THE EFFECT OF TAURINE ON METHOTREXATE INDUCED HEPATORENAL TOXICITY IN RATS.

The aim of the work: Hepatorenal toxicity is a major side effect for methotrexate. Taurine is a natural compound with multiple pharmacological activities such as antioxidant, antiapoptotic and anti-inflammatory effects. This study investigated the effect of taurine on methotrexate-induced toxic effects in male albino rats. Methods: 40 male albino rats were divided into 4 groups (10 rats each): (control group): Rats injected with1ml saline intraperitoneally for 6 days, (methotrexate treated group): Rats injected daily with 1ml i.p for days, the th day they were injected i.p with a single of methotrexate (20 mg/kg), treated Rats injected with taurine mg/kg daily i.p for 6 days, (taurine+methotrexate treated group): Rats injected i.p with taurine 100 mg/kg daily for 5 days at the 6 th day after the last dose of taurine, rats were injected i.p with a single dose of methotrexate (20 mg/kg). Results: Methotrexate administration resulted in hepatorenal toxicity, oxidative stress, lipid peroxidation and apoptosis. These results were confirmed by histopathology and by detection of DNA fragmentation by gel electrophoresis. Taurine administration before methotrexate improved hepatorenal functions caused a reduction in oxidative stress, lipid peroxidation, and elevation in the activity of antioxidant enzymes. This was confirmed by the histopathological findings of hepatic and renal tissue and by the reduction in DNA fragmentation. Conclusion: Taurine supplementation had beneficial effects on liver and kidney functions, with marked reductions in oxidative stress and apoptosis induced by methotrexate. as demonstrated by elevation of serum AST, ALT, creatinine, urea and BUN. It caused elevation of serum TNFα. Also, MTX treatment caused oxidative tissue damage, as assessed by increased lipid peroxidation (MDA), caspase, and nitrite and decreased GSH, CAT levels in the liver and kidney. These results were confirmed by DNA fragmentation as well as by histopathological findings.

then transferred into clean cuvette tube stored at -80 o C and used for measurements of the following: Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) activities were assayed by using commercial kit that was supplied by Egyptian Company for Biotechnology kit number 264 001 according to the method described by (Reitman and Frankel, 1957). Creatinine was measured according to the method described by (Bartles et al.,1972) it was obtained from Biodiagnostic CO. Egypt kit number CR 1251.Urea was assayed by Modified Urease -Berthlot Method described by (Tiffany et al.,1972), it was obtained from Biodiagnostic CO. Egypt kit number UR2110. Blood urea nitrogen (BUN) was estimated by the equation described by (Deacon, 2009). Tumor necrosis factor alpha (TNFα) was assayed by Rat TNFα ELISA kit (Shanghai Sunred Biological Technology Co. Ltd, China. Catalog no 201-11-0765) according to manufacturer's protocol (Maskos et al., 1998).
After animals were sacrificed part of the liver and kidney were instantly removed, washed three times in ice-cold saline and blotted on filter paper, and homogenized in 50mM potassium phosphate (pH 7.4). The homogenate was centrifuged in 7000×g for 10 min at 4°C and supernatant were stored at -80 o C and used for measurement of oxidative stress by determination of tissue reduced glutathione (GSH) levels which was measured using Biodiagnostic Kit No. GR 25 11(BiodiagnosticCo.,Egypt) that is based on the spectrophotometric method of (Beutler, 1963). Malondialdehyde (MDA) was assayed by using Biodiagnostic Kit No MD 25 29 based on the spectrophotometric method of (Ohkawa et al., 1979). Catalase (CAT) was measured using Biodiagnostic Kit No. CA25 17 which is based on the spectrophotometric method described by (Aebi, 1984). Caspase-3 was assayed using RayBio Kit No. 68CL-Casp3-S100 which is based on a spectrophotometric method described by (Porter and Janicke, 1999). Nitrite was assayed using Biodiagnostic Kit No. NO 25 33 which is based on the spectrophotometric method described by ( Montgomery and Dymock, 1961).
Another part of liver and kidney was separated for detection of DNA damage by gel electrophoresis. DNA extraction kits (EZ-10 Spin Column Animal Genomic DNA Miniprep Kit) was obtained from Bio Basic Inc Co. Canda, NY, USA kit code 41105504m DNA which is based on the protocol described by (Kasibhatla et al., 2006). Another part from liver and kidney from all rats of all groups were fixed in 10% neutral buffered formalin (pH 7.2) and embedded in paraffin for histopathological examination. Paraffin-embedded tissues were sectioned into 4µm thickness slices by microtone and stained with hematoxylin-eosin (H&E) stain.
Then, the sacrificed animals were packed in a special package according to safety precautions and infection control measures.

