Environmental Monitoring and Assessment (2005) 104: 437–444 DOI: 10.1007/s10661-005-2281-5

c Springer 2005


NICKEL AND CADMIUM CONCENTRATIONS IN PLASMA AND Na+ /K+ ATPase ACTIVITIES IN ERYTHROCYTE MEMBRANES OF THE PEOPLE EXPOSED TO CEMENT DUST EMISSIONS ˙ AKYUZ ¨ 2, TEM˙IR AL˙I DEM˙IR1 , TAMER AKAR1,∗ , FAHRETTIN 3 2 ¨ ¨ KANBAK BURHANETT˙IN IS¸IKLI and GUNG OR 1

Department of Chemistry, Faculty of Arts and Sciences, Osmangazi University, Eskis¸ehir, Turkey; 2 Department of Biochemistry, Faculty of Medicine, Osmangazi University, Eskis¸ehir, Turkey; 3 Department of Public Health, Faculty of Medicine, Osmangazi University, Eskis¸ehir, Turkey (∗ author for correspondence; e-mail: [email protected])

(Received 21 January 2004; accepted 15 June 2004)

Abstract. The aim of this study was to determine the concentrations of nickel and cadmium in blood plasma of the people exposed to cement dust emissions and to investigate the effects of exposure period on the activities of Na+ /K+ ATPase enzymes in their erythrocyte membranes. The study was carried out on people living in Eski¸sehir C ¸ ukurhisar rural area, located near a cement factory. Blood samples of the individuals residing in this area were taken from 80 subjects (30 for control) following a physical examination. The analysis of plasma samples showed that nickel concentrations in subject group were found to be significantly higher than those of the control group ( p < 0.001). Cadmium concentrations were found to be within the reference values for both group and no difference was found between the subjects and controls ( p > 0.05). Furthermore, no correlation was observed between the levels of Na+ /K+ ATPase activity in erythrocyte membranes of the subject group and the ages of people living in the region ( p > 0.05, r = 0.133). It was also observed that nickel concentrations increased by age ( p < 0.001, r = 0.646) while no effect was observed in means of cadmium. Na+ /K+ ATPase activities in the erythrocyte membranes were not affected. In conclusion although there was no difference between the Na+ /K+ ATPase activity in means of age, there was an environmental pollution and may be it was due to the industrial plant. Keywords: blood plasma, cadmium, cement, Na+ /K+ ATPase, nickel

1. Introduction Environmental pollution is one of the ever surging problems receiving careful attention in our country as well as in the world. As a result of industrial development many chemical substances have generated pollution in air, water and soil. A considerable amount of heavy metals remained inert in the deeper parts of the earth until 20th century. When these metals began to be used in industries, they spread and condensed more in the environment. Heavy metals are more or less toxic elements. They can be carried through soil–plant– animal–human cycle in increasing concentrations (I¸sıklı et al., 2003). Some metals such as nickel and cadmium are found in polluted atmosphere and known to be hazardous to human health (Manahan, 1994). Solid particles can

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negatively affect the environmental quality. Cement factory is one of the causes of particle pollution. Although cement manufacturing units are generally established far from city centers, local areas are affected negatively. Cement dust spreads along a large area through wind, rain, etc. and are accumulated in and on plants, ani¨ mals and soil and can have negative effects on human health (Bayhan and Ozbay, 1992). As it is known that heavy metals are emitted from various sources including fossil fuels (Spiegel, 2002). Metals can enter the cement manufacturing process in three ways. These are through raw materials, primary fuels (i.e. coal) and waste fuels (Woodford et al., 1992). Nickel and cadmium are also among these metals. In recent years, some studies have indicated that living organisms are affected from elements present in the environment. Increasing or decreasing levels of these elements in living tissues cause important effects on metabolism (Kızıler and Barut¸cu, 1997) and in some studies it is reported that both cadmium (Waalkes, 2003) and nickel (Kasprzak et al., 2003; Cavallo et al., 2003) are toxic and carcinogenic for human. It is reported that nickel (WHO, 1991) and cadmium (WHO, 1992) could play a role in reducing the activity of ATPase enzymes in some biological membranes. In this study we aimed to determine nickel and cadmium concentrations in plasma of the people residing near a cement factory and the relationship between Na+ /K+ ATPase activities in erythrocyte membranes and the ages of people living in the region. 2. Materials and Methods The study was carried out between July 1999 and March 2000 in Eski¸sehir, Turkey (Figure 1). C ¸ ukurhisar, the town where the study was conducted, is located in an area that is 500 m from the Eski¸sehir Cement Factory. The cement factory had been operating in the area since 1954.

Figure 1. Map of Turkey, Eski¸sehir.

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The subject group included (n = 80) people who had lived in the town since their birth and who were over 15 years of age. These residents were classified into three groups on the basis of age, and we also divided the village into zones in order to exlude selection bias. Individuals in each group were randomly chosen from each zone. Three of the subjects refused to give blood samples so new ones were selected from the neighborhood. Individuals employed at the cement factory were not included in the study; instead, their neighbors were chosen. The control group, on the other hand (n = 30) lived in city center of Eski¸sehir and had neither been exposed to cement dust nor to nickel or cadmium due to work place situations. The venous blood samples of the subjects and controls were taken into two heparinized tubes and transferred to the laboratory in iceboxes and the first samples were centrifuged and plasma was taken out into other tubes and stored at −20 ◦ C until analysis.

2.1. A NALYSIS

OF NICKEL AND CADMIUM

Following the preparation of the samples as described by Que Hee and Boyle (1988) analysis of nickel and cadmium in plasma samples were carried out by using Graphite Furnace Hitachi (180–70) Polarized Zeeman Atomic absorption spectrophotometer. Nickel and cadmium atomic absorption standard solutions prepared by (980 µgNi/mL in 1% HNO3 and 1000 µgCd/mL 1% HNO3 , Sigma, respectively) were used for the calibration of the atomic absorption spectrophotometer for every 15th reading. Cement samples were taken from cement produced in Eski¸sehir Cement Factory in three different periods and prepared as described by G¨und¨uz (1993) to determine the concentrations of nickel and cadmium, and analyzed by using Hitachi (180–70) Polarized Zeeman Atomic Absorption Spectrophotometer.

2.2. P REPARATION

OF ERYTHROCYTE GHOSTS

We also studied the levels of Na+ /K+ ATPase of the subjects. The second samples taken into the heparinized tubes were centrifuged for 30 min at ×600 g (4 ◦ C). Packed erythrocytes were washed three times in 0.9% NaCl with a 5-min centrifugation at 600g after each wash. Erythrocytes were hemolyzed in 10 mmol/L Tris/1 mmol/L EDTA pH 7.4, 4 ◦ C, and ghosts were sedimented at 27,000g for 20 min (4 ◦ C). The pellet was washed 2–4 times in 10 mmol/L Tris pH 7.4, with a 10-min centrifugation at ×27,000g after each wash. Membranes were suspended in buffer (Dodge et al., 1963; Golik et al., 1996).

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2.3. Na+ /K+ ATPASE

ACTIVITIES

Na+ /K+ ATPase activities were measured using a method that couples ATP hydrolysis with NADH oxidation, which was recorded spectrometrically at 340 nm with a Schimadzu (1201) UV spectrophotometer (37 ◦ C). In the assay of Na+ /K+ ATPase, the final composition of the reaction mixture was Tris–HCl 50 mmol/L pH 7.4; NaCl 130 mmol/L; KCl 5 mmol/L; MgCl2 3 mmol/L; vanadium-free TrisATP 3 mmol/L; NADH 0.1 mmol/L; phosphoenolpyruvate 0.83 mmol/L; pyruvate kinase 1 U/mL; lactate dehydrogenase 1 U/mL. The reaction started on adding 60– 100 µg membrane protein. Mg2+ -activated ATPase was measured in an identical medium also containing 1 mmol/L oubain to inhibit Na+ /K+ ATPase. The difference between the two reactions represents as Na+ /K+ ATPase activity (Schoner et al., 1967; Golik et al., 1996). Results were expressed as U/mg protein. Protein values were determined according to the Biuret’s Method (Silverman and Christenson, 1994).

