Abstract
Aim:
To avert the health problems induced by many environmental pollutants, available antioxidants have been evaluated. The present study was aimed to investigate whether α-tocopherol could protect the hexavalent chromium (Cr VI)-induced peroxidation in the liver and kidney and to explore the underlying mechanism of the same.
Materials and Methods:
A total of 24 Wistar adult female rats were equally divided into four groups. Group 1 served as control while Groups 2 and 3 were administered K2Cr2O7(10 mg/kg b.wt. s.c. single dose). In addition to (Cr VI), Group 3 also received α-tocopherol (125 mg/kg, daily) by oral gavage for 14 days. Group 4 was maintained as α-tocopherol control (dose as above). At the end of 14 days, blood samples were drawn for hematology. Subsequently, all the rats were sacrificed to collect liver and kidney samples for assay of tissue peroxidation markers, antioxidant markers and functional markers and histopathology.
Results:
Administration of chromium (Cr VI) in Group 2 significantly (P < 0.05) reduced the antioxidant markers such as superoxide dismutase and reduced glutathione along with significant (P < 0.05) increase in peroxidation markers such as malondialdehyde and protein carbonyls in the liver and kidney as compared with other groups. The functional markers in serum such as total protein was decreased significantly (P < 0.05), whereas other functional markers viz. alanine transaminase, blood urea nitrogen and creatinine were increased significantly (P < 0.05) in Group 2 as compared with the other groups. Significant (P < 0.05) decrease in hemoglobin, packed cell volume, total erythrocyte count, mean corpuscular volume, mean corpuscular hemoglobin and total leukocyte count were observed in Cr VI treated Group 2 rats. Prominent pathological changes were observed in the liver and kidney of Group 2. Co-treatment with α-tocopherol in Group 3 rats significantly (P < 0.05) reversed the Cr VI induced changes. The parameters in the study in Group 4 did not differ as compared with Group 1.
Conclusions:
α–tocopherol exhibited protective effect against Cr VI-induced damage to the liver and kidney by inhibition of lipid peroxidation owing its antioxidant activity.
KEY WORDS: α-tocopherol, chromium (Cr VI), kidney, lipid peroxidation, liver
Introduction
Chromium is abundant in many substances that are commercially used in wood preservation, leather industries and industrial welding.[1] Chromium (Cr VI) is found in the environment in two valence states: trivalent Cr (III) and hexavalent Cr (VI). Chromium (III) compounds have been reported to be less toxic than Cr (VI) compounds because the latter can cross the cell membrane easily. Reduction of Cr (VI) to Cr (III) results in the formation of reactive oxygen species (ROS) that induce oxidative damage.[2] This in turn is responsible for defective hematopoiesis[3] and a cascade of cellular events including modulation of apoptosis regulatory gene p53 and contribute to the cytotoxicity, genotoxicity and carcinogenicity.[4]
The potential role of oxidative stress in injury associated with Cr6+ exposure suggests that antioxidant supplementation may mitigate chromate-induced toxicity. Vitamin E (α-tocopherol) is an important component in the human diet and considered the most effective liposoluble antioxidant found in the biological system. It reacts with peroxy radicals 10,000-fold faster than do polyunsaturated lipids.[5] Therefore, vitamin E is potentially useful as a therapeutic agent in the treatment of several disorders associated with oxidative damage.[6] It might diminish lipid peroxidation (LPO) induced by heavy metals, including dichromate and protects the body's biological systems.[7] Because of the health problems induced by many environmental pollutants, much effort has been expended in evaluating the relative antioxidant potency of vitamin E.[8]
Therefore, the present study was designed to investigate the possibility that the administration of α-tocopherol would have a beneficial effect on Cr-induced hepatic and renal injuries.
Materials and Methods
Chemicals
All chemicals were of analytical grade and obtained from Qualigens Pvt. Ltd., Mumbai, India.
Animals
Adult Wistar rats aged about 60 days with an average body weight of 140 ± 10 g were obtained from National Institute of Nutrition, Hyderabad. The animals kept in polypropylene cages were maintained under standard conditions prescribed by the committee for the purpose of control and supervision on experiments on animals.
Experimental Design
A total of 24 rats were randomly divided into four groups with six rats in each. Group 1 was maintained as normal while Group 2 rats acted as Cr toxicity control. These rats were given Cr VI as K2Cr2O7 dissolved in sterile saline (Nacl: 0.9%) @ 10 mg/kg b.wt. as a single s.c. injection. Group 3 received Cr VI as above, but along with α-tocopherol, daily for 14 days by oral gavage. Group 4 was maintained as α-tocopherol control and was given α-tocopherol daily for 14 days by oral gavage. The study was approved by Institutional Animal Ethics Committee (Approval No. I/7/2012).
In this experiment, the dose of Cr VI to induce oxidative stress was based on a report by Biber et al.[9] The selected dose of α-tocopherol was as per Arreola-Mendoza et al.[7] who stated that α-tocopherol at a dose of 125 mg/kg b.wt. for 14 days effectively protected the kidney against Cr VI-induced alteration in lipid patterns.