Results:-
Results were expressed as Mean ± SD and all statistical comparisons were made by means of one-way ANOVA test, followed by Tukey's post hoc analysis, and p values less than 0.05 were considered statistically significant. The analysis was performed by statistical package for the social science software (SPSS version 22.0.).
Effect of taurine on MTX hepatotoxicty:-AST, ALT are markers of liver dysfunction. As shown in Fig. 1, levels of AST& ALT were significantly (p<0.05) increased in the MTX group compared to the control group. Administration of taurine to normal rats did not change the levels of AST& ALT compared to rats in control group. In addition, pretreatment with taurine prevented MTXinduced hepatotoxicity and induced a significant (p<0.05) decrease in AST and ALT levels in taurine+MTX treated group when compared with MTX group (Fig. 1A and B).
Effect of taurine on MTX renal toxicity:-Creatinine, urea, and BUN are markers of renal dysfunction. As shown in Fig. 2, levels of creatinine, urea and BUN were significantly (p<0.05) increased in the MTX group compared to the control group. Administration of taurine to normal rats did not change the levels of creatinine, urea, and BUN compared to rats in control group.
In addition, pretreatment with taurine significantly prevented MTX-induced renal toxicity as it caused significant decrease (p<0.05) in creatinine, urea, and BUN levels in taurine+MTX group compared to MTX group ( Fig. 2A, B and C).

Effect of taurine on MTX induced inflammation:-
MTX induced elevation of inflammatory markers as shown in Fig. 3, levels of TNFα was significantly (p<0.05) increased in the MTX group compared to the control group. Administration of taurine to normal rats did not change 1781 the levels of TNFα compared to rats in control group. In addition, pretreatment with taurine significantly decreased (p<0.05) TNFα in taurine+MTX group when compared with MTX group (Fig. 3).

Effect of taurine on oxidative stress induced by MTX:-Effects of taurine on antioxidant enzymes:-
The effect of taurine on the activity of antioxidant enzymes GSH and CAT content is illustrated in Fig. 4. The GSH content and activity of CAT were significantly (p<0.05) decreased in the MTX group compared to the control group in both hepatic and renal tissue (Fig. 4A, B, C and D). Administration of taurine to normal rats caused significant elevation (p<0.05) of GSH in both hepatic and renal tissue but did not change hepatic and renal tissue CAT compared to rats in control group as shown in (Fig. 4A and B).
Pretreatment with taurine significantly increased (p<0.05) GSH content and CAT activity in taurine+MTX group when compared to the MTX group in both hepatic and renal tissue (Fig. 4A, B, C and D).

Effects of taurine on oxidative stress parameters:-
The effect of taurine on oxidative stress parameters such as MDA, caspase, and nitrite levels is illustrated in Fig.5. As shown in Fig. 5A&B the concentration of MDA, an end product of lipid peroxidation, was significantly (p<0.05) increased in hepatic and renal tissue of the MTX group compared to the control group. Administration of taurine to normal rats didn't change tissue MDA. In contrast, pretreatment with taurine significantly (p<0.05) decreased hepatic and renal MDA concentration in Taurine+MTX group compared to the MTX group.
As shown in Fig. 5C&D administration of MTX significantly increased hepatic and renal tissue caspase-3 level compared to the control group (p<0.05). Administration of taurine to normal rats did not change tissue caspase-3 level. Pretreatment with taurine significantly decreased (p<0.05) hepatic and renal caspase-3 level in taurine+MTX group compared to the MTX group.
As shown in Fig. 5E&F administration of MTX significantly increased (p<0.05) hepatic and renal tissue nitrite concentration compared to the control group (p<0.05). Administration of taurine to normal rats did not change tissue nitrite level compared to control group. Pretreatment with taurine significantly decreased (p<0.05) hepatic and renal nitrite level in taurine+MTX compared to the MTX group.