2.4. STATISTICAL

ANALYSIS

In this study all the samples were analyzed in triplicate and the mean values were calculated. Mean correlations were given as means ± S.E. SPSS version 10.0 for Windows was used to evaluate the data and t-test and correlation analyses were used to determine significance. Rejection of the null hypothesis was set at p < 0.05.

3. Results and Discussion During cement manufacturing, nickel is emitted either as a component of the clays, limestones and shales used as raw materials or as an oxide formed in the high temperature process kilns (WHO, 1991). In this study the analysis of the three different cement samples showed that they contained 19.2 ± 0.17 mg/kg nickel and 0.09 mg/kg cadmium. In a study conducted by same researchers in C ¸ ukurhisar, it was found that the mean nickel concentration of the soils taken from the residential area was 88.416 ± 1.31 and 73.424 ± 0.79 mg/kg for plant specimens. The mean nickel concentration of the inner circle (the factory as the center with a 500 m of semi-diameter) was higher than that of the outer circle (the factory as the center, with a radius of 2500 m) with regard to soil (t = 7.432, p < 0.001) and plants (t = 7.408, p < 0.001). The mean cadmium concentration of the soil samples taken from the residential area was 0.277 ± 0.01 mg/kg and it was 0.369 ± 0.01 mg/kg for plants. The mean cadmium concentration of the inner circle (the factory as the center, with a radius of 500 m) was higher than that of the outer circle (the factory as the center, with

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TABLE I The mean concentration of nickel and cadmium in plasma of the subject and control group Subject (n = 80)

Control (n = 30)

Metal

Mean

S.E.

Range

Mean

S.E.

Range

t

p

Nickel (µg/L) Cadmium (µg/L)

9.29 0.47

0.35 0.009

3.45–17.95 0.21–0.58

6.26 0.47

0.32 0.01

3.10–10.60 0.37–0.56

5.029 0.263

<0.001 >0.05

a 2500 m of semi-diameter) with regard to soil (t = 4.429, p < 0.001) and plants (t = 6.853, p < 0.001). Nickel is normally present in human tissues and under conditions of high exposure, these levels may increase significantly. The levels of nickel in biological fluids increase remarkably in persons with increased occupational exposure and decline rapidly when exposure is reduced or stopped. Thus, measurement of nickel, particularly in biological fluids may serve as induces of exposure (WHO, 1991). Reference interval for nickel in blood plasma of healthy persons is 0.6–7.5 µg/L (Milne, 1994). For the subject group the value was 9.29 ± 0.35 µg/L and was significantly higher than that of the control group (6.26 ± 0.32 µg/L; t = 5.029, p < 0.001; Table I). In a study reported by Rondia (1978) the mean nickel plasma levels of the workers exposed to soluble dust was reported as 7.4 and 6.0 µg/L for workers exposed to insoluble dust and 4.2 µg/L for the control group. It was reported in the last decades that normal nickel values in urine, blood plasma and serum vary widely. In the research conducted during the last 30 years, it was reported that normal values of the individuals who are healthy and not exposed to cement dust were changeable (WHO, 1991). For such people, Linden et al. (1985) found blood nickel levels as 0.05–1.05 µg/L with an average of 0.34 ± 0.28 µg/L. However, these values can be said to increase because of environmental pollution. Indeed Matilla and Kilpelainen (2001) report that nickel sensitivity for female university students in 1986 (13%) increased up to 39% in 1995. The percentage increases also depend on exposure level (WHO, 1991). In our study the average cadmium concentrations of the subject group were 0.47 ± 0.009 and 0.47 ± 0.01 µg/L for the control group and no significant difference was observed between the two groups (t = 0.263, p > 0.05; Table I). These values were lower than the reference values of plasma cadmium (0.5 µg/L) given by Martinsen and Thomassen (1983) and were similar to their results. Average nickel concentrations of the plasma samples of the subject group were 4.89 ± 0.35, 9.11 ± 0.41 and 10.71 ± 0.47 µg/L, respectively, for the first, second and third age groups (Table II). The lower value was in under-31 age group and the highest value was in the ≥51 age group. Average cadmium concentrations of the

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TABLE II Concentration of nickel and cadmium in plasma of the subject for three different age group Plasma nickel levels (µg/L)

Plasma cadmium levels (µg/L)

Age group

n

Mean

S.E.

