Comprehensive analysis of the effect of phytoestrogen, daidzein, on a testicular cell line, using mRNA and protein expression profile

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Abstract

In this study, we examined the effects of exposure to phytoestrogen (daidzein), 17β-estradiol (E2), diethylstilbestrol (DES) and staurosporin on the TM4 testicular cell line, using comprehensive analysis, such as cDNA microarray and two-dimension polyacrylamide gel electropholesis (2D-PAGE) analysis, and we demonstrated if these toxicogenomic analyses could classify the chemical compounds. First, RNA was extracted from TM4 cells that had been treated with daidzein (80 μM), DES, E2 (40 μM) and stauroporin (100 nM) for 30 min. We performed cDNA microarray analysis, and the expression ratio data thus obtained were then analyzed using hierarchical clustering. This hierarchical clustering showed that daidzein exposure induced a different effect on gene expression change from that of E2, DES and staurosporin. Next, protein extracted from TM4 cells also underwent cDNA microarray analysis for 3 h. We performed 2D-PAGE analysis, and the spot intensity ratio data thus obtained were analyzed using hierarchical clustering. As with cDNA microarray, the hierarchical clustering of protein spot ratios showed that daidzein exposure induced a different effect on gene expression change from that of the other substances. In conclusion, we have demonstrated for the first time that classification of these chemicals can be performed by clustering analysis, using data from cDNA microarray and 2D-PAGE analyses, and that exposure to daidzein induces effects different from those of E2, DES and staurosporin.

Introduction

Phytoestrogens, such as daidzein, genistein and coumestrol, are defined as estrogenic compounds. They are found in plants (Kurzer and Xu, 1997), and are known to bind to estrogen receptors α (ERα) and β (ERβ) (Kuiper et al., 1998). Phytoestrogens reportedly have beneficial effects on cancer (including reproductive cancer), cardiovascular disease, brain function, alcohol abuse, osteoporosis and menopausal symptoms (Hirayama, 1984, Tajima and Tominaga, 1985, Koo, 1988, Seveson, 1989, Lee et al., 1991, Goodman et al., 1997). However, it has been reported that neonatal exposure to genistein in animal experiments also causes uterine adenocarcinomas (Newbold et al., 2001). Moreover, as reported in our previous study, mRNA expression changes were detected in neonatal genistein-treated mouse testes using a cDNA microarray (Adachi et al., 2004). These compounds also have adverse effects on reproductive systems (Kurzer and Xu, 1997, Bingham et al., 1998).

As discussed above, it is not been clear by what mechanism phytoestrogen exerts these effects on reproductive organs. Therefore, it is necessary to develop methods of estimating the effects of phytoestrogen on reproductive organs. The most applicable and efficient approaches to developing such methods are comprehensive analysis using DNA microarray and two-dimension polyacrylamide gel electrophoresis (2D-PAGE). DNA microarray is known to be a powerful and high-throughput method for monitoring the expression of thousands of genes simultaneously (Duggan et al., 1999, Rockett and Dix, 1999, Hossain et al., 2000, Adachi et al., 2002, Komiyama et al., 2003). Moreover, 2D-PAGE is the only method currently available that is capable of simultaneously separating and quantifying hundreds or thousands of proteins (Garrels, 1989). Many reports of studies using 2D-PAGE have recently been published.

In this study, we examined the effects of TM4 Sertoli cells on exposure to phytoestrogen, daidzein, using DNA microarray and 2D-PAGE. Diethylstilbestrol (DES) and 17β-estradiol (E2), which are known to be estrogenic compounds, and staurosporin, which is known to be a tyrosine kinase inhibitor, were used as controls. Daidzein is known to be an estrogenic compound, and an inactive form of tyrosine kinase inhibitor (Magee and Rowland, 2004). We also conducted a clustering analysis, using data from the DNA microarray and 2D-PAGE, in order to determine the degree of similarity of the mRNA and protein expression of phytoestrogen to that following treatment with DES, E2 and staurosporin. Moreover, we demonstrated if these toxicogenomic analyses could classify the chemical compounds.

Section snippets

Chemicals

Daidzein (minimum 98%), DES (minimum 99%), E2 (minimum 98%) and staurosporin (minimum 95%) were purchased from Sigma–Aldrich (St Louis, MO). Dimethyl sulfoxide (DMSO), which was used as solvent, was also purchased from Sigma–Aldrich.

Cell cultures

TM4 cells, a mouse Sertoli cell line, were obtained from American Type Culture Collection (Manassas, VA). Cells were cultured in 1:1 mixture of Ham’s F12 medium and Dulbecco’s modified Eagle’s medium (Invitrogen Corp., Carlsbad, CA) with 1.2 g/l sodium bicarbonate

Cell viability following the treatment with daidzein, E2, DES and staurosporin

We first examined the effects of treatment with daidzein, E2, DES and staurosporin on cell viability. Daidzein treatment for 24 and 48 h at a concentration of 80 μM reduced cell viability; it also reduced cell viability after a 24-h treatment at a concentration of 40 μM (Fig. 1A). Treatment with E2 and DES for 24 and 48 h at a concentration of 40 μM reduced cell viability, as did E2 treatment for 6 h (Fig. 1B and C). Staurosporin treatment for 24 and 48 h at concentrations of 10 and 100 μM also reduced

Discussion

It has been reported that phytoestrogens have beneficial effects on cancer (including reproductive cancer), cardiovascular disease, brain function, alcohol abuse, osteoporosis and menopausal symptoms (Hirayama, 1984, Tajima and Tominaga, 1985, Koo, 1988, Seveson, 1989, Lee et al., 1991, Goodman et al., 1997). However, these compounds also have adverse effects on reproductive systems (Kurzer and Xu, 1997, Bingham et al., 1998). Phytoestrogen displays estrogenic and tyrosine kinase inhibition

Acknowledgments

We are grateful to the Dr. Hitoshi Tainaka (Asahi Techno Glass Co. Funabashi, Japan) for the donation cDNA microarrays. This study was supported by grants-in-aid from the Ministry of Education, Science, Sports, Culture and Technology of Japan, as part of the 21st Century COE program “Knowledge Information Infrastructure for Genome Science”. It was supported by the Fund for Endocrine Disrupters provided by the Ministry of the Environment, Japan.

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