Does alfa lipoic acid prevent liver from methotrexate induced oxidative injury in rats ? 1

PURPOSE: To determine the antioxidant and anti-inflammatory effects of alfa lipoic acid (ALA) on the liver injury induced by methotrexate (MTX) in rats. METHODS: Thirty two rats were randomly assigned into four equal groups; control, ALA, MTX and MTX with ALA groups. Liver injury was performed with a single dose of MTX (20 mg/kg) to groups 3 and 4. The ALA was administered intraperitonealy for five days in groups 2 and 4. The other rats received saline injection. At the sixth day the rats decapitated, blood and liver tissue samples were removed for TNF-α, IL-1β, malondialdehyde, glutathione, myeloperoxidase and sodium potassium-adenosine triphosphatase levels measurement and histological examination. RESULTS: MTX administration caused a significant decrease in tissue GSH, and tissue Na+, K+ ATPase activity and which was accompanied with significant increases in tissue MDA and MPO activity. Moreover the pro-inflammatory cytokines (TNF-α, ILβ) were significantly increased in the MTX group. On the other hand, ALA treatment reversed all these biochemical indices as well as histopathological alterations induced by MTX. CONCLUSION: Alfa lipoic acid ameliorates methotrexate induced oxidative damage of liver in rats with its anti-inflammatory and antioxidant effects.


Introduction
Methotrexate (MTX) is an effective cytotoxic drug and has been widely used in chemotherapeutic based treatments for malignancies primarily in leukaemias 1,2 as well as inflammatory diseases including psoriasis and rheumatoid arthritis [3][4][5][6] .Longterm methotrexate use, or its use in high doses, may cause hepatic steatosis, cholestasis, fibrosis and cirrhosis 7 .Accordingly the dose of methotrexate should be lowered or the drug should be discontinued in case of hepatic toxicity which causes delay in the treatment of the disease.On the other hand much attention is now being paid to factors that may enhance the effectiveness of existing drugs while reducing their unwanted side effects.
Alfa-lipoic acid (ALA) is described as a therapeutic agent in a number of conditions related to liver disease, including alcohol-induced damage, mushroompoisoning, metal intoxification, CCl4 poisoning, and hyperdynamic circulation in biliary cirrhosis [8][9][10] .The effect of ALA against methotrexate toxicity on the liver is not yet clearly.
Furthermore the effect of ALA on MTX induced liver injury has not been studied before.Thus, in this present study we aimed to investigate whether the ALA has any effect on treatment against MTX induced oxidative injury on the liver in rats.

Methods
The experimental protocols were approved by the animal care and use committee of İnönü University Faculty of Medicine.
Thirty two Wistar albino rats of both sexes of 200-250 g were used in this experiment.The rats maintained at a constant temperature (22°C) with a 12-h light-dark cycle and randomly divided into four groups.Group 1 (control group): rats in this group received only physiological saline.Group 2 (α-lipoic acid group): rats in this group received α-lipoic acid (Sigma, St Louis, USA) for five days intraperitoneally (60 mmol/kg).Group 3 (Methotrexate group): rats received a single dose of MTX (Onco-Tain; Faulding Pharmaceutics Plc, Leamington Spa, UK) intraperitoneally (20 mg/ kg).Group 4 (Methotrexate group-αlipoic acid group): rats received a single dose of MTX and also received α-lipoic acid for five days.Alfa-lipoic acid was dissolved in 0.1% dimethyl sulfoxide (DMSO).At the end of the experiment rats were decapitated and blood samples were obtained for the measurement of tumour necrosis factor-alpha (TNF-α) and interleukin-1-beta (IL-1β).The levels of malondialdehyde (MDA) and glutathione (GSH), as well as myeloperoxidase (MPO) and sodiumpotassium adenosine triphosphatase (Na+/K+-ATPase) activity were alo analysed in the liver tissues.Furthermore the degree of inflammation and histopathologic damage (necrosis, inflammation, vacuolization and vascular congestion) were evaluated via histological examination under a light microscope.

Measurement of malondialdehyde and glutathione levels
To determine the MDA and GSH levels, liver tissue samples were homogenized in ice cold 150mm KCl.The MDA levels (nmol MDA/g tissue) were assayed for the products of lipid peroxidation 11 .The GSH levels (mg GSH/g tissue) were measured by spectrophotometric method using Ellman's reagent 12 .

