Comprehensive analysis of the effect of phytoestrogen, daidzein, on a testicular cell line, using mRNA and protein expression profile
Department of Genomic Drug Discovery Science, Graduate School of Pharmaceutical Sciences, Kyoto University, Yoshida-Shimoadachicho, Sakyo-ku, Kyoto 606-8501, Japan.Food and Chemical Toxicology (Impact Factor: 2.9). 05/2005; 43(4):529-35. DOI: 10.1016/j.fct.2004.12.006
In this study, we examined the effects of exposure to phytoestrogen (daidzein), 17beta-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 microM), DES, E2 (40 microM) 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.
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ABSTRACT: Two-dimensional gel electrophoresis (2DGE) continues to be a useful approach to study protein expression. Although liquid chromatographic and mass spectrometric approaches that overcome some of the limitations and labour intensity of 2DGE are increasingly popular, this electrophoretic approach still has exceptional relevance in toxicology. Despite the technical challenges, pharmacologists/toxicologists continue to use gel-based proteomics to assess the biological and health effects of chemical treatment and exposure. This brief review addresses the use of 2DGE-based proteomics in drug development and toxicology, emphasising its unique strengths and weaknesses, and considers recent developments in this strategy that have evolved to directly confront the issues of dynamic range and reproducibility that have previously limited the overall use of 2D electrophoresis.Expert Opinion on Drug Metabolism & Toxicology 03/2006; 2(1):103-11. DOI:10.1517/17425255.2.1.103 · 2.83 Impact Factor
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ABSTRACT: Effect of running exercise on fibre-type distributions of the slow soleus and fast plantaris muscles was investigated in male Otsuka Long-Evans Tokushima fatty rats (OLETF) as an animal model of spontaneous type 2 diabetes mellitus. Five-week-old OLETF rats were allowed to exercise voluntarily in running wheels for 32 days and the data were compared with those of age-matched non-exercised OLETF and non-diabetic Long-Evans Tokushima Otsuka rats (LETO). In the soleus muscle, a higher percentage of type I fibres was observed in non-exercised OLETF rats compared with LETO rats, and there were no type IIA fibres in non-exercised OLETF rats. In the plantaris muscle, a higher percentage of type IIB fibres and a lower percentage of type I and type IIA fibres were observed in non-exercised OLETF rats compared with LETO rats. In contrast, there were no differences in the fibre-type distribution of soleus and plantaris muscles between exercised OLETF and LETO rats. The body weight and type I fibre percentage of the soleus muscle were related to the running distance in exercised OLETF rats. White adipose tissue weight, HbA(1c) and blood insulin and glucose concentrations were lower in exercised OLETF rats than in non-exercised OLETF rats, irrespective of the running distance. There was a difference in the gene-expression pattern of the soleus muscle among LETO rats, non-exercised OLETF and exercised OLETF rats. Running exercise can inhibit diabetes-associated type shifting of fibres, which is more apparent with postnatal growth, in skeletal muscles of diabetic OLETF rats, as a result of mRNA expression change in muscle.Diabetes Obesity and Metabolism 06/2006; 8(3):311-21. DOI:10.1111/j.1463-1326.2005.00507.x · 6.36 Impact Factor
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ABSTRACT: Epidemiological studies have shown that Asian populations display a lower incidence of hormone-dependant cancers, cardiovascular disease, osteoporosis, and menopausal ailments compared to Western societies. Available data support the proposal that lower incidence is associated with the high dietary consumption of isoflavones, such as genistein. This study used two-dimensional electrophoresis to characterize the effect of genistein on the proteome of an endometrial tumor cell model, namely the Ishikawa cell line. Proteome maps displaying approx 1800 proteins were obtained from cells treated with vehicle or genistein at physiologically attainable concentrations of 0.5, 5, or 50 μM or supra-physiological concentration, 500 μM. The effects of genistein on protein expression were characterized using image analysis software. A total 65 protein spots displayed a significant decrease in expression and 32 proteins displayed a significant increase in expression. Of these protein spots, 29 were randomly selected for characterization by matrix assisted laser desorption/ionization tandem mass spectrometry, yielding 18 different proteins. This type of analysis enabled the characterization of a wide range of cellular proteins and allowed for the identification of functional and biochemical pathways that may be regulated or affected by genistein, including cellular transcription, cell proliferation, stress response, or modulation of oncogenic pathways.Clinical Proteomics 07/2006; 2(3):153-167. DOI:10.1007/BF02752498
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