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DOI: 10.1039/C4RA10213D (Paper) RSC Adv., 2014, 4, 60397-60403

Effect of enteral administration of α-linolenic acid and linoleic acid against methotrexate induced intestinal toxicity in albino rats

Prince Raj, Manjari Singh, Jitendra Kumar Rawat, Swetlana Gautam, Shubhini A. Saraf and Gaurav Kaithwas*
Department of Pharmaceutical Sciences, School of Biosciences and Biotechnology, Babasaheb Bhimrao Ambedkar University (A Central University), Vidya vihar, Raebareli road, Lucknow-226 025, India. E-mail: gauravpharm@gmail.com; gauravpharm@hotmail.com; Tel: +91-522-2998129 Tel: +91-9670204349

Received 11th September 2014 , Accepted 29th October 2014

First published on 30th October 2014

Abstract

The present study was conducted to show the effect of α-linolenic acid (ALA) (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and linoleic acid (LA) (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) on experimental intestinal toxicity induced by methotrexate (MTX). The groups of albino rats received: Group I: normal saline (2 ml kg−1, i.p. sham control); Group-II: MTX (2.5 mg kg−1, i.p. toxic control); Group-III: ALA (2 ml kg−1, i.p.); Group-IV: LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) (2 ml kg−1, i.p.), Group-V: ALA (2 ml kg−1, i.p.) and Group-VI: LA (2 ml kg−1, i.p.) with MTX (2.5 mg kg−1, i.p.). Animals were sacrificed after 7 days treatment schedule and appraised for intestinal pH, total acidity, free acidity and colonic mucosal disease index (CMDI). Intestinal tissues were further evaluated for oxidative stress parameters (TBARS, SOD, protein carbonyl and catalase), and morphological modulation using scanning electron microscopy. The intestinal tissues were further graded for the enzymatic activities of COX-1, COX-2 and 15-LOX. Both ALA and LA demonstrated momentous protection against MTX induced intestinal toxicity, which could be attributed to their prooxidant nature.

Introduction

Methotrexate (MTX) is a robust anticancer drug, anatomically similar to folic acid, and impedes the dihydrofolate reductase enzyme which reduces dihydrofolic acid to tetrahydrofolic acid. The competence of MTX is limited by relentless side effects and toxic effects including intestinal injury and enterocolitis.1 MTX causes the devastation of intestinal mucosa and perturbs the barrier against intravascular bacteria leading to relentless inflammation followed by degradation and ulceration of the intestine and colon.2 MTX induced inflammatory reactions motivate impairment of the antioxidant defence mechanisms and make the tissue more receptive to oxidative damage due to fructification of reactive oxygen species (ROS).3 One of the most prevalent pharmacological approaches to countervailing the MTX induced intestinal toxicity is to combat the inflammatory pathway by consolidating down the biosynthesis of pro-inflammatory eicosanoids, particularly derived from arachidonic acid (AA) (20[thin space (1/6-em)]:[thin space (1/6-em)]4, ω-3).