Measurement of myeloperoxidase activity
Tissue-associated MPO (U/g tissue) activity was measured according to the procedure reported by Hillegas et al. 13 Liver tissue samples were homogenized in 50mm potassium phosphate buffer (PB, pH 6.0) and homogenates were centrifuged at 41 400g for 10 min; pellets were suspended in 50mm PB containing 0.5% hexadecyltrimethylammonium bromide.After three cycles of freezing and thawing, with sonication between the cycles, the samples were centrifuged at 41 400g for 10 min.Volumes of 0.3 ml were added to 2.3 ml of reaction mixture containing 50mm PB, o-dianisidine, and 20mm H2O2 solution.
One unit of enzyme activity was defined as the amount of MPO that caused a change in the absorbance measured at 460 nm for 3 min.

Measurement of Na+/K+-ATPase activity
The measurement of Na+/K+-ATPase activity was based on the measurement of inorganic phosphate produced from 3mm disodium adenosine triphosphate added to the incubation medium 14 .The medium (containing in mm: 100 NaCl, 5 KCL, 6 MgCl2, 0.1 EDTAand 30 Tris HCL (pH 7.4)) was incubated at 37°C in water bath for 5 min.Following this preincubation period, Na2ATP, at a final concentration of 3mm, was added into each tube and incubated at 37°C for 30 min.After the incubation, the tubes were placed in an ice bath to stop the reaction.The mixture was then centrifuged at 3500g, and Pi in the supernatant fraction was determined by the method of Fiske and Subarrow 15 .The specific activity of the enzyme was expressed as nmol Pi mg-1 protein h-1.The protein concentration of the supernatant was measured by the Lowry method 16 .

Biochemical analysis
The plasma TNF-α and IL-1β were analysed using the enzyme-linked immunosorbent assay (ELISA) kits (Biosource International, Nivelles, Belgium).These kits were particularly selected because of their high degree of sensitivity, specificity and inter-assay and intra-assay precision, and due to requiring a small amount of plasma sample.

Histological evaluation
Each liver samples were processed for light microscopic examination.The samples were placed in 10% neutral formalin for 48 h and prepared for routine parafin embedding.Tissue samples were cut into 5 µm thick sections, mounted on slides and stained with hematoxylin-eosin (H&E).
The degree of inflammation and histopathologic damage (necrosis, inflammation, vacuolization and vascular congestion) was expressed within each liver section (Table 1), classified on a scale of 0-3 (0, absent; 1, mild; 2, moderate; 3, severe) with a maximum score of 12 17 .acid treatment.Similarly IL-1 β proinflammatory cytokine, was also increased in the MTX group (p<0.001),however when rats were treated with α-lipoic acid following MTX administration, these cytokines were back to control levels (Table 2).

Statistical analysis
Statistical analysis was performed by GraphPad Prism 3.0 (GraphPad Software, San Diego, USA).The data were expressed as mean±standard error of the mean (SEM).Group comparisons were performed with the analysis of variance followed by Tukey's tests.The p<0.05 was considered as statistically significant.

Results
In the MTX group, TNF-α levels were significantly increased (p<0.001) when compared to control group, while this MTX-induced rise in serum TNF-α level was abolished (p<0.001) with α-lipoic  In accordance with these findings, levels of the major cellular antioxidant GSH of liver samples in MTX group were significantly lower than those of the group (p<0.001).On the other hand, α-lipoic acid treatment to MTX group restored the GSH levels in all tissues (p<0.01, Figure 1).The mean level of MDA, which is a major degradation product of lipid peroxidation, was increased in all tissues after MTX administration when compared with the control group (p<0.001),while α-lipoic acid treatment to the MTX group caused a marked decrease in MDA levels (p<0.01, Figure 2).Myeloperoxidase activity, which is accepted as an indicator of neutrophil infiltration, was significantly higher in the liver tissues of the MTX group when compared to control group (p<0.001).On the other hand, α-lipoic acid treatment in MTX group significantly decreased all tissues MPO level (p<0.001, Figure 3), which was found to be not different than that of the control group.The activity of Na + -K + ATPase was shown to be significantly decreased in the liver tissue of saline treated MTX group compared with control group; however, α-lipoic acid treatment in MTX group significantly increased all tissues Na + -K + ATPase activity (p<0.001, Figure 4).

