European Journal of General Medicine Medical Investigations Society ISSN: 1304-3897 Vol. 1, Num. 4, 2004, pp. 22-28

European Journal of General Medicine, Vol. 1, No. 4, 2004, pp. 22-28

ORIGINAL ARTICLE

EVALUATION OF ERYTHROCYTE Na⁺, K⁺ -ATPASE AND SUPEROXIDE DISMUTASE ACTIVITIES AND MALONDIALDEHYDE LEVEL ALTERATION IN COAL MINERS

Ahmet Gürel¹, Ferah Armutçu¹, Şeyda Damatoğlu¹, Murat Unalacak², Nejat Demircan²

Karaelmas University Faculty of Medicine, ¹Department of Biochemistry and Clinical Biochemistry, ²Department of Family Medicine
Correspondence: Dr. Ahmet Gürel Karaelmas University, Faculty of Medicine, Department of Biochemistry, 67600 Kozlu, Zonguldak, Turkey Tel: +90 372 261 0169 Fax: +90 372 261 0155 e-mail:dragurel@yahoo.com

Code Number: gm04040

The purpose of the present study was to determine the structural integrity of red blood cells in coal miners by assessing the concentration of malondialdehyde and the activities of superoxide dismutase and, Na⁺, K⁺ -ATPase in erythrocytes. Occupational exposure to coal mine dust can result in a wide range of lung diseases, which include pneumoconiosis, bronchitis, and emphysema. Pathophysiological mechanisms of the diseases are not fully understood; however, cytotoxicity is related to reactive oxygen substances which are produced by coal dust. The study population consisted of 40 coal workers previously known not to have any pulmonary disease and 34 healthy subjects who were randomly selected from the population register or recruited from the hospital staff. The activities of Na⁺, K⁺ -ATPase in the erythrocyte membrane was significantly decreased in the coal workers as compared to the control group. Serum potassium and iron concentrations were significantly higher whereas serum sodium was moderately decreased in coal workers as compared to control. MDA levels of all samples were significantly increased in the coal workers as compared to the control group. SOD activity in serum and erythrocyte was significantly lower in the coal miner group as compared to the control group. The present study demostrated that the elevated MDA and iron levels and insufficiency of antioxidant potential in serum and erythrocytes cause a decrease in erythrocyte Na⁺, K⁺ -ATPase enzyme activity in coal miners as compared to normal subjects.

Key words: Coal miners, eythrocyte, Na⁺, K⁺ -ATPase, MDA, SOD, iron, ROS, and coal dust

Occupational exposure to coal mine dust can result in a wide range of lung diseases, which include pneumoconiosis, bronchitis, and emphysema (1). Pathophysiological mechanisms of the diseases are not fully understood. However, cytotoxicity is related to reactive oxygen substances (ROS) which are produced by coal dust (2). Basically, two mechanisms by which coal dust expose causes formation of ROS have been proposed, (i) the generation of ROS by intrinsic properties of particles and iron content of coal dust, noncellular mechanism, and (ii) the excessive formation of ROS by the oxidative burst of macrophages and neutrophils activated during phagocytosis and persistent inflammation (3). In addition to these mechanisms, coal dust causes production of ROS via increaced metabolism of arachidonic acid in cell membranes (4). It has long been recognized that ROS are harmful for cells, because they injure lipids, thiol proteins or nucleic acids, which leads to structural and functional impairments (5,6). An extensive system of cytosolic and mitochondrial enzymatic and nonenzymatic antioxidants function to scavenge ROS in physiologycal processes and in pathological conditions (7).

The Na⁺, K⁺ -ATPase is the intrinsic membrane protein responsible for pumping Na⁺ and K⁺ against their membrane gradient (8). In almost all animal cells, including human erythrocytes, Na⁺, K⁺ -ATPase is an essential protein transporter under normal physiological conditions. Na⁺, K⁺ -ATPase catalyzes the hydrolysis of ATP that is coupled to the active transport of Na⁺/K⁺ across the cell membrane (9). It mediates the transport of three Na ⁺ ions out of the cell and two K⁺ ions into the cell per ATP molecule. The purpose of the present study was to determine the structural integrity of red blood cells in coal miners by assessing the concentration of malondialdehyde and the activities of superoxide dismutase and Na⁺, K⁺ -ATPase in erythrocytes.

