Effect of chromium(VI) on the status of plasma lipid peroxidation and erythrocyte antioxidant enzymes in chromium plating workers

Chemico-Biological Interactions 164 (2006) 192–199

Effect of chromium(VI) on the status of plasma lipid peroxidation and erythrocyte antioxidant enzymes in chromium plating workers Ravi Babu Kalahasthi a,∗ , Rajmohan Hirehal Raghavendra Rao a , Rajan Bagalur Krishna Murthy a , M. Karuna Kumar b a

Regional Occupational Health Centre (Southern), Indian Council of Medical Research, Bangalore Medical College campus, Bangalore 560002, India b Department of Studies in Biochemistry, University of Mysore, Mysore 570006, India

Received 30 June 2006; received in revised form 12 September 2006; accepted 29 September 2006 Available online 6 October 2006

Abstract Objectives: The present study was carried out to determine the effect of chromium(VI) on the status of plasma lipid peroxidation and erythrocyte antioxidant enzymes in workers exposed to chromium during chromium plating process. Methods: Fifty subjects working in chromium plating process formed the study group. An equal number of age–sex matched subjects working in administrative units formed the control group. The control subjects were residing in the same city but away from the work place of study group subjects. Urinary chromium levels were determined by using a graphite furnace atomic absorption spectrophotometer. The plasma lipid peroxidation and erythrocyte antioxidant enzymes were determined by using spectrophotmetric methods. Results: A significant increase of plasma lipid peroxidation and a significant decrease of superoxide dismutase and glutathione peroxidase levels were noted in the study group as compared with the controls. The level of plasma lipid peroxidation was positively and erythrocyte antioxidant enzymes were negatively and significantly correlated with chromium levels in urine. Multiple regression analysis was assessed the oxidative stress associated with chromium and life style confounding factors such as BMI, coffee, tea, alcohol and smoking. The multiple regression analysis showed that the urine chromium levels >10 ␮g/g of creatinine, smoking, consumption of green vegetables and BMI variables were significantly associated with the levels of oxidative stress. Conclusion: The results show that the increased plasma lipid peroxidation and decreased antioxidant enzymes (superoxide dismutase and glutathione peroxidase) observed in chromium-exposed workers could be used as biomarkers of oxidative stress. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Urine chromium; Plasma lipid peroxidation; Erythrocyte antioxidant enzymes; Chromium plating

1. Introduction

∗

Corresponding author. Tel.: +91 80 26705037; fax: +91 80 26703359. E-mail address: [email protected] (R.B. Kalahasthi).

Electroplating is the process of oxidation of metal articles by the use of electrolytes containing acids or bases. The process of electroplating involves three steps: cleaning, plating and post-treatment of articles. Chromium is used as chromic acid, in electroplating of

