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

• Published in: Food and chemical toxicology 2005-04, Vol.43 (4), p.529-535
• Main Authors: Adachi, Tetsuya, Okuno, Yasushi, Takenaka, Shigeo, Matsuda, Kazuyuki, Ohta, Naoki, Takashima, Kyoka, Yamazaki, Koji, Nishimura, Daisuke, Miyatake, Kazutaka, Mori, Chisato, Tsujimoto, Gozoh
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; cDNA microarray; Cell Line; cell lines; daidzein; diethylstilbestrol; Diethylstilbestrol - pharmacology; Electrophoresis, Gel, Two-Dimensional; estradiol; Estradiol - pharmacology; Estrogens, Non-Steroidal - pharmacology; Gene Expression Profiling; gene induction; hormone receptors; Humans; Isoflavones - pharmacology; Leydig cells; Male; Medical sciences; messenger RNA; microarray technology; nutrition-genotype interaction; Oligonucleotide Array Sequence Analysis; Phytoestrogen; Phytoestrogens - pharmacology; plant estrogens; protective effect; protein synthesis; RNA, Messenger - analysis; Sertoli Cells; soybean products; Testicular cell line; Testis - cytology; Testis - drug effects; TM4; toxicity testing; Toxicology; trypsin inhibitors; Two-dimension polyacrylamide gel electrophoresis
• ISSN: 0278-6915; 1873-6351
• DOI: 10.1016/j.fct.2004.12.006

In this study, we examined the effects of exposure to phytoestrogen (daidzein), 17β-estradiol (E 2), 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, E 2 (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 E 2, 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 E 2, DES and staurosporin.