Subchronic exposure to a mixture of groundwater-contaminating metals through drinking water induces oxidative stress in male rats

Environmental Toxicology and Pharmacology 23 (2007) 205–211

Subchronic exposure to a mixture of groundwater-contaminating metals through drinking water induces oxidative stress in male rats Sachin Hanmantrao Jadhav a,1 , Souvendra Nath Sarkar a,∗ , Meena Kataria b , Harish Chandra Tripathi a a

Division of Pharmacology and Toxicology, Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India b Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar 243122, Uttar Pradesh, India Received 17 May 2006; received in revised form 23 August 2006; accepted 29 September 2006 Available online 5 October 2006

Abstract The current study examines the oxidative stress-inducing potential of a mixture of metals, representative of groundwater contamination in different areas of India. Male albino rats were exposed to the mixture through drinking water for 90 days at 0, 1, 10 and 100 times the mode concentrations of the metals in contaminated waters and at concentrations equal to their WHO maximum permissible limit (MPL) in drinking water. The endpoints evaluated were lipid peroxidation (LPO), GSH content and activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase in heart, liver, kidney and brain. MPL and 1× levels did not induce any alterations. The mixture at 10× and 100× doses increased LPO and decreased GSH level and activities of the antioxidases in kidney, liver and brain, but no alterations were observed in heart. An inverse correlation between LPO and GSH or antioxidaes and a positive correlation between GSH and glutathione peroxidase or glutathione reductase were found in the affected organs. The findings suggest that the mixture induces oxidative stress and decreases antioxidant status in 10× and 100× the mode concentrations of the metals in drinking water. © 2006 Elsevier B.V. All rights reserved. Keywords: Groundwater; Metal contaminants; Mixture; Oxidative stress; Rats

1. Introduction Chronic low-level environmental and occupational exposures to hazardous metals are important global toxicological concerns. Environmentally relevant metals seldom occur alone, rather they most often occur in hazardous waste sites or groundwater supplies along with other contaminants. This substantially complicates the risk assessment process for these elements. Many reports reveal that groundwater contamination with various metals is widespread in India. Groundwater meets India’s 80% of the drinking water requirement (Nagarathna, 2001), however, information on potential adverse health effects associated with

∗

Corresponding author. Tel.: +91 581 2300291; fax: +91 581 2303284. E-mail address: [email protected] (S.N. Sarkar). 1 Present address: Department of Pharmacogenomics, School of Pharmacy, 3307 N. Broad Street, Philadelphia, PA 19104, USA. 1382-6689/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2006.09.004

low-level intake of mixture of most frequently found metal contaminants in groundwater sources of India is not known. As each geologically contaminated groundwater source or hazardous site has a unique set of conditions, it is virtually impossible to find a representative sample of the various sites. It was presumed that a chemically defined mixture of groundwater-contaminating metals could be used to simulate a worst real-life scenario. We designed a mixture of eight metals (viz., arsenic, cadmium, lead, mercury, chromium, manganese, iron and nickel) with a relative concentration similar to that detected in the groundwater sources of India. Oxidative stress could be an important component of the mechanism of toxicity of the metal mixture (Fowler et al., 2004). The generation of reactive oxygen species (ROS), viz., hydrogen peroxide (H2 O2 ), superoxide anion radical (• O− ) and hydroxyl radical (OH• ) can lead to oxidative stress. A hallmark of the oxidative stress is LPO. Out of the eight metals selected, six, viz., chromium, manganese, iron, nickel cadmium and

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mercury are transition metals. It is known that transition metal ions can directly generate ROS and reactive nitrogen species (RNS; Gregus and Klaassen, 2001). Reductive homolytic fission of H2 O2 to OH• and OH− (Fenton reaction) is catalyzed by transition metals, which is a major toxication mechanism for H2 O2 and its precursor • O− as well as for transition metals. While iron, chromium, manganese and nickel can undergo redox-cycling reactions, mercury, cadmium, arsenic, lead and nickel are sulfhydryl-reactive metals and cause depletion of reduced glutathione (GSH) and bonding to sulfhydryl groups of many proteins (Quig, 1998; Valko et al., 2005). Pi et al. (2002) demonstrated that chronic exposure of Chinese residents to arsenic contaminated drinking water increased LPO and induced oxidative stress. Subchronic to chronic exposure of rats to arsenic (Chaudhuri et al., 1999; Santra et al., 2000), cadmium (Bagchi et al., 1997; Shukla et al., 1996) and lead (Adonaylo and Oteiza, 1999) through water caused escalation of LPO in brain and liver tissues. Several studies suggest that mercury (Huang et al., 1996; Hussain et al., 1997), chromium (Bagchi et al., 1997, 2002), nickel (Chen et al., 1999, 2003), iron (Day et al., 2003; Zang et al., 2003) and manganese (Brenneman et al., 1999) also induce LPO and exert their toxicity through the generation of ROS. Thus, the unifying factor in determining toxicity of these metals appears to be the generation of ROS and RNS. Metal-induced formation of free radicals causes enhanced LPO, modifications to DNA bases and altered calcium and sulfhydryl homeostasis (Valko et al., 2005). There are a number of enzymatic and non-enzymatic antioxidant defense systems to control excessive levels of ROS. Superoxide dismutase (SOD), an antioxidase catalyzing the dismutation of • O− into H2 O2 and O2 is thought be essential for the protection of cells against ROS and is used as an antioxidant drug (Kumagai et al., 1994). Catalase (CAT) exerts its antioxidant action by reductive degradation of H2 O2 into water and O2 . GSH, the most abundant nonprotein sulfhydryl in most cells, acts as a nucleophilic scavenger for various electrophiles and free radicals, thereby plays an important role against oxidative damage. It can react directly with ROS and metals and can act as a substrate in the glutathione peroxidase (GPX)mediated destruction of H2 O2 . Protection against toxic heavy metals, including arsenic and antimony has been shown to be associated with GSH efflux from the cell through multidrug resistance (MDR) protein transporter (Vernhet et al., 1999). Metal (mercury, cadmium)–GSH complex can desirably result in the excretion of the toxic metals into the bile, but simultaneously depletes the intracellular GSH also (Quig, 1998). GSH depletion can impair cellular defense against toxic onslaught and lead to cell injury and death. The oxidation–reduction cycling of GSH, i.e., oxidation of GSH to GSSG and the rapid reduction of GSSG by glutathione reductase (GR) contributes to the maintenance of a cellular GSH:GSSG ratio of about 300:1 (Alpert and Gilbert, 1985; Chung et al., 1991). Inhibition of GR reduces intracellular GSH level. The prooxidative properties the metals are exacerbated by the fact that the metals also inhibit antioxidative enzymes and deplete intracellular GSH (Hussain et al., 1997; Pi et al., 2002; Quig, 1998; Sandir and Gill, 1995; Shukla et al., 1996; Stohs et al., 2000). However, no information seems

available on the relationship between oxidative stress in vital organs and repeated exposure of animals and humans to a mixture of frequently found groundwater-contaminating metals at real-life exposure concentrations. The purpose of the present study was to investigate whether simultaneous subchronic exposure through drinking water to a mixture of frequently occurring metals in groundwater sources at environmentally realistic concentrations as well as at a concentration equal to WHO maximum permissible limit (MPL) can cause oxidative stress in different visceral tissues of rats and can alter the different antioxidant systems. We also sought to elucidate the possible underlying mechanism of oxidative stress induced by the metal mixture. 2. Materials and methods 2.1. Selection of metals Information on metal contamination of groundwater in India was gathered through literature survey. Data collected were utilized to formulate a mixture of metals that broadly represents the most frequently found metal contaminants with a relative concentration similar to that detected in contaminated groundwater sources. Therefore, it does not represent any particular contaminated water resource. The selection of these metals was based primarily on the frequency of their occurrence and contamination level above WHO maximum permissible limit in drinking water. A minimum of 15 to a maximum of 30 reports were considered for selection of an individual metal. The metals selected, viz., arsenic, cadmium, lead, mercury, chromium, nickel, manganese and iron are widely found in our environment and highly relevant to animal and human exposure. Reported concentrations of each metal were recorded. A frequency table of these concentrations was prepared by group frequency distribution and from this the mode concentration (the most frequently occurring concentration) of each metal was calculated following the method described by Hoshmand (1998) for grouped data.

