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

ArticleinFood and Chemical Toxicology 43(4):529-35 · May 2005with4 Reads
DOI: 10.1016/j.fct.2004.12.006 · Source: PubMed
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.
    • "In contrast, the expression of only five genes was affected by daidzein with respect to E2 in TM4 Sertoli cells. These five genes were related to cell signaling, cell proliferation, and apoptosis, suggesting a possible correlation with the inhibition of cell viability reported after treatment with daidzein [126]. A variety of cell types respond to estrogens in a very short time, making the classical ER-mediated mechanism of action unlikely. "
    [Show abstract] [Hide abstract] ABSTRACT: Phytochemicals (PhC) are a ubiquitous class of plant secondary metabolites. A "recommended" human diet should warrant a high proportion of energy from fruits and vegetables, therefore providing, among other factors, a huge intake of PhC, in general considered "health promoting" by virtue of their antioxidant activity and positive modulation, either directly or indirectly, of the cellular and tissue redox balance. Diet acts through multiple pathways and the association between the consumption of specific food items and the risk of degenerative diseases is extremely complex. Recent literature suggests that molecules having a chemical structure compatible with a putative antioxidant capacity can actually "perform" activities and roles independent of such capacity, interacting with cellular functions at different levels, such as affecting enzyme activities, binding to membrane or nuclear receptors as either an elective ligand or a ligand mimic. Inductive or signaling effects may occur at concentrations much lower than that required for effective antioxidant activity. Therefore, the "antioxidant hypothesis" is to be considered in some cases an intellectual "shortcut" possibly biasing the real understanding of the molecular mechanisms underlying the beneficial effects of various classes of food items. In the past few years, many exciting new indications elucidating the mechanisms of polyphenols have been published. Here, we summarize the current knowledge of the mechanisms by which specific molecules of nutritional interest, and in particular polyphenols, play a role in cellular response and in preventing pathologies. In particular, their direct interaction with nuclear receptors and their ability to modulate the activity of key enzymes involved in cell signaling and antioxidant responses are presented and discussed.
    Full-text · Article · Sep 2008
    • "Real-time PCR was performed using Opicon2 (Bio-Rad, Tokyo, Japan) to quantify mRNA expression levels (Adachi et al. 2005). RNA without reverse transcription was used as a negative control. "
    [Show abstract] [Hide abstract] ABSTRACT: We have reported that a change in muscle fibre type distribution is present in two strains of diabetic rats (Otsuka Long-Evans Tokushima Fatty and Goto-Kakizaki rats). In this study, we determined whether the change in soleus muscle fibre type distribution was caused by diabetes, using obese, diabetic (Zucker diabetic fatty, ZDF), obese, non-diabetic (Zucker fatty, ZF) and non-diabetic, non-obese rats (Zucker lean, ZL). Moreover, we investigated whether the gene expression levels of metabolic key molecules, namely the transcriptional factors of metabolic genes, exemplified by peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), and the oxidative enzymes in mitochondria, exemplified by succinate dehydrogenase (SDH), were changed in type I and II muscle fibres in each type of rat, using the new technique of laser capture microdissection (LCM). Both plasma glucose and glucosylated haemoglobin levels were significantly higher in ZDF than in ZL and ZF rats. A lower percentage of type IIA fibres was observed in the muscles of ZDF rats than in those of ZL and ZF rats. The mRNA expression levels of SDH in type II fibres and of PGC-1alpha in type I fibres were significantly lower in ZDF than in ZL and ZF rats as assessed by LCM and real-time PCR analysis. We have shown, for the first time, that a lower percentage of type IIA fibres was observed in ZDF rats. We have also discovered that the expression levels of the oxidative metabolism-related genes, PGC-1alpha and SDH, decreased in type I and type II fibres, respectively, of ZDF rats.
    Article · Apr 2007
    • "This analysis revealed that only the expression of 5 genes changed after daidzein exposure with respect to estradiol. The 5 genes were related to cell signaling, cell proliferation, and apoptosis suggesting a probable correlation with the inhibition of TM4 cell viability reported after exposure to daidzein (Adachi et al., 2005). To facilitate the study of ligand-dependent ER genomic actions in reproductive and non-reproductive organs, the genome of a mouse model has been recently engineered with a transgene containing an exogenous reporter gene, luciferase, driven by an ERE-thymidine kinase promoter. "
    [Show abstract] [Hide abstract] ABSTRACT: Flavonoids are a large group of nonnutrient compounds naturally produced from plants as part of their defence mechanisms against stresses of different origins. They emerged from being considered an agricultural oddity only after it was observed that these compounds possess a potential protective function against several human degenerative diseases. This has led to recommending the consumption of food containing high concentrations of flavonoids, which at present, especially as soy isoflavones, are even available as overthecounter nutraceuticals. The increased use of flavonoids has occurred even though their mechanisms are not completely understood, in particular those involving the flavonoid impact on estrogen signals. In fact, most of the human health protective effects of flavonoids are described either as estrogenmimetic, or as antiestrogenic, while others do not involve estrogen signaling at all. Thus, the same molecule is reported as an endocrine disruptor, an estrogen mimetic or as an antioxidant without estrogenic effects. This is due in part to the complexity of the estrogen mechanism, which is conducted by different pathways and involves two different receptor isoforms. These pathways can be modulated by flavonoids and should be considered for a reliable evaluation of flavonoid, both estrogenicity and antiestrogenicity, and for a correct prediction of their effects on human health.
    Full-text · Article · Oct 2006
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