0009-2797/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2006.09.012

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different articles used in the automobile manufacturing industry. The workers engaged in this process are exposed to chromium through inhalation, ingestion and dermal contact. Inhalation is the primary route of occupational exposure to metals [1]. Chromium is a Fenton metal and generates free radicals by itself. Fenton reactions are associated with membranous fractions including mitochondria, microsomes and peroxisomes. The generation of free radicals occurs when chromium undergoes redox-cycling reaction [2]. Chromium(VI) is reduced to short-lived chromium intermediates such as chromium(V), chromium(IV) and ultimately kinetically stable form of chromium(III). This reduction process generates reactive oxygen species (ROS), such as superoxide (O− ), hydroxyl (OH− ) and (H2 O2 ), is a source of hydroxyl radicals. The formation of free radicals is reported to cause oxidative damage to DNA, lipids and proteins [3,4]. An increased level of lipid peroxidation and decreased levels of enzymatic and non-enzymatic antioxidants were noticed in liver, kidney, brain and testicular tissues of animals [5–10]. The studies relating to occupational exposure to chromium during chromium plating process has reported nasal dysfunction, chromosome abnormality, oxidative injury to DNA, immunological effects and renal tubular dysfunction [11–15]. Goulart et al. [16] and Elis et al. [17] have reported increased levels of lipid peroxidation and decreased levels of antioxidants in workers exposed to chromium(VI) during metal arc welding and chromium(III) during leather tanning process. Several studies have reported increased levels of lipid peroxidation in plasma, blood and urine samples of workers exposed to chromium(VI) during chromium plating process [12,18]. However, both the studies did not assess oxidative stress with life style confounding factors. Therefore, the present study was undertaken to investigate the generation of free radicals involved in plasma lipid peroxidation and their potential effects on erythrocyte antioxidant enzymes (superoxide dismutase and glutathione peroxidase) in workers exposed to chromium during chromium plating process with life style confounding factors. The enzymatic and non-enzymatic antioxidant provides defense against the reactive oxygen species. The most important enzymatic antioxidants are superoxide dismutase, which catalyses dismutation of the superoxide anion (O2 − ) into H2 O2 , which is then deactivated to H2 O by glutathione peroxidase. Glutathione peroxidase uses glutathione as a cofactor for breakdown of H2 O2 . The present study has chosen these two marker enzymes in order to find the inactivation capacity of superoxide (O2 − ) and hydrogen peroxide (H2 O2 ) in chromium(VI) exposed workers.

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2. Materials and methods The study was carried out in 100 male subjects working in chromium plating industry located in Bangalore (India). These subjects were divided into two groups. The first group (study group) consisted of 50 workers involved in chromium plating process with an exposure period ranging from 15 to 20 years. Fifty subjects from administrative unit with no exposure to chromium formed the control group. Control subjects were matched regarding age and socio-economic status as that of study group subjects. Demographic details, work history and habits of the subjects were collected by using a questionnaire. Subjects with a history of diabetes or hypertension were excluded from the study. 2.1. Urine chromium Urine samples were collected from each subject in metal-free polyethylene bottles and used for determination of chromium according to the method of Claude et al. [19]. Chromium in urine samples was determined by using a flameless atomic absorption spectrophotometer equipped with graphite furnace (GF-3000) and auto-sampler (PAL-3000). This method has been recognized as a specific method for direct determination of chromium in human urine and hence is suitable for routine clinical use. Determination of chromium as internal standard added to urine and showed a recovery rate of 98.4%. The levels of urine chromium were expressed as ␮g/g of creatinine. The urinary chromium was standardized with urinary creatinine concentration measured by the Jaffe reaction method of Husdan and Rapoport [20]. From each of the subjects, 5 ml of whole blood was collected in heparinzed tubes and centrifuged at 3000 × g for 10 min at 4 ◦ C. The plasma was used for the estimation of lipid peroxidation and erythrocytes were used for determination of antioxidant enzymes. 2.2. Plasma lipid peroxidation The plasma lipid peroxidation (LPO) was determined by thiobarbituric acid method of Keosatoh [21]. This is also known as the thiobarbituric acid method since it involves the measurement of thiobarbituric acid reactive substances (TBARAS) released as a result of plasma LPO. In this method plasma is heated with thiobarbituric acid (TBA) at low pH. The malondialdehyde (MDA) formed reacts with TBA and the pink color developed is measured at 530 nm by using a spectrophotometer (Schimadazu, UV-1601PC, Japan). The stan-

