Thimerosal induces oxidative stress in HeLa S epithelial cells

Environmental Toxicology and Pharmacology 22 (2006) 194–199

Thimerosal induces oxidative stress in HeLa S epithelial cells Seunghwan Lee a , Md. Firoz Mian b , Hu-Jang Lee a , Chung-boo Kang a , Jong-Shu Kim a , Sung Ho Ryu b , Pann-Ghill Suh b , Euikyung Kim a,∗ a

Laboratory of Toxicology, Institute of Animal Medicine, College of Veterinary Medicine, Gyeongsang National University, Gajwa-Dong, Jinju 660-701, Republic of Korea b Department of Life Science, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea Received 11 November 2005; accepted 14 March 2006 Available online 28 March 2006

Abstract Thimerosal is one of the most widely used preservatives and is found in a variety of biological products, including vaccines, contact lens cleaning solutions, and cosmetics. It has been reported to have harmful effects on epithelial tissues, such as causing conjunctivitis or contact dermatitis. However, the molecular mechanism of its toxicity has not been characterized using epithelial tissues. In the present study, we report that reactive oxygen species play a key role in thimerosal-induced cytotoxicity in HeLa S epithelial cells. Thimerosal significantly reduced HeLa S cell viability and it was associated with a decrease in intracellular glutathione levels. Flow cytometric cell cycle analysis showed a marked increase in the hypodiploidic cell population, indicating apoptosis of thimerosal-treated cells. The apoptotic cell death of epithelial cells was confirmed by observing a significant increase of caspase-3 activity in the cytosolic fraction of the treated cells. Thimerosal also induced a concentration-dependent increase of genomic DNA fragmentation, a biochemical hallmark of apoptosis. Hoechst 33342 nuclear staining demonstrated apoptotic-fragmented multinuclei in thimerosal-treated cells. All the thimerosal-mediated toxic responses observed in the present study were almost completely suppressed by pretreating cells with N-acetyl-l-cysteine, a radical scavenger. Taken together, these results suggest for the first time that epithelial cytotoxicity of thimerosal is mediated by oxidative stress. © 2006 Elsevier B.V. All rights reserved. Keywords: Thimerosal; Epithelial toxicity; Reactive oxygen species; Glutathione; Caspase-3

1. Introduction The antiseptic and antimicrobial activities of thimerosal have led to its use as a preservative in biological products, including vaccines, cleaning solutions for contact lenses, and cosmetics since the 1930s. However, there have been several reports that thimerosal has potential side effects, including causing inflammatory diseases (Clarkson, 2002; Lopez Bernal and Ubels, 1991). In particular, it has been speculated that thimerosal preservative in juvenile vaccines is a causative factor of autism in the vaccinated children (van’t Veen, 2001); however, this idea is still the subject of much debate. Recently, the mechanisms of this potential side effect have been illustrated in neuronal cells (Ball et al., 2001; Geier and Geier, 2004; James et al.,

∗

Corresponding author. Tel.: +82 55 751 5812; fax: +82 55 751 5803. E-mail address: [email protected] (E. Kim).

1382-6689/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2006.03.003

2005) and T-lymphocytes (Lebrec et al., 1999; Makani et al., 2002). In a previous study, we have directly demonstrated for the first time that thimerosal can generate reactive oxygen species (ROS), including hydrogen peroxide in cultured mammalian cells (Kim et al., 2002a). The ROS generation stimulates focal adhesion kinase and cytoskeletal rearrangement, resulting in the typical morphological changes observed in thimerosal treatment. It appears to generate not only hydrogen peroxide but also other species of ROS (E. Kim, unpublished data). Apparently, thimerosal-induced intracellular calcium upregulation is also dependent on the generation of ROS by thimerosal (Kim et al., 2002b). The side effects of thimerosal on epithelial tissues, such as conjunctivitis and contact dermatitis, have been previously reported (Garner, 2004; Belsito, 2002; Pratt et al., 2004; Wantke et al., 1994; Lebrec et al., 1999). However, the molecular mechanism of its toxicity has not been clearly elucidated in epithelial tissue. While the adverse effects of thimerosal on an epithe-