2.2. Chemicals Cadmium chloride, mercuric chloride, chromium trioxide, lead acetate and nickel chloride were procured from E. Merck (India) Ltd., Mumbai. Sodium arsenite, manganese chloride and ferric chloride were purchased from Sigma Chemicals, USA; s.d. fine chemicals, Mumbai and Himedia, Mumbai, respectively. All other chemicals used in the study were of analytical grade from Sigma Chemicals, USA; E. Merck, Germany/India; SRL Chemicals, India.

2.3. Mixture formulation The mixture was prepared following the protocol reported by Wade et al. (2002). All the metal salts, which are soluble in water, were weighed into a glass bottle in amounts appropriate to make the 100× stock solution of the mode concentration. Lower doses (1× and 10×) of the dosing solution were prepared by 10-fold serial dilution of the 100× stock. Dosing solution for the concentrations equivalent to the MPL (WHO) was prepared separately. Dosing solutions were prepared daily to minimize possible instability of the chemicals in the mixture.

2.4. Animals and experimental protcol Male Wistar rats (75–100 g) procured from the Laboratory Animal Resources Section of the Institute were used. They were caged in pairs in clean plastic cages containing wheat straw chips for bedding and maintained under standard management conditions. All animals were given standard pellet diet (Amrut Inbred Rat and Mice Feed, Pranav Agro Industries, Delhi) and deionized water ad libitum. Prior to dosing, the animals were kept in laboratory conditions for 7 days. Animal care and handling were in accordance with the Institute Animal

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Table 1 The mixture of metals and its dose levels for subchronic toxicity studies in male rats Metal salt

Sodium arsenite Cadmium chloride Lead acetate Mercuric chloride Chromium trioxide Nickel chloride Manganese chloride Ferric chloride

Mode concentration (ppm)a

0.200 (15) 0.055 (30) 0.120 (23) 0.044 (17) 0.180 (15) 0.100 (23) 0.560 (15) 0.700 (29)

Dose levels (ppm)b Control

1×

10×

100×

MPL

0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0

0.380 0.098 0.220 0.060 0.346 0.810 2.026 2.033

3.80 0.98 2.20 0.60 3.46 8.10 20.26 20.33

38.0 9.8 22.0 6.0 34.6 81.0 202.6 203.3

0.096 0.005 0.018 0.008 0.141 0.081 1.800 0.870

a

Values in parentheses indicate the number of reports studied to derive the mode concentration. The baseline dose (1×) for each chemical was adjusted to give the representative elemental concentration of mode concentration and then safety factors (i.e. 10× and 100×) were incorporated for dose selection because of probable interspecies and interindividual variations as per the standard protocol (Yang and Rauckman, 1987). WHO maximum permissible limit (MPL) in drinking water was also adjusted to give elemental concentration. b

Ethics Guidelines. Rats were exposed daily to the mixture of metal components through drinking water (deionized) for 90 days. Five groups of rats (Groups I–V) comprising 10 animals each were used. Table 1 shows the mixture of metals and its dose levels for subchronic toxicity studies in male rats. Group I was given only deionized water and served as control. Group II, the baseline dose group (1×), was given mode concentrations of individual metals. Groups III and IV were, respectively, set at 10- and 100-fold concentrations of those of the baseline group. Rats of Group V received metals at concentrations equivalent to the MPL (WHO) for drinking water. During the course of the 90-day long experiment, 2–3 rats died in each group, including control, however, data for six animals have been presented. None of the animals died showed any visible signs of toxicity.

by 0.2 ml of DTNB (0.01 M) and absorbance was read at 412 nm within 5 min. The level of GSH has been expressed as mmol GSH/g wet tissue.

2.8. Determination of SOD activity Activity of SOD was measured following the procedure of Madesh and Balasubramanian (1998). Briefly, the reaction mixture contained 0.65 ml PBS saline (pH 7.4), 30 ␮l 3-(4-5 dimethyl thiazol 2-xl) 2,5-diphenyl tetrazolium bromide (MTT; 1.25 mM), 75 ␮l pyrogallol (100 ␮M) and 10 ␮l homogenate. The mixture was incubated at room temperature for 5 min and the reaction was stopped by adding 0.75 ml of dimethyl sulfoxide. The absorbance was read at 570 nm against DDW and the activity has been expressed as Unit (U). One U is the ␮g of protein required to inhibit MTT reduction by 50%.

