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This study is the first to examine the increased apoptosis in the adult rat ovary after lactational exposure to coumestrol (COU), a potent phytoestrogen. Lactating dams were gavaged at doses of 0.01, 0.1, 1, and 10 mg/kg COU during the lactation period and the reproductive effects of female pups were investigated in young adults. Rats were sacrificed at postnatal days (PND) 81-84. Ovarian weights were reduced significantly at 0.1 and 1.0 mg/kg COU. The reduction in the ovarian weight occurred in parallel with an increase in the apoptosis at PND 135-140. A marked dose-dependent increase in the expressions of active caspase-3 and -7 was observed in ovarian granulosa cells. Immunostaining for active caspase-3 and the TUNEL staining of apoptotic cells were also increased in ovaries exposed to COU in a dose-dependent manner. These results suggest new sights into the effect of lactational exposure to COU on the female reproductive health.

Di(2-ethylhexyl) phthalate (DEHP) is a well-known hepatic and reproductive toxicant whose toxicity may be mediated by peroxisome proliferators-activated receptor (PPAR). This study examined the effects of DEHP on the expression of PPAR-regulated genes involved in testicular cells apoptosis. Sprague-Dawley male rats were treated orally with 250, 500, or 750 mg/kg/d DEHP for 28 d, while control rats were given corn oil. The levels of cell cycle regulators (pRb, cyclins, CDKs, and p21) and apoptosis-related proteins were analyzed by Western blot analysis. The role of PPAR-gamma (PPAR-gamma), class B scavenger receptor type 1 (SR-B1), and ERK1/2 was further studied to examine the signaling pathway for DEHP-induced apoptosis. Results showed that the levels of pRB, cyclin D, CDK2, cyclin E, and CDK4 were significantly lower in rats given 500 and 750 mg/kg/d DEHP, while levels of p21 were significantly higher in rat testes. Dose-dependent increases in PPAR-gamma and RXRalpha proteins were observed in testes after DEHP exposure, while there was a significant decrease in RXRgamma protein levels. In addition to PPAR-gamma, DEHP also significantly increased SR-B1 mRNA and phosphorylated ERK1/2 protein levels. Furthermore, DEHP treatment induced pro-caspase-3 and cleavage of its substrate protein, poly(ADP-ribose) polymerase (PARP), in a dose-dependent manner. Data suggest that DEHP exposure may induce the expression of apoptosis-related genes in testes through induction of PPAR-gamma and activation of the ERK1/2 pathway.

Efforts have been made to develop a chemoprevention strategy that selectively triggers apoptosis in malignant cancer cells. Previous studies showed that capsaicin, the major pungent ingredient of red pepper, had differential effect between normal and transformed cells. As an approach to unveil the molecular mechanism by which capsaicin selectively induces apoptosis in transformed cells, we investigated the effect of capsaicin in nontransformed and ras-transformed cells of a common origin: parental (MCF10A) and H-ras-transformed (H-ras MCF10A) human breast epithelial cells. Here, we show that capsaicin selectively induces apoptosis in H-ras-transformed cells but not in their normal cell counterparts. The capsaicin-induced apoptosis, which is dependent on ras transformation, involves the activity of DEVDase (caspase-3 like). In H-ras MCF10A cells, capsaicin treatment markedly activated c-Jun N-terminal protein kinase (JNK)-1 and p38 matigen-activated protein kinase (MAPK) while it deactivated extracellular signal-regulated protein kinases (ERKs). The use of kinase inhibitors and overexpression of dominant-negative forms of MAPKs demonstrated a role of JNK-1 and p38, but not that of ERKs, in apoptosis induced by capsaicin in H-ras-transformed MCF10A cells. Based on the present study, we propose that capsaicin selectively induces apoptosis through modulation of ras-downstream signaling molecules in ras-activated MCF10A cells. Taken in conjunction with the fact that uncontrolled ras activation is probably the most common genetic defect in human cancer cells, our finding may be critical to the chemopreventive potential of capsaicin and for developing a strategy to induce tumor cell-specific apoptosis., (Copyright 2002 Wiley-Liss, Inc.)

The aim of the present study was to determine whether hexabromocyclododecane (HBCD) or 4-nonylphenol (NP) may induce prostatic cancer progression in LNCaP cells. Androgenic effects of HBCD and NP were examined in LNCaP prostate cancer cells expressing androgen receptors (ARs). HBCD and NP increased LNCaP cell viability similar to dihydrotestosterone (DHT) by MTT assay. This phenomenon was reversed by treatment with Casodex, an AR antagonist, suggesting that they act as xenoandrogens via AR signaling pathway. In cell migration assay, HBCD and NP also enhanced LNCaP cell migration similar to DHT. To elucidate underlying mechanisms of their actions on LNCaP, transcriptional levels of cell cycle- and apoptosis-related markers, including cyclin D1, cyclin E, p27, bcl-2, and bax, were determined by reverse transcription (RT)-PCR. An increase in expression cyclin D1 and cyclin E and reduction in p27 and bax mRNA levels were observed by their treatments. Western blot assay showed their alterations in translational levels of cyclin D1, cyclin E, p21, bax, and cathepsin D. Expressions of genes related to a G1/S transition of cell cycle and cathepsin D were elevated, while expression of p21 and bax was decreased. Taken together, these results indicate that HBCD and NP may enhance progression of prostate cancer by modulating growth and migration of LNCaP prostate cells by acting on cell cycle, apoptosis, and metastasis. [ABSTRACT FROM AUTHOR]