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lial tissue are generally due to chronic or repetitive exposure, most biochemical studies of thimerosal have reported results of the short-term observations obtained within an hour of the exposure. For the present study, we examined the potentially harmful effects of thimerosal using a model of 24 h of exposure in HeLa S epithelial cells. Using biochemical methods, we determined the extent of apoptotic cell death in epithelial cells that resulted from a prolonged treatment of thimerosal. The cytotoxicity was directly related to the cellular depletion of reduced glutathione, which is presumably caused by thimerosal-induced ROS generation. We believe these results suggest oxidative stress is a biochemical mechanism causing chronic cytotoxicity of thimerosal in epithelial tissues. 2. Materials and methods 2.1. Chemicals Thimerosal (mercury-[(o-carboxyphenyl)thio]ethyl sodium salt), N-acetyll-cysteine (NAC), Hoechst 33342, sodium phosphate, o-phthalaldehyde (OPT), and thiosalicylic acid were purchased from Sigma (St. Louis, MO). BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N ,N -tetraacetic acid tetrakis(acetoxymethyl ester)) was obtained from Molecular Probes Inc. (Eugene, OR). Trypan blue was purchased from Gibco BRL (Grand Island, NY). Z-VAD-FMK (n-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone) was obtained from Enzyme Systems Products (Livermore, CA).

2.2. Cell culture and thimerosal treatment HeLa S cells were maintained in Dulbecco’s Modified Eagle’s Medium (Biowhittaker; Walkersville, MD) supplemented with 10% heat-inactivated bovine calf serum (HyClone; Logan, UT) containing 100 U/ml of penicillin and 100 ␮g/ml of streptomycin at 37 ◦ C in a humidified, CO2 -controlled (5%) incubator. HeLa S cells cultured overnight in P60 dishes were treated with vehicle alone or thimerosal for the indicated period of times. Effects of inhibitors were examined by the preincubation of cells with an inhibitor for 1 h before thimerosal treatment.

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100,000 × g supernatant was added into 0.9 ml of the phosphate–EDTA buffer for dilution. The final assay mixture (2.0 ml) contained 100 ␮l of the above diluted supernatant, 1.8 ml of phosphate–EDTA buffer, and 100 ␮l of the OPT solution (containing 100 ␮g of OPT in reagent-grade absolute methanol). After thorough mixing and incubation at room temperature for 15 min, the fluorescence was determined (excitation of 350 nm, emission at 420 nm) using a fluorometer.

2.5. Flow cytometry analysis Exponentially growing HeLa S cells (1 × 106 ) were exposed to thimerosal (10 ␮M) for 24 h, then harvested by trypsinization and washed with PBS (pH 7.4). The cells were fixed with ice-cold 70% ethanol for 30 min. The fixed cells were washed with PBS and treated with 1 ml (5 ␮g/ml) of RNase A solution at 37 ◦ C for 30 min. The cells were harvested by centrifugation at 400 × g for 5 min and stained with 250 ␮l (5 ␮g/ml) of propidium iodide (Molecular Probes) at room temperature for 30 min in the dark. After adding 750 ␮l of PBS, the DNA content in each cell cycle phase was analyzed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). DNA histograms were obtained using a CellQuest apparatus and CellQuest software (Becton Dickinson, San Jose, CA). Electronic gates were set for viable and apoptotic cells with 2–4N DNA and subnormal DNA contents, respectively, and for exclusion of debris. Data on 10,000 cells were collected for analysis and the results presented as a percentage of the total cell counts.

2.6. Caspase-3 activity assay Exponentially growing HeLa S cells (1 × 106 ) were incubated for 1 h in the absence or presence of various concentrations of Z-VAD-FMK, then treated for an additional 24 h with thimerosal (10 ␮M). The cells were harvested and washed with PBS, then sonicated in assay buffer (100 mM Hepes, 10% sucrose, 5 mM dithiothreitol, 10−6 % NP-40, and 0.1% CHAPS at pH 7.25). Following centrifugation at 20,000 × g for 10 min, 20 ␮g (Bradford protein assay) of the supernatant protein was added to each well of a 96-well plate along with 50 ␮M N-acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (DEVD-AMC; Biomol, Plymouth Meeting, PA). After incubation at 37 ◦ C for 1 h, cleaved free AMC (excitation of 355 nm, emission of 460 nm) was detected using a fluorometer.

2.7. DNA fragmentation measurement 2.3. Cell viability Following the indicated periods of incubation with thimerosal, the percentage of cell death was determined by a trypan blue exclusion assay. Briefly, the detached cells were harvested by centrifuging the media and the attached cells on culture dishes were harvested by trypsinization and centrifugation. The detached cells and the attached cells from each treatment were washed with phosphate buffered saline (PBS), then these two populations from each treatment were pooled together for assay. Equal (50 ␮l) volumes of cell suspension and trypan blue solution were mixed together and the cells were counted using a hemacytometer under a light microscope.

2.4. Determination of cellular glutathione The level of cellular glutathione was determined by the method of Hissin and Hilf (1976), with a modification made to the assay for its use in cell culture studies. Briefly, HeLa S cells in P60 dishes (about 50–70% confluency) were incubated for 24 h with either vehicle alone or thimerosal in the presence or absence of 20 mM NAC. The treated cells were harvested by trypsinization and centrifugation followed by washing with PBS. The cells were resuspended in 375 ␮l of ice-cold phosphate-EDTA buffer (0.1 M sodium phosphate, 0.005 M EDTA, pH 8.0, prepared daily) and sonicated (30 s) on ice for three times in 1 min intervals. The protein in the cell extract was precipitated by the addition of 100 ␮l of 25% HPO3 . The total homogenate was centrifuged at 100,000 × g at 4 ◦ C for 30 min and the resulting supernatant was used for the determination of cellular glutathione content. For the glutathione assay, 100 ␮l of the

Exponentially growing HeLa S cells were incubated for 24 h with indicated concentrations of thimerosal, then harvested and washed with PBS. Following gentle lysis in a buffer containing 5 mM Tris–HCl (pH 7.4), 20 mM EDTA, and 0.5% Triton X-100, the cell lysates were centrifuged at 20,000 × g for 5 min to collect the supernatant containing the soluble fragmented DNA. The isolated DNA was cleaned up with phenol, phenol:chloroform:isoamyl alcohol (25:24:1), and chloroform, then incubated with 50 ␮g/ml RNase A at 37 ◦ C for 30 min. The enzyme reaction was stopped with phenol:chloroform:isoamyl alcohol (25:24:1). The DNA fragments were precipitated with ethanol in the presence of 0.3 M sodium acetate, pH 5.2, then resuspended and subjected to 1.8% agarose gel electrophoresis.

2.8. Staining of nuclei Exponentially growing HeLa S cells (5 × 105 ) were treated with thimerosal, then harvested by trypsinization and washed with PBS. The cells were stained with the DNA-specific dye Hoechst 33342 for 5 min. The stained cells were mounted on glass slides and the fluorescent nuclei viewed through a Zeiss fluorescence microscope.

2.9. Statistical analysis The results are expressed as means ± standard deviation (S.D.). A paired Student’s t-test was used to assess the significance of differences between two mean values. P < 0.05 was considered to be statistically significant.

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3. Results 3.1. Epithelial cell toxicity We have previously reported that thimerosal can acutely (within a range of minutes) generate hydrogen peroxide and other ROS in HeLa S epithelial cells (Kim et al., 2002a). In the present study, we have examined if prolonged (24 h) exposure to thimerosal results in oxidative stress-associated cytotoxicity in epithelial cells. For assessing the potency of its cytotoxicity, HeLa S cells were treated with indicated concentrations of thimerosal and thiosalicylic acid for 24 h. Thiosalicylic acid is a structural analogue of thimerosal and lacks the ethylmercury moiety (Fig. 1A). Compared with thiosalicylic acid, which exhibited no evident cytotoxicity up to 100 ␮M, thimerosal significantly increased cell death in a concentration-dependent manner starting at a low micromolar concentration (Fig. 1B). In light microscopic examinations, thimerosal-treated cells displayed marked changes in morphological features with increased rounding up of cells and fragmented cell debris in the whole culture dish (Fig. 1C). These cytotoxic effects of thimerosal were almost completely prevented by the pretreatment of cells with N-acetyl-l-cysteine (NAC), a membarane-permeable radical scavenger. 3.2. Cellular glutathione Glutathione is the most common low molecular weight sulfhydryl-containing compound in mammalian cells and is present in millimolar amounts in most cells (Sen, 1997). It

