Elsevier

Toxicology Letters

Volume 160, Issue 1, 30 December 2005, Pages 76-83
Toxicology Letters

Nonylphenol-induced Ca2+ elevation and Ca2+-independent cell death in human osteosarcoma cells

https://doi.org/10.1016/j.toxlet.2005.06.007Get rights and content

Abstract

The effect of the environmental toxicant nonylphenol on cytosolic free Ca2+ concentration ([Ca2+]i) and proliferation has not been explored in human osteoblast-like cells. This study examined whether nonylphenol alters Ca2+ levels and causes cell death in MG63 human osteosarcoma cells. [Ca2+]i and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. Nonylphenol at concentrations above 3 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 90% by removing extracellular Ca2+. The nonylphenol-induced Ca2+ influx was insensitive to blockade of L-type Ca2+ channel blockers. After pretreatment with 10 μM nonylphenol, 1 μM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) failed to induce [Ca2+]i rises. Inhibition of phospholipase C with 2 μM U73122 did not change nonylphenol-induced [Ca2+]i rises. The nonylphenol-induced [Ca2+]i rises were enhanced or inhibited by phorbol myristate acetate or GF 109203X, respectively. At concentrations of 10 and 20 μM nonylphenol killed 55% and 100% cells, respectively. The cytotoxic effect of 10 μM nonylphenol was unaltered by pre-chelating cytosolic Ca2+ with BAPTA. Collectively, in MG63 cells, nonylphenol induced [Ca2+]i rises by causing Ca2+ release from intracellular stores and Ca2+ influx from extracellular space. Furthermore, nonylphenol can cause Ca2+-unrelated cytotoxicity in a concentration-dependent manner.

Introduction

Endocrine disrupting chemicals are exogenous compounds that mimic or inhibit the action of estrogens or other hormones. Among these chemicals, nonylphenol, an environmental contaminant from various sources including food (Guenther et al., 2002) has been reported to act as a weak estrogen (Ekelund et al., 1990); however, many effects of nonylphenol appear to be via non-estrogenic pathways (Watanabe et al., 2004). In vitro, nonylphenol exerts diverse effects on different cells. This chemical was shown to affect the immune system, such as altering T cell function (Iwata et al., 2004), induce apoptosis (Aoki et al., 2004) and inhibit sarcoplasmic/endoplasmic reticulum-type Ca2+ pumps (Thomson et al., 1994).

The present study was aimed to explore the effect of nonylphenol on [Ca2+]i in osteoblast-like cells by using human MG63 osteosarcoma cells. The effect of nonylphenol on [Ca2+]i and growth in cells has not been studied previously. A regulated rise in cytosolic free Ca2+ levels ([Ca2+]i) is an important message in all cell types, and can evoke various physiopathological processes (Berridge, 1993, Berridge, 1997, Bootman et al., 2002); but abnormal fluctuations in [Ca2+]i may be cytotoxic (Annunziato et al., 2003). Thus, it is pivotal to investigate the effect of an reagent on cellular Ca2+ handling in order to understand its in vitro action. In this study, MG63 human osteosarcoma cells were applied because previous reports implicate that this line of cell produces robust [Ca2+]i rises upon stimulation of different reagents. MG63 osteosarcoma cells have characteristics similar to human osteoblasts and have been used as a model for research on human osteoblasts (Rezzonico et al., 2002). Many endogenous and exogenous reagents can stimulate MG63 cells by evoking a [Ca2+]i rise, such as 2,2′-dithiodipyridine (Kuo et al., 2003), carvedilol (Liu and Jan, 2004), and desipramine (Jan et al., 2003). The inositol-1,4,5-trisphosphate-sensitive Ca2+ store is an important store that releases Ca2+ into the cytosol when cells are stimulated by endogenous reagents such as histamine (Lee et al., 2001). But exogenous reagents can release Ca2+ from inositol 1,4,5-trisphosphate-insensitive stores (Kuo et al., 2003, Liu and Jan, 2004, Jan et al., 2003).

Using fura-2 as a fluorescent Ca2+ indicator, this study shows that nonylphenol induced [Ca2+]i rises in a concentration-dependent manner in MG63 cells. The time course and the concentration–response relationship, the Ca2+ sources of the Ca2+ signal, the role of phospholipase C and protein kinase C in the signal were explored. The effect of nonylphenol on cell growth and its Ca2+-dependence was examined.

Section snippets

Cell culture

MG63 cells obtained from American Type Culture Collection (CRL-6253) were cultured in Dulbecco's modified Eagle medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin.

Solutions

Ca2+-containing medium contained 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM Hepes, 5 mM glucose, pH 7.4. In Ca2+-free medium, Ca2+ was substituted with 0.1 mM EGTA. Nonylphenol was dissolved in water as a stock solution. The other agents were dissolved in water, ethanol or

Results

Nonylphenol at concentrations above 3 μM increased [Ca2+]i in a concentration-dependent manner in Ca2+-containing medium. Fig. 1A shows the effects of 3–10 μM nonylphenol. At a concentration of 1 μM, nonylphenol had no effect (=baseline). The [Ca2+]i increase induced by 10 μM nonylphenol expressed a rapid initial rise followed by a slower rise that reached 255 ± 2 nM at the time point of 250 s. In the presence of 20 μM nonylphenol, all cells were lysed. Fig. 1C (filled circles) shows the

Discussion

Our study is the first to demonstrate that the environmental toxicant nonylphenol induced a [Ca2+]i rise and Ca2+-independent cell death in MG63 human osteosarcoma cells. The data suggest that nonylphenol increased [Ca2+]i by depleting intracellular Ca2+ stores and causing Ca2+ influx from extracellular milieu because removing extracellular Ca2+ reduced the major portion of nonylphenol-induced [Ca2+]i rise, and that addition of Ca2+ back to cells pretreated with nonylphenol under Ca2+-free

Acknowledgments

This work was supported by grants from Veterans General Hospital-Kaohsiung (VGHKS94G-11) (VGHKS94-054) to CR Jan, and VGHKS94-088 to JL Wang.

References (25)

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