Environmental chemicals may affect human health by disrupting endocrine function. Many of the endocrine disrupting chemicals (EDCs) are estrogens or estrogen-like molecules that have been classified as environmental estrogens or xenoestrogens (XEs). XEs include endosulfan, chlordance, nonylphenol, bisphenol A, octylphenol, and coumestrol, etc. Although these compounds have wide structural diversity, but all have in common the and/or other hydrophobic components. Many studies have shown that XEs affect cell viability. For instance, Nonylphenol is used in surfactants or plasticizers and bisphenol A (4, 4¡¦-isopropylidene-2-diphenol) is used as protective coatings on food containers and for composites and sealants in dentistry. Most previous studies have focused on the toxicity of XEs on development process and reproductive system, especially in aquatic ecosystems. Thus, the effects of these two environmental chemicals on the toxicological effect are still controversial. The aim of this study is to investigate the molecular mechanisms of nonylphenol and bisphenol A in induction of cell death in human gastric cancer (SCM-1) cells and Madin Darby canine renal tubular (MDCK) cells. First, WST-1 reduction assays and propidium iodide-staining assay were used to determine cell viability and apoptosis in the present of nonylphenol and bisphenol A. Furthermore, we will use immunoblotting to measure the activity of apoptotic markers caspase-3, mitogen-activated protein kinases (MAPKs) to survey how nonylphenol affects apoptotic pathways. Besides, I will explore bisphenol A whether induces cell death and the mechanisms underlying the [Ca2+]i rise in MDCK cells. The results may be helpful for understanding the pharmacological and toxicological effects of these two environmental chemicals in cells from important organs. Results showed that nonylphenol caused apoptosis via the activation of caspase-3 in cultured human gastric cancer (SCM-1) cells. Although nonylphenol could activate the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38 MAPK inhibitor) partially prevented cells from apoptosis. Nonylphenol was also found to induce [Ca2+]i increases and pretreatment with BAPTA/AM, a Ca2+ chelator, prevented nonylphenol-induced [Ca2+]i increases, and protect cells from death. These results suggest that nonylphenol induced apoptosis via a Ca2+- and p38 MAPK-dependent pathway. On the other hand, the effect of the environmental contaminant bisphenol A on cytosolic free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells is unclear. This study explored whether bisphenol A changed basal [Ca2+]i levels in suspended MDCK cells by using fura-2 as a Ca2+-sensitive fluorescent dye. Bisphenol A at concentrations between 50-300 £gM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Bisphenol A induced Mn2+ influx, leading to quench of fura-2 fluorescence suggesting Ca2+ influx. This Ca2+ influx was inhibited by phospholiapase A2 inhibitor aristolochic acid, store-operated Ca2+ channel blockers nifedipine and SK&F96365; and protein kinase C inhibitor GF109203X. In Ca2+-free medium, pretreatment with the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) and the endoplasmic reticulum Ca2+ pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited bisphenol A-induced Ca2+ release. Conversely, pretreatment with bisphenol A abolished thapsigargin (or BHQ)- and CCCP-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 abolished bisphenol-induced [Ca2+]i rise. Bisphenol A caused concentration-dependent decrease in cell viability via apoptosis in a Ca2+-independent manner. Collectively, in MDCK cells, bisphenol A induced [Ca2+]i rises by causing phospholipase C-dependent Ca2+ release from the endoplasmic reticulum and mitochondria and Ca2+ influx via phospholipase A2-protein kinase C-sensitive store-operated Ca2+ channels. Key words: calcium, apoptosis, human gastric cancer cells (SCM-1), Madin Darby canine kidney (MDCK), nonylphenol, bisphenol A.

Although synthetic analogs of alkylglycerol (AG), such as dodecylglycerol, possess potent biological activities, their mechanism of action has not been determined. We recently detected substantial amounts of AG in unstimulated MDCK cells (Warne, T.R. and Robinson, M. (1991) Anal. Biochem. 198, 302-307) raising the possibility mediator. In this study, we examined the effects of synthetic AG on the release of arachidonic acid and arachidonate metabolites (AA) from Madin Darby canine kidney (MDCK) cells in response to 12-O-tetradecanoylphorbol-13-acetate (TPA) in order to characterize its effects on this signalling pathway. Treatment of MDCK with AG potently inhibited the release of AA during subsequent stimulation with TPA. Dodecylglycerol, the most effective of a series of alkylgycerols tested, was active at concentrations as low as 3 μM. The sn-1 and sn-3 forms of AG were found to be equally potent inhibitors. The effects of AG on AA release were not the result of arachidonic acid redistribution among cellular lipids and were independent of the phospholipid source of the released AA. AG did not inhibit the release of AA from MDCK cells when bradykinin was used as a stimulus, indicating selectivity for the effects produced by phorbol esters. These results show that AG can function as a potent and specific inhibitor of TPA-mediated AA release. The ability of AG to regulate this signalling pathway in intact MDCK cells, together with its natural occurrence, suggests a potential bioregulatory role for the endogenous compound as an inhibitor of protein kinase C.