Fig. 2. Thimerosal depletes the intracellular glutathione in HeLa S epithelial cells. HeLa S cells were incubated for 1 h in the presence or absence of NAC (20 mM), then treated for an additional 24 h with vehicle alone (control) or 3, 10, or 30 ␮M of thimerosal, respectively. The cellular levels of GSH were determined as described in Section 2. * P < 0.05 vs. control.

protects cells from ROS-mediated cellular damage that can be induced by various kinds of toxic agents. Prolonged (24 h) exposure of HeLa S cells to thimerosal dramatically depleted cellular glutathione in a concentration-dependent manner (Fig. 2). 3.3. Apoptosis These results prompted us to examine if thimerosal-treated cells undergo apoptotic cell death. From the cell cycle analyses, we found that thimerosal induced a decrease in G1 -phase cells and an increase in hypodiploid cells (Fig. 3; Table 1). These results suggest that thimerosal-induced apoptotic cell

Fig. 1. The cytotoxicity of thimerosal in HeLa S epithelial cells. (A) Chemical structures of thimerosal and thiosalicylic acid. (B) HeLa S cells were incubated for 24 h in the absence (vehicle alone) or the presence of various concentrations of thimerosal (TM) or thiosalicylic acid (TSA), respectively. Then, cell viability was determined by a trypan blue dye exclusion assay. (C) HeLa S cells were preincubated for 1 h in the absence or presence of BAPTA-AM (10 ␮M) or NAC (20 mM), then treated for an additional 24 h with vehicle alone (control) or 10 ␮M of thimerosal. The treated cells were examined and photographed using light microscopy. The results shown are representative of four independent experiments.

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Fig. 3. Cell cycle analyses of treated HeLa S epithelial cells. HeLa S cells were incubated for 1 h in the presence or absence of 20 mM NAC, then treated for additional 24 h with vehicle alone (control) or 10 ␮M of thimerosal, respectively. The stage of the cell cycle found in treated cells was determined using a fluorescence cell sorter as described in Section 2. The results shown are representative of three independent experiments. The results from these experiments are also shown in Table 1, presented as a percentage of the total cells. Table 1 Cell cycle analysis of treated cells Cell cycle

Treatment (24 h) Vehicle, %

Hypodiploid G1 S G2 , M

This result suggests that caspase-3 activation plays an important role in thimerosal-induced apoptosis of epithelial cells.

6 46 8 40

± ± ± ±

2 6 3 6

Thimerosal, % 21 19 11 49

± ± ± ±

5* 4* 2 8

NAC + thimerosal, % 7 51 9 33

± ± ± ±

2 6 3 5

Results show the relative percent of the total cell population in a particular stage of the ell cycle analyzed after 24 h of each treatment represented in Fig. 3. See Fig. 3 for experimental details. Results are reported as mean ± S.D. of three independent experiments. * P < 0.05 vs. respective vehicle.

death with a marked increase in fragmented cell nuclei. Generally, procaspase activation is a key step in the process of cellular apoptosis, with the activation of procaspase-3 to caspase-3 being especially crucial. To confirm the presence of apoptosis, we examined if procaspase-3 is activated by the prolonged thimerosal treatment (Fig. 4). The increase in caspase-3 activity was suppressed by the preincubation of cells with Z-VAD-FMK, a pan-caspase inhibitor, in a concentration-dependent manner.

Fig. 4. Thimerosal treatment induces caspase-3 activation in HeLa S epithelial cells. HeLa S cells were incubated for 1 h in the absence or presence of various concentrations of Z-VAD-FMK, then treated for an additional 24 h with vehicle alone or 10 ␮M of thimerosal, respectively. The caspase-3 activities of the cells were determined as described in Section 2. The results are expressed as mean ± S.D. for three independent experiments. * P < 0.05 vs. thimerosal alone.