MATERIAL AND METHODS

The study population consisted of 40 coal workers previously known not to have any pulmonary disease and 34 healthy subjects who were randomly selected from the population register or recruited from the hospital staff. All cases were male and Caucasians. Healthy subjects were totally normal and had no pulmonary disease or complaints. The coal and coal mine characteristics Zonguldak Coal Basin has five areas for coal production. All coal workers were from the same area, namely Karadon. The workers had been working for at least 5 years (range 5-21 years). Mean coal chemical features of Karadon mines are as follows; 55% carbon, 26% volatile substances, 11% ash, 8% damp. All coal workers selected were from sections (coal face, mining, stope) that had coal dust exposure changing from low to high concentrations (0.5 to 12.3 mg/m³) from time to time. All data were obtained from the Turkish Coal Company. Informed written consent was obtained from all subjects.

Blood samples were taken from 40 coal miners working in the Zonguldak coal mining industry and 34 healthy subjects. Ten ml of blood samples were taken from each donor after 8 hr work shift. All samples were taken by venapuncture without and with anticuagulant (EDTA) evacuated tubes. Sample tubes were transferred to our laboratory within 2 hours for evaluation. Centrifugation was performed at 4 ⁰C (10 min, 3000 rpm) and measurements were performed in serum, ertyhrocyte and erytrocyte membrane.

The blood was centrifuged at 2,000 g for 15 minutes to separate plasma. The layer of white blood cells above the packed red blood cells was removed, and discarded. The red blood cells were washed two times with Trisbuffered saline (TBS), i.e., 20 mM Tris-HCl, pH 7.5 containing 145 mM NaCl by spinning at 2,000 g for 15 minutes. The washed packed red blood cells were lysed by diluting 10 fold in 10 mM Tris-HCl buffer.The lysed cells were kept on ice for 15 minutes, followed by centrifugation at 12,000 g for 20 minutes. The supernatant was discarded, and the pellet was washed with lysis-buffer until the membrane became white (It usually required 3-4 washes).

The activity of Na⁺, K⁺ -ATPase in the erthrocyte membrane was measured by the method of Kitao-Hattori (10). The reaction mixture contained 200 µ ghost, and 800 µL medium fluid which included NaCl, KCl, EDTA, and MgCl. The reaction was started at 37 ^oC by the addition ATP. After 60 minutes, the reaction was stoppered by the addition of SDS. The samples were centrugated at 1,000 g for 15 minutes. The phosphrus content in the supernatant was measured using the inorganic phosphors estimation kit from Roch Diagnostica.

In the samples, which are serum, erythrocyte , and eyhrocyte membrane, malondialdehyde MDA levels were determined using the method of Draper and Hadley (11) based on the reaction of MDA with thiobarbituric acid (TBA) at 95^oC. In the TBA test reaction, MDA and TBA react to form a pink pigment with an absorption maximum at 532 nm. The reaction was performed at pH 2-3 at 95^oC for 15 min. The sample was mixed with 2.5 volumes of 10% (w/v) trichloroacetic acid to precipitate the protein. The precipitate was pelleted by centrifigation and an aliquot of supernatant was reacted with 0.67% TBA in a boiling water-bath for 15 min. After cooling, the absorbance was read at 532 nm. Arbitrary values obtained were compared with a series of standard solutions (1,1,3,3 tetramethoxypropane). Results were expressed as nanomole per gram hemoglobin, mg protein, and mililiter.

Protein determination

The protein content of erythrocyte membrane was determined by the method of Lowry et al. (12).

Total (Cu-Zn and Mn) SOD (EC 1.15.1.1) activity was determined according to the method of Sun et al (13) and a slightly modified method by Durak et al (14). The principle of the method is based on the inhibition of NBT reduction by the xanthine-xanthine oxidase system as a superoxide generator. Activity was assassed in the ethanol phase of the lyzate after 1.0 ml ethanol/chloroform mixture (5/3, v/v) was added to the same volume of sample and centrifuged. One unit of SOD was defined as the enzyme amount causing 50% inhibition in the NBT reduction rate. SOD activity was also expressed as Units per gram hemoglobine or milliliter.