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dard curve prepared by using 1,1,3,3-tetraethoxypropane dissolved in 0.05 M H2 SO4 and the calibration curve showed linearity up to 21.1 nmol/ml malondialdehyde. Plasma LPO was expressed as nm MDA formed/ml of plasma. 2.3. Superoxide dismutase (EC 1.15.1.1) The activity of superoxide dismutase was determined by the method of McCord and Fridovich [22]. In this method, xanthine and xanthine oxidase are used to generate the superoxide radicals, which react with 2(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride to form a red formazan dye. Superoxide dismutase inhibits the formation of formazan dye. The activity of superoxide dismutase was measured as percentage of inhibition. The unit of enzyme activity is defined as that amount which inhibits formazan formation by 50%. The results were expressed as ␮katal/g of heamoglobin. 2.4. Glutathione peroxidase (EC 1.11.1.9) The activity of glutathione peroxidase was determined by the method of Pagilia and Valentine [23]. Glutathione peroxidase catalyses the oxidation of glutathione in the presence of cumene hydroperoxide and glutathione reductase. The oxidized glutathione is converted into reduced form during the oxidation of NADPH to NADP+ . The unit of enzyme activity was defined as ␮m NADPH oxidized/s. The results were expressed as ␮katal/g of heamoglobin. 2.5. Heamoglobin The heamoglobin content in different samples were measured by using the method developed by Drabkin and Austin [24]. In this method, blood is mixed with Drabkin’s solution that contains ferricyanide and cyanide. The ferricyanide oxidizes hemoglobin into methemoglobin. Methemoglobin then unites with the cyanide to form cyanmethemoglobin. Cyanmethemoglobin produces a color that is measured at 530 nm using a spectrophotometer (Schimadaz, UV-1601PC, Japan). The level of hemoglobin is expressed as g/ml of blood. 2.6. Statistical analysis SPSS package, Version 7.5 for windows, was used for the statistical analysis of the data. The χ2 -test was used to compare the frequency distribution of life style confounding factors between study and control groups. The

Z-test was used to compare the differences in mean values among study and control group for variables age, duration of exposure, BMI, urine chromium, plasma lipid peroxidation and antioxidant enzymes. Pearson’s correlation coefficient was used to find out the correlation between urinary chromium and plasma lipid peroxidation and antioxidant enzymes. ANOVA was used to compare oxidative stress parameters among variables. Stepwise multiple regression analysis was used to assess the effect of the variables on oxidative stress. 3. Results Table 1 shows the demographic details of the study and control group. The average age and body mass index of study and control groups were suitably matched. The frequency distribution of life style confounding factors (such as consumption of vegetables, coffee, tea, smoking and alcohol) showed that there were no significant differences between the two groups. The average levels of urine chromium, plasma lipid peroxidation and erythrocyte antioxidant enzymes in study and control group subjects are presented in Table 2. The levels of urine chromium and plasma lipid peroxidation were significantly increased in study group subjects. The levels of erythrocyte antioxidant enzymes Table 1 Demographic data for the study and control group Variables

Study group (n = 50)

Control group (n = 50)

Age (years) BMI (kg/m2 ) Work duration (years)

41.4 ± 3.5a 26.4 ± 2.83 16.0 ± 2.6

42.0 ± 3.2 26.5 ± 2.96 15.5 ± 4.0

Consumption of vegetable (g/day) Beans 11 (22)b Lady finger 12 (24) Green leafy vegetables 17 (34) Cabbage 10 (20)

12 (24) 12 (24) 11 (22) 15 (30)

Consumption of coffee or tea (cups/day) Tea 16 (32) Coffee 24 (48) None 10 (20)

19 (38) 22 (44) 9 (18)

Smoking (no. of cigarettes smoked/day) No 39 (78) Yes 11 (22)

41 (82) 9 (18)

Alcohol consumption (drink/week) Usually 2 (4) Sometimes 6 (12) Never 42 (84)

4 (8) 2 (4) 44 (88)

Figure in parenthesis are percentages. a Mean and standard deviation. b Number of persons.

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Table 2 Urine chromium, plasma lipid peroxidation and antioxidant enzymes concentration in study and control group Variables

Study group (n = 50)

Control group (n = 50)

Urine chromium (␮g/g of creatinine) Plasma lipid peroxidation (nmol/ml) Superoxide dismutase (␮katal/g of Hb) Glutathione peroxidase (␮katal/g of Hb)

10.42 ± 8.34** (1.3–28.0) 4.01 ± 1.05** (2.70–8.00) 19.5 ± 0.59** (18.1–20.3) 0.75 ± 0.11** (0.47–1.17)

3.16 ± 0.84 (1.3–6.0) 2.63 ± 0.68 (1.0–4.0) 20.0 ± 0.63 (18.3–20.6) 0.89 ± 0.17 (0.68–1.18)

Values are mean ± S.D., values in parentheses are ranges. ** p < 0.001.