2.5. Preparation of tissue homogenates 2.9. Determination of CAT activity On the day following the final dose, rats were killed under light ether anaesthesia by cervical dislocation. Prior to dissection of the organs, the body surface was wiped with 70% ethanol. The liver, kidneys, heart and brain were removed, cleaned of connective tissues and a piece of adequate amount from each tissue was stored at −80 ◦ C for assessment of oxidative stress. Frozen tissue samples were partially thawed and 200 mg of each sample was taken in 2 ml ice-cold normal saline. Another 200 mg was taken in 2 ml of 0.02 M EDTA for GSH estimation. Tissue homogenates (10%) were prepared in normal saline with IKA Homogenizer, Germany, under ice-cold condition and centrifuged for 10 min at 3000 rpm. The supernatant was used for biochemical estimations.

2.6. Assay of tissue LPO The level of LPO in tissue homogenates was determined by measuring thiobarbituric acid-reactive substances by the method of Shafiq-Ur-Rehman (1984). Briefly, 1 ml of tissue homogenate was incubated at 37 ± 0.5 ◦ C for 2 h. To each sample, 1 ml of 10% (w/v) trichloroacetic acid was added. After thorough mixing, the mixture was centrifuged at 2000 rpm for 10 min. To 1 ml of supernatant, an equal volume of 0.67% thiobarbituric acid was added and kept in boiling water bath for 10 min. After cooling, it was diluted with 1 ml of double-distilled water (DDW). The absorbance was read at 535 nm and the LPO level has been expressed as nmol malondialdehyde (MDA)/g wet tissue.

2.7. Assay of tissue GSH level GSH level was determined in the visceral tissues by estimating free sulfhydryl groups, using 5,5 -dithiobis-2-nitrobenzoic (DTNB) acid method of Sedlak and Lindsay (1968). In brief, to 1 ml supernatant, 0.8 ml of DDW and 0.2 ml of 50% trichloroacetic acid solution were added and incubated at room temperature for 15 min. This was centrifuged at 3000 rpm for 15 min. From this, 0.4 ml of supernatant was added to 0.8 ml of 1 M Tris buffer (pH 8.9) followed

Activities of CAT in heart, liver, kidney and brain tissue homogenates were estimated by the method of Aebi (1983). Briefly, 2 ml PBS (pH 7.4) and 10 ␮l tissue homogenate were taken in a cuvette. Reaction was started by adding 1 ml H2 O2 (kidney 2 mM; liver, heart and brain 20 mM) and the absorbance was recorded at every 10 s for 1 min at 240 nm against water blank. The activity of CAT has been expressed as k/g wet tissue. One k stands for nmol H2 O2 utilized/min.

2.10. Determination of GPX activity GPX activity was determined by the method of Paglia and Valentine (1967). The reaction mixture contained 2.48 ml PBS (pH 7.0, containing 5 mM EDTA), 0.1 ml NADPH (8.4 mM), 0.1 ml GSH (150 mM), 0.01 ml sodium azide (112.5 mM), 4.6 U glutathione reductase (Type III; Sigma Chemicals, USA) and 10 ␮l of homogenate. The reaction was initiated by adding 0.1 ml of H2 O2 (2.2 mM) to the mixture containing 500–1000 ␮g protein. The change in absorbance was read at 540 nm for 4 min. The enzyme activity has been expressed as unit/mg protein. One unit (U) is the amount (nmol) of NADPH utilized/min mg protein−1 at 25 ◦ C.

2.11. Determination of GR activity The enzyme activity was assayed as per the procedure of Goldberg and Spooner (1983). The 3 ml of reaction mixture contained 2.6 ml PBS (0.12 M, pH 7.2), 0.1 ml EDTA (15 mM), 0.1 ml GSSG (65.3 mM). To this 10 ␮l of homogenate was added and the volume was made up to 2.95 ml with DDW. After incubation at room temperature for 5 min, 0.05 ml of NADPH (9.6 mM) was added. Decrease in absorbance/min was recorded immediately at 340 nm for 3 min. Control was run without GSSG. The activity of GR has been expressed as U/mg protein. One unit (U) is nmol NADPH utilized/min mg protein−1 at 25 ◦ C.

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2.12. Protein estimation

GSH or different antioxidant enzymes as well as between GSH and GPX or GR were analyzed by using GraphPad Prism software.

