Elsevier

Toxicology

Volume 156, Issues 2–3, 2 January 2001, Pages 93-100
Toxicology

Interaction of metals and thiols in cell damage and glutathione distribution: potentiation of mercury toxicity by dithiothreitol

https://doi.org/10.1016/S0300-483X(00)00331-0Get rights and content

Abstract

In the present study, we have investigated the effects of extracellular redox status and metal/thiol interactions on glutathione distribution in HeLa cell cultures. No effects were seen on glutathione distribution after the addition of different thiols, whereas the pro-oxidant copper ions affected glutathione distribution in several ways. The addition of dithiothreitol (DTT) but not the other thiols potentiated the effects of mercury ions on glutathione distribution and cell toxicity. In the presence of DTT, increased intra- and extracellular glutathione concentrations were noted already at 0.05 μmol/l, which was below the previously reported toxicity threshold for mercury ions in blood. Likewise DTT potentiated the effects of copper ions on glutathione distribution and cell toxicity, whereas the addition of DTT to cell cultures with a non-metal thiol reactive agent (hydroquinone) or an oxidative agent (hydrogen peroxide) did not affect glutathione distribution or cell toxicity. Thus, it seems as the synergistic effects between DTT and thiol reactive agents only apply to metal ions.

Introduction

The tripeptide glutathione is found in virtually all the aerobic life forms and is present in mammalian tissues at millimolar concentrations and hence accounts for more than 90% of the total non-protein sulphur (Meister, 1988). Its various roles in cellular function and metabolic regulation have been reviewed extensively (Larsson et al., 1987, Meister, 1988, Meister, 1994, Meister and Andersson, 1983).

Gluthathione is synthesized from glutamate, cysteine and glycine by the sequential action of γ-glutamylcysteine synthetase and glutathione synthetase (Meister, 1988, Meister and Andersson, 1983). The availability of cysteine is the limiting factor in glutathione synthesis. The concentration of cellular glutathione may be modulated by a variety of compounds (Larsson et al., 1987, Meister, 1988, Meister, 1994, Meister and Andersson, 1983). It is well recognized that depletion of glutathione is caused by several factors, such as inhibition of its synthesis (e.g. by buthionine sulfoximine, BSO), conjungation to electrophilic xenobiotics (e.g. diethylmaleate), and oxidative stress. In the absence of additional stress factors, even severe loss of glutathione due to BSO or diethylmaleate treatment does not markedly influence the important cell functions (Toborek et al., 1995).

Ions of metals such as mercury and copper are known to exhibit a high affinity for thiol groups (Li and Manning, 1955, Jocelyn, 1972, Vallee and Ulmer, 1972, Cooper, 1983). Interaction of metals with glutathione metabolism is an integral part of the toxic response of many metals. Glutathione forms complexes with several heavy metal ions and thus functions in the protection of cells against metal toxicity.

We have recently (Hultberg et al., 1999) shown that glutathione production is stimulated in the presence of agents that form complex/adducts with glutathione (e.g. thiol reactive metals such as mercury ions or quinones), whereas no increase of glutathione production was observed after addition of agents, which preferentially exerted an oxidative stress (hydrogen peroxide, homocysteine). The aim of the present study was to extend the previous findings (Hultberg et al., 1999) by investigating the impact of different thiols or oxidative agents (which cause a change of extracellular redox status) on glutathione metabolism after long-term (3-day) exposure. The thiols used as reductive agents were homocysteine, N-acetylcysteine and dithiothreitol (DTT). Copper and selenite ions are reported to be excellent catalysts (Jocelyn, 1972) for thiol oxidation, and both these ions were used as oxidative agents in the present study. Since the effect of metal ions on cell toxicity and glutathione metabolism has been reported to be influenced by the presence of thiols (Bohets et al., 1995, Quig, 1998, Divine et al., 1999), we have in the present study also investigated the effect on glutathione metabolism exerted by mercury and copper ions in the presence of thiols.

Section snippets

Cell culture

The established HeLa cell culture (epitheloid cells) was obtained from American Type Culture Collection, Rockville, MD, USA. The cells were cultured at 37°C in 75-cm2 flasks, in 12-ml of RPMI 1640 (Gibco Laboratories, Santa Clara, CA, USA) containing 10% fetal calf serum (FCS) (Gibco Laboratories). The cells were grown in a humidified air with 5% CO2. The cells were routinely screened for and shown to be free from mycoplasma using a kit with a specific DNA probe from Gen-Probe Inc., San Diego,

Recovery of extracellular glutathione

Reduced glutathione (50 μmol/l) was added to medium containing 10% FCS and incubated for 24 h without contact with HeLa cells. Reduced and total glutathione were measured. Added glutathione was recovered in the assay of total glutathione and no glutathione was present in its reduced form. The addition of copper ions (10 and 100 μmol/l), mercury ions (1 μmol/l), homocysteine (100 and 500 μmol/l), hydroquinone (5 μmol/l) or N-acetylcysteine (2000 μmol/l) under these conditions did not change the

Recovery of extracellular glutathione

The experiments with glutathione and different agents added to medium (without cells) showed that the concentration of DTT (6000 μmol/l) used in the assay of total thiols (Andersson et al., 1993, Hultberg et al., 1997a, Hultberg et al., 1997b) is able to break all types of complexes and disulphide bonds between glutathione and the agents tested. Therefore, the effects observed on glutathione distribution after addition of the different agents are valid and not disturbed by the interference from

Acknowledgements

This work was supported by grants from the Swedish National Association against Heart and Chest Disease and the Albert Påhlsson Foundation.

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