3.4. DNA and nuclei staining For further proof of the presence of apoptosis, we examined the thimerosal-treated HeLa S cells for genomic DNA fragmentation, a biochemical hallmark of apoptosis. We observed a concentration-dependent increase of DNA fragmentation in thimerosal-treated cells (Fig. 5). In addition, Hoechst 33342 nuclear staining showed the apoptotic-fragmented multinuclei in thimerosal-treated cells (Fig. 6). All the thimerosal-mediated cytotoxic responses observed in the present study were almost completely suppressed by pretreating cells with the radical scavenger NAC. Taken together, these results suggest that epithelial cytotoxicity of thimerosal is likely to be mediated by thimerosalinduced oxidative stress in epithelial cells.

Fig. 5. Apoptotic features of thimerosal-induced cell death in HeLa S epithelial cells. HeLa S cells were incubated for 1 h in the absence or presence of 20 mM NAC, then treated for an additional 24 h with indicated concentrations of thimerosal. The DNA was prepared from the treated cells as described in Section 2 and subjected to 1.8% agarose gel electrophoresis. SM indicates DNA molecular weight size markers. The results shown are representative of three independent experiments.

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Fig. 6. Apoptotic features of thimerosal-induced cell death in HeLa S epithelial cells. HeLa S cells were incubated for 1 h in the absence or presence of 20 mM NAC, then treated for additional 24 h with 10 ␮M of thimerosal. The cells were washed with PBS and stained with Hoechst 33342. The fluorescent nuclei were viewed through a Zeiss fluorescence microscope. Arrows indicate the apoptotic-fragmented multinuclei. The results shown are representative of three independent experiments.

4. Discussion As an ophthalmic preservative, thimerosal has been clinically used at the concentration of 0.001–0.004%, which is equal to 25–100 ␮M in molarity. In vaccine products, thimerosal is currently used as a preservative at concentrations of 0.0002–0.01%, which is equal to 5–250 ␮M in molarity. Thimerosal is also used in cosmetics at a concentration of 0.0007%, which is equal to 17.5 ␮M in molarity (Suneja and Belsito, 2001). Therefore, the concentrations of thimerosal used in the present study are within the relevant range so as to make meaningful comparisons to thimerosal effects in its practical applications. Taken together, the present study’s results suggest that a prolonged exposure to thimerosal can induce oxidative stress and related side effects in its practical use. Thimerosal is an ethylmercury-containing thiosalicylic acid, and this mercurial moiety represents 49.9% of the molecular weight of thimerosal. Most mercurial compounds are strongly suspected to cause neurotoxicity in early developmental stages (Costa et al., 2004) as well as contact allergies (Garner, 2004; Belsito, 2002; Pratt et al., 2004). It has been previously proposed that thimerosal is metabolized by cellular enzymes and releases ethylmercury inside cells. In addition, thimerosal allergy patients demonstrated positive patch-test reactions to ethylmercuric chloride, suggesting that ethyl mercury is the responsible agent in allergic responses associated with thimerosal use (Pirker et al., 1993). When compared with methylmercury, tissue accumulation is greater for ethylmercury, which may result in more detrimental effects in individuals exposed to ethylmercury for a long period of time (Magos et al., 1985). Recently, thimerosal has been reported to trigger caspase-3 activation and apoptosis in human neuronal cells and fibroblasts (Baskin et al., 2003). The harmful side effects of thimerosal on epithelial tissues, such as inducing conjunctivitis and contact dermatitis as a contact allergen, have been well-documented; however, the molecular mechanism responsible for the side effects has not previously been properly examined in epithelial tissue. In the present study, we examined if thimerosal-induced ROS generation could result in epithelial cell death. From our results