Protein content in different tissue homogenates was measured by the method of Lowry et al. (1951), using bovine serum albumin as the standard.

2.13. Statistical analysis

3. Results 3.1. Effects of MPL and 1× dose levels

Results have been expressed as mean ± S.E.M. One-way ANOVA followed by Dunnett’s post hoc test was used to find out the differences between mean values at the significance level of P < 0.05. Correlations between the LPO and

Results presented in Tables 2 and 3 demonstrate that the MPL and 1× doses of the mixture of eight metals given to male rats

Table 2 Changes in the levels of lipid peroxidation and reduced glutathione in different visceral tissues of male rats exposed daily to the mixture of metals for 90 days through drinking water Treatment

Heart

Brain

Lipid peroxidation (nmol malondialdehyde/g wet tissue) Control 16.47 ± 2.00 MPL 17.56 ± 1.95 1× 17.90 ± 1.29 10× 18.56 ± 1.80 100× 20.05 ± 1.90 Reduced glutathione (mmol/g wet tissue) Control 0.070 ± 0.002 MPL 0.068 ± 0.003 1× 0.067 ± 0.004 10× 0.065 ± 0.003 100× 0.062 ± 0.003

Liver

Kidney

54.89 58.59 65.45 80.39 109.22

± ± ± ± ±

2.91 a 3.38 a 3.00 a 5.16 b 7.82 c

27.91 30.23 35.03 48.20 66.53

± ± ± ± ±

1.09 a 2.12 a 3.99 a 2.77 b 7.52 c

29.23 32.69 37.85 51.04 73.07

± ± ± ± ±

2.05 a 2.31 a 2.36 a 3.46 b 6.27 c

0.111 0.108 0.102 0.090 0.068

± ± ± ± ±

0.005 a 0.004 a 0.005 ab 0.003 b 0.004 c

0.094 0.090 0.085 0.072 0.050

± ± ± ± ±

0.005 a 0.004 a 0.004 a 0.003 b 0.004 c

0.084 0.082 0.075 0.064 0.040

± ± ± ± ±

0.004 a 0.003 a 0.004 a 0.003 b 0.003 c

MPL: maximum permissible limit in drinking water set by the WHO. Values (mean ± S.E.M; n = 6) in the same column bearing no letters (a, b or c) common vary significantly (P < 0.05).

Table 3 Changes in the activities of different antioxidative enzymes in different visceral tissues of male rats exposed daily to the mixture of metals for 90 days through drinking water Treatment Superoxide dismutase (Unit) Control MPL 1× 10× 100× Catalase (k/g wet tissue) Control MPL 1× 10× 100×