As a phytoestrogen, kaempferol (Kaem) is one of bioflavonoids, which are found in a variety of vegetables including broccoli, tea and tomato. In this study, the antiproliferative effects of Kaem in triclosn (TCS)-induced cell growth were examined in MCF-7 breast cancer cells. TCS promoted the cell viability of MCF-7 cells via estrogen receptor α (ERα) as did 17β-estradiol (E2), a positive control. On the other hand, Kaem significantly suppressed E2 or TCS-induced cell growth. To elucidate the molecular mechanisms of TCS and Kaem, alterations in the expressions of cell cycle, apoptosis and metastasis-related genes were identified using western blot assay. The treatment of the cells with TCS up-regulated the protein expressions of cyclin D1, cyclin E and cathepsin D, while down-regulated p21 and bax expressions. Kaem reversed TCS-induced gene expressions in an opposite manner. The phosphorylation of IRS-1, AKT, MEK1/2 and ERK was increased by TCS, indicating that TCS induced MCF-7 cell proliferation via nongenomic ER signaling pathway associated with IGF-1R. Kaem presented an antagonistic activity on this signaling by down-regulating the protein expression of pIRS-1, pAkt and pMEK1/2 promoted by E2 or TCS. In an in vivo xenografted mouse model, tumor growth was induced by treatment with E2 or TCS, which was identified in the measurement of tumor volume, hematoxylin and eosin staining, bromodeoxyuridine and immunohistochemistry assay. On the contrary, E2 or TCS-induced breast tumor growth was inhibited by co-treatment with Kaem, which is consistent with in vitro results. Taken together, these results revealed that Kaem has an anticancer effect against procancer activity of E2 or TCS, a xenoestrogen, in breast cancer and may be suggested as a prominent agent to neutralize breast cancer risk caused by TCS. [ABSTRACT FROM AUTHOR]

Abstract: The non-animal in vitro test methods, especially for assessment of kidney toxicity, have become invaluable tools due to the target organ-selective nature of many nephrotoxic xenobiotics. In vitro evaluation of biomarkers for nephrotoxicity assessment using human cell lines, which can provide more reliable information for toxicological risk evaluation in humans than animal cells, has not been well established to date. The present study investigated the potential use of biomarkers for cisplatin-induced nephrotoxicity assessment in vitro using HK-2 cells derived from human kidney proximal tubule epithelial cells. Cisplatin induced apoptosis of HK-2 cells in which down-regulation of Bcl-2 and activation of caspase-3 were possibly involved. We investigated the effect of cisplatin on the protein levels of kidney injury molecule (KIM)-1, clusterin, calbindin, tissue inhibitor of metalloproteinase (TIMP)-1, cystatin C (CysC), β2-microglobulin (β2-M) and neutrophil gelatinase associated lipocalin (NGAL), which have been recently identified as in vivo biomarkers of nephrotoxicity. The protein levels of KIM-1, calbindin and TIMP-1 were significantly increased in the conditioned media of HK-2 cells treated with cisplatin, while β2-M, CysC, NGAL and clusterin were not affected by cisplatin treatment. The mRNA levels of KIM-1, calbindin and TIMP-1 were increased by cisplatin, indicating that cisplatin-induced up-regulation involves transcriptional activation. The levels of KIM-1, calbindin and TIMP-1 were significantly increased in urine of cisplatin-treated rats, providing in vivo validation of the in vitro results. Taken together, our results clearly demonstrate that among the known in vivo nephrotoxic biomarkers, KIM-1, calbindin and TIMP-1 can be effectively used as in vitro biomarkers for cisplatin-induced nephrotoxicity using a HK-2 human kidney cell system. [Copyright &y& Elsevier]

Abstract: Acrylamide (ACR) is a well-known neurotoxin in mammalian species that causes neuropathy characterized by ataxia and skeletal muscle weakness. Therefore, ACR-mediated axon damage in the central and peripheral nervous systems is considered to be central-peripheral axonopathy. However, the molecular mechanisms underlying ACR''s toxicity to neural progenitor cells are unknown. This study investigated the adverse effects of ACR on mouse multipotent neural progenitor cells and adult hippocampal neurogenesis. ACR significantly reduced the proliferation of neural progenitor cells, and high ACR concentrations induced apoptotic and necrotic cell death. We found that elevated intracellular levels of reactive oxygen species were involved in ACR-mediated cytotoxicity. Interestingly, the administration of ACR to young mice resulted in a significant decrease in the number of newly generated cells in the dentate gyrus of the hippocampus, suggesting an impairment of adult neurogenesis. These results suggest that ACR''s deleterious effects on the central nervous system are due to the death of neural progenitor cells and impaired adult neurogenesis. [Copyright &y& Elsevier]