Essential fatty acids (EFA) are fatty acids that humans and other animals cannot synthesize and instead obtain from their diet. α-Linolenic acid (ALA) (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and linoleic acid (LA) (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) are two such fatty acids, paramount for humans, with an array of roles in the physiological system.4 LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) is converted into gamma-linolenic acid (GLA) (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-6) in the body, which is further converted to AA (20[thin space (1/6-em)]:[thin space (1/6-em)]4, ω-3). AA (20[thin space (1/6-em)]:[thin space (1/6-em)]4, ω-3) sits on the top of the inflammatory cascade with more than 20 different signalling pathways and governs a wide array of body functions including the inflammatory cascade.5 In contrast to the fact that GLA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-6) is one of the intermediate molecules in the synthesis of AA (20[thin space (1/6-em)]:[thin space (1/6-em)]4, ω-3), previous research has proposed that GLA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-6) plays an important role in allocating inflammation.6 Recently, it was ascertained that GLA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-6) precludes the switching of inflammatory cytokines by reconciling nuclear factor kappa β (NF-κβ). GLA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-6) also brings to bear its anti-inflammatory effects by promoting the pervasive peroxisome proliferator activated receptor (PPAR) system.7 GLA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-6) has also shown great effects in overcoming symptoms of rheumatoid arthritis.8 Two other essential fatty acids integrate a cascade that runs alongside and emulates with the AA (20[thin space (1/6-em)]:[thin space (1/6-em)]4, ω-3) cascade, eicosapentanoic acid (EPA) (20[thin space (1/6-em)]:[thin space (1/6-em)]5 ω-3) and decosahexanoic acid (DHA) (22[thin space (1/6-em)]:[thin space (1/6-em)]6 ω-3). EPA (20[thin space (1/6-em)]:[thin space (1/6-em)]5 ω-3) provides the most conspicuous competing cascade. EPA (20[thin space (1/6-em)]:[thin space (1/6-em)]5 ω-3) and DHA (22[thin space (1/6-em)]:[thin space (1/6-em)]6 ω-3) are ingested from fish oils or derived from dietary ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) by a series of desaturation and elongation reactions.9 Previous studies have taken account that the EPA (20[thin space (1/6-em)]:[thin space (1/6-em)]5 ω-3) cascade softens the inflammatory effects of the AA (20[thin space (1/6-em)]:[thin space (1/6-em)]4, ω-6) cascade, hence, suggesting it as an anti-inflammatory agent.10 As outlined above, GLA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-6)/AA (20[thin space (1/6-em)]:[thin space (1/6-em)]4, ω-6) and EPA (20[thin space (1/6-em)]:[thin space (1/6-em)]5 ω-3)/DHA (22[thin space (1/6-em)]:[thin space (1/6-em)]6 ω-3) are the products of LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) and ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) metabolism respectively and exhibit uncertain pharmacological actions.6 Therefore a scientific predicament exists against delineating the anti-inflammatory potential/mechanism of ω-3 and/or ω-6 fatty acids. Recently our laboratory has reported the significant in vitro and in vivo anti-inflammatory activity of LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) and ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3).6 Moreover, the anti-inflammatory and anti-arthritic along with anti-ulcer activity of Linum usitatissimum fixed oil has been reported and the aforesaid was thought to act through dual inhibition of AA (20[thin space (1/6-em)]:[thin space (1/6-em)]4, ω-3) metabolism by ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) (a major constituent in oil).11 A similar series of work has also shown the presence of a large amount of LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) (precursor for AA (20[thin space (1/6-em)]:[thin space (1/6-em)]4, ω-6) synthesis) in the oil as well. In view of the reports from our laboratory as well as from others, one can derive that there is a paucity of scientific evidence towards this aspect of PUFA research. Considering the above and with the aim of delineating the physiological role of ω-3 and ω-6 EFA, the present work has been undertaken to investigate the effect of LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) and ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) against MTX induced intestinal toxicity in albino rats.

Materials and methods

Drug and chemicals

ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3), LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) (Rolex Chemical Industries, Mumbai, India) and MTX (Folitrax-15, Ipca Pharmaceuticals Ltd. Mumbai, India) were purchased from the local market. The ELISA kits for COX-1, COX-2 (catalogue no. 760111) and 15 LOX (catalogue no. 760700) were procured from Cayman Chemicals Ltd USA. All other chemicals were procured from Hi-media Mumbai, India and were of analytical grade.