Heart

Brain

Liver

Kidney

28.87 27.70 26.03 25.30 22.99

± ± ± ± ±

2.49 3.32 3.97 2.85 2.56

11.96 11.63 10.61 9.13 7.49

± ± ± ± ±

0.55 a 0.66 a 0.39 a 0.62 b 0.34 c

34.25 32.06 30.32 25.00 18.17

± ± ± ± ±

1.70 a 2.07 a 1.80 a 1.40 b 1.35 c

31.75 30.19 26.60 19.94 10.90

± ± ± ± ±

2.50 a 2.72 a 1.60 ab 2.11 b 1.19 c

237.75 232.74 227.94 215.85 207.45

± ± ± ± ±

10.73 12.43 12.02 6.63 5.13

87.45 84.56 79.93 70.09 56.50

± ± ± ± ±

5.58 a 3.87 a 3.04 a 2.65 b 3.29 c

479.96 457.77 436.11 368.05 282.10

± ± ± ± ±

19.91 a 15.35 a 12.97 a 14.90 b 15.83 c

349.01 337.38 309.94 270.28 164.63

± ± ± ± ±

15.10 a 13.08 a 10.85 ab 12.93 b 21.88 c

354.82 349.92 329.93 284.17 214.45

± ± ± ± ±

10.87 a 9.79 a 8.34 a 9.19 b 10.05 c

217.98 210.19 198.03 177.11 139.49

± ± ± ± ±

7.85 a 6.09 a 7.85 a 7.15 b 5.18 c

165.48 154.00 143.32 118.66 85.14

± ± ± ± ±

5.56 a 11.34 a 7.20 a 5.91 b 7.12 c

94.07 91.59 87.99 79.03 65.36

± ± ± ± ±

3.21 a 5.13 ab 5.41 ab 4.99 b 6.00 c

74.65 71.03 67.53 55.78 39.49

± ± ± ± ±

4.17 a 6.30 a 4.45 ab 6.28 b 4.63 c

125.48 120.66 109.98 90.14 51.81

± ± ± ± ±

11.16 a 9.40 a 10.24 ab 6.13 b 8.05 c

Glutathione peroxidase (Unit/mg protein) Control 85.10 ± 4.28 MPL 83.45 ± 4.13 1× 82.27 ± 3.34 10× 80.72 ± 3.56 100× 79.01 ± 3.55 Glutathione reductase (Unit/mg protein) Control 35.31 ± MPL 34.32 ± 1× 33.67 ± 10× 32.93 ± 100× 31.25 ±

2.77 4.46 2.47 3.10 3.52

MPL: maximum permissible limit in drinking water set by the WHO. Values (mean ± S.E.M; n = 6) in the same column bearing no letters common vary significantly (P < 0.05).

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daily in drinking water for 90 days failed to induce any significant alterations in all the toxicological endpoints studied in heart, liver, kidney and brain. 3.2. Effect of 10× and 100× exposure levels The mixture produced dose-dependent and significant effects on all the attributes in rats exposed to 10× and 100× doses. None of the biochemical parameters in the heart was significantly altered with these doses. LPO was significantly increased by 74.6 and 150% in kidney, 72.7 and 138.4% in liver, and 46.4 and 99% in brain of rats exposed to 10× and 100× dose levels, respectively, compared to the control rats (Table 2). Rats exposed to these two doses showed significant decrease in GSH levels in kidney (23.8 and 52.2%), liver (23.0 and 46.3%) and brain (19.8 and 35.2%) in comparison to control animals (Table 2). With 10× and 100× doses, the activities of SOD were decreased significantly by 37.2 and 65.7% in kidney, 26.0 and 47.2% in liver and 23.7 and 37.4% in brain (Table 3), while of CAT decreased by 22.6 and 53.0% in kidney, 23.2 and 41.1% in liver and 19.9 and 35.4% in brain (Table 3), respectively. In these doses, the mixture significantly decreased the activities of both the enzymes of glutathione system, viz., GPX and GR. Compared to control values, the respective decreases in case of GPX were 28.3 and 48.5 in kidney, 19.9 and 39.5% in liver and 18.7 and 36.1% in brain (Table 3). With respect to GR, the respective percentages of reduced enzyme activity for these organs were 28.2 and 58.2; 25.3 and 47.9; 16.0 and 30.5 (Table 3).

Table 4 Correlation of lipid peroxidation (LPO) with GSH content and activities of different antioxidant enzymes, and of GSH with glutathione peroxidase and glutathione reductase (n = 30) r value/P value