(superoxide dismutase and glutathione peroxidase) were significantly decreased in study group subjects. The correlation coefficients (r) between urine chromium, plasma lipid peroxidation and erythrocyte antioxidant enzymes in subjects are presented in Table 3. A positive correlation coefficient was noticed between urine chromium levels and plasma lipid peroxidation. The correlation coefficient was significant at p < 0.01. Negative correlation coefficients (r) were found between urine chromium levels and erythrocyte antioxidant enzymes: superoxide dismutase and glutathione peroxidase. These correlation coefficients were significant at p < 0.01 Table 4 shows the results of univariate analysis of variables that affect plasma lipid peroxidation and antioxidant enzymes. The plasma lipid peroxidation and activities of superoxide dismutase and glutathione peroxidase were found to be affected significantly in subjects who had urinary chromium levels >10 ␮g/g of creatinine. The consumption of green vegetables was significantly affected on plasma lipid peroxidation and glutathione peroxidase but on the levels of superoxide dismutase. However, no significant differences were observed for variables such as age, BMI, consumption of coffee, tea, smoking, alcohol and in subjects who had urine chromium levels <10 ␮g/g of creatinine. Table 5 shows the results of stepwise multiple regression analysis of variables that affect plasma lipid peroxidaton and erythrocyte antioxidant enzymes. The variables, included in the regression model, were age (1: ≤45years and 2: >45 years), body mass index (1: 18.5–24.9 kg/m2 , 2: 25.0–29.9 kg/m2 and 3:

≥30 kg/m2 ), consumption of vegetables (1: metallic vegetables (green leafy vegetables and green vegetables such as beans and lady finger) and 2: low metallic vegetables (cabbage)), coffee and tea (1: coffee, 2: tea and 3: none), smoking (0: no, 1: yes), alcohol (1: usually, 2: sometimes and 3: never). The level of urine chromium was categorized into two groups (1: ≤10 ␮g/g of creatinine and 2: >10 ␮g/g of creatinine) as per the recommendation of international standard of ACGIH-2005 [25]. The multiple regression analysis showed that the body mass index of 18.5–24.9 kg/m2 had a significant influence (70%) on the levels of plasma lipid peroxidation, whereas subjects with body mass index of 25.0–29.9 kg/m2 had a significant association (59%) with plasma lipid peroxidation and superoxide dismutase. Smokers appeared to have a significant influence (62%) on the levels of plasma lipid peroxidation and superoxide dismutase. Subjects who had urine chromium levels beyond 10 ␮g/g of creatinine appeared to have significant influence (27%) on levels of plasma lipid peroxidation, superoxide dismutase and glutathione peroxidase. The consumption of green leafy and green vegetables was showed a significant influence (61%) on plasma lipid peroxidation and glutathione peroxidase. The other confounding factors such as age, tea, coffee, alcohol and urine chromium levels <10 ␮g/g of creatinine had no influence on the levels of plasma lipid peroxidation or antioxidant enzymes. 4. Discussion The study assessed the status of plasma lipid peroxidation and antioxidant enzymes in subjects exposed to

Table 3 Correlation coefficient (r) between urine chromium, plasma lipid peroxidation and antioxidant enzymes (n = 100) Variables

Urine chromium

Plasma lipid-peroxidation

superoxide dismutase

Glutathione peroxidase

Urine chromium (␮g/g of creatinine) Plasma lipid peroxidation (nmol/ml) Superoxide dismutase (␮katal of Hb) Glutathione peroxidase (␮katal/g of Hb)

1.000 0.744** −0.449** −0.526**

– 1.000 −0.444** −0.469**

– – 1.000 0.398**

– – – 1.000

**

Correlation is significant at the p < 0.01.

