Elsevier

Toxicology in Vitro

Volume 18, Issue 5, October 2004, Pages 563-569
Toxicology in Vitro

Property of thimerosal-induced decrease in cellular content of glutathione in rat thymocytes: a flow cytometric study with 5-chloromethylfluorescein diacetate

https://doi.org/10.1016/j.tiv.2004.01.002Get rights and content

Abstract

There is a concern on the part of public health community that adverse health consequences by thimerosal, a preservative in vaccines for infants, may occur among infants during immunization schedule. Therefore, the effect of thimerosal on cellular content of glutathione was examined on thymocytes obtained from 4-week-old rats using a flow cytometer and 5-chloromethylfluorescein diacetate. Thimerosal at concentrations ranging from 1 to 10 μM reduced the cellular content of glutathione in a concentration-dependent manner, and the complete depletion of cellular glutathione was observed when the cells were treated with 30 μM thimerosal. L-Cysteine significantly attenuated the actions of thimerosal to reduce the glutathione content and to increase the intracellular Ca2+ concentration. Prolonged incubation (24 h) with 1–3 μM thimerosal induced the apoptosis. The cytotoxic action of thimerosal was greatly augmented when the cells suffered oxidative stress induced by H2O2. It may be unlikely that thimerosal exerts potent cytotoxic action under the in vivo condition because the blood concentration of thimerosal after receiving vaccines does not seem to reach micromolar range and nonprotein thiols at micromolar concentrations are present in the blood.

Introduction

Thimerosal has been used as an organomercurial preservative in vaccines to prevent contamination with harmful microbes since early 1930s. There is a recent concern on the part of public health community that adverse health consequences by thimerosal may occur among infants during immunization schedule (Ball et al., 2001; van't Veen, 2001; Westphal and Hallier, 2002; Westphal et al., 2003). In laboratory in vitro studies, thimerosal has been used as a sulfhydryl reagent to modify some of membrane and cellular functions (Bootman et al., 1992; Cai and Sauve, 1997; Marengo et al., 1998; Lang et al., 2000). The action of thimerosal to decrease content of cellular glutathione is probably one of basic actions related to its toxicity because of a following reason. Change in cellular redox status modulates channel and receptor activities in several types of cells (Elliott and Koliwad, 1997; Lipton et al., 1998; Tanaka et al., 1999; Choi and Lipton, 2000; Pessah, 2001). Furthermore, the cell growth and death are related to cellular redox state (Powis et al., 1995; Buttke and Sandstrom, 1995; Hampton and Orrenius, 1998; Mates et al., 2002). Therefore, to elucidate the property of thimerosal-induced action on cellular content of glutathione, we have examined the effect of thimerosal on lymphocytes obtained from rat thymic glands using a flow cytometer with fluorescent dyes.

Section snippets

Reagents

Thimerosal was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Ethylmercury chloride was obtained from Wako Pure Chemical (Osaka, Japan). These were initially dissolved in dimethyl sulfoxide (DMSO) (Wako Pure Chemical, Osaka, Japan). The DMSO solution containing thimerosal or ethylmercury chloride was added into the cell suspension. Maximum test concentration was 30 μM for thimerosal and ethylmercury chloride. DMSO at final concentrations (0.1–0.3%) did not affect viability of rat

Effect of thimerosal on 5-CMF fluorescence of rat thymocytes

To see if thimerosal decreases cellular content, the effect of thimerosal on 5-CMF fluorescence of rat thymocytes was examined. As shown in Fig. 1, the histogram of 5-CMF fluorescence monitored from 2500 cells was shifted toward a direction of lower intensity in the presence of 3 or 10 μM thimerosal when the cells were incubated with the agent for 60 min. Results indicate that thimerosal decreased the cellular content of nonprotein thiol, glutathione, in rat thymocytes. Thimerosal seemed to

Toxicity of thimerosal suggested by in vitro studies

It is likely that thimerosal at micromolar concentrations increases the vulnerability of cells to oxidative stress because of following reasons. First, micromolar thimerosal concentration-dependently decreased the intensity of 5-CMF fluorescence (Fig. 1, Fig. 2, Fig. 3), indicating the thimerosal-induced decrease in cellular content of glutathione. The complete depletion of cellular glutathione seemed to be observed when the concentration of thimerosal was 10 μM or more. Second, L-cysteine, one

Acknowledgements

This study was partly supported by the research grant awarded to Y. Oyama from the Nippon Life Insurance Foundation, Tokyo, Japan.

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