In vitro antioxidant assay

DPPH radical scavenging activity. A methanol solution accommodating ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) (20–120 μg ml−1 for each separately) was mixed with DPPH solution (100 μM in methanol) and incubated at 37 °C for 30 min. After incubation, the absorbance of the reaction mixture was read at 517 nm using a UV-visible spectrophotometer (Labtronics – LT – 2910 Double Beam).12 The experiment was performed in triplicate.
H2O2 scavenging activity. Spectrophotometric methods were used to resolve the competency of ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) to quench H2O2. Divergent concentrations of ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) (20–120 μg ml−1 for each separately) were dissolved in 0.1 M, pH 7.4 phosphate buffer and mixed with 40 mM solution of H2O2. Absorption of H2O2 at 230 nm was determined 10 min later using a UV-visible spectrophotometer (Labtronics – LT – 2910 Double Beam). A separate blank sample was used for background subtraction. The experiment was performed in triplicate.12

Animals

Albino Wistar rats (120–150 gm) of both sexes were retrieved from the central animal house facility. The albino rats were kept in polypropylene cages under standard conditions of temperature (22 ± 5 °C) with a 12 h light/dark cycle and with free access to a commercial pellet diet and water. The experimental protocol was endorsed by the Institutional Animal Ethics Committee (IAEC) (approval no. UIP/IAEC/2014/Feb/08). Animals were randomized and divided into 6 groups of 6 animals each. Group I (sham control, 0.9% normal saline i.p.); Group II (toxic control, MTX 2.5 mg kg−1 i.p.); Group III (ALA 2 ml kg−1 i.p.); Group IV (LA 2 ml kg−1 i.p.); Group V (MTX + ALA 2.5 mg kg−1 + 2 ml kg−1 i.p.); Group VI (MTX + LA 2.5 mg kg−1 + 2 ml kg−1 i.p.).1,6 Toxicity was induced by single i.p. injection of MTX followed by ALA and LA supplementation therapy for seven days at the dose prescribed above. Animals were sacrificed on the 8th day and subjected to estimation.

Evaluations

Estimations of pH, free acidity and total acidity. After the respective treatment animals were euthanized with cervical dislocation and the intestinal tissue was collected. The content of the intestinal tissue was collected and evaluated for intestinal pH using a pen type pH meter (Hanna Instrument HI 98107). Free acidity and total acidity were appraised using the procedure described previously. Total acidity and free acidity are expressed as mEq l−1.13,14
Assessment of CMDI. The colon tissue of approximately 10 cm to anus was taken, opened longitudinally and washed in normal saline buffer and fixed on a wax block. The scoring was done and evaluated by using the formula of CMDI represented as follows. 0 = normal mucosa, 1 = mild hyperemia, no erosion or ulcers on the mucosa surface, 2 = moderate hyperemia, erosion or ulcers appear on the mucosa surface, 3 = sever hyperemia, necrosis and ulcers on the mucosa surface with the ulcerative area less than 40%, 4 = severe hyperemia, necrosis and ulcers on the mucosa surface with the ulcerative area more than 40%.15
Biochemical estimation. The distal part of the intestinal tissues (10% w/v) was homogenised in 0.15 M KCl at 10[thin space (1/6-em)]000 rpm (4 °C). The supernatants were scrutinized for biochemical parameters including TBARS,16 SOD,17 protein carbonyl18 and catalase19 using the methods established at our laboratory.20,21
COX-1, COX-2 and 15-LOX. The supernatants as collected above were further appraised for the enzymatic activities of COX-1, COX-2 and 15 LOX using commercial ELISA kits from Cayman Chemicals Ltd USA, as per the method described by the manufacturer using a microplate reader (Alere Microplate Reader AM, 2100).
Morphological evaluation. The intestinal tissues from all the groups were evaluated for their morphological changes using scanning electron microscopy. Samples were fixed in 2.5% glutaraldehyde for 6 h at 4 °C and washed with 0.1 M phosphate buffer, for 3 changes each of 15 min at 4 °C. 1% osmium tetraoxide was used as a post fixative for 2 h at 4 °C and samples were washed in 0.1 M phosphate buffer for 3 changes each of 15 min at 4 °C to remove the unreacted fixative. Specimens were dehydrated by using increasing concentrations of acetone viz. 30%, 50%, 70%, 90%, 95%, 100% (dry acetone) to remove water at 4 °C for 30 min periods. After that, samples were air dried (critical point i.e. at 1100 psi). The specimens were mounted on to the aluminium stub with adhesive tape and the specimens were observed using a scanning electron microscope (JEOL-JSM-6490LV).
Statistical analysis. All data were presented as mean ± SD and analyzed by one way ANOVA followed by Bonferroni test for the possible significance identification between the various groups. *P < 0.05, **P < 0.01, ***P < 0.001 were considered statistically significant. Statistical analysis was carried out using Graph pad prism (3.2), San Diego, California.

Results

The intraperitonial administration of ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) incomparably inhibited the intestinal toxicity in the experimental animals illustrated through conspicuous reduction in the free acidity (26.15%), total acidity (22.35%) and CMDI (83.25%) in comparison to a control (Table 1). Treatment with LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) also afforded momentous protection in contrast to MTX induced toxicity; however the same was perceived to be inconsiderable in comparison to ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3).
Table 1 Effect of ALA and LA therapy on intestinal pH, free acidity, total acidity and CMDIa
Group Treatment (i.p.) Intestinal pH Free acidity (mEq l−1) Total acidity (mEq l−1) CMDI
a Each group contains six animals; data are represented as mean ± SD, statistical significance compared to toxic control using one-way ANOVA followed by Bonferroni test. *P < 0.05, **P < 0.01, ***P < 0.001 were considered statistically significant. Values in parentheses represent percentage inhibition.
Group I Sham control (normal saline, 2 ml kg−1) 5.28 ± 0.23*** 10.59 ± 0.69*** 13.58 ± 0.61*** 0.00 ± 0.00***
Group II MTX (toxic control) (2.5 mg kg−1) 4.55 ± 0.27 17.32 ± 2.23 20.71 ± 0.81 4.00 ± 0.00
Group III ALA (2 ml kg−1) 5.20 ± 0.17*** 10.59 ± 0.49*** (38.85%) 14.29 ± 0.46*** (30.99%) 0.00 ± 0.00*** (100%)
Group IV LA (2 ml kg−1) 5.47 ± 0.14*** 12.20 ± 1.08*** (29.56%) 15.01 ± 0.35*** (27.52%) 0.50 ± 0.84*** (87.5%)
Group V MTX + ALA (2.5 mg kg−1 + 2 ml kg−1) 5.25 ± 0.19*** 12.79 ± 0.78*** (26.15%) 16.08 ± 0.28*** (22.35%) 0.67 ± 0.81*** (83.25%)
Group VI MTX + LA (2.5 mg kg−1 + 2 ml kg−1) 5.37 ± 0.15*** 13.96 ± 0.78*** (19.39%) 16.05 ± 0.77*** (22.50%) 1.00 ± 0.89*** (75%)

MTX made evident a compelling upsurge in fructification of MDA (9.70 ± 0.37 nM of MDA per mg of protein). The treatment group with LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) and ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) bestowed a momentous protection from the same, just about uniformly (Table 2). When scrutinized for protein oxidation, the MTX treatment evidenced a compelling increase in protein carbonyl levels in toxic groups (116.81 ± 0.68 nM per ml) in resemblance to the normal control (50.60 ± 4.17 nM per ml). Concomitant administration of LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) and ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) re-established the protein carbonyl to a significant level. Similarly, an outstanding increase in SOD was contemplated in the toxic control (43.99 ± 7.86 SOD per mg of protein) in counterpart to the sham control (29.63 ± 2.51 SOD per mg of protein) (Table 2). Discordant compelling subsidence in the enzymatic activity of catalase was perceived in the toxic control (8.27 ± 1.45 nM of H2O2 per min per mg of protein); it is noteworthy that treatment with ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) helped to restore the enzymatic activities of SOD and catalase synchronously.

