Elsevier

Food and Chemical Toxicology

Volume 41, Issue 12, December 2003, Pages 1711-1717
Food and Chemical Toxicology

Effects of environmental estrogenic compounds on growth of a transplanted estrogen responsive pituitary tumor cell line in rats

https://doi.org/10.1016/S0278-6915(03)00198-4Get rights and content

Abstract

The modifying effects of three endocrine disrupting chemicals on the promotion phase of estrogen dependent tumor development were investigated with a transplantable rat pituitary cell line, MtT/E-2. The EDCs examined were genistein (Gen), a phytoestrogen, p-nonylphenol (NP), a surfactant and atrazine (Atz), a herbicide. Since potential exposure is through food and drinking water, oral administration was examined in the present study. Gen and NP stimulated in vitro MtT/E-2 growth at concentrations of 10−8 and 10−6 M, respectively, while Atz did not show any effects. The estrogenic activity was further confirmed by measuring transcription of an ERE-luciferase reporter transiently transfected in the cells. When MtT/E-2 cells are inoculated into ovariectomized female F344 rats, estrogen dependent tumors develop providing a simple and sensitive model to examine modulation effects of estrogenic compounds in the promotion phase. Ovariectomized F344 rats implanted with MtT/E-2 were fed diet containing NP or Gen at 25 and 250 mg/kg, or Atz at 5, 50 and 500 mg/kg. NP and Atz did not exert any modifying effects on pituitary tumor development, while Gen at 250 mg/kg exhibited a promotion influence. These results indicate that Gen might facilitate the estrogen responsive tumor development if only the promotion phase is concerned, while NP and Atz at doses used in the present study were without effect.

Introduction

There have been many concerns regarding possible adverse effects of endocrine disrupting chemicals (EDCs) that are capable of altering the functions of the endocrine system by interfering with the synthesis, metabolism, binding or cellular responses of natural hormones (Cooper and Kavlock,1997, Kavlock et al., 1996, Sonnernschein and Soto, 1998). For human health, the reproduction system, neurobehavior, the immune system and cancer development might be affected in particular. In general, elevated incidences of certain cancers in many industrialized countries have been cited as evidence of widespread exposure to chemical pollutants. Possible relationships may exist between EDCs and the observed increase in hormone responsive cancers such as breast, prostate and testicular cancers (Adami et al., 1995, Hunter et al., 1997, Morton et al., 1997, Russo and Russo, 1996, Toppari et al., 1996). Carcinogenic chemicals are categorized as tumor initiators, tumor promoters and compounds with both activities under the multistage model of carcinogenesis (Russo and Russo, 1996). In this context, EDCs are likely to act primarily as tumor promoters.

In the rat, estrogen is known to induce anterior pituitary hyperplasia, and prolonged treatment results in mammo/somatotrophic tumors (Ueda et al., 1968). From one such tumor in a F344 rat, we have established a pituitary tumor cell line, MtT/E-2, which is characterized by high sensitivity to estrogen regarding proliferation (Fujimoto et al., 1999). It provides a relatively sensitive way to examine the potency of EDCs to promote development of estrogen responsive tumors in rats (Maruyama et al., 1999). In the present study, we examined genistein (Gen), p-nonylphenol (NP) and atrazine (Atz) in the diet, which might found as components or contaminants in food.

Section snippets

Chemicals

NP and Atz were purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan) and Wakojunyaku (Osaka, Japan), respectively. 17β-estradiol (E2) was from Sigma Chemicals, St. Louis, MO, USA. Gen was synthesized as followed by Chang et al., 1994 and confirmed to have a purity of more than 97%.

Animals

Female F344 rats, 4 weeks old (purchased from Charles River Japan Co., Kanagawa) were used. Modified CRF-1, a powdered basal diet from which soy constituents had been eliminated, was purchased from Oriental Yeast

Agent dependent cell proliferation

MtT/E-2 cells grew in response to E2 at as low as 10−12 M (Fig. 1). Significant growth was noted with Gen and NP at 10−8 and at 10−6 M, respectively, while Atz had no effect on growth between 10−7 and 10−4 M. Growth suppression with Gen was apparent at concentrations over 10−5 M.

ERE reporter assay

The (ERE)3-SV40-luciferase reporter in MtT/E-2 responded well to E2 with a maximal 20-fold induction at 10−9 M (Fig. 2). Gen exhibited strong estrogenic activity, inducing significant increase in ERE-luciferase activity

Discussion

In the present study, chemicals which might be present as components or contaminants in food were examined. Regarding effects of EDCs on carcinogenesis, the primary question is what hormonal action the chemical possesses and how it might modify tumor formation. Since three chemicals examined here have been clamed as estrogen mimicking compounds, we examined the effect on the tumor formation process with an estrogen responsive pituitary cell line, MtT/E-2. In the pituitary gland of rats,

Acknowledgments

We thank Mr. U. Mizuno for his expert technical assistance and Dr. M.A. Moore for reading the manuscript and English clarification. This work was supported in part by Grants-in-Aid (H11-Seikatsu-018 and H13-Seikatsu) from the Ministry of Health, Labor and Welfare, Japan and a Grant-in-Aid (No. 14042241) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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