Enzymatic antioxidant defense in isolated rat hepatocytes exposed to cadmium

Open access

Enzymatic antioxidant defense in isolated rat hepatocytes exposed to cadmium

The aim of the study was the evaluation of cadmium effects on the activity of antioxidant enzymes in rat hepatocytes. The studies were conducted with isolated rat hepatocytes incubated for 1 or 2 hours in a modified (deprived of carbonates with phosphates) Williams' E medium (MWE) in the presence of cadmium chloride (25, 50 and 200 μM). Hepatocytes incubated in the MWE medium without cadmium chloride were used as a control. The application of the modified Williams' E medium allowed for the appearance of cadmium compounds in a soluble form that is indispensable for suitable estimation of its toxic action. There were evaluated markers of the oxidative stress such as: concentration of thiobarbiturate reactive substances (TBARS) - proportional to the level of lipid peroxidation, concentration of reduced glutathione (GSH), and the activity of antioxidant enzymes, including superoxide dismutase (SOD1 and SOD2), catalase (CAT), total glutathione peroxidase (GSHPx), selenium - dependent glutathione peroxidase (SeGSHPx), glutathione transferase (GST) and glutathione reductase (GSHR). Alterations of antioxidant enzymes activity, the level of TBARS and GSH in isolated rat hepatocytes caused by cadmium in vitro, were shown to depend on the concentration and time of exposure of cells to this metal. The increased level of TBARS and GSH was observed as well as changes in the activity of antioxidant enzymes. The activity of SOD isoenzymes and CAT was increased, whereas GSHPx and GST were decreased. These results indicate that cadmium induces oxidative stress followed by alterations in the cellular antioxidant enzyme system in isolated rat hepatocytes.

Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44: 276-287.

Bojakowska K (2006) Evaluation of cytotoxicity of cadmium compounds: in vitro studies on rat and equine isolated hepatocytes. Doctoral thesis, Warsaw, Warsaw University of Life Sciences, Poland.

Borzęcka K, Bąkała A, Grono D, Głowala A, Wiechetek M (2004) Evaluation of cadmium cytotoxicity in isolated hepatocytes: effect of plating time, medium composition and cadmium chemical compounds. 13th Workshop of the European Society of Toxicology In Vitro "INVITOX", Zegrze, pp 32.

Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye-binding. Anal Biochem 72: 248-254.

Brzóska MM, Moniuszko-Jakoniuk J (1998) The influence of calcium content in the diet on accumulation and toxicity of cadmium in the organism. Arch Toxicol 72: 63-73.

Casalino E, Calzaretti G, Sblano C, Landriscina C (2002) Molecular inhibitory mechanisms of antioxidant enzymes in rat liver and kidney by cadmium. Toxicology 179: 37-50.

European Food Safety Authority (2009) Cadmium in food. Scientific Opinion of the panel on contaminants in the Food Chain. EFSA J 980: 1-139.

Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys 82: 70-77.

Fariss MW (1991) Cadmium toxicity: unique cytoprotective properties of alpha tocopheryl succinate in hepatocytes. Toxicology 69: 63-77.

Golberg DM, Spooner RJ, Bregmayer HV (1983) Glutathione reductase. Methods Enzymatic Analysis. Verlag Chemie, Weinheim 3: 258-265.

Góth L (1999) A simple method for determination of serum catalase activity and revision of reference range. Clin Chim Acta 196: 143-151.

Habig WH, Pabst M, Jaoby WB (1974) Glutathione S-transferase: The first step in mercapturic acid formation. J Biol Chem 249: 7130-7139.

Hayes JD, McLellan LI (1999) Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress. Free Radic Res 31: 273-300.

International Agency for Research on Cancer (IARC) (1993) Beryllium, cadmium, mercury, and exposure in the glass manufacturing industry. Monographs on the evaluation of the carcinogenic risks to humans. IARC Scientific Publications, Lyon, 58, pp 119-237.

Invitox Protocol Number 20. Isolation of Rat Hepatocytes. Invitox, Protocol ERGATT/FRAME Data bank of In Vitro Techniques in Toxicology (1991)

Järup L, Berglund M, Elinder CG, Nordberg G, Vahter M (1998) Health effects of cadmium exposure: a review of the literature and a risk estimate. Scand J Work Environ Health 24: 1-51.

