Interaction of metals and thiols in cell damage and glutathione distribution: potentiation of mercury toxicity by dithiothreitol
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.
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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.
References (32)
- et al.
Cytotoxicity of mercury compounds in LLC-PKI, MDCK and human proximal tubular cells
Kidney Int.
(1995) - et al.
The role of glutathione in copper metabolism and toxicity
J. Biol. Chem.
(1989) - et al.
Mechanisms of the reaction of some copper complexes in the presence of DNA with superoxide anion, hydrogen peroxide and molecular oxygen
J. Free Radic. Biol. Med.
(1986) - et al.
Copper ions differ from other metal ions in their effects on the concentration and redox status of thiols in HeLa cell cultures
Toxicology
(1997) - et al.
The cell-damaging effects of low amounts of homocysteine and copper ions in human cell line cultures are caused by oxidative stress
Toxicology
(1997) - et al.
Alteration of thiol metabolism in human cell lines induced by low amounts of copper mercury of cadmium ions
Toxicology
(1998) - et al.
Thiol and redox reactive agents exert different effects on glutathione metabolism in HeLa cell cultures
Clin. Chim. Acta
(1999) Export pumps for glutathione S-conjugates
Free Radic. Biol. Med.
(1999)- et al.
Protein measurement with folin phenol reagent
J. Biol. Chem.
(1951) Glutathione metabolism and its selective modification
J. Biol. Chem.
(1988)