1. Background
Cyclosporine A (CsA) is an important immunosuppressive agent which is an essential part of drug regimen in transplant patients and in the treatment of diseases involving immune system (1). However, its clinical and experimental use is limited by several side effects such as nephrotoxicity, cardiotoxicity, hypertension and hepatotoxicity (2). Different authors suggest that reactive oxygen species (ROS) production, oxidative stress, depletion of hepatic antioxidant system, and increase in malondialdehyde (MDA) be possible mechanisms of CsA hepatotoxicity (3-6). Fortunately, there are several antioxidant mechanisms that can neutralize free radicals in living organisms. Antioxidant defense mechanisms can be grouped by enzymatic antioxidants (mainly superoxide dismutase, glutathione peroxidase and catalase) and non-enzymatic antioxidants (e.g. tocopherols, carotenoids, ascorbic acid and others) that can neutralize free radicals (7). From non-enzymatic antioxidants, vitamin C (ascorbic acid) is a very important, and powerful, antioxidant, and protects membranes against oxidation (8). Recent in vitro and ex-vivo studies have revealed that Vitamin C in plasma increases dose-dependent resistance to lipid peroxidation (9). Majority of in vivo studies have showed reduction in markers of oxidative DNA, lipid and protein damage after supplementation with vitamin C (10).
2. Objectives
The aim of this study was to study protective role of vitamin C against CsA-induced hepatotoxicity through studying changes in some plasma biochemical factors.
3. Materials and Methods
Thirty male Wister strain rats weighting 230 - 260 g were randomly divided in to 3 groups (n = 10); besides, they were kept in accordance with the “Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals”. They had free access to commercial rat food and water. The CsA (Sigma-Aldrich Corp. St. Louis, MO, U.S.A.) oral solution (100 mg/mL, 15 mg/kg/day) was delivered by oral gavage. Vitamin C (Osveh, Tehran, IR Iran) (100mg/mL, 200 mg/kg/day) solution was given orally by gavage to CsA + Vitamin C group. Group A was the control group and received placebo (Normal Saline), group B was CsA-treated group and received 15mg/kg/day CsA for 21 days. Furthermore, group C was CsA + vitamin C group and was received 200mg/kg/day vitamin C orally by gavage 3 hours before receiving 15mg/kg/day CsA. Treatment of oral vitamin C was started since 3 days before initiation of CsA therapy and continued during CsA treatment. Blood samples were obtained on day 22th under light ether anesthesia to study changes of bilirubin, total protein, albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride, alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) on commercial kits (Pars Azmoon, Karaj , IR Iran) by auto analyzer.
Values were expressed as mean ± SD. Statistical evaluation of significant difference between means was performed with one-way ANOVA test followed by Tukey post hoc test (P < 0.05 was considered significant).
4. Results
The results are shown in Table 1. Bilirubin Level was significantly higher in the group B (0.179 ± 0.0144 mg/dL) than those of the group A (0.120 ± 0.008) and the group C (0.154 ± 0.014, P < 0.05). Also, there was a significant difference between the group B and the group C (P < 0.05). Alkaline phosphatase level in the group B (183.740 ± 6.361 IU/L) was significantly higher than those of the group A (109.690 ± 7.018) and the group C (160.360 ± 6.565, P < 0.05). Also, there was a significant difference between group B and group C (P < 0.05). AST level in group B (131.690 ± 5.466 IU/L) was significantly higher than those of the group A (73.920 ± 5.463) and the group C (107.200 ± 4.885, P < 0.05). Furthermore, there was a significant difference between the group B and the group C (P < 0.05). ALT level in the group B (43.770 ± 3.391 IU/L) was significantly higher than those of the group A (25.460 ± 3.312) and the group C (33.523 ± 3.046, P < 0.05). Also, there was a significant difference between the group B and the group C (P < 0.05). Lactate Dehysrogenase level in the group B (1750.00 ± 37.392 IU/L) was significantly higher than those of the group A (1156.700 ± 27.252) and the group C (1521.200 ± 33.891, P < 0.05). Besides, there was a significant difference between the group B and the group C (P < 0.05). Total protein level in the group B (5.334 ± 254 g/dL) was significantly lower than those of the group A (7.114 ± 154) and the group C (5.930 ± 201, P < 0.05). Also there was a significant difference between the group B and the group C (P < 0.05). Albumin level in the group B (3.012 ± 0.181 g/dL) was significantly lower than those of the group A (4.264 ± 0.251) and the group C (3.546 ± 234, P < 0.05). Moreover, there was a significant difference between the group B and the group C (P < 0.05). Triglyceride level in the group B (119.900 ± 6.279 mg/dL) was significantly higher than those of the group A (71.314 ± 4.516) and the group C (91.266 ± 5.046, P < 0.05). Also, there was a significant difference between the group B and the group C (P < 0.05).
Table 1.Serum levels of bilirubin, Total protein, Albumin, ALT, AST, Triglyceride, ALP, and LDM in control, CsA and CsA + Vit C groups. Results expressed as mean ± SD.
| Control | CsA a | CsA + Vit C | |
|---|---|---|---|
| Bilirubin, mg/dL | 0.120 ± 0.008 | 0.179 ± 0.144 | 0.154 ± 0.014 |
| ALP a, IU/L | 109.690 ± 7.018 | 183.740 ± 6.361 | 160.360 ±6.565 |
| LDH a, IU/L | 1156.700 ± 27.252 | 1750.00 ± 37.392 | 1521.20 ± 33.891 |
| ALT a, IU/L | 25.460 ± 3.312 | 43.770 ± 3.391 | 33.523 ± 3.046 |
| AST a, IU/L | 73.920 ± 5.463 | 131.690 ± 5.466 | 107.200 ± 4.885 |
| Total proteina, g/dL | 7.114 ± 0.154 | 5.334 ± 0.254 | 5.930 ± 0.201 |
| Albumina, g/dL | 4.264 ± 0.251 | 3.012 ± 0.181 | 3.546 ± 0.234 |
| Triglyceridea, mg/dL | 71.314 ± 4.516 | 119.900 ± 6.279 | 91.266 ± 5.046 |
a Abbreviations: ALT, Alanine Aminotransferase; AST, Aspartate Aminotransferase; ALP, Alkaline Phosphatase; CsA, Cyclosporine A; LDH, Lactate Dehydrogenase
5. Discussion
The process of CsA-induced hepatotoxicity begins with degradation of arachidonic acid, synthesis of eicosanoids and release of free radicals (as hydroxyl radical) that initiate lipid peroxidation and the secondary activation of physiologic antioxidant defenses. In prior studies, changes in the activities of antioxidant enzymes (lowered SOD, and increased CAT activities) and observed increase in peroxidation products in the liver tissues from cyclosporine-treated rabbits have been established (11).
Our study shows that there were significant increases in ALT, AST, bilirubin, LDM, ALP and triglyceride levels in plasma, and significant decrease in total protein and albumin level of the plasma in the group B and the group C but the amount of increase between group B and C was also significant. Vitamin C treatment significantly decreased liver toxicity against CsA-induced plasma biochemical changes. The results in this experiment recommend that vitamin C be effective in decreasing the CsA-induced hepatotoxicity; however, level of ALT, AST, bilirubin, LDM, ALP and triglyceride in the group C was higher than that of the group A and the level of total protein and Albumin was lower than that of the group A significantly.
Our results approve the results of Benito B (12) report in which studied effect of vitamin C in liver protein pattern of CsA-treated rats. Although this study shows the beneficial effect of vitamin C in decreasing CsA-induced liver toxicity it has been suggested that antioxidant supplementation is beneficial only when elevated oxidative stress is present (13) because some studies show that the co-administration of antioxidants like vitamin C may decrease CsA dose in the body and interfere with the aim of using CsA regimen in patients (14). In conclusion, we have shown that vitamin C administration provides protection against CsA-induced injury in rat liver function and it may have hepatoprotective role in patients experiencing CSA treatment.
