25. HURNÁ,Edita, SKALICKÁ,Magdalena, HURNÁ;,Sylvia: The effect of antioxidants on cytotoxicity of heavy metals (Antioxidánsok hatása nehézfémek citotoxicitására)

25. Hurná

érk: 97.10.16.

UEVM, 04001 Kosice, Hlinkova 1/A, Slovak Republic (first author's address)

ABSTRACT

The aim of our study was to investigate Cd toxicity after pretreatment of L-ascorbic acid (100M) on Anr 4 cell line (rat liver). The cell viability, proliferation and release of lactate dehydrogenase were investigated. Partial protection of L-ascorbic acid in via-bility of cell against 25, 50, 100 M of Cd and against 5 and 10 M of Cd by meas-urement of total protein was observed. Protective effect of ascorbic acid was not ob-served by lactate dehydrogenase release assay.

INTRODUCTION

Certain metals such as chromium, nickel, cadmium and mercury and their compounds have been reported to be potent carcinogenic and/or toxic agents in human and ani-mals. Although these metals produce DNA damage and lipid peroxide in vitro and/or in vivo, the molecular mechanisms of their toxicity have not been adequately studied. Acute exposure to inorganic cadmium produced hepatotoxicity. In contrast, chronic exposure to Cd produces nephrotoxic effects. Recent studies have increasingly implied that the generation of free radical such as active oxygen species, which have well known to produce a number of toxic effects, may at least in part, be involved in the process of metal-induced carcinogenicity and/or toxicity. Thus, the cellular antioxidant defense system, which removes free radicals, may play an important role in the geno-toxicity and toxic effects of metal compounds (Ollinger and Brunk, 1995).
Every antioxidant, including vitamin antioxidants is in fact a redox (reduction-oxidation) agent, protecting against free radicals in some circum-stances, promoting free radical generation in others. tocoferol (vitamin E) and ascorbate (vitaminC) are components of the antioxidant defenses of cells. Vitamin E is found in biological membranes, where it is believed to react with and thereby, detoxify lipid radicals. By contrast, vitamin C is a hydrophilic anti-oxidant that has two potential biological actions. In addition to the direct reaction with lipid radicals, it has been proposed that vitamin C is also a reduc-tant of the tocopheroxyl radical, and action that would regenerate the reduced form of vitamin E. The data indicated that vitamins E and C act as independent antioxidants and that vitamin C does not appear to act as a reductant of the tocopheroxyl radical in the intact cell (Glascott et al., 1996)
The aim of our study was to investigate Cd cytotoxicity and toxicological effects of Cd after the pretreatment of ascorbic acid. Cell viability, proliferation and cell damage were investigated in rat liver cells (Anr 4).

MATERIALS AND METHODS

Chemicals
Tissue culture reagent and ascorbic acid were purchased from Sigma, CdCl2 were obtained from Merck.
Cell culture Anr 4 cell line, rat liver epithelial cells were obtained from ECACC (Salis-bury UK) and maintained as continuous monolayer cultures in tissue culture flasks at 37°C in a humidified atmosphere of % CO2, 95% air. Cells were grown in Williams E medium, supplemented with 10% fetal calf serum, 100 IU/ml penicillin-G and 100 g/ml streptomycin.
Toxicant exposure
Approximatelly 1 x 104 cells were plated in 96 well tissue culture plates. After 4 hours, when the cells were attached on the surface, we influenced them with 100 M ascorbic acid on another 20 hours. After 24 hours, the culture medium was replaced with 100 l medium containing various concentration of cadmium in solution (5-100 M) and incu-bated for a further 24 hours.
Cytotoxicity assessment
The medium containing cadmium chloride was removed after incubation with the cells for 24 hr and replaced with 100 l medium containing 50 g NR/ml. Tuhe assay plate was returned to the incubator for another 3 hr to allow for the uptake of the vital dye into the lysosomes of viable uninjured cells. Thereafter, the medium was removed and cells were rapidly washed with fixative, followed by addition of 150 l of mixture of 1% acetic-50% ethanol to extract the dye from the cells. After 10 min at room temperature and rapid agitation on a microtitre plate shaker, the plates were transfered to a micro-plate reader equipped with a 540 nm filter to measure absorbance of the extracted dye. Results are expressed as a percentage of the optical density determined with extracts from control culturres.
A procedure for determination of total cell protein/well is based on the direct relation-ship between protein content and binding of a dye (kenacid Blue R). After removal of the culture medium, the cells are washed with physiological saline before being fixed in 3% glutaraldehyd for 10-12 minutes. Tuhe cells are then stainded with Kenacid Blue R solution. Excess stain is removed by a series of rinses involving agitation in a destain solution and precise amount of desorb solution (1M potassium acetate in 70% ethanol) is added, and the plate is agitated for 15 minutes. Tuhe colour of the desorb solution is then read at 570 nm, and values obtained for test wells are compared with those for control wells.
Lactate dehydrogenase release in cell cultures
LDH released into the incubation medium was measured by collecting the culture medium after 24 hours of exposure to the cadmium chloride, centrifugated and super-natant was analysed by determining disappearance of beta -NADH at 340 nm in a spectrophotometer. Reagent kits for clinical screening of LDH were used in this assay.

RESULTS AND DISCUSSION

Cadmium chloride decreased the viability of Anr 4 cells in a concentration dependent manner. Partial protection of L-ascorbic acid against 25, 50 and 100 M of Cd was ob-served. At 25 M concentration, the viability was 62,27 + 2,79% and after the pretreat-ment of L-ascorbic acid 69,08 + 1,58%, at 50 M Cd 15,38 + 2,25% and 50,11 + 3,23 %. At the highest concentrarionm the viability was reduced (0,29 + 0,33% and 8,75 + 1,04%).
Partial protection of L-ascorbic acid at and 10 M Cd was observed. The proliferation of cells had been decreased at 5 M Cd 93,13 + 1,61% and after the pretreatment of L-ascorbic acid 94,07 + 6,73%. At 10 M concentration, the amount of total protein was 87,62 + 3,12% and after L-ascorbic acid pretreatment 92,76 + 4,63%.
Leakage of LDH into the medium was used to assess Cd induced cell membrane damage. Tuhe protection of L-ascorbic acid against cadmium has not been observed by this assay.
Shiraishi et al.(1993) investigated the effect of L-ascorbic acid on cadmium letality. Ascorbic acid clearly decreased the toxicity of cadmium but had no effect on the dis-tribution of cadmium in kidney, liver and testes in rats.

REFERENCES

Glascott, P.A., Glifor, J.E., Serroni, A, Farber, J.L.: Biochemical Pharmacology, 52, 1996, p.1245-1252

Ollinger, K, Brunk, U.T.: Free Radical Biology and Medicine, 19, 5, 1995, p.565-574.

Shiraishi, N., Hiroshi, U, Waalkes,M.P.: Toxicology, 85, p.85-100