Discussion
Findings from our study revealed that MTX administration causes oxidative tissue damage, while with its free radical scavenging effect the ALA prevented lipid peroxidation and neutrophil infiltration of the rat liver tissues.Furthermore, ALA treatment decreased the plasma cytokines and improved the liver tissue morphological changes caused by methotrexate.
Methotrexate is an antimetabolite that competitively inhibite the folic acid metabolism thus impairs the DNA synthesis.
7-hydroxymethotrexate (7-OH-MTX) is the major extracellular metabolite of MTX that is metabolized in the liver by an enzymatic system 18 .In the cell MTX store in a polyglutamate form.With the use of MTX intracellular amount of polyglutamate increases on the other hand folic acid levels decreased, that leads to necrosis of hepatocyte 19 .Hepatotoxic effect of methotrexate was caused by an increase of its polyglutamate form intracellularly.The hepatotoxic effects of MTX have been reported in many studies 1,20,21 .
ALA is found in mitochondria as cofactor of pyruvate dehydrogenase and α-ketoglutarate dehydrogenase 22,23 and is an effective free radical scavenger 24 .
Lipid peroxidation by free oxygen radicals is an important causes of destruction and oxidative damage to cell membranes these containing unsaturated fatty acids, nucleic acids and proteins 25 .It has contribute to develope methotrexate associated tissue damage 10,11,18,21 .
With the attack of free oxygen radicals lipid peroxidation increase and fail the Na+/K+-ATPase activity 26 .Na+/K+-ATPase is the other target of cellular oxidative tissue damage 25 .In this present study, MTX administration caused to a significant liver tissue damage since MDA which was the end product of lipid peroxidation is increased while Na+/K+-ATPase activity is depressed due to damage of cell membrane.
Liver tissue injury was also observed microscopically.
On the other hand, following MTX administration, treatment with ALA was significantly reduce the MDA levels and increased the Na+/K+-ATPase enzyme activity, while normal histological appearance was observed in liver tissue.Glutathione (GSH) plays a particularly important role in the maintenance and regulation of the thiol-redox status of the cell 27 .Tissue GSH depletion is one of the primary factors permitting liver tissue damage is associated with oxidative stress caused by MTX in our study.
It was expected that free radicals plays an important role in MTX induced liver toxicity 28,29 .The reactive oxygen metabolites play a role in mediating liver toxicity of some xenobiotics and pathogenesis of organ failure 10,11,18,21,27 .It was reported that ALA protect the nuclear DNA, cell membrane lipids and intracellular proteins from oxidative tissue damage 30 .
Free oxygen radicals trigger the leukocytes accumulation in tissue and activate the enzyme (including MPO, elastase and protease) secretion of neutrophils thus leads to further tissue damage.Therefore, MPO plays role in oxidant production by neutrophils 31,32 .In our study MPO level which is an index of polimorphonuclear leukocyte infiltration was increased.Systemic inflammatory response indicators; TNF-α and IL-1β levels were also found increased.Increased levels of MPO indicate that neutrophil accumulation contributes to MTX induced oxidative injury in liver tissues.Treatment with ALA decreased the MPO activity and plasma TNF-α and IL-1β.

Conclusion
Alfa lipoic acid can be capable of reducing the methotrexate induced liver oxidative injury through its anti inflammatory and antioxidative effects.

TABLE 1 -
The degree of inflammation and histopathologic damage (necrosis, inflammation, vacuolization and vascular congestion) was expressed as means within each liver section of groups was shown.

FIGURE 1 -
FIGURE 1 -Glutathione (GSH) levels in the liver tissues of control, methotrexate (MTX), MTX-ALA (α-lipoic acid) groups.Each group consists of eight animals.Groups of data were compared with an analysis of variance (ANOVA) followed by Tukey's multiple comparison tests.*** p<0.001; compared to control group.++ p<0.01; compared to MTX group.

FIGURE 2 -
FIGURE 2 -Malondialdehyde (MDA) levels in the liver tissues of control, methotrexate (MTX), MTX-ALA (α-lipoic acid) groups.Each group consists of eight animals.Groups of data were compared with an analysis of variance (ANOVA) followed by Tukey's multiple comparison tests.***p<0.001;compared to control group.++ p<0.01; compared to MTX group.