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Table 4 Univariate analysis of the factors that affect plasma lipid peroxidation and antioxidant enzymes (n = 100) Variables

n

Plasma lipid peroxidation (nmol/ml)

Superoxide dismutase (␮katal/g Hb)

Glutathione peroxidase (␮katal/g Hb)

Age (years) ≤45 >45

92 8

3.30 ± 1.10 3.77 ± 1.33

19.85 ± 0.67 19.81 ± 0.54

0.83 ± 0.16 0.77 ± 0.18

BMI (kg/m2 ) 18.5–24.9 25.0–29.9 >30

26 62 12

3.08 ± 1.13 3.46 ± 1.16 3.08 ± 0.76

19.85 ± 0.71 19.85 ± 0.61 19.80 ± 0.83

0.85 ± 0.17 0.81 ± 0.17 0.83 ± 0.15

75

3.51 ± 1.19**

19.78 ± 0.70

0.80 ± 0.17**

25

2.80 ± 0.69

20.03 ± 0.50

0.88 ± 0.11

Consumption of coffee and tea (cups/day) Tea Coffee None

35 46 19

3.42 ± 1.05 3.30 ± 1.25 3.19 ± 0.94

19.80 ± 0.73 19.82 ± 0.61 20.00 ± 0.65

0.85 ± 0.19 0.80 ± 0.14 0.84 ± 0.18

Smoking (cigarettes/day) Yes No

20 80

3.19 ± 0.89 3.35 ± 1.17

19.83 ± 0.67 19.93 ± 0.60

0.85 ± 0.11 0.81 ± 0.18

Alcohol consumption (drink/week) Usually Sometimes Never

6 8 86

3.81 ± 1.13 3.10 ± 0.70 3.29 ± 1.14

20.07 ± 0.50 19.82 ± 0.50 19.83 ± 0.68

0.89 ± 0.07 0.86 ± 0.22 0.82 ± 0.16

Urine chromium ≤10.0 >10.0

81 19

2.94 ± 0.74 4.91 ± 1.10**

20.00 ± 0.60 19.22 ± 0.53**

0.87 ± 0.13 0.64 ± 0.16**

Consumption of vegetable (g/day) Metallic vegetables (green leafy vegetables, beans, lady finger) Low metallic (cabbage)

Values are mean ± S.D. ** p < 0.001.

chromium during chromium plating process. The levels chromium noted in urine, plasma and organs reflect the body burden of chromium. The determination of urine chromium was considered as an indicator for chromium exposure [26]. The absorption of chromium is quantified in the urine samples of chromium exposed and non-exposed subjects. The levels of urine chromium in chromium-exposed subjects noted in this study closely agree with the ones reported by Lukanova et al. [27]. During the present study urine chromium levels showed a high degree of variation among chromium-exposed subjects. This is similar to the findings of Kuo et al. [28] who noticed a higher variation (seven times) in chromiumexposed subjects as compared to control subjects. During the present study it was noted that plasma lipid peroxidation was significantly increased and the levels of antioxidant enzymes were significantly decreased in chromium-exposed subjects. The level of plasma lipid peroxidation was positively and levels of antioxidant enzymes were negatively and significantly correlated with the levels of chromium in urine. Since the oxidative

stress is related to life style confounding factors such as body mass index, the consumption of vegetables, coffee, tea, alcohol and smoking, the present study assessed the correlation between the oxidative stress and the life style confounding factors. Fehmi et al. [29] reported a significant association between the mean age of 65 years and the levels of plasma lipid peroxidation and superoxide dismutase. In this study, the age of subjects ranged from 40 to 55 years and evaluation using both univariate and multiple regression analysis showed no significant association. Ozata et al. [30] and Ohmori et al. [31] reported association between body mass index (BMI) and the levels of plasma lipid peroxidation, superoxide dismutase and glutathione peroxidase. During the present study, the univariate analysis did not find any such association. However, multiple regression analysis indicated that body mass index significantly influenced the plasma lipid peroxidation and superoxide dismutase levels. A large number of studies reported that there is reduction of oxidative stress in subjects who consumed vegetables [32–35]. Xie et al.