(Fig. 1C), thimerosal-induced cell death was completely inhibited by NAC, a ROS scavenger, suggesting ROS play a pivotal role in the thimerosal-mediated cell death mechanism. Another potential mechanism of thimerosal toxicity in epithelial cells is the alteration of intracellular calcium homeostasis, which has been previously observed in various cell types (Gould et al., 1987; Elferink, 1999; Kim et al., 2002b). As shown in Fig. 1C, an intracellular calcium chelator, BAPTA-AM could not block thimerosal-mediated cell death in epithelial HeLa S cells. Our findings indicate that ROS generation but not calcium overload might play a major role in thimerosal cytotoxicity in epithelial tissue. In general, mercury exhibits high-affinity binding to the thiol (-SH) group, which is found in glutathione and thiol-containing proteins (Patrick, 2002). Thus, it can be speculated that preincubation with NAC may bind and scavenge thimerosal added into the medium, resulting in the decrease of free thimerosal. To examine this possibility, we tested the effects of posttreatment with NAC after the incubation of cells in the presence of thimerosal. We also examined the protective effects of NAC by replacing the NAC-containing medium before adding thimerosal. From the both tests, we observed that NAC still had significant protective effects for the thimerosal-mediated cell death in a time-dependent manner (data not shown). These data suggested that the protective effects of NAC in the present study were not the result of the scavenging of extracellular thimerosal, but mainly by the protection mechanism against ROS generated by thimerosal inside cells. These results were also confirmed by observing the cellular level of glutathione, in which thimerosal-treated cells showed a significant depletion of cytosolic glutathione in a concentrationdependent manner (Fig. 2). The thimerosal-induced cytosolic glutathione depletion was completely prevented by preincubation with NAC. Glutathione has specific roles in protecting the body from mercury toxicity (Patrick, 2002). Briefly, glutathione binds with mercury and consequently forms a complex that prevents mercury from binding to cellular proteins and causing damage to enzymes and tissues, then it is transported and eliminated from the body (Kromidas et al., 1990; Zalups, 2000). Glutathione also increases the anti-oxidant capacity of the cell,

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providing a defense against hydrogen peroxide, singlet oxygen, hydroxyl radicals, and lipid peroxides produced by mercury (Kromidas et al., 1990). As a source of a cellular cysteine pool, NAC can be converted to glutathione by glutathione synthetase inside cells. Therefore, either NAC or glutathione may work as an anti-oxidant (Kelly, 1998). In the present study, the pretreatment with NAC is likely to protect cells either by directly forming a complex with the mercurial component or by increasing the cellular anti-oxidant capacity against ROS generation. The present study, we believe, proposes a possible mechanism of action for thimerosal, which may play an important role in causing its side effects in epithelial tissues. These results also suggest some pharmacological intervention approaches for preventing the thimerosal-mediated adverse effects in epithelial tissue. References Ball, L.K., Ball, R., Pratt, R.D., 2001. An assessment of thimerosal use in childhood vaccines. Pediatrics 107 (5), 1147–1154. Baskin, D.S., Ngo, H., Didenko, V.V., 2003. Thimerosal induces DNA breaks, caspase-3 activation, membrane damage, and cell death in cultured human neurons and fibroblasts. Toxicol. Sci. 74 (2), 361–368. Belsito, D.V., 2002. Thimerosal: contact (non)allergen of the year. Am. J. Contact. Dermat. 13 (1), 1–2. Clarkson, T.W., 2002. The three modern faces of mercury. Environ. Health Perspect. 110 (Suppl. 1), 11–23. Costa, L.G., Aschner, M., Vitalone, A., Syversen, T., Soldin, O.P., 2004. Developmental neuropathology of environmental agents. Annu. Rev. Pharmacol. Toxicol. 44, 87–110. Elferink, J.G., 1999. Thimerosal: a versatile sulfhydryl reagent, calcium mobilizer, and cell function-modulating agent. Gen. Pharmacol. 33 (1), 1–6. Garner, L.A., 2004. Contact dermatitis to metals. Dermatol. Ther. 17 (4), 321–327. Geier, D., Geier, M.R., 2004. Neurodevelopmental disorders following thimerosal-containing childhood immunizations: a follow-up analysis. Int. J. Toxicol. 23 (6), 369–376. Gould, G.W., Colyer, J., East, J.M., Lee, A.G., 1987. Silver ions trigger Ca2+ release by interaction with the (Ca2+ -Mg2+ )-ATPase in reconstituted systems. J. Biol. Chem. 262 (16), 7676–7679. Hissin, P.J., Hilf, R., 1976. A fluorometric method for determination of oxidized and reduced glutathione in tissues. Anal. Biochem. 74 (1), 214–226. James, S.J., Slikker III, W., Melnyk, S., New, E., Pogribna, M., Jernigan, S., 2005. Thimerosal neurotoxicity is associated with glutathione

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