Jurczuk M, Brzóska MM, Moniuszko-Jakoniuk J, Gałażyn-Sidorczuk M, Kulikowska-Karpińska E (2004) Antioxidant enzymes activity and lipid peroxidation in liver and kidney of rats exposed to cadmium and ethanol. Food Chem Toxicol 42: 429-438.

Kehrer JP (1993) Free radicals as mediators of tissue injury and disease. Crit Rev Toxicol 23: 21-48.

Kołacz R, Dobrzański Z, Bodak E (1996) Bio-accumulation of Cd, Pb and Hg in animal tissues. Veterinary Medicine (Polish) 52: 686-691.

Oberley LW, Spitz DR (1984) Assay of superoxide dismutase activity in tumor tissue. Methods Enzymol 105: 457-464.

Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95: 351-358.

Paglia DE, Valentine WN (1967) Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 70: 158-169.

Pastore A, Federici G, Bertini E, Piemonte F (2003) Analysis of glutathione: implication in redox and detoxification. Clin Chim Acta 33: 19-39.

Pourahmad J, O'Brien PJ (2000) A comparison of hepatocyte cytotoxic mechanisms for Cu+2 and Cd+2. Toxicology 143: 263-273.

Pourahmad J, O'Brien PJ, Jokar F, Daraei B (2003) Carcinogenic metal induced reactive oxygen species formation in hepatocytes. Toxicol in Vitro. 17: 803-810.

Robertson JD, Orrenius S (2000) Molecular mechanisms of apoptosis induced by cytotoxic chemicals. Crit Rev Toxicol 30: 609-627.

Sedlak J, Lindsay RH (1968) Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Anal Biochem 25: 192-205.

Sen CK (2000) Cellular thiols and redox-regulated signal transduction. Curr Top Cell Regul 36: 1-30.

Shaikh ZA, Vu TT, Zaman K (1999) Oxidative stress as a mechanism of chronic cadmium-induced hepatotoxicity and renal toxicity and protection by antioxidants. Toxicol Appl Pharmacol 154: 256-263.

Sies H (1999) Glutathione and its role cellular functions. Free Radic Biol Med 27: 916-921.

Stohs SJ, Bagchi D, Hassoun E, Bagchi M (2001) Oxidative mechanisms in the toxicity of chromium and cadmium ions. J Environ. Pathol Toxicol Oncol 20: 77-88.

Tennant RJ (1964) Evaluation of trypan blue technique for determination of cell viability. Transplantation 2: 685-694.

Ungar K (1991) Invitox Protocol Number 20. Isolation of Rat Hepatocytes. Invitox, Protocol ERGATT/FRAME Data bank of In Vitro Techniques in Toxicology.

Waalkes MP (2003) Cadmium carcinogenesis. Mutat Res 533: 107-120.

Waisberg M, Joseph P, Hale B, Beyersmann D (2003) Molecular and cellular mechanisms of cadmium carcinogenesis. Toxicology 192: 95-117.

Wang Y, Fang J, Leonard SS, Rao KM (2004) Cadmium inhibits the electron transfer chain and induces reactive oxygen species. Free Radic Biol Med 11: 1434-1443.

Wendel A (1981) Glutathione peroxidase. Methods Enzymol 77: 325-333.

Williams GM, Jeffrey AM (2000) Oxidative DNA damage: endogenous and chemically induced. Regul Toxicol Pharmacol 32: 283-292.

Polish Journal of Veterinary Sciences

The Journal of Committee of Veterinary Sciences of Polish Academy of Sciences and University of Warmia and Mazury in Olsztyn

Journal Information


IMPACT FACTOR 2016: 0.697
5-year IMPACT FACTOR: 0.773

CiteScore 2016: 0.73

SCImago Journal Rank (SJR) 2016: 0.315
Source Normalized Impact per Paper (SNIP) 2016: 0.486

Cited By

Metrics

All Time Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 202 201 13
PDF Downloads 51 51 2