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Table 5 Multiple regression analysis of variables that affect the plasma lipid peroxidation and antioxidant enzymes (n = 100) Plasma lipid peroxidation (nmol/ml), β (p-value)

Superoxide dismutase (␮katal/g Hb), β (p-value)

Glutathione peroxidase (␮katal/g Hb), β (p-value)

R2

5.246 (0.000)*,a 3.654 (0.000)*

−0.188 (0.155)b −0.199 (0.008)*

−0.188 (0.113) −0.052 (0.600)

0.70 0.59

4.190 (0.000)*

−0.128 (144)

−9.668 (0.011)*

0.61

5.080 (0.00)*

−0.077 (0.647)

−0.294 (0.089)

0.63

Smoking Yes No

3.927 (0.000)* 3.039 (0.012)*

−0.035 (0.673) −3.859 (0.028)*

−0.158 (0.006)* −0.111 (0.514)

0.62 0.57

Alcohol consumption Never

3.800 (0.000)*

−0.078 (0.347)

−7.486 (0.018)*

0.56

Urine chromium >10.0

2.801 (0.034)*

−0.001 (0.805)*

−0.138 (0.409)*

0.27

Variables BMI (kg/m2 ) 18.5–24.9 25.0–29.9 Consumption of vegetable Metallic vegetables (green leaves, beans, lady finger) Coffee or tea None = 3

β (p-values) = regression coefficient (p-values of regression coefficients). a Regression coefficient is significant. b Regression coefficient is not significant. * Indicated significant p-value.

[36] have reported that the green vegetable has higher accumulation coefficient for metals. Khader and Rama [37,38] reported that the consumption of green leafy vegetables provide the greatest amount of minerals. Hence in the present study the consumption of vegetables by the subjects were separated into two groups, viz., (1) high metallic (green leafy vegetables, beans and lady finger) and (2) low metallic vegetables (cabbage) and effects on antioxidant enzymes and plasma lipid peroxidation were assessed. Univariate and multiple regression analysis indicated that the consumption of ‘metallic’ vegetables significantly influenced plasma lipid peroxidation and glutathione peroxidase but not superoxide dismutase levels. Mursu et al. [39] found that the long term or short term consumption of coffee did not have any detectable effect on lipid peroxidation and the antioxidant enzymes level in healthy non-smoking men. Rietveld and Wiseman [40] reported that tea (which contains high levels of flavonoids) could protect cells and tissues from oxidative damage by scavenging oxygen-free radicals. The present study did not find any association between oxidative stress and consumption of tea or coffee. Nalini et al. [41] reported oxidative stress in patients with alcoholic cirrhosis. Lecomte et al. [42] reported increased levels of plasma lipid peroxidation in chronic alcoholics than in groups with low and moderate alcohol consumption. During the present study it was noted that subjects, who consumed alcohol usually had increased

level of plasma lipid peroxidation and decreased level of superoxide dismutase. The univariate and multiple regression analysis showed no association between consumption of alcohol and oxidative stress (perhaps due to the subjects taking low or moderate amounts of alcohol). Koylu Halis et al. [43] and Nielsen et al. [44] reported a significant association between smoking habit and levels of plasma lipid peroxidation, superoxide dismutase and glutathione peroxidase. In the present study, the univariate analysis found no such association. However, multiple regression analysis showed that the smoking habit significantly influenced the levels of plasma lipid peroxidation and superoxide dismutase but had no effect on the levels of glutathione peroxidase. The increased plasma lipid peroxidation and decreased antioxidant enzymes were associated with chromium exposure and life style confounding factors such as BMI and smoking. In the present study, the univariate and multiple regression analysis showed that subjects who had urine chromium levels beyond 10 ␮g/g of creatinine appeared to have significant influence on plasma lipid peroxidation and antioxidant enzymes (superoxide dismutase and glutathione peroxidase). 5. Conclusion The plasma lipid peroxidation and antioxidant enzymes levels were significantly altered in the chromium exposed subjects. The level of plasma lipid

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