After completion of 14 days, the blood samples were collected from retro-orbital plexus of experimental rats for studying hematological (total erythrocyte count [TEC], total leukocyte count [TLC], differential leukocyte count [DLC], hemoglobin [Hb], packed cell volume [PCV], mean corpuscular volume [MCV], mean corpuscular hemoglobin [MCH], mean corpuscular hemoglobin concentration [MCHC]) and serum biochemical profile (alanine transaminase [ALT], blood urea nitrogen [BUN], creatinine and total protein). Then all the rats were euthanized. Liver and kidney tissues were collected immediately and kept in ice cold phosphate buffer. A portion of the organs was homogenized with tissue homogenizer individually to make 10% homogenate to assay the tissue antioxidants such as superoxide dismutase (SOD), reduced glutathione (GSH) and tissue peroxidation markers such as thiobarbituric acid reacting substances (TBARS/malondialdehyde [MDA]) and protein carbonyls. Pieces of tissues from liver and kidney were immediately kept in 10% of formalin fixative to study histological alterations, if any.
Biochemical Analysis
Hematology
Hematological parameters viz., TEC, TLC, DLC, Hb, PCV, MCV, MCH, and MCHC were analyzed by Auto Blood Analyzer Merck Specialties Pvt. Ltd, Mumbai.
Antioxidant Markers
SOD was estimated by the method that involved inhibition of superoxide-dependant reduction of tetrazolium dye methyl thiazolyl tetrazolium to its formazan.[10] GSH was estimated based on a reaction of reduced GSH with 5-5 ditiobis-2-nitrobenzoic acid.[11]
Peroxidation Markers
MDA, the product of lipid peroxidation, was estimated by reaction with thiobarbituric acid as per the method prescribed by Balasubramanian et al.[12] Protein carbonyls were estimated based on the reaction of amino carbonyls with 2, 4-dinitrophenyl hydrazine to form hydrazones, which can be detected spectrophotometrically at 372 nm.[13]
Functional Marker Enzymes
Total protein, ALT, BUN and creatinine were estimated in serum by using the Erba Diagnostic Kits, Germany.
Total Protein
Total protein in the liver and kidney tissue was quantified as per Lowry et al.'s[14] method.
Histology
For light microscopy examination, the formalin fixed tissues were dehydrated through ascending grades of alcohol, cleared in three changes of xylene and were embedded in paraffin. Serial sections, each of four-micron thickness, were cut and stained with H and E.
Statistical Analysis
Data were subjected to statistical analysis by applying one-way analysis of variance using the statistical package for social sciences (SPSS) version 12.0. Differences between means were tested using Duncan's multiple comparison tests and significance was set at P < 0.05.
Results
The average body weight gain was significantly (P < 0.05) reduced in Group 2 as compared with other groups. However, co-administration of α-tocopherol with Cr VI (Group 3) showed a significant (P < 0.05) increase in weights as compared with Group 2. The average body weight in Group 4 rats was comparable with that Group 1 [Figure 1].
Figure 1.
Mean weights of different groups of rats
In Cr VI-treated Group 2 group, a significant (P < 0.05) decrease in hemoglobin and TEC values were seen. Similarly, significant (P < 0.05) decrease in mean values of PCV, MCV, and MCH were seen. For MCHC value, no significant decrease was noticed in all groups. Leucocyte picture reveals significant (P < 0.05) decrease in TLC along with neutrophilia, eosinophilia and lymphopaenia in Group 2 rats as compared with other groups. Administration of α-tocopherol along with Cr VI significantly reversed the above alterations in Group 3 [Table 1].
Table 1.
Effect of α-tocopherol on haematological parameters in female wistar rats
In Group 2, the peroxidation markers in the liver and kidney such as MDA and protein carbonyls were significantly (P < 0.05) increased and the levels of antioxidants such as SOD and reduced GSH were reduced significantly (P < 0.05) as compared with other groups. Administration of α-tocopherol significantly (P < 0.05) reversed the above values in Group 3 as compared with Group 2 [Table 2].
Table 2.
Antioxidant defenses and peroxidation biomarkers in liver and kidney homogenates of different groups of rats
The functional markers of liver in serum such as total protein were significantly (P < 0.05) decreased while the ALT levels were significantly (P < 0.05) increased following Cr VI administration in Group 2. Kidney functional markers such as serum creatinine and BUN were also significantly (P < 0.05) increased in Group 2 compared with Group 1. The above altered functional markers were significantly (P < 0.05) reduced with administration of α-tocopherol in Group 3 [Table 3].
Table 3.
Effect of α-tocopherol on liver and kidney functional markers
Liver of chromium-treated (Cr VI) group showed vascular congestion, degenerative changes and dilatation of sinusoids [Figure 2a]. In addition, periportal areas showed infiltration of mononuclear cells in the large numbers [Figure 2b]. Kidney sections from Group 2 revealed degeneration of tubular epithelial cells, cystic dilatation of tubules, hyaline casts, congestion of blood vessels [Figure 2c and d]. Recovery from histological injury was observed in α-tocopherol co-administered Group 3 rats, with mild congestion of hepatocytes ]Figure 3a] and mild swelling of glomerular tufts [Figure 3b]. In Group 4, treatment with α-tocopherol alone, revealed normal architecture [Figure 3c and d].
