Cancer Letters

Cancer Letters

Volume 165, Issue 1, 10 April 2001, Pages 95-101
Cancer Letters

Suppression of CYP1A1 expression by 4-nonylphenol in murine Hepa-1c1c7 cells

https://doi.org/10.1016/S0304-3835(01)00407-4Get rights and content

Abstract

This study investigated the effects that 4-nonylphenol (NP) has on CYP1A1 expression in Hepa-1c1c7 cell cultures. NP alone did not affect CYP1A1-specific 7-ethoxyresorufin-O-deethylase (EROD) activity. In contrast, the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible EROD activities were markedly reduced upon concomitant treatment with TCDD and NP in a dose-dependent manner. Treatment with tamoxifen, an anti-estrogen that acts through the estrogen receptor, did not affect the suppressive effects that NP has on TCDD-inducible EROD activity. The TCDD-inducible CYP1A1 mRNA levels were markedly suppressed upon concomitant treatment with TCDD and NP that is consistent with their effects on EROD activity. A transient transfection assay using dioxin-response element (DRE)-linked luciferase and an electrophoretic mobility shift assay revealed that NP reduced the transformation of the aryl hydrocarbon (Ah) receptor to a form capable of binding specifically to the DRE sequence of the CYP1A1 gene promoter. These results suggest that the down-regulation of CYP1A1 gene expression by NP in Hepa-1c1c7 cells might be an antagonism of the DRE-binding potential of the nuclear Ah receptor, but is not mediated through the estradiol receptor.

Introduction

4-Nonylphenol (NP) is a degradation product of a widely used non-ionic surfactant group, alkylphenol polyethoxylates, which are found mainly as an intermediate in the chemical manufacturing industry. NP is used, for example, to label tax-favored light fuel oil, as a preservative agent in the tanning industry, and in pesticide formulation, etc. NP is approximately ten times more toxic than the parent compound, and because of its hydrophobic nature, it persists in the environment [1]. NP has been shown to possess estrogenic properties in both in vivo and in vitro assays [2], [3]. Therefore, NP is an ‘endocrine disrupter’ that has a significant influence on sexual and reproductive development [4]. Human exposure to environmental compounds with estrogenic activity and their potential effects on human health is the subject of an ongoing scientific debate. There is evidence that both xenobiotic and biogenic environmental estrogens can influence the human endocrine system, and accordingly, both reproductive and tumor development [5]. However, the mechanism by which NP causes these adverse effects is unclear.

Cytochrome P450s (CYP) are a superfamily of heme-containing monooxygenase enzymes that metabolize foreign chemicals, such as drugs and environmental chemicals, in addition to endogenous compounds, such as steroids and fatty acids [6]. CYPs activate, inactivate, and facilitate the excretion of most xenobiotics. Thus, the CYPs modulate both the duration and intensity of their respective toxicity. The level of gene expression of these enzymes is influenced by a number of endogenous regulatory factors, such as hormones, as well as by xenobiotic substrates including natural and synthetic chemicals [6], [7]. The effects of both natural and synthetic chemicals on the CYP enzymes are currently of considerable interest as they may indicate a possible mechanism by which they affect the toxicity of environmental chemicals. It has been shown that CYP3A is normally responsible for steroid metabolism [8]. There is also indirect evidence suggesting that CYP1A is similarly involved [9]. Conversely, estradiol has been shown to inhibit CYP1A activity in vitro [10]. A previous study reported that NP decreases hepatic CYP1A activity and the steady-state hepatic CYP1A mRNA levels in rats. The competitive nature of NP-induced inhibition on hepatic microsomal CYP1A-specific 7-ethoxyresorufin-O-deethylase (EROD) activity indirectly suggests that NP is a possible substrate of the CYP1A enzyme [11].

CYP1A1 oxidatively biotransforms various polyaromatic hydrocarbons, such as benzo(a)pyrene. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent environmental contaminant and has been used as a model compound for investigating the mechanism of aryl hydrocarbon (Ah) action. The control and regulation of CYP1A1 gene expression have been extensively investigated. It is currently believed that TCDD and its related Ah receptor agonist induce the CYP1A1 gene expression through Ah receptor-mediated signal transduction. After binding with the ligand, the Ah receptor forms a heterodimer with the Ah receptor nuclear translocator and binds to specific DNA recognition sequences known as dioxin-response elements (DREs), which are located upstream of the CYP1A1 transcription start site [12], [13]. Binding to these enhancer sequences causes a change in the chromatin structure that facilitates the binding of the transcription factors to the CYP1A1 promoter [14].

Although NP is widely used in the chemical industry, little is known about its effects on CYP expression. Studies on CYP expression with regard to understanding NP toxicity on both wildlife and human health are attracting increasing interest. However, the molecular basis for CYP regulation by NP has not been elucidated. In chemical structure, NP is very similar to the synthetic estrogen, diethylstilbestrol. Moreover, NP has been reported to have estrogenic activity [2], [3]. It was recently reported that estradiol suppressed CYP1A1 in Hepa-1c1c7 cells [15]. In contrast, the effects of NP on CYP1A1 regulation have not been described.

In the present study, the effect of NP on TCDD-inducible CYP1A1 gene expression was investigated in mouse hepatoma Hepa-1c1c7 cells. The involvement of the estrogen receptor in this process was also investigated using an estradiol receptor antagonist, such as tamoxifen. Evidence is provided that NP down-regulates TCDD-induced CYP1A1 gene expression in Hepa-1c1c7 cells, but does not act through the estrogen receptor in these cells.

Section snippets

Materials

All chemicals and cell culture materials were obtained from the following sources: NP (>99% pure: Alderich); 7-ethoxyresorufin and resorufin (Pierce Chemical Co.); TCDD (Chemsyn Science Lab.); LipofectAMINE Plus, αMEM, fetal bovine serum, penicillin–streptomycin solution, and trypsin (Life Technologies, Inc.); pCMV-β-gal (Clontech).

Cell culture and treatment

The mouse hepatoma Hepa-1c1c7 cells were obtained from the American Type Culture Collection (Rockville, MD). The cells were cultured in an α-MEM supplemented with

Results and discussion

The effects of NP on Ah receptor-mediated induction of CYP1A1 gene expression in Hepa-1c1c7 cells were investigated. Consequently, TCDD, a prototypical inducer of the CYP1A1 that binds to the Ah receptor with a high affinity, was used to induce CYP1A1 gene expression. EROD activity is considered as a convenient measure of CYP1A1 gene activity [20] and is commonly used to monitor CYP1A1 induction [15], [19], [21]. Subsequent to treating the cells with 0.5 nM TCDD, there was markedly increased

Acknowledgements

This work was supported by the Ministry of Science and Technology, Korea and the KOSEF through the Research Center for Proteineous Materials.

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