Effect of taurine on MTX induced apoptosis:-
These results were confirmed by DNA gel electrophoresis with the typical ''ladder'' pattern of DNA fragmentation in hepatic and renal tissue in group treated with MTX. Pretreatment with taurine abrogated MTX induced DNA fragmentation while taurine alone had no effect (Fig. 6).

liver and kidney histopathology:-
The control group presented livers with a normal architecture of the liver cells (Fig. 7A). In the MTX group, liver tissue from all of rats showed portal congestion, infiltration of inflammatory cells and apoptotic bodies ( Fig. 7 B& C). In addition, administration of taurine did not cause any detectable alteration in the liver structure ( Fig 7D). In the taurine+MTX treated group (Fig. 7E), the histopathological lesions were effectively attenuated.
As regard to the kidney, control rats also presented kidney with normal renal architecture as regard to renal tubules and glomeruli (Fig. 8A). Renal tissue from all of the MTX treated rats showed degeneration of renal tubules and inflammatory cellular infiltration (Fig. 8B). In addition, administration of taurine did not cause any detectable alteration in the renal structure ( Fig 8C). In the taurine+MTX treated group (Fig. 8D), the histopathological lesions were effectively attenuated.

Discussion:-
The results of the present study revealed that MTX induced hepatorenal toxicity as demonstrated by elevation of serum AST, ALT, creatinine, urea and BUN. It caused elevation of serum TNFα. Also, MTX treatment caused oxidative tissue damage, as assessed by increased lipid peroxidation (MDA), caspase, and nitrite and decreased GSH, CAT levels in the liver and kidney. These results were confirmed by DNA fragmentation as well as by histopathological findings.          The apoptotic effect of MTX was explained by Czarnecka-Operacz and Sadowska-Przytocka, (2014) who stated that MTX is folic acid antagonist which is retained within the cell as a polyglutamate, it binds with an affinity greater than that of folic acid to dihydrofolate reductase and inhibits it. MTX also inhibits AICAR (5aminoimidazole-4-carboxamide ribonucleotide) transformylase and thymidylate synthase. Inhibition of these enzymes limits the conversion of folic acid to tetrahydrofolate which is essential for DNA synthesis. Inhibition of synthesis of purine and pyrimidine thymidine by MTX results in improper DNA synthesis and subsequent apoptosis. In addition, MTX affects MTHFR (methylenetetrahydrofolat ereductase) and hence the generation of methionine from homocysteine. Excess homocysteine can generate oxidative stress and increases cell sensitivity to the cytotoxic effect of ROS. Homocysteine may activate proinflammatory cytokines (Pandit et al, 2012).
So, several mechanisms could be attributed to the hepatorenal toxic effect of MTX; cell inflammation, neutrophil migration and increased cytokine concentration that may lead to hepatorenal cell apoptosis and necrosis and release of toxic agents that induce cellular damage. Also, increase in tissue NO and MDA together with decrease GSH levels indicates an imbalance of oxidant and antioxidant systems.
However, the results of the present work showed significant improvement of hepatic and renal function in rats injected with taurine. This improvement was evidenced by the significant reduction in serum level of AST, ALT, creatinine, urea, BUN and TNFα, tissue level of MDA, caspase, nitrite and significant increase of tissue level of GSH and CAT in taurine+MTX treated group when compared with MTX group. There was also a significant reduction in DNA fragmentation in the hepatic and renal tissue of rats in taurine+MTX treated group.
These results were confirmed by histopathological findings of hepatorenal tissue in taurine+MTX group.

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These results are in agreement with El Kader et al., (2015) who investigated the protective role of taurine, against oxidative stress induced by gamma irradiation and found that taurine, significantly improved radiation-induced injury in hepatic, cardiac, and renal tissues.
Moreover, El-Sayed et al., (2011) studied the protective effectt of taurine against aluminum-induced hepatotoxicity. Also, Zhang et al., (2014) studied whether taurine could reduce the hepatotoxicity of iron overload. They found that taurine can reduce hepatic oxidative stress, preserve liver function and inhibit hepatocyte apoptosis.
In addition, Demircioglu et al., (2011) reported that taurine has renoprotective effect as pretreatment with taurine can prevent oxidative changes in renal tissue caused by ischemia reperfusion.
In agreement with histopathological finding, Al-Asmari et al., (2016) studied the ameliorative effect of taurine on hepatorenal damage induced by 5 fluorouracil and observed that infiltration of inflammatory cells, necrosis, and renal tubular degeneration were attenuated by taurine.
Cysteine is the common precursor of taurine and GSH biosynthesis, so treatment with taurine might increase the GSH levels as a result of directing more amount of cysteine into GSH biosynthesis, so this mechanism explains how taurine increases antioxidant cellular capacity (El Kader et al., 2015).

Ahmad et al., (2015)
stated that taurine enhances the synthesis of GSH and as it stimulates the activity of G6PD, an enzyme that generates NADPH which is required by glutathione reductase to convert oxidized glutathione into GSH.
Another mechanism of antioxidant effect of taurine was explained by El-Sayed et al., (2011) who suggested that taurine stimulate the nitrosylation of GSH into nitrosoglutathionen which is much potent antioxidant than GSH itself.
Taurine has the ability to conjugate with MDA, the end product of lipid peroxidation, so it stabilizes the lipid bilayer and decreases the vulnerability of the membranes lipids to toxic insult induced by ROS (Ahmad et al., 2015).

Ahmad et al., (2015)
stated that taurine itself does not directly quench classical ROS and free radicals but its metabolic precursor, hypotaurine, has been shown as an efficient radical scavenger.
The antiapoptotic effect of taurine may be due to the reduction in ROS which in turn prevents opening of mitochondrial permeability transition pore (mPTPs) and subsequent mitochondrial swelling (Zhang et al., 2014).
Taurine ensures an efficient flux of reducing equivalents through the respiratory chain, thereby preventing the diversion of electrons to the acceptor oxygen; so it inhibits the generation of superoxide anion (Jong et al., 2012).
Taurine has antiapoptotic effect as it reduces caspase-3 activation and subsequent DNA fragmentation. This could be due to the ability of taurine to inhibit oxidative stress, which in turn reduces pro-apoptotic pathway activation 1789 (Bax, Bcl-XS) and prevents the loss of the anti-apoptotic pathway (Bcl-2, Bcl-XL) and prevents subsequent caspase activation (Depboylu et al., 2008).
Taurine antioxidant activity suppresses extrinsic apoptotic pathway by decreasing in the gene expression of Fas receptors (FasR, apoptosis antigen 1or tumor necrosis factor receptor superfamily member) and subsequent caspase activation (Nagai et al., 2016).
Taurine also suppresses intrinsic apoptotic pathway by stabilizing mitochondrial membrane and elevating level of GSH since high levels of cytoplasmic GSH maintain cytochrome c in a reduced or inactive state. This prevents DNA damage and reduces apoptosis (Devi &Anuradha, 2010).

Ahmed et al., (2015)
demonstrated that excess Ca 2+ during oxidative stress conditions cause opening of mPTPs. Taurine prevents Ca 2+ overload via Na/Ca exchanger so, it is cytoprotective that protects cells from injury and subsequent necrosis.
So, taurine could improve the hepatic and renal functions by its antioxidant effect which increase the CAT activity and GSH level which are powerful antioxidant, Consistent with the antioxidant properties, taurine could reduce lipid peroxidation and MDA level, it also has anti-inflammatory effect by reducing TNFα and it has anti-apoptotic effect through reduction of tissue caspase and DNA fragmentation.

Conclusion:-
We conclude that taurine has ability to reduce MTX-induced hepatorenal oxidative injury through its antiinflammatory and antioxidant and antiapoptotic effects, which were evaluated both biochemically and histologically.

Recommendation:-
Thus, our data suggest that taurine may be used therapeutically in patients receiving other toxic chemotherapeutic agents to prevent hepatic and renal toxicity. Significant improvement of anti-cancer drugs side effects can increase the tolerance of these drugs and increase the therapeutic effect of these drugs for oncology or rheumatology patients.

Disclaimer:-
The review authors report no conflict of interest. This study was self-funded.