Figure 2a.
Light microscopic study of (a) chromium treated group rat liver showing vascular congestion, degenerative changes and dilatation of sinusoids (H and E, ×400)
Figure 2b.
Chromium treated group rat liver showing mononuclear cell infiltration (H and E, ×100)
Figure 2c.
Chromium treated group rat kidney showing degeneration of tubular epithelial cells, cystic dilatation of tubules and hyaline casts (H and E, ×100)
Figure 2d.
Chromium treated group rat kidney showing dilation and congestion of blood vessels, degenerative changes and dilatation of bowmans space (H and E, ×100)
Figure 3a.
Chromium and vitamin E treated rat liver showing congestion (H and E, ×200)
Figure 3b.
Chromium and vitamin E treated rat kidney showing mild swelling of glomerular tufts (H and E, ×100)
Figure 3c.
Vitamin E treated group rat liver showing normal architecture (H and E, ×100)
Figure 3d.
Vitamin E treated group rat kidney showing normal histoarchitecture (H and E, ×100)
Discussion
Chromium (Cr VI) compounds are widely recognized as human carcinogens.[15] From the epidemiological studies, it is suggestive that hexavalent chromium causes increased risk of bone, prostate lymphomas etc. reflecting the ability of hexavalent chromium to penetrate all tissues in the body.[16] Due to their extensive use in the industry, there is a need to investigate their combined toxicity in organ system and mitigative role of vitamin on their toxicity. Previous studies have shown that dichromate exposure increases the concentration of ROS,[17] and provokes oxidative damage in hepatocytes[18] and kidney.[19]
Administration of Cr revealed a significant (P < 0.05) decrease in body weight in Group 2 indicating its ability to induce oxidative stress. Cr administration also revealed a significant (P < 0.05) decrease in hemoglobin, TEC, TLC, PCV, MCV, and MCH values in Group 2. For MCHC value, no significant decrease was noticed in all groups. Leucocyte picture revealed neutrophilia, eosinophilia and lymphopenia in Group 2. Similar observations of hematological parameters were reported earlier in rats on exposure to dichromate.[20] The decrease in MCH, Hb and PCV values could be attributed to the intracellular reduction of hexavalent chromium to trivalent chromium and subsequent binding of trivalent chromium to various intra-cellular molecules and proteins including hemoglobin.[21]
Administration of Cr VI resulted in oxidative stress in the liver and kidney that was reflected by altered histoarchitecture, with degenerative changes and dilatation of sinusoids in liver. Kidney sections revealed degeneration of tubular epithelial cells, cystic dilatation of tubules, hyaline casts, congestion of blood vessels and dilatation of bowmans space. Severe histological changes in the liver and kidney of Cr treated rats were earlier reported by Acharya et al.[22] and Da Silva et al.[23] Cr VI induces free radical production by multiple mechanisms leading to peroxidation, which in the present study was revealed by a significant increase in peroxidation markers such as MDA and protein carbonyls and decrease in antioxidant markers such as SOD and GSH. Peroxidative damage causes reduction in hepatic and kidney function, which was reflected by a significant decrease in total protein with a significant increase in ALT activity indicating hepatotoxicity and significant increase in serum levels of BUN and creatinine suggesting nephrotoxicity. The results of the present study are in agreement with earlier findings of reduction in the antioxidant markers with simultaneous increase in peroxidation markers and functional markers in rats under Cr VI influence.[24]
α-tocopherol, is a chain breaking antioxidant that exists in cell membranes[25] and plasma.[26] It eliminates lipid peroxyl and alkoxyl radicals, suppresses the chain reaction of LPO and promotes the production of scavenger antioxidant enzymes.[27] In our in vivo study, co-administration of α-tocopherol along with Cr VI exhibited significant protective effect in all aspects as evident from reversal of histological alterations, lowered tissue peroxidation, elevated antioxidant activity, reversal of functional markers and hematological parameters. This ability might be related to the fact that lipid peroxyl radicals react more rapidly (by four orders of magnitude) with α-tocopherol than with membrane lipids as was suggested by Halliwell and Gutteridge.[6] Our results are consistent with previous studies by Susa et al.,[28] who reported that pre-treatment with α-tocopherol normalized the level of non-enzymatic antioxidants such as GSH by dichromate.
Conclusion
In conclusion, chromium caused the formation of free radicals in the liver and kidney by reducing the antioxidant indices. However, α-tocopherol supplementation to chromium fed rats exhibited no adverse effects indicating its protective antioxidant property. Thus, present investigation confirmed the role of α-tocopherol as a scavenger of free radicals, probably preserving structural and functional integrity of subcellular organelles.
Footnotes
Source of Support: Nil
Conflict of Interest: None declared
References
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