Table 2 Effect of ALA and LA therapy on TBARS, protein carbonyl, SOD and catalase on intestinal tissuea
Group Treatment (i.p.) TBARS (nM of MDA/mg of protein) Protein carbonyl (nM per ml) SOD (SOD per mg of protein) Catalase (nM of H2O2 per min per mg of protein)
a Each group contains six animals; Data are represented as mean ± SD, statistical significance compared to toxic control using one-way ANOVA followed by Bonferroni test. *P < 0.05, **P < 0.01, ***P < 0.001 were considered statistically significant.
Group I Sham control (normal saline, 2 ml kg−1) 8.18 ± 0.13*** 50.60 ± 4.17*** 29.63 ± 2.51*** 12.60 ± 2.15**
Group II MTX (toxic control) (2.5 mg kg−1) 9.70 ± 0.37 116.81 ± 0.68 43.95 ± 7.86 8.27 ± 1.45
Group III ALA (2 ml kg−1) 8.32 ± 0.17*** 50.45 ± 1.60*** 29.68 ± 1.85*** 10.18 ± 2.62
Group IV LA (2 ml kg−1) 8.33 ± 0.39*** 37.57 ± 5.34*** 30.68 ± 1.85*** 8.22 ± 0.92
Group V MTX + ALA (2.5 mg kg−1 + 2 ml kg−1) 8.72 ± 0.17*** 50.84 ± 4.17*** 38.66 ± 1.50*** 8.25 ± 0.73
Group VI MTX + LA (2.5 mg kg−1 + 2 ml kg−1) 8.71 ± 0.27*** 49.85 ± 0.35*** 37.31 ± 2.21*** 8.38 ± 0.86

The intestinal tissue showed a compelling rise in the enzymatic activity of COX-1 and COX-2 after the concomitant administration of ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) with MTX; discordantly 15-LOX activity was seen to decrease in ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and was markedly up in LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) treated groups when administered concomitantly with MTX (Table 3).

Table 3 Effect of ALA and LA therapy on cyclooxygenase and lipoxygenase activity in intestinal tissuea
Group Treatment (i.p.) COX-1 (μmol ml−1 min) COX-2 (μmol ml−1 min) 15-LOX (μmol ml−1 min)
a Each group contains six animals; data are represented as mean ± SD, statistical significance compared to toxic control using one-way ANOVA followed by Bonferroni test. *P < 0.05, **P < 0.01, ***P < 0.001 were considered statistically significant.
Group I Sham control (normal saline, 2 ml kg−1) 29.40 ± 2.00 24.22 ± 1.99** 13.54 ± 2.26***
Group II MTX (toxic control) (2.5 mg kg−1) 34.32 ± 3.35 28.99 ± 0.74 37.08 ± 7.88
Group III ALA (2 ml kg−1) 36.86 ± 8.68 18.42 ± 0.07*** 14.69 ± 1.13***
Group IV LA (2 ml kg−1) 47.79 ± 5.97* 24.05 ± 1.13*** 17.47 ± 3.90**
Group V MTX + ALA (2.5 mg kg−1 + 2 ml kg−1) 52.55 ± 2.28** 25.37 ± 0.00* 18.64 ± 3.73**
Group VI MTX + LA (2.5 mg kg−1 + 2 ml kg−1) 56.32 ± 3.00** 23.72 ± 0.28*** 15.02 ± 0.00***

When monitored morphologically abnormalities in the mucosa of treated rats were detected including hyperproliferation, progressive distortion of the crypts, mucosal surface irregularities suggesting degradation and formation of focal protuberances in the MTX treated groups. Consequent administration of ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) gave rise to a pronounced protection against the same in a dose dependent manner (Fig. 1).


image file: c4ra10213d-f1.tif
Fig. 1 (A) Sham control (normal saline 2 ml kg−1 i.p.); (B) toxic control (MTX 2.5 mg kg−1 i.p.); (C) ALA (2 ml kg−1 i.p.); (D) LA (2 ml kg−1 i.p.); (E) MTX + ALA (2.5 mg kg−1 + 2 ml kg−1 i.p.); (F) MTX + LA (2.5 mg kg−1 + 2 ml kg−1 i.p.).