Kidney

Liver

Brain

LPO with GSH r P

0.764 <0.0001

0.791 <0.0001

0.716 <0.0001

LPO with superoxide dismutase r 0.786 P <0.0001

0.764 <0.0001

0.686 <0.0001

LPO with catalase r P

0.783 <0.0001

0.817 <0.0001

0.716 <0.0001

LPO with glutathione peroxidase r 0.731 P <0.0001

0.782 <0.0001

0.748 <0.0001

LPO with glutathione reductase r 0.738 P <0.0001

0.573 ≤0.0009

0.560 ≤0.0013

GSH with glutathione peroxidase r 0.723 P <0.0001

0.735 <0.0001

0.709 <0.0001

GSH with glutathione reductase r 0.724 P <0.0001

0.560 <0.0013

0.616 <0.0003

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3.3. Correlations between the oxidative stress parameters Table 4 shows that there was a significant inverse correlation between the LPO and GSH levels in liver, kidney and brain. Similar correlation was also observed in these visceral organs between LPO and the activities of different antioxidant enzymes such as SOD, CAT, GPX and GR (Table 4). However, a significant positive correlation was found when the levels of GSH were correlated with the activities of GPX or GR (Table 4) in these tissues. 4. Discussion The present study reveals that the subchronic exposure of rats to the mixture of eight metals in MPL and 1× levels via drinking water did not produce any significant alterations in any of the endpoints examined. This offers some support to the assumption that derivation of safe levels (MPL) of exposure from single agent toxicity studies could provide an adequate protection for adult male rats. However, it is noteworthy that the current 90day toxicity study is not adequate for toxicological assessment of chronic/lifetime exposure. Fowler et al. (2004) also emphasized the importance of duration of exposure in assessing the toxic potential of lead, cadmium and arsenic mixtures at low dose levels. They observed that cellular protective systems, which protected against oxidative damage at the 90-day time point, failed to protect the molecular manifestations of oxidative stress that became statistically significant at the 180-day time point for several of the combination exposure groups. The mixture caused dose-dependent increase in LPO and decreases in enzymatic and non-enzymatic antioxidant systems in kidney, liver and brain with 10× and 100× doses. Among the tissues, kidney was the most affected one. Chronic exposure of Chinese residents to arsenic contaminated drinking water increased LPO and induced oxidative stress (Pi et al. (2002). Several studies indicated oxidative damage and LPO in brain, liver and kidney of rats by repeated exposure to arsenic (Chaudhuri et al., 1999; Santra et al., 2000), cadmium (Bagchi et al., 1997; Shukla et al., 1996) and lead (Adonaylo and Oteiza, 1999; AykinBurns et al., 2003) through water was triggered by the generation of ROS. Literature reveals that other metals in the mixture such as mercury (Huang et al., 1996; Hussain et al., 1997), chromium (Bagchi et al., 1997, 2002), nickel (Chen et al., 1999, 2003), iron (Day et al., 2003; Zang et al., 2003) and manganese (Brenneman et al., 1999) also induce LPO and exert their toxicity through generation of ROS. Valko et al. (2005) suggested that the unifying factor in determining toxicity of these metals could be the generation of ROS and RNS. Thus, oxidative damage to these structurally and functionally dissimilar tissues through increased LPO could be the result of combined effect of accumulation of ROS, resulting from dysfunction GSH and antioxidases, and overproduction of free radicals during subchronic exposure to the metal mixture. There are many reports, which reveal that the prooxidative effect of the metals is compounded by their inhibitory effect on enzymatic and non-enzymatic antioxidant processes (Hussain et al., 1997; Pi et al., 2002; Quig, 1998; Sandir and Gill, 1995;

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Shukla et al., 1996; Stohs et al., 2000; Valko et al., 2005). The strong inverse correlations between the levels of GSH and LPO in liver, kidney and brain suggest that depletion of GSH was a reasonable cause of LPO. Conversely, the overproduction of ROS could cause the depletion of GSH. Significant positive correlations between GSH and GPX or GR also substantiate the involvement of GSH in the mixture-induced LPO. It is known that SOD can be up-regulated by overproduction of ROS, and the lack of its expression in SOD knock-out animals results in oxidative stress (Mates, 2000). The glutathione redox cycle is a major source of protection against low levels of oxidative stress, whereas CAT becomes more significant in protecting against severe oxidant stress (Yan and Harding, 1997). The negative correlations of LPO with SOD or GPX or CAT in liver, kidney and brain suggest that these antioxidant enzymes contribute to the oxidative stress-dependent toxicity caused by the metal mixture. Decrease in the activities of antioxidant enzymes may be due to their depletion in response to metal mixture-induced oxidative stress. The decreased GPX activity in tissues may be attributed to the observed diminished level of GSH and this could be substantiated by the positive correlation between GSH and GPX. The metal mixture decreased the activity of GR. Inhibition of GR leads to accumulation of the prooxidant GSSG by preventing reduction of GSSG to GSH, suggesting that the consumption of GSH may not be compensated by GR. One important aspect of the antioxidative enzymes is their synergistic functioning, which may partly explain the mechanism of their reduced activity. A decrease in SOD activity increases the level of • O− , which is known to inactivate CAT activity (Kono and Fridovich, 1982); when CAT or GPX fails to eliminate H2 O2 from the cell, the accumulated H2 O2 causes inactivation of SOD (Sinet and Garber, 1981). The metal mixture decreased the levels of GSH in liver, kidney and brain with higher doses in a dose-related manner. It is known that the metals such as arsenic (Ochi et al., 1994; Pi et al., 2002), cadmium (Shukla et al., 1996; Stohs et al., 2000), lead (Gong and Evans, 1997; Sandir and Gill, 1995), mercury (Quig, 1998) and nickel (Valko et al., 2005) deplete intracellular GSH. The principal mechanisms by which xenobiotics decrease the level of GSH are conjugation of GSH with the prooxidants (electrophiles, including metals and oxyradicals), inhibition of GR activity and inhibition of GSH synthesis. Conjugation of arsenic, mercury and cadmium with GSH is desirable in that it results in their excretion into the bile (Gyurasics et al., 1991; Quig, 1998), however, it can deplete the cell of GSH and thereby decrease the antioxidant capacity. In several instances, protection against toxic heavy metals, including arsenic and antimony has been shown to be associated with GSH efflux from the cell through MDR protein transporter (Vernhet et al., 1999). In the present study, the magnitude of reduction in GR activity in all the tissues was almost proportionate to the extent of fall in GSH level. This suggests that inhibition of GR activity by the metal mixture could be an important mechanism for reducing the level of GSH. Inhibition of the activities of GPX and CAT implies that H2 O2 remains accumulated in the cells of these visceral organs, while reduced SOD activity indicates decreased dismutation of

• O−

to H2 O2 . It is known that • O− can also be spontaneously converted to H2 O2 (Gregus and Klaassen, 2001). These observations suggest possible cellular accumulation of H2 O2 by the metal mixture. One of the important biological consequences of • O− production is the formation of H O , which is converted 2 2 by Fenton reaction to OH• , the ultimate toxicant for transition metals and H2 O2 or for xenobiotics that form • O− (Gregus and Klaassen, 2001). Therefore, the metal mixture-induced oxidative stress may relate to conversion of accumulated H2 O2 to OH• . In summary, the current study of potential toxicity associated with the consumption of the mixture of eight metals representative of groundwater contamination in different parts of India demonstrates induction of oxidative stress in rats receiving the mixture in drinking water in 10× and 100× the mode concentrations of the individual components determined by literature survey of groundwater contamination with metals and suggests that MPLs of the metals in drinking set by the WHO could provide adequate protection for adult male rats. Our data suggest that the oxidative stress induced by subchronic exposure to the mixture may be related to the Fenton reaction and was associated with the diminution in antioxidative capacity of the rats. Depletion of GSH by the mixture could be a consequence of combined effects of conjugation of GSH with the prooxidants and inhibition of GR activity and GSH synthesis. As the components of the mixture were selected based on the known metal contaminants in ground water, the relationship between the levels of exposure used in the current study and the levels to which the general populations are exposed is relevant to public health. Acknowledgements The Senior Research Fellowship awarded to the first author is gratefully acknowledged. The authors are thankful to the Director of the Institute for providing all necessary facilities for carrying out the present work. References Adonaylo, V.N., Oteiza, P.I., 1999. Lead intoxication: antioxidant defenses and oxidative damage in rat brain. Toxicology 135, 77–85. Aebi, H.E., 1983. Catalase. In: Bergmeyer, H.U., Bergmeyer, J., Graßl, M. (Eds.), Methods of Enzymatic Analysis, vol. III, 3rd ed. Verlag Chemie, Weinheim, pp. 273–286. Alpert, A.J., Gilbert, H.F., 1985. Detection of oxidized and reduced glutathione with a recycling post column reaction. Anal. Biochem. 144, 553–562. Aykin-Burns, N., Laegeler, A., Kellog, G., Ercal, N., 2003. Oxidative effects of lead in young and adult fisher 344 rats. Arch. Environ. Contam. Toxicol. 44, 417–420. Bagchi, D., Stohs, S.J., Downs, B.W., Bagchi, M., Preuss, H.G., 2002. Cytotoxicity and oxidative mechanisms of different forms of chromium. Toxicology 180, 5–22. Bagchi, D., Vuchetich, P.J., Bagchi, M., Hassoun, E.A., Tran, M.X., Tang, L., Stohs, S.J., 1997. Induction of oxidative stress by chronic administration of sodium dichromate (chromium VI) and cadmium chloride (cadmium II) to rats. Free Radic. Biol. Med. 22, 471–478. Brenneman, K.A., Cattley, R.C., Ali, S.F., Dorman, D.C., 1999. Manganeseinduced developmental neurotoxicity in the CD rat: is oxidative damage a mechanism of action? Neurotoxicology 20, 477–487.

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