Hemoglobin concentration in erythrocytes was determined with the haemoglobin cyanide procedure (15).

The serum concentrations of sodium, potassium and iron are measured with Rohe diagnostica kit by using the same otoanalyzer (Roche Diagnostica).

All values were expressed as means and standard deviations (SDs). SPSS for windows 11.0 was used for statistical analysis. Student's t-test was used to estimate the significance between parameters. The differences were considered to be significant when p was less than 0.05.

The table 4 showes erythrocyte Na⁺, K⁺ ATPase activity in the coal miner and control groups. The activities of Na⁺, K⁺ -ATPase in the erythrocyte membrane was significantly decreased in the coal workers as compared to the control group.

Table 2 indicates the mean values and standard deviations (SDs) of the concentrations of serum sodium, potassium, and iron in the coal miner and control groups. Serum potassium and iron concentrations were significantly higher, whereas serum sodium was moderately decreased in coal workers as compared to controls.

Table 3 indicates lipid peroxidation in erythrocyte, erythrocyte membrane and serum of control and coal miner goups. MDA levels of all samples were significantly increased in the coal workers as compared to the control group. The table 4 shows SOD activities of serum and erythrocytes. SOD activity in serum and erythrocyte were significantly lower in coal miner group as compared to the control group. (Table 1)

DISCUSSION

Lipid peroxidation is thought to be involved in a number of pathlogical processes. ROS have been implicated in the pathogenesis of various conditions including lung disease (16). The involvement of free radical induced lipid peroxidation and the role of antioxidants in several diseases are well established. MDA is an end product of fatty acid oxidation, and is often used as an indicator of lipid peroxidation.

The present study demonstrates that erythrocyte and erthrocyte membrane MDA levels are increased in a group of coal miners when comparated to a control group. Some animal studies have indicated that lipid peroxidation is enhanced in different tissues of rats exposed to coal dust and its derivates. For example, Srivastava et al showed that fly ash and fly ash residue increased the formation of conjugated dienes and the levels of oxidized glutathione (GSSG) and reduced the levels of reduced glutathione (GSH) in lung and liver, whereas fly ash extract administration had no effect on the formation of conjugated dienes and glutathione levels in lung and liver (17). Our findings are in agreement with the results obtained from some other studies (18, 19). The observed increase in lipid peroxidation in the present study might be due to increased oxidative stress caused by the coal dust-induced generation of free radicals and increased formation of lipid hydroperoxides or due to other products, as reported by others (3,20).

Erytrocytes and erytrocyte membrane are more vulnerable to lipid peroxidation due to constant exposure to high oxgen tension and richness in polyunsaturated fatty acid (21). By-products of lipid peroxidation have been shown to cause profound alterations in the structural organization and functions of the cell membrane including decreased membrane fluidity, increased membrane permeability, inactivation of membrane-bound enzymes and loss of essential fatty acids (22).

Peroxidation of membrane lipids not only alters the lipid milieu and structural as well as functional integrity of cell membranes, but also affects the activity of various membrane-bound enzymes, including Na⁺, K⁺ -ATPase (23). Since the Na⁺, K⁺ -ATPase is an essential enzyme of the plasma membrane of animal cells, it has been suggested to represent an important target of ROS induced membrane damage. ROS are known as the to inhibit Na⁺, K⁺ -ATPase activity. Previous studies have shown that the activity of the enzyme is strongly and irreversibly reduced in the presence of ROS such as hydroxyl radicals, superoxide anion radical, or singlet oxygen (24). Jamme et al. (25) reported that the inhibition of Na⁺, K⁺ -ATPase activitiy by generated ^.OH and peroxy radicals is also related to lipid peroxidation induced disruption of membrane integrity that results in conformational changes, leading to inactivation of membrane bound proteins. It has also been suggested that oxidant agent induced inactivation of Na⁺, K⁺ -ATPase activitiy is due to lipid peroxidation-induced reduction in affinity for Na and K (26). The pump also appeared to be sensitive to changes in membrane phospholipids. Because phospholipid molecules, which are components of both types of Na⁺, K⁺ ATPase subunits, have a significant role in modulation of enzyme activity, changes in their composition are expected to alter Na⁺,K⁺ -ATPase function (27).