Range

Mean

S.E.

Range

Under 31 31–50 ≥51

11 31 38

4.89 9.11 10.71

0.35 0.41 0.47

3.45–7.00 3.95–14.16 3.80–17.95

0.46 0.47 0.48

0.03 0.01 0.01

0.33–0.58 0.31–0.58 0.21–0.62

TABLE III Concentration of nickel and cadmium in plasma of the subject according to sex Plasma nickel levels (µg/L)

Plasma cadmium levels (µg/L)

Sex

n

Mean

S.E.

Range

Mean

S.E.

Range

Male Female

40 40

9.76 8.82

0.52 0.45

3.95–17.95 3.45–13.60

0.47 0.48

0.01 0.01

0.21–0.59 0.32–0.62

Total

80

t = 1.356

p > 0.05

t = 0.360

p > 0.05

plasma samples of the subject group were 0.46 ± 0.03, 0.47 ± 0.01 and 0.48 ± 0.01 µg/L, respectively, for the first, second and third age groups (Table II). Nickel concentrations in the plasma samples of the subject group were increasing with the increase in the age (r = 0.646, p < 0.001) while cadmium concentrations in plasma samples were similar for all age groups and no significant change with age (r = 0.107, p > 0.05) was observed. Furthermore, there was no significant correlation in means of Ni and Cd concentrations in the plasma samples and the ages of the control group (r = 0.347, p > 0.05 and r = 0.36, p > 0.05, respectively). No significant differences were observed between both the nickel ( p > 0.05, t = 1.356) and cadmium ( p > 0.05, t = 0.360) concentrations of the plasma samples in means of sex in the subject group (Table III). Na+ /K+ ATPase activity in erythrocyte membranes of the subjects were similar in means of age groups ( p > 0.05, t = 0.213; p > 0.05, t = 0.223 and p > 0.05, t = 0.225, respectively; Table IV) and there was no correlation between the enzyme activity and age (r = 0.133, p > 0.05). Also there was no correletion between Na+ /K+ ATPase activity in erythrocyte membranes and the nickel (r = 0.006, p > 0.05) and cadmium (r = 0.111, p > 0.05) concentrations of the plasma samples. According to these results, our study had also showed that the levels of Na+ /K+ ATPase activity in erythrocyte membranes were not affected from the environment. In conclusion, a higher and increased plasma nickel concentration with age was observed in the plasma of the individuals living near a cement factory. Although there was no difference between the Na+ /K+ ATPase activities in means of age,

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TABLE IV The distribution of Na+ /K+ ATPase activity in erythrocyte membrane of the subjects according to age groups Levels of Na+ /K+ ATPase activity (u/mg protein) Age group

n

Mean

S.E.

Range

Under 31 31–50 ≥51

11 31 38

0.014 0.013 0.013

0.0018 0.0015 0.0013

0.009–0.022 0.009–0.021 0.008–0.080

Total

80

∗

p > 0.05, t = 0.213; ∗∗ p > 0.05, t = 0.223; ∗∗∗ p > 0.05, t = 0.225. Comparing the age groups under 31 and 31–50. ∗∗ Comparing the age groups under 31 and ≥51. ∗∗∗ Comparing the age groups 31–50 and ≥51. ∗

there was an environmental pollution which might be due to the industrial plant releasing cement and coal combustion particles and nickel content of food stuffs grown in the area. The study also underlines the need for further studies in this rural area in terms of environment and human health.

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