The environmental estrogen, nonylphenol, activates the constitutive androstane receptor

Abstract

Nonylphenol (NP) and its parent compounds, the nonylphenol ethoxylates are some of the most prevalent chemicals found in U.S. waterways. NP is also resistant to biodegradation and is a known environmental estrogen, which makes NP a chemical of concern. Our data show that NP also activates the constitutive androstane receptor (CAR), an orphan nuclear receptor important in the induction of detoxification enzymes, including the P450s. Transactivation assays demonstrate that NP increases murine CAR (mCAR) transcriptional activity, and NP treatment can overcome the inhibitory effects of the inverse agonist, androstanol, on mCAR activation. Treatment of wild-type (CAR +/+) mice with NP at 50 or 75 mg/kg/day increases Cyp2b protein expression in a dose-dependent manner as demonstrated by Western blotting, and was confirmed by quantitative reverse transcription-PCR of Cyp2b10 transcript levels. CAR-null (CAR -/-) mice show no increased expression of Cyp2b following NP treatment, indicating that CAR is required for NP-mediated Cyp2b induction. In addition, NP increases the translocation of CAR into the nucleus, which is the key step in the commencement of CAR's transcriptional activity. NP also induced CYP2B6 in primary human hepatocytes, and increased Cyp2b10 messenger RNA and protein expression in humanized CAR mice, indicating that NP is an activator of human CAR as well. In conclusion, NP is a CAR activator, and this was demonstrated in vitro with transactivation assays and in vivo with transgenic CAR mouse models.

FIG. 1

NP activates both the rPXR and hPXR. HepG2 cells were transfected with either the rPXR (A) or hPXR (B), and activation of PXR by NP determined using a luciferase-coupled reporter containing CYP3A23 PXRE. PCN (10μlM) and rifampicin (10μM) were used as positive controls for activation of rPXR and hPXR, respectively. The results are shown as fold induction compared to the solvent control, mean ± SD (n = 3 rPXR, n = 4 hPXR). Asterisk indicate a significant difference from the DMSO control (UT) cells by ANOVA followed by Fisher's PLSD as the post hoc test (*p < 0.05, **p < 0.01).

FIG. 2

NP activates mCAR. HepG2 cells were cotransfected with an expression plasmid for mCAR and the luciferase reporter plasmid, containing CYP2B6 PBREM, were then treated with an increasing amount of NP (A). In graph (B) all mCAR-transfected cells were cotreated with the inverse agonist androstanol (An) to reduce mCAR's constitutive activity and increase the sensitivity of the assay. The potent mCAR agonist, TCPOBOP (TC) at 250nM, was used as a positive control. Data are presented as mean ± SD. Statistical analysis was performed by ANOVA followed by Fisher's PLSD as the post hoc test (n = 3). An asterisk indicates statistical significance from the androstanol control (p < 0.01).

FIG. 3

NP activation of CAR. NP increased the translocation of CAR into the nucleus as determined by Western blotting of nuclear fractions from wild-type (+/+) and CAR-null (−/−) mice. (A) CAR is detected in the nucleus of TCPOBOP (TC)–treated wild-type, but not TC-treated CAR-null mice. (B) Increased translocation of CAR into the nucleus following treatment with TC at 3 mg/kg/day. (C) Increased translocation of CAR into the nucleus following treatment with NP at 75 mg/kg/day (UT = untreated). Q-PCR of hepatic Cyp2b10 after treatment with NP or TC in (D) wild-type or (E) CAR-null mice demonstrates CAR-dependent induction of Cyp2b10. Data are presented as mean ± SD. Western blots were used to confirm changes in Cyp2b protein concentrations in wild-type (F) and CAR-null (G) mice treated with NP. A Western blot of Cyp2b levels in TC-treated wild-type and CAR-null mice are shown in (H). Below each Cyp2b Western blot picture is the housekeeping gene β-actin. Relative protein induction is denoted by the values above the Western blots. An asterisk indicates a statistical difference (p < 0.05) determined by an ANOVA followed by Fisher's PLSD (n = 3; Western blots, n = 3-4 Q-PCR).

FIG. 4

ZOX-induced paralysis in wild-type (WT) and CAR-null (CAR KO) mice treated with 50 mg/kg/day NP or 3 mg/kg/day TCPOBOP (TC). The number of mice treated in each group is provided above the error bars. An (a) indicates a statistical difference from the untreated wild-type mice (WT 0). A (b) indicates a statistical difference from the untreated CAR-null mice (CAR KO 0), and (c) indicates a significant difference from the wild-type mice treated with NP (WT NP) as determined by ANOVA followed with Fisher's PLSD as the post hoc test (p < 0.05). Data are presented as mean ± SEM.

FIG. 5

NP induces Cyp2b in humanized mice. CAR-null mice containing a transgene for human CAR (hCAR) on the albumin promoter were treated with 0, 50, or 75 mg/kg/day NP or 30 mg/kg/day CITCO. Q-PCR (A) and Western blots (B) of hepatic Cyp2b10 in hCAR mice indicate that NP activates hCAR in addition to mCAR. Data are presented as mean ± SD. An asterisk indicates a statistical difference (p < 0.05) determined by an ANOVA followed by Fisher's PLSD (n = 3; Western blots, n = 3–4 Q-PCR).

FIG. 6

Induction of CYP2B6 in human hepatocytes by NP. Primary hepatocytes from three donors obtained from CellzDirect were treated with NP or PB for 24 h and CYP2B6 induction measured by Q-PCR. Data are presented as mean ± SD. An asterisk indicates a statistical difference (p < 0.05) as determined by a Student's t-test (n = 4 wells, Hu420, Hu435; n = 3 wells, Hu504).