This study demonstrated that Na⁺, K⁺ ATPase activity in erythrocyte membranes was lower in coal miners compared to a control group, whereas MDA level in erythrocyte membrane in coal workers group was was found to be higher than the control group. Decrease in erythrocyte Na⁺, K⁺ ATPase activity associated with increased lipid peroxidation, which disturbs the phospholipids moiety that is essential for the functioning of the enzyme, could be related to an impairment in the optimal interaction of Na⁺, K⁺ -ATPase with membrane phospholipids, considering that its activity is modulated by the microenvironment given by the physicochemical properties of the membranes into which it is inserted. The alteration of phospholipid composition of the membrane due to coal dust might be an important factor in the decrease of enzyme activity. Also activation of lipooxygenase by coal dusts, which results in enzyme inhibition, might have added to the decrease in Na⁺, K⁺ -ATPase activity (28).

It has been suggested that serum Na and K concentrations reflect inhibition of Na⁺, K⁺ ATPase (29). The observed increase in plasma potassium and decline in sodium accompanied by lowered activity of erythrocyte membrane Na⁺, K⁺ -ATPase can disturb cellular ion homeostasis. ROS-induced oxidative damage to membrane Na⁺, K⁺ -ATPase and increase in potassium efflux from the cell have been assumed to be crucial for cell lysis. Hence, the disturbance in ionic homeostasis in the group of coal miners may result from elevated lipid peroxidation of erythrocytes and erythrocyte membrane and decreased Na⁺, K⁺ -ATPase activity, resulting in increased membrane permeability.

Increased serum iron concentrations reflected that coal miners are exposed to excessive iron, which is a transition metal ion, and appears to be an important mediator of oxidative damage in vivo/in vitro together with coal dust. High serum iron concentration shows that workers are exposed to high concentration of this metal, together with coal dust. Transition metal ions, especially iron, appear to be important mediators of oxidative damage in vivo. The iron present in medium may be involved in the formation of hydroxyl radicals via the Fenton-reaction. The iron content may also play an important role in the toxicity of coal dust (30). Fenton reaction type formation of hydroxy radicals was found to be positively correlated with the iron content of coal dust (31). The hydroxyl radical that may be produced by this reaction at the surface of the coal is a potent oxidant, which can initiate lipid peroxidation of cell membranes and oxidatively inactivate essential cell proteins (32).

The antioxidant defense system is known to inhibit lipid peroxidation in erythrocytes by destroying some ROS that have an important role in the initiation of lipid peroxidation processes. The antioxidant defense system operates through enzymatic and nonenzymatic components. The system is affected by coal dust and its derivates.

The studies show that reports about antioxidan parametres in coal miners are contradictory. While, coal dust and its derivates lead to decreased GSH levels and selenium concentration which is strongly an antioxidant mineral, and SOD activity (17,33,34). The other antioxidant enzymes are affected differently by coal dust. Both increase (35) and decrease (33) in activity has been reported. SOD activity was reported to be significantly reduced in coal miners with pneumoconiosis as compared to those without pneumoconiosis (34,35). Our results support these findings. We observed a significant reduction in erythrocyte SOD activities in coal miners. Reduction in SOD activity as observed by us may be due to an increased endogenous production of ROS as evidenced by increased MDA and products of ROS. This decrease in antioxidant enzyme may be related to the consumption of activated enzymes against oxidative stress. This inhibition is probably achieved through the process of lipid peroxidation, which disturbs the phospholipid moiety that is essential for the functioning of Na⁺, K⁺ -ATPase. Depletion of antioxidants in coal workers may also be a contributing factor to the decrease in Na⁺, K⁺ -ATPase activity.

Plasma MDA level has been used in many diseases as an indirect indicator of tissue lipid peroxidation and general oxidant stress. High serum plasma levels in our study suggest that coal dust, by exerting oxidant effect on tissues, especially the lungs, causes an increase in lipid peroxidation.

This agrees with the suggestion of Halliwell and Gutteridge (16) who proposed that lipid peroxidation in human diseases can be better explanained by the following reaction sequence: diseases or toxins induce cell damage or death; and lipid peroxidation then increases because disrupted tissues undergo peroxidation more quikly than healthy ones. Plasma concentration of antioxidants also has been used as a biomarker of oxidative stress in several diseases (36). A lower level of antioxidant as a consequence of a past situation of oxidative stress will correspond to a higher membrane lipid peroxide and MDA. Both are products of the lipid peroxidation process and presumably the result of ROS action (37). An increased serum MDA level in our group of coal miners may indicate a general oxidant effect of coal dust. Also, decreased SOD activty in coal miner might be a marker of diminished antioxidant defense system which was caused by dust.

Increased lipid peroxidation products in circulation, for instance MDA, may interact with erythrocyte membran components such as lipid and protein, and demolish membrane bound enzyme activities and cellular functions.

The present study, thus, demostrated that the elevated MDA and iron levels and insufficiency of antioxidant potential in serum and erythrocytes cause a decrease in erythrocyte Na⁺, K⁺ -ATPase enzyme activity in coal miners as compared to normal subjects.

1. Bowden DH. Macrophages, dust, and pulmonary diseases. Exp Lung Res 1987; 12(2): 89-107
2. Vallyathan V, Shi X, Castranova V. Reactive oxygen species: their relation to pneumoconiosis and carcinogenesis. Environ Health Perspect 1998;106 Suppl 6:1595.
3. Schins RP, Borm PJ. Mechanisms and mediators in coal dust induced toxicity: a review. Ann Occup Hyg 1999; 43(1): 7-33
4. Vanhee D, Gosset P, Boitelle A, Wallaert B, Tonnel AB. Cytokines and cytokine network in silicosis and coal workers'pneumoconiosis. Eur Respir J 1995; 8(5): 834-42
5. Freeman BA, Crapo JD. Biology of disease: free radical and tissue injury. Laboratory Investigation 1982; 47: 412–
6. Mantle D, Preedy VR. Free radicals as mediators of alcohol toxicity. Adverse Drug Reactions 1999; 18: 235–52
7. Halliwel B.reactive oxygen species in living systems; source, biochemistry and role in human disease. Am J Med 1991;91: 145-225
8. Bonilla S, Goecke IA, Bozzo S, Aluo M, Michea L, Marusie ET. Effects of Chronic Renal Failure on Na; K-ATPase 21 and 22 mRNA Transcription in Rat Skeletal Muscle. Journal of Clinical Investigation 1991; 88: 2137–41
9. Skou JC. Enzymatic basis for active transport of Na+ and K+ across cell membrane. Physiol Rev 1965; 45: 596–617
10. Kitao T, Hattori K. Inhibition of erythrocyte ATPase activity by aclacinomycin and reverse effects of ascorbate on ATPase activity. Experientia 1983; 39(12): 1362-4
11. Draper H, Hadley M. Malondialdehyde determination as index of lipid peroxidation. Methods Enzymol 1990; 186: 421-431
12. Lowry O, Rosenbraugh N, Farr L, Rondall R. Protein measurement with the Folin-phenol reagent. J Biol Chem 1951; 183: 265-275
13. Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide dismutase. Clin Chem 1988; 34: 497-500
14. Durak I, Yurtarslanl Z, Canbolat O, Akyol O. A methodological approach to superoxide dismutase (SOD) activity assay based on inhibition of nitroblue tetrazolium(NBT)reduction.Clin Chim Acta 1993; 214(1): 103-4
15. Van Kampen EJ, Zijlstra WG. Determination of hemoglobin and its derivatives. Adv Clin Chem 1965; 8: 141 87.
16. Halliwell B, Gutteridge JM.Lipid peroxidation, oxygen radicals, cell damage, and antioxidant therapy. Lancet 1984; 1(8391): 1396-7
17. Srivastava PK, Singh Y, Misra UK. Pulmonary and hepatic glutathione levels, glutathione shuttle enzymes and lipid peroxidation in rats exposed intratracheally to coal fly ash. Biochem Int 1988; 17(3): 509-16