The results from the DPPH and H2O2 scavenging assay depict no antioxidant properties of ALA and LA. Rather the results reflect a significant prooxidant nature of both the test compounds (Fig. 2).


image file: c4ra10213d-f2.tif
Fig. 2 In vitro antioxidant activity of ALA and LA using DPPH and hydrogen peroxide assay. Data represented as mean ± SD (n = 3).

Discussion

MTX is an anticancer drug with anti-metabolite action and is used as an anticancer and anti-rheumatic agent. The use of MTX is accompanied by a sizable number of toxicities, not to mention intestinal toxicity, cardiotoxicity, hepatotoxicity, nephrotoxicity and a few more, restricting its suitability for use in the diverse maladies.22 In the present work we validated a remarkable protection by ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) against MTX induced intestinal toxicity.

Treatment with ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) decidedly impeded the intestinal toxicity by regularizing the pH, decreasing the free acidity and total acidity in contrast to a toxic control. The concomitant administration of ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) with MTX also slackened the CMDI to a convincing level. The above perceived effects of ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) are in agreement with the antecedent reports and the same could be attributed to the anti-histaminergic (anti-secretary) and anti-cholinergic (anti-secretary and vasodilator) effects of PUFA. The anti-secretary effects of ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) could be ascribed to the muscarinic 3 and histaminergic 2 antagonistic actions as outlined previously.23 ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) displayed exceptional protection against intestinal toxicity in relation to LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6).

The heightened production of MDA and protein carbonyl are unambiguous markers for oxidative damage to lipids and proteins respectively.24 Although there is no solitary universal marker for protein oxidation, protein carbonyl appraisal is extensively used as a marker for protein oxidation.25 We saw a convincing upsurge in the protein carbonyl content in the toxic group which was re-established after the concomitant administration of ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6). It is worth mentioning that ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) showed imperceptible conservation of protein oxidation in analogy to LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6). MTX treatment in the toxic control group showed a compelling increase in the creation of MDA products and thereby the concurrence of lipid peroxidation in the MTX toxicity, which is in corroboration with previous work.26 Concomitant administration of ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) depreciated the levels of MDA products to a significant level and by that curtailed oxidative stress.

SOD and catalase together complement an extensive defence team against ROS; SOD abrogates the superoxide free radical to form hydrogen peroxide which after a while is neutralized by heme protein, catalase.20 Catalase decomposes the hydrogen peroxide to give rise to water and molecular oxygen. Both the enzymes work in tandem to protect the tissue from highly reactive free radicals.27 In the present experiment we observed a momentous increase in the SOD enzyme, further affirming the concurrence of ROS and accompanying administration of ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) significantly restored the enzymatic activity of SOD. The synchronic increase in the enzymatic activity of catalase is expected with an increase in SOD and is reported extensively as well.21 However, this was not replicated in our experiment and a momentous decrease in tissue catalase was evidenced after MTX administration. Notwithstanding, the therapeutic regimen of ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) dwindled to rehabilitate the slackened levels of catalase.

MTX associated chemotherapy has been proclaimed to cause mucositis directly by provoking DNA strand breaking through the generation of ROS; ROS may outrage other cell and tissues and prompt secondary mediators including NF-κβ and pro-inflammatory cytokines.28,29 The rousing of transcription factor (NF-κβ) in counter to ROS, further results in gene upregulation for TNF-α, interleukins (IL-1β, IL-6) leading to injury and apoptosis not beyond the submucosal and basal epithelium.30 The inflamed intestine manifests oxidative stress leading to oxidation of lipids, proteins and DNA damage.3 Therefore, the MTX induced toxicity is conspicuous by the increased enzymatic activity of COX-1, COX-2 and 15-LOX as experienced in the current experiment. ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) proceeded to farther increase the COX-1 and COX-2 activity, whereas 15-LOX was re-established to routine levels. This could be interpreted with certainty that ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) are subsequently metabolized to AA (20[thin space (1/6-em)]:[thin space (1/6-em)]4, ω-6) and EPA (20[thin space (1/6-em)]:[thin space (1/6-em)]5, ω-3). Both ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) are the substrates for COX-1, COX-2 and 15-LOX.6 Moreover, ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) have forthright inhibitory effects on COX-1 and COX-2 only, as reported in previous studies.6 Thus, we conclude that due to increased substrate availability for COX-1 and COX-2 followed by the direct inhibitory effect of ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6), the rebuttal mechanism could have initiated the increased enzymatic activity of COX-1 and COX-2 as ascertained in our experiment. It would be pertinent to mention that a previous report has suggested the direct inhibitory activity of ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) against COX, without too much affect on LOX.4 Due to paucity of a forthright inhibitory effect of ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and/or LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) on LOX, we observed the restoration in the enzymatic level of 15-LOX.

