (IL)-1β, IL-6 and IL-10, and CXCL chemokines in white epididymal adipose tissue from both wt and AhRR Tg mice. However, the expression of IL-1β, CXCL2 and CXCL3 were significantly lower in AhRR Tg versus wt mice following TCDD treatment. Exposure to TCDD caused a rapid accumulation of neutrophils and macrophages in white adipose tissue of wt and AhRR Tg mice. Furthermore we found that male AhRR Tg mice were protected from high-dose TCDD-induced lethality associated with a reduced inflammatory response and liver damage as indicated by lower levels of TCDD-induced alanine aminotransferase and hepatic triglycerides. Females from both wt and AhRR Tg mice were less sensitive than male mice to acute toxicity induced by TCDD.Conclusion:In conclusion, the current study identifies AhRR as a previously uncharacterized regulator of specific inflammatory cytokines, which may protect from acute toxicity induced by TCDD.Citation:Vogel CF, Chang WL, Kado S, McCulloh K, Vogel H, Wu D, Haarmann-Stemmann T, Yang GX, Leung PS, Matsumura F, Gershwin ME. 2016. Transgenic overexpression of aryl hydrocarbon receptor repressor (AhRR) and AhR-mediated induction of CYP1A1, cytokines, and acute toxicity. Environ Health Perspect 124:1071–1083; http://dx.doi.org/10.1289/ehp.1510194

Adipose Tissue as a Site of Toxin Accumulation

“…In addition to BPA (404) (32) (433), gene expression and secretion of inflammatory factors from adipocytes has been reported to increase in response to TCDD (195) (235) (34) (292) (203), PCB-77 (34) PCB-126 (203), and DEHP (62). Moreover, infiltration of macrophages into AT, a pathway associated with obesity-induced insulin resistance, has been reported as a result of exposure to DEHP (a phthalate) (62) HBCD (a BFR) (431), and TCDD (415). …”

Regulation of Human Cytochrome P4501A1 (hCYP1A1): A Plausible Target for Chemoprevention?

“…AHRR has been described as a negative tissue-specific regulator of mCYP1A1 expression [43, 44]. Its overexpression in transgenic mice suppresses the mCYP1A1 induction in lung, spleen, and adipose tissue [45]. Moreover, it has been suggested that rat CYP1A1 regulates its own expression because it catalyzes the removal of AHR agonists and thus decreases the activation of this pathway [46, 47].…”

Suppressive effects of aryl-hydrocarbon receptor repressor on adipocyte differentiation in 3T3-L1 cells

“…On the other hand, CYP1A1 induction by AhR activation is not always enhanced in AhRR KO mice [11], and thus, the function of AhRR has remained a subject of dispute. Interestingly, in AhRR transgenic mice, which we produced previously, the basal expression of CYP1A1 in the kidney was increased compared to wild-type mice [14], suggesting that AhRR may have a positive regulatory function in murine kidneys. AhRR has been reported not to suppress CYP1A1 expression in human skin fibroblasts [16].…”

“…Equal amounts of protein were loaded, separated via SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The blocked membranes were incubated with the following primary antibodies: anti-AhRR [14], anti-C/EBPα (Santa Cruz Biotechnology, Dallas, TX, USA), anti-C/EBPβ (Santa Cruz Biotechnology), anti-PPARγ (Santa Cruz Biotechnology) and anti-α-tubulin (Sigma-Aldrich). Then, the membrane was incubated in solutions with peroxide-conjugated secondary antibodies (Thermo Fisher Scientific) and was visualized using peroxide substrates (SuperSignal West Dura, Thermo Fisher Scientific).…”

“…Two control siRNAs (12935–300 and 12935–200, Invitrogen) were used as the control. The cloning and generation of the mouse AhRR expression vector (pAhRR) has been described recently [14]. A mixture of 3 AhRR siRNAs or 2 control siRNAs, pAhRR and the empty vector were transfected into 3T3-L1 cells using Lipofectamine 2000 reagent (Invitrogen) as recommended by the manufacturer.…”