18. Rebrov BA, Lymar'SP. The functional characteristics of the immune system, of lipid peroxidation and of the endogenous antioxidant system in miners working in deep coal mines. Lik Sprava 1999; 4: 162-4
19. Vallyathan V, Goins M, Lapp LN, et al. Changes in bronchoalveolar lavage indices associated with radiographic classification in coal miners. Am J Respir Crit Care Med 2000; 162(3 Pt 1): 958-65
20. Vallyathan V. Generation of oxygen radicals by minerals and its correlation to cytotoxicity. Environ Health Perspect 1994;102 Suppl 10:111-5
21. Eritsland J. Safety considerations of polyunsaturated fatty acids. Am J Clin Nutr 2000; 71: 197S–201S
22. van Ginkel G, Sevanian A. Lipid peroxidation induced membrane structural alterations. Methods Enzymol 1994; 233: 273–88
23. Rauchova H, Ledvinkova J, Kalous M, Drahota Z. The effect of lipid peroxidation on the activity of various membrane bound ATPase in rat kidney. Int J Biochem Cell Biol 1995;27;251-5
24. Kim MS, Akera T. O₂ free radicals: cause of ischemia-reperfusion injury to cardiac Na+-K+-ATPase. Am J Physiol 1987; 252(2 Pt 2): H252-7
25. Jamme I, Petit E, Divoux D, Gerbi A, Maixent JM, Nouvelot A. Modulation of mouse cerebral Na+,K(+)-ATPase activity by oxygen free radicals. Neuroreport 1995; 7(1): 333-7
26. Mishra OP, Delivoria-Papadopoulos M, Cahillane G, Wagerle LC. Lipid peroxidation as the mechanism of modification of the affinity of the Na+, K+-ATPase active sites for ATP, K+, Na+, and strophanthidin in vitro. Neurochem Res 1989; 14(9): 845-51
27. Roelofsen B, van Meer G, Op den Kamp JA . The lipids of red cell membranes. Compositional, structural and functional aspects. Scand J Clin Lab Invest Suppl 1981; 156: 111-5
28. Kourie JI. Interaction of reactive oxygen species with ion transport mechanisms. Am J Physiol 1998; 275(1 Pt 1): C1-24
29. Kolanjiappan K, Manoharan S, Kayalvizhi M. Measurement of erythrocyte lipids, lipid peroxidation, antioxidants and osmotic fragility in cervical cancer patients. Clin Chim Acta. 2002; 326(1-2): 143-9
30. Tourmann JL, Kaufmann R. Biopersistence of the mineral matter of coal mine dusts in silicotic human lungs: is there a preferential release of iron? Environ Health Perspect 1994; 102Suppl5: 265-8
31. Dalal NS, Newman J, Pack D, Leonard S, Vallyathan V. Hydroxyl radical generation by coal mine dust: possible implication to coal workers' pneumoconiosis (CWP). Free Radic Biol Med 1995; 18(1): 11-20
32. Halliwell B, Gutteridge JM. Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J 1984; 219(1): 1-14
33. Nadif R, Oryszczyn MP, Fradier-Dusch M, et al. Cross sectional and longitudinal study on selenium, glutathione peroxidase, smoking, and occupational exposure in coal miners. Occup Environ Med 2001; 58(4): 239-45
34. Nadif R, Bourgkard E, Dusch M, et al. Relations between occupational exposure to coal mine dusts, erythrocyte catalase and Cu⁺⁺/Zn⁺⁺ superoxide dismutase activities, and the severity of coal workers'pneumoconiosis. Occup Environ Med 1998; 55(8): 533-40
35. Perrin-Nadif R, Auburtin G, Dusch M, Porcher JM, Mur JM. Blood antioxidant enzymes as markers of exposure or effect in coal miners. Occup Environ Med. 1996 Jan; 53(1): 41-5
36. Buffon A, Santini SA, Ramazzotti V, et al. Large, sustained cardiac lipid peroxidation and reduced antioxidant capacity in the coronary circulation after brief episodes of myocardial ischemia. J Am Coll Cardiol 2000; 35: p. 633
37. FJ. Oliveira, R. Cecchini. Oxidative stress of liver in hamsters infected with Leishmania (L)chagasi. J Parasitol. 2000 ;86(5):1067-72

Copyright 2004 - Medical Investigations Society

The following images related to this document are available:

Photo images