α-Lipoic acid ameliorates oral mucositis and oxidative stress induