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Article

Ameliorative Effect of Gallic Acid on Methotrexate-Induced Hepatotoxicity and Nephrotoxicity in Rat

by
Ebenezer Olayinka
,
Ayokanmi Ore
*,
Oluwatobi Adeyemo
and
Olaniyi Ola
Biochemistry Unit, Department of Chemical Sciences, Ajayi Crowther University, Oyo, Oyo State, Nigeria
*
Author to whom correspondence should be addressed.
J. Xenobiot. 2016, 6(1), 6092; https://doi.org/10.4081/xeno.2016.6092 (registering DOI)
Submission received: 14 June 2016 / Revised: 13 August 2016 / Accepted: 15 August 2016 / Published: 26 August 2016
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Abstract

:
We investigated the protective effect of gallic acid (GA) against methotrexate (MTX)-induced hepatotoxicity and nephrotoxicity. Male Wistar rats were randomized into five groups (n = 6/group): I, control; II, MTX-treated for seven days; III, pre-treated with GA for seven days, fol- lowed by MTX for seven days; IV, co-treated with MTX and GA for seven days and V, GA for seven days. MTX caused a significant increase (P<0.05) in plasma biomarkers of nephrotoxic- ity (urea, creatinine) and hepatotoxicity (Bilirubin, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma glutamyl transferase) when compared with control. Furthermore, MTX caused a signif- icant decrease in the activities of hepatic enzymic antioxidants (superoxide dismutase, catalase, glutathione S-transferase) and nonen- zymic antioxidants (Vitamin C and glu- tathione), followed by a significant increase in hepatic malondialdehyde content. However, pre- treatment and co-treatment with gallic acid ameliorated the MTX-induced biochemical changes observed. Taken together, GA protected against MTX-induced hepatotoxicity and nephrotoxicity in rats, by reducing the impact of oxidative damage to tissues.

Introduction

Methotrexate (MTX), (Figure 1A) is an antimetabolite and a 4-amino, 10-methyl ana- logue of folate that inhibit dihydrofolate reduc- tase (DHFR), involved in the formation of tetrahydrofolate from dihydrofolate and conse- quently inhibiting DNA synthesis and cell pro- liferation.1 MTX is a cytotoxic agent used in the treatment of leukaemia and various malig- nancies, as well as in non-neoplastic diseases such as psoriasis, rheumatoid arthritis and as anti-inflammatory and immunosuppressive agent.[1,2,3] Earlier studies have revealed that MTX is converted mainly to metabolites con- nected to glutamate (MTX-glu) in cells and tis-sues.[4] The complex so formed is referred to as polyglutamated forms which is responsible for most biochemical activities of MTX.[5]
MTX is known to generate reactive oxygen species (ROS) in both normal and cancer cells resulting in oxidative damage.[6] The anti- cancer, anti-inflammatory and immunosup- pressive actions of MTX has been shown to occur via ROS generation and induction of apoptosis.[7] MTX has been reported to induce renal and hepatic toxicity via oxidative stress and its efficacy has been limited by severe organ toxicity.[8,9]
In the regulation of oxidative processes, cel- lular systems have been equipped with several antioxidant defense mechanisms, and there are several phytochemicals of plant origin with inherent antioxidant properties that are capa- ble of boosting cellular enzymic and non- enzymic antioxidant indices thus making them excellent scavengers of free radicals.[10] The chemoprotective properties of plant extracts have been extensively studied and are attributed to the presence of flavonoids, antho- cyanins and phenolic compounds.[11] Gallic acid (GA) (3,4,5-trihydroxybenzoic acid) (Figure 1B) is a naturally occurring phenolic compound present in green tea, gall nut, grapes, red wine, hops, oak bark etc.[12] Several authors have reported that GA possess strong antioxidant properties and a wide array of biological and pharmacological activities such as free radical scavenging, anti-apoptotic and anti-inflamma- tory.[13] Other reported biological effects include protection against doxorubicin-induced myocardial toxicity and cyclophosphamide- induced oxidative stress.[14]
The present study is therefore aimed at evaluating the protective effect of gallic acid pre-treatment and co-treatment on methotrex- ate-induced hepatotoxicity, nephrotoxicity and oxidative stress in rats. In this study, the role of GA in MTX induced hepatotoxicity and renal toxicity was evaluated by assessing plasma biomarkers of hepatic and renal function as well as selected oxidative stress markers (level of lipid peroxidation, enzymic and non enzymic antioxidants).

