Nonylphenol activates the constitutive androstane receptor and causes sexually dimorphic changes in p450 expression
Nonlyphenols (NP) are becoming one of the most ubiquitous contaminants in U.S. waterways. NP was previsouly shown to increase the incidence while decreasing the latency of breast cancer in FVB/NJ MMTV neu mice. During these breast cancer studies, P450 induction was measured. We therefore sought to determine cytochrome P450 induction by NP in wild-type FVB/NJ mice; however, in our pilot study with male mice we saw nominal induction of P450s by Quantitative Real-Time PCR (Q-PCR), and downregulation of the Cyp3a subfamily by Western blot. Basal sexual dimorphic expression of P450s in mice and rats was previsouly published. The first aim of this study was to determine if FVB/NJ mice had sexual dimorphic expression of P450s measured in other mouse strains, and to determine the sexual dimorphic induction of P450s by NP. Mice treated with honey vehicle control, 25 mg/kg/day (males only), 50 mg/kg/day, or 75 mg/kg/day NP, or 100 μg/kg/day estradiol (E2) (females only) for one week. No significant P450 induction was measured at 25 mg/kg/day or with 100 ug/kg/day estradiol, suggesting the threshold for P450 induction by NP in vivo is greater than 25 mg/kg, and P450 alterations were not due to NP's estrogenicity. Eight of the ten P450s were female predominant, and sexual dimorphic induction of P450s by NP treatment was measured. These results were confirmed by Western blot and testosterone hydroxylation enzyme assays. Higher basal/induction of P450s in females, resulted in increased Zoxazolamine (ZOX) paralysis and decreased survival of ZOX-challenged males mice. The preferential induction of the Cyp2b subfamily compared to the Cyp3a subfamily, induction of Cyp2b10 in both sexes, and v the decreased paralysis time in males and females by TCPOBOP (a known CAR-agonist), suggested activation of the constitutive androstane receptor (CAR). The second aim of this study was to determine whether NP can activate mouse and human CAR. Luciferase reporter assays utilizing HepG2 cells, a liver cancer cell line, was used to determine NP activation of mouse CAR (mCAR) in vitro. NP activated mCAR at 1 μM, and was able to overcome androstanol inhibition of CAR’s basal activity; on the other hand human CAR (hCAR) assays were inconclusive. Wild-type (BL6129) and CAR-null mice were treated with honey vehicle control, 50 mg/kg/day, 75 mg/kg/day NP, or 3 mg/kg TCPOBOP. Cyp2b10, the biomarker for CAR activation, was induced in a CAR-dependent manner by NP measured by Q-PCR and Western blot. These results suggest NP activates mCAR in vivo. Cyp2b10 induction by NP was measured by Q-PCR and Western blot in transgenic human CAR mice. Induction of CYP2B6, the only CYP2B isoform expressed in humans, was measured in primary human hepatocytes from three donors. The induction of Cyp2b10 in humanized mice and primary hepatocytes suggested NP activates hCAR in vivo. We determined NP activates mouse and human CAR, and causes sexual dimorphic induction of P450s. The final aim of this study was to determine CAR’s role in the maintenance of basal sexual dimorphic expression and induction of P450s by NP. Male and female wild-type (B6129) and CAR-null mice were treated as previsouly described in aim two. Basal P450 expression in this mouse strain was similar to FVB/NJ mice, though female P450 expression was stunted in the BL6129 strain. The BL6129 strain mimiced human P450 sexual dimorphic patterns, as the P450s measured are female predominant but not at the exaggerated levels seen in rats and FVB/NJ mice. CAR regulated the expression of Cyp2b13 and Cyp2c29 in male mice, while Cyp2b10 and Cyp2c29 were regulated in females to a lesser degree. NP induced Cyp2b10, Cyp2c29, and Cyp3a11 in a CAR-mediated female specific vi manner; however TCPOBOP induced these P450s in both males and females. TCPOBOP and NP exhibited similar P450 induction profiles in females but differed in males, suggesting a differential sensitivity of males to weak or moderate CAR activators such as NP. The ubiquitous environmental estrogen, NP, altered P450s in male and female mice, activated mouse and human CAR, and the sexual dimorphic sensitivity to NP is mediated in part through CAR. We hypothesize that NP exposure may have enocrine-independent effects mediated through the pregnane X receptor (PXR) and CAR in the liver. These results suggesr drug-drug interactions and decreased pharmaceutical efficacy could be more prevelant in women exposed to NP and other CAR partial agonists. Men may respond poorly to toxicant insult due to lower basal P450 expression and P450 induction of genes transcriptionally regulated by CAR. Two possible mechanisms for decreased sensitivity in men could be lower CAR expression, previsouly observed in male mice, and increased levels of androgens which repress CAR basal activity. (Abstract shortened by UMI.)^
Health Sciences, Toxicology|Biology, Physiology
Hernandez, Juan Pablo, "Nonylphenol activates the constitutive androstane receptor and causes sexually dimorphic changes in p450 expression" (2008). ETD Collection for University of Texas, El Paso. AAI3310687.