When contemplated microscopically, considerable hyperproliferation and mucosal degeneration was seen in MTX treated experimental animals, which is in agreement with previous studies. Both ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) manifested significant microscopic protection contrary to the MTX induced intestinal toxicity in experimental animals.

The in vitro anti-oxidant activities of ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) were appraised via DPPH and H2O2 assay. DPPH is a steady free radical, whereas H2O2 is highly reactive and consequently short lived. Every H2O2 molecule brought together with DPPH has the ability to destroy almost every molecule in a living cell.31 ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) demonstrated robust pro-oxidant activity in the DPPH and H2O2 assay suggesting their ability to interact with a wide range of free radicals. The pro-oxidant activity of ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6), as perceived in the present experiment, can be attributed to the high degree of unsaturation present in ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6).32

As discussed above, the metabolic products of LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) are pro-inflammatory, whereas those of ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) are anti-inflammatory. However, both contributed physiological, biochemical and morphological protection against MTX induced toxicity. It could also be found that the exogenic supplementation of ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) (pro-oxidant unsaturated FA), would have made them susceptible for incursion by ROS, engendered through MTX toxicity. Henceforth, we postulate/hypothesize that both ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) demonstrate significant protection due to their competence as prooxidants, which could be attributed to their polyunsaturated nature, making them susceptible to ROS attack. It would be worth remarking that ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) demonstrated somewhat sharpened protection in comparison to LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) and the aforesaid could be attributed to the generation of anti-inflammatory mediators in comparison to pro-inflammatory mediators from LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6). The authors would also like to comment that the preservation demonstrated by LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) may be lost in long term therapeutic regimens due to the generation of pro-inflammatory metabolites of AA (20[thin space (1/6-em)]:[thin space (1/6-em)]4, ω-6).

It can be concluded that ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) could be used as an adjuvant with MTX chemotherapy and/or clinical management of arthritis. These therapeutic effects as uncovered through the current experimental evidence can be attributed to their action on oxidant–antioxidant systems and the inflammation process. However, the clinical significance of ALA (18[thin space (1/6-em)]:[thin space (1/6-em)]3, ω-3) and LA (18[thin space (1/6-em)]:[thin space (1/6-em)]2, ω-6) in various clinical pathologies has been a matter of debate and different scientists across the world have opined differently on this issue. Therefore further experimental and clinical studies are required to ascertain these findings.

Conflict of interest

No conflicts of interest are declared.

Abbreviations

AAarachidonic acid
ALAα-linolenic acid
CMDIcolonic mucosal disease index
DAIdisease activity index
DHAdocosahexanoic acid
EPAeicosapentanoic acid
EFAessential fatty acid
LAlinoleic acid
MTXmethotrexate
PUFApolyunsaturated fatty acid

Acknowledgements

The authors would like to acknowledge Dr V. Elongovan, Coordinator, University Scientific Instrument Centre for extending his help in SEM analysis. The authors would like to thank the University Grants Commission for providing fellowships to PR, SG, MS and JKR. The authors would also like to thank the University Grants Commission for providing partial funding for the work. [Letter no. 20-1/2012 (BSR)/20-9/2012 (BSR)].

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