Materials and Methods

Drug, chemicals and reagents

Methotrexate tablets is a product of West Coast Pharmaceutical Works Ltd, Gota, Ahmedabad, India; Gallic acid, Glutathione, 1- chloro-2,4-dinitrobenzene (CDNB),5,5 -dithio bis-2-nitrobenzoic acid (DTNB), epinephrine, and hydrogen peroxide (H2O2), were obtained from Sigma® Chemical Company, London, UK; Assay kits for alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), alkaline phos-phatase (ALP), urea, Creatinine, total bilirubin were purchased from Randox® Laboratories Ltd. (Antrim, UK). All other reagents used were of analytical grade and of highest purity.

Animals

Male rats (Wistar strain) weighing between 160-180 g were obtained from the animal housing unit, in the Department of Chemical Sciences, Ajayi Crowther University, Oyo, Nigeria. The rats were acclimatized under lab- oratory conditions prior to experiment. The animals were housed in wire-meshed cages and provided with water and food ad libitum. They were fed with commercial rat diet (Ladokun® Feeds, Nigeria Ltd Ibadan, Nigeria). The study was approved by the ethi- cal committee of the Faculty of Natural Sciences, Ajayi Crowther University, Oyo, Nigeria. Handling of the experimental animals was done in accordance with international guidelines on the care and use of experimental animals (National Research Council).[15]

Experimental design

Thirty rats were randomly assigned into five experimental groups (I–V) of six animals each. The animals of each group were treated as presented in Table 1. The dose for MTX (0.2 mg/kg bw) was selected based on the recom- mended adult dose for rheumatoid arthritis and other inflammatory diseases while the dose for GA (20 mg/kg bw) was arrived at based on available literature.[16] The respective doses were prepared based on the average weight of animals in each treatment groups and admin- istered in one mL of distilled water. The drug doses were administered once daily by oral intubation.

Plasma and tissue preparation

Blood samples were collected from each ani- mal, via retro orbitals plexus in heparinized sample tubes (Li heparin). Animals were sac- rificed and liver was collected from each ani- mal for preparation of the post-mitochondrial fraction (PMF).
Centrifugation of blood samples were done at 4000 rpm for 5 minutes in a bench cen-trifuge (Analytica, Athens, Greece). The plas-ma obtained were stored at –4°C for subse- quent plasma assays. Liver samples were rinsed in ice-cold 1.15% KCl and homogenized in 4 volumes of 0.01 M potassium phosphate buffer (pH 7.4). The homogenates obtained were subjected to centrifugation at 12,500×g for 15 min at –4°C in a refrigerated centrifuge (Eppendorf UK Ltd., Stevenage, UK) and supernatants (PMF) were collected in sample tubes and used for subsequent biochemical assays.

Biochemical analysis

Total protein

The protein concentration in the liver PMF was determined according to the biuret method of Gornall et al. [17]

Biomarkers of renal function

Plasma level of creatinine and urea were determined with RANDOX® diagnostic kits fol- lowing the manufacturer’s protocol. The method for creatinine assays was based on Jaffe[18] and the method of Tietz[19] was employed for plasma urea determination.

Biomarkers of hepatic function

Plasma total bilirubin (TBILI) level, and activities of alkaline phosphatase (ALP), ala- nine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma glutamyl transferase (-GT) were assayed using RAN- DOX® diagnostic kits based on the manufactur- er’s procedure. Assay of TBILI level was based on the method of Tietz et al.[20] Activity of ALP was determined according to Tietz et al. [20] Plasma activities of ALT and AST were meas- ured according to the method of Reltman and Frankel.[21] The plasma activity of -GT was determined following the method described by Szasz.[22]

