|Year : 2013 | Volume
| Issue : 5 | Page : 490-495
An evaluation of the protective role of α-tocopherol on free radical induced hepatotoxicity and nephrotoxicity due to chromium in rats
Rajendran Balakrishnan, Chitturi Sree Venkat Satish Kumar, Matukumalli Usha Rani, Mylaram Kistaiah Srikanth, Gopu Boobalan, Alla Gopala Reddy
Department of Pharmacology and Toxicology, College of Veterinary Science, Delhi, India
|Date of Submission||09-Nov-2012|
|Date of Decision||23-Jan-2013|
|Date of Acceptance||30-Jun-2013|
|Date of Web Publication||6-Sep-2013|
Alla Gopala Reddy
Department of Pharmacology and Toxicology, College of Veterinary Science, Delhi
Source of Support: None, Conflict of Interest: None
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 K 2 Cr 2 O 7 (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.
Keywords: α-tocopherol, chromium (Cr VI), kidney, lipid peroxidation, liver
|How to cite this article:|
Balakrishnan R, Satish Kumar CS, Rani MU, Srikanth MK, Boobalan G, Reddy AG. An evaluation of the protective role of α-tocopherol on free radical induced hepatotoxicity and nephrotoxicity due to chromium in rats. Indian J Pharmacol 2013;45:490-5
|How to cite this URL:|
Balakrishnan R, Satish Kumar CS, Rani MU, Srikanth MK, Boobalan G, Reddy AG. An evaluation of the protective role of α-tocopherol on free radical induced hepatotoxicity and nephrotoxicity due to chromium in rats. Indian J Pharmacol [serial online] 2013 [cited 2019 Jun 17];45:490-5. Available from: http://www.ijp-online.com/text.asp?2013/45/5/490/117778
| » Introduction|| |
Chromium is abundant in many substances that are commercially used in wood preservation, leather industries and industrial welding.  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.  This in turn is responsible for defective hematopoiesis  and a cascade of cellular events including modulation of apoptosis regulatory gene p53 and contribute to the cytotoxicity, genotoxicity and carcinogenicity. 
The potential role of oxidative stress in injury associated with Cr 6+ 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.  Therefore, vitamin E is potentially useful as a therapeutic agent in the treatment of several disorders associated with oxidative damage.  It might diminish lipid peroxidation (LPO) induced by heavy metals, including dichromate and protects the body's biological systems.  Because of the health problems induced by many environmental pollutants, much effort has been expended in evaluating the relative antioxidant potency of vitamin E. 
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|| |
All chemicals were of analytical grade and obtained from Qualigens Pvt. Ltd., Mumbai, India.
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.
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 K 2 Cr 2 O 7 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.  The selected dose of α-tocopherol was as per Arreola-Mendoza et al.  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|| |
Hematological parameters viz., TEC, TLC, DLC, Hb, PCV, MCV, MCH, and MCHC were analyzed by Auto Blood Analyzer Merck Specialties Pvt. Ltd, Mumbai.
SOD was estimated by the method that involved inhibition of superoxide-dependant reduction of tetrazolium dye methyl thiazolyl tetrazolium to its formazan.  GSH was estimated based on a reaction of reduced GSH with 5-5 ditiobis-2-nitrobenzoic acid. 
MDA, the product of lipid peroxidation, was estimated by reaction with thiobarbituric acid as per the method prescribed by Balasubramanian et al. 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. 
Functional Marker Enzymes
Total protein, ALT, BUN and creatinine were estimated in serum by using the Erba Diagnostic Kits, Germany.
Total protein in the liver and kidney tissue was quantified as per Lowry et al.'s  method.
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.
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].
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|
Click here to view
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|
Click here to view
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].
Liver of chromium-treated (Cr VI) group showed vascular congestion, degenerative changes and dilatation of sinusoids [Figure 2]a. In addition, periportal areas showed infiltration of mononuclear cells in the large numbers [Figure 2]b. Kidney sections from Group 2 revealed degeneration of tubular epithelial cells, cystic dilatation of tubules, hyaline casts, congestion of blood vessels [Figure 2]c and d. Recovery from histological injury was observed in α-tocopherol co-administered Group 3 rats, with mild congestion of hepatocytes [Figure 3]a and mild swelling of glomerular tufts [Figure 3]b. In Group 4, treatment with α-tocopherol alone, revealed normal architecture [Figure 3]c and d.
| » Discussion|| |
Chromium (Cr VI) compounds are widely recognized as human carcinogens.  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.  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,  and provokes oxidative damage in hepatocytes  and kidney. 
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.  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. 
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. and Da Silva et al.  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. 
α-tocopherol, is a chain breaking antioxidant that exists in cell membranes  and plasma.  It eliminates lipid peroxyl and alkoxyl radicals, suppresses the chain reaction of LPO and promotes the production of scavenger antioxidant enzymes.  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.  Our results are consistent with previous studies by Susa et al.,  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.
| » References|| |
|1.||Wang XF, Xing ML, Shen Y, Zhu X, Xu LH. Oral administration of Cr(VI) induced oxidative stress, DNA damage and apoptotic cell death in mice. Toxicology 2006;228:16-23. |
|2.||Manerikar RS, Apte AA, Ghole VS. In vitro and in vivo genotoxicity assessment of Cr(VI) using comet assay in earthworm coelomocytes. Environ Toxicol Pharmacol 2008;25:63-8. |
|3.||Bainy AC, Saito E, Carvalho PS, Junqueira VB. Oxidative stress in gill, erythrocytes, liver and kidney of nile tilapia (orechromis miloticus) from a polluted site. Aquat Toxicol 1996;34:151. |
|4.||Bagchi D, Bagchi M, Stohs SJ. Chromium (VI)-induced oxidative stress, apoptotic cell death and modulation of p53 tumor suppressor gene. Mol Cell Biochem 2001;222:149-58. |
|5.||Traber MG, Atkinson J. Vitamin E, antioxidant and nothing more. Free Radic Biol Med 2007;43:4-15. |
|6.||Halliwell B, Gutteridge JM. Free radicals, other reactive species and disease. In: Free Radicals in Biology and Medicine. 3 rd ed. Oxford: Clarendon Press; 1999. p. 617-783. |
|7.||Arreola-Mendoza L, Reyes JL, Melendez E, Martín D, Namorado MC, Sanchez E, et al. Alpha-tocopherol protects against the renal damage caused by potassium dichromate. Toxicology 2006;218:237-46. |
|8.||Arreola-Mendoza L, Del Razo LM, Mendoza-Garrido ME, Martin D, Namorado MC, Calderon-Salinas JV, et al. The protective effect of alpha-tocopherol against dichromate-induced renal tight junction damage is mediated via ERK1/2. Toxicol Lett 2009;191:279-88. |
|9.||Biber TU, Mylle M, Baines AD, Gottschalk CW, Oliver JR, MacDowell MC. A study by micropuncture and microdissection of acute renal damage in rats. Am J Med 1968;44:664-705. |
|10.||Madesh M, Balasubramanian KA. Microtiter plate assay for superoxide dismutase using MTT reduction by superoxide. Indian J Biochem Biophys 1998;35:184-8. |
|11.||Moron MS, Depierre JW, Mannervik B. Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochim Biophys Acta 1979;582:67-78. |
|12.||Balasubramanian KA, Manohar M, Mathan VI. An unidentified inhibitor of lipid peroxidation in intestinal mucosa. Biochim Biophys Acta 1988;962:51-8. |
|13.||Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz AG, et al. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol 1990;186:464-78. |
|14.||Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-75. |
|15.||Gibb HJ, Lees PS, Pinsky PF, Rooney BC. Lung cancer among workers in chromium chemical production. Am J Ind Med 2000;38:115-26. |
|16.||Costa M. Toxicity and carcinogenicity of Cr(VI) in animal models and humans. Crit Rev Toxicol 1997;27:431-42. |
|17.||O'Brien TJ, Ceryak S, Patierno SR. Complexities of chromium carcinogenesis: Role of cellular response, repair and recovery mechanisms. Mutat Res 2003;533:3-36. |
|18.||Patlolla AK, Barnes C, Hackett D, Tchounwou PB. Potassium dichromate induced cytotoxicity, genotoxicity and oxidative stress in human liver carcinoma (HepG2) cells. Int J Environ Res Public Health 2009;6:643-53. |
|19.||Boºgelmez II, Güvendik G. Effects of taurine on oxidative stress parameters and chromium levels altered by acute hexavalent chromium exposure in mice kidney tissue. Biol Trace Elem Res 2004;102:209-25. |
|20.||Priti DV, Jatin P, Rasesh DV, Jignesh MP, Ghodasara DJ, Joshi BP, et al. Effects of sodium dichromate on haemato-biochemical parameters in Wistar rats. J Pharmacol Toxicol 2012;7:58-63. |
|21.||Goyer RA. Toxic effects of metals. In: Klaassen CD, editor. Casarett and Doull's Toxicology: The Basic Science of Poisons. 5 th ed. New York: McGraw-Hill; 1995. p. 696-8. |
|22.||Acharya S, Mehta K, Krishnan S, Rao CV. A subtoxic interactive toxicity study of ethanol and chromium in male Wistar rats. Alcohol 2001;23:99-108. |
|23.||Da Silva RF, Lopes RA, Sala MA, Vinha D, Regalo SC, De Souza AM, et al. Action of trivalent chromium on rat liver structure, histometric and haematological studies. Int J Morphol 2006;24:197-203. |
|24.||Patlolla AK, Barnes C, Yedjou C, Velma VR, Tchounwou PB. Oxidative stress, DNA damage, and antioxidant enzyme activity induced by hexavalent chromium in Sprague-Dawley rats. Environ Toxicol 2009;24:66-73. |
|25.||Palamanda JR, Kehrer JP. Involvement of vitamin E and protein thiols in the inhibition of microsomal lipid peroxidation by glutathione. Lipids 1993;28:427-31. |
|26.||Bursell SE, King GL. Can protein kinase C inhibition and vitamin E prevent the development of diabetic vascular complications? Diabetes Res Clin Pract 1999;45:169-82. |
|27.||Ernster L, Forsmark P, Nordenbrand K. The mode of action of lipid-soluble antioxidants in biological membranes: Relationship between the effects of ubiquinol and vitamin E as inhibitors of lipid peroxidation in submitochondrial particles. Biofactors 1992;3:241-8. |
|28.||Susa N, Ueno S, Furukawa Y, Sugiyama M. Protective effect of vitamin E on chromium (VI)-induced cytotoxicity and lipid peroxidation in primary cultures of rat hepatocytes. Arch Toxicol 1996;71:20-4. |
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]