Biomarkers of oxidative stress

The level of hepatic reduced glutathione (GSH) was determined following the method of Jollow et al. [23] Ellman’s reagent reacts with reduced glutathione, and the chromophoric product resulting from the reaction has a molar absorption at 412 nm. The level of vita- min c in the liver PMF was determined accord- ing to Jagota and Dani.[24] Vitamin C in samples reacts with Folin-phenol reagent resulting in blue chromophore which has maximum absorption at 760 nm. Hepatic GST activity was measured following the method of Habig et al.[25] The procedure described by Misra and Fridovich[26] was used for the determination of hepatic superoxide dismutase activity. Hepatic catalase activity was determined by the method described by Singha.[27] Hepatic level of lipid peroxidation (LPO) was determined by the method described by Vashney and Kale.[28] This assay involved the reaction between thio- barbituric acid and malondialdehyde (MDA) a product of lipid peroxidation to yield a stable pink chromophore which absorbs maximally at 532 nm.

Statistical analysis

Data are presented as the mean ± standard deviation (SD) of six replicates. Statistical sig- nificance was determined by one-way analysis of variance (ANOVA) followed by Duncan’s mul- tiple comparison between control and treated rats in all groups using Sigma plot® statistical package (Systat Software Inc., San Jose, CA, USA). P-values less than 0.05 (P<0.05) were considered statistically significant.

Results

Protective effects of gallic acid on MTX-induced changes in markers of hepatic and renal toxicity

As presented in Table 2, administration of MTX caused a significant (P<0.05) increase in plasma creatinine and urea levels (124% and 68.5% respectively) compared to control. However, pre-treatment and co-treatment of GA with MTX attenuated the observed elevated plasma urea and creatinine levels when com- pared with the MTX-treated group.
The plasma level of total bilirubin (TBILI) and alkaline phosphatase (ALP) activity (bio- markers of hepatobiliary damage) increased significantly (P<0.05) in rats by 138.8% and 158% respectively following MTX treatment (Table 2). However, the levels of TBILI and ALP activity were significantly ameliorated in the plasma of animals pre-treated or co-treated with GA when compared with the MTX group.
MTX treatment also caused a significant increase in the activities of alanine amino- transferase (ALT), aspartate aminotransferase (AST), and gamma glutamyl transferase (- GT) (biomarkers of hepatocellular toxicity) in the plasma of rats by 102.8%, 93% and 106.7% respectively compared to values in control (Table 2). Pre-treatment and co-treatment of GA with MTX significantly ameliorated the ele- vated activities of plasma ALT, AST, and -GT when compared to MTX-treated group.
Protective effects of gallic acid on MTX- induced changes in markers of oxidative stress. Hepatic SOD and catalase activity (Table 3) were significantly reduced in the MTX-treated group by 34.0% and 47.8% when compared with control (P<0.05). Also, hepatic GST activity (Figure 2) was also significantly reduced by 38.3% in the MTX-treated rats when compared with the control. However, GA pre-treatment and co-treatment significantly ameliorated the MTX-induced decrease in hepatic activities of SOD, CAT, and GST rela- tive to the MTX-treated group (P<0.05).
Furthermore, hepatic ascorbic acid (AA) and GSH level (Figure 3 and Figure 4) were significantly (P<0.05) decreased following treatment with MTX by 37.9% and 38.9% when compared with the control. Conversely, pre-treatment and co- treatment with GA significantly (P<0.05) pro- tected against the MTX-induced decrease in hepatic AA and GSH levels when compared with the MTX group. In addition, the hepatic MDA level rose significantly (P<0.05) in the MTX-treated rats by 58.8% when compared with the control (Figure 5). However, GA pre- treatment and co-treatment attenuated the increase in hepatic MDA relative to the MTX-treated group.

Discussion and Conclusions

Methotrexate (MTX) is a chemotherapeutic agent indicated in conditions such as autoim- mune diseases, inflammatory myopathies, leukemia etc. However it is known to be asso- ciated with side effects including hepatotoxic- ity and nephrotoxicity; often mediated by reac- tive oxygen species (ROS).[29,30,31] Recent studies have demonstrated the protective roles of phy- tochemicals in drug-induced organ toxicity. Gallic acid employed in the present study is a potent natural antioxidant with numerous bio- logical activities including hepatoprotective activity.[32,33,34]
Biomarkers of renal function: urea and cre- atinine were considered in this study. Urea and creatinine are metabolic products removed by the kidney from circulation. Increase in their plasma level is an indication of loss in renal function.[35] The increase in plasma urea and creatinine observed in this study is in agreement with previous report on MTX.31 The attenuation of the level of plasma urea and creatinine by GA is an indication of improved renal function and the nephroprotec- tive role of GA.[36]
The liver is the target of several xenobiotics including chemotherapeutic agents such as MTX. MTX is stored in the liver cells as MTX- polyglumates and has been linked to MTX- induced hepatotoxicity.[37] The plasma level of bilirubin and activities of AST, ALT, ALP and - GT are reliable indices of hepatotoxicity.[38] Elevation of the plasma activities of AST and ALT have been linked to structural damage to the liver.[39] Bilirubin is present in the liver, bile, intestine and the reticuloendothelial cells of the spleen. High plasma bilirubin, ALP and - GT levels are indicators of hepatobiliary injury.[38,39] However, pre-treatment and co- treatment with GA ameliorated the MTX- induced cellular damages which agrees with previous finding on the hepatoprotective activ- ity of GA.[37]
Oxidative stress has been identified as a toxicological mechanism of most chemothera- peutic drugs. Free radicals release and ROS plays a significant role in the MTX-induced hepatotoxicity and nephrotoxicity.[30,31] In this study, MTX caused a significant decrease in the hepatic SOD, CAT, GST, GSH and vitamin C. SOD catalyses the rapid dismutation of super- oxide anions to hydrogen peroxide (H2O2) and molecular oxygen, while CAT converts the H2O2 formed to water and molecular oxygen. The low molecular weight antioxidants, vitamin C and GSH play an important role in cellular redox balance. They are the first line of defense against oxidation damages.[40] AA is involved in the preservation of tocopherol in membranes and lipoproteins, while GSH act as a substrate for several antioxidants enzymes such as glutathione peroxidase (GPx), glu- tathione –S – transferase (GST).[41] GSTs are a family of proteins primarily involved in the detoxification of highly reactive electrophiles including drugs by it combined action with GSH as a conjugating agent.[42] The reduction in the levels of the antioxidant defense system occasioned by MTX may predispose the liver to oxidative injury. However, GA significantly improved the antioxidant defense systems in the liver of rats in a similar manner to previous findings.[43] Lipid peroxidation induced by MTX is an indication of the involvement of free rad- icals in MTX-mediated toxicity. The ameliora- tion of hepatic LPO by GA in the study may be related to the free radical scavenging proper- ties of gallic acid.[43,44]
In summary, current findings suggest that gallic acid has the potential to protect against Methotrexate-induced hepatotoxicity and nephrotoxicity. In addition, the mechanism of protection by gallic acid may involve free – rad- ical scavenging. Therefore, gallic acid may be employed as a co-therapy in MTX chemothera- py as a protection against chemotherapy-asso- ciated oxidative damage to tissues.

Research highlights

-
Chemotherapy-associated oxidative stress is a relevant side effect of most anticancer agents.
-
Methotrexate (MTX) acts as an antimetabo- lite as its anticancer mechanism.
-
Gallic acid is a plant derived antioxidant.
-
Hepatotoxicity, nephrotoxicity and oxidative stress was observed following exposure to methotrexate.
-
Administration of gallic acid as a pre-treat- ment or co-administered with MTX amelio- rated MTX induced toxicity in rats.

Author Contributions

ETO designed the study; AO, OAA and OSA were involved in the animal treatments, laboratory and statistical analysis. OAA prepared the manuscript. AO handled all correspondence regarding the manuscript. The final version was read and approved by all authors.

Conflicts of Interest

Conflict of interest: the authors declare no potential conflict of interest.

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Jox 06 06092 g001
Figure 1. Chemical structure of methotrex- ate (A) and gallic acid (B).
Figure 1. Chemical structure of methotrex- ate (A) and gallic acid (B).
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Figure 2. Ameliorative effect of gallic acid pre-treatment and co- treatment on methotrexate-induced reduction in hepatic glu- tathione S-transferase (GST) activity in rats. Data are expressed as mean ± S.D for six rats in each group. *Significantly different from the control (P<0.05); asignificantly different from the methotrexate group (P<0.05).
Figure 2. Ameliorative effect of gallic acid pre-treatment and co- treatment on methotrexate-induced reduction in hepatic glu- tathione S-transferase (GST) activity in rats. Data are expressed as mean ± S.D for six rats in each group. *Significantly different from the control (P<0.05); asignificantly different from the methotrexate group (P<0.05).
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Figure 3. Ameliorative effect of gallic acid pre-treatment and co- treatment on methotrexate-induced reduction in hepatic vitamin C level in rats. Data are expressed as mean ± S.D for six rats in each group. *Significantly different from the control (P<0.05); asignificantly different from the methotrexate group (P<0.05).
Figure 3. Ameliorative effect of gallic acid pre-treatment and co- treatment on methotrexate-induced reduction in hepatic vitamin C level in rats. Data are expressed as mean ± S.D for six rats in each group. *Significantly different from the control (P<0.05); asignificantly different from the methotrexate group (P<0.05).
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Figure 4. Ameliorative effect of gallic acid pre-treatment and co- treatment on methotrexate-induced reduction in hepatic glu- tathione (GSH) level in rats. Data are expressed as mean ± S.D for six rats in each group. *Significantly different from the con- trol (P<0.05); asignificantly different from the methotrexate group (P<0.05).
Figure 4. Ameliorative effect of gallic acid pre-treatment and co- treatment on methotrexate-induced reduction in hepatic glu- tathione (GSH) level in rats. Data are expressed as mean ± S.D for six rats in each group. *Significantly different from the con- trol (P<0.05); asignificantly different from the methotrexate group (P<0.05).
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Figure 5. Ameliorative effect of gallic acid pre-treatment and co-treat- ment on methotrexate-induced increase in hepatic level of lipid per- oxidation (MDA) in rats. Data are expressed as mean ± S.D for six rats in each group. *Significantly different from the control (P<0.05); asignificantly different from the methotrexate group (P<0.05).
Figure 5. Ameliorative effect of gallic acid pre-treatment and co-treat- ment on methotrexate-induced increase in hepatic level of lipid per- oxidation (MDA) in rats. Data are expressed as mean ± S.D for six rats in each group. *Significantly different from the control (P<0.05); asignificantly different from the methotrexate group (P<0.05).
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Table 1. Experimental design.
Table 1. Experimental design.
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CTRL, control; MTX, methotrexate; GA, gallic acid.
Table 2. Ameliorative effect of gallic acid pre-treatment and co-treatment on methotrexate-induced changes in plasma biomarkers of renal and hepatic function in rat.
Table 2. Ameliorative effect of gallic acid pre-treatment and co-treatment on methotrexate-induced changes in plasma biomarkers of renal and hepatic function in rat.
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Data are expressed as mean ± S.D for six rats in each group. *Significantly different from the control (P<0.05); asignificantly different from the methotrexate group (P<0.05); values in parenthesis represent percentage (%) increase.

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Olayinka, E.; Ore, A.; Adeyemo, O.; Ola, O. Ameliorative Effect of Gallic Acid on Methotrexate-Induced Hepatotoxicity and Nephrotoxicity in Rat. J. Xenobiot. 2016, 6, 6092. https://doi.org/10.4081/xeno.2016.6092

AMA Style

Olayinka E, Ore A, Adeyemo O, Ola O. Ameliorative Effect of Gallic Acid on Methotrexate-Induced Hepatotoxicity and Nephrotoxicity in Rat. Journal of Xenobiotics. 2016; 6(1):6092. https://doi.org/10.4081/xeno.2016.6092

Chicago/Turabian Style

Olayinka, Ebenezer, Ayokanmi Ore, Oluwatobi Adeyemo, and Olaniyi Ola. 2016. "Ameliorative Effect of Gallic Acid on Methotrexate-Induced Hepatotoxicity and Nephrotoxicity in Rat" Journal of Xenobiotics 6, no. 1: 6092. https://doi.org/10.4081/xeno.2016.6092

APA Style

Olayinka, E., Ore, A., Adeyemo, O., & Ola, O. (2016). Ameliorative Effect of Gallic Acid on Methotrexate-Induced Hepatotoxicity and Nephrotoxicity in Rat. Journal of Xenobiotics, 6(1), 6092. https://doi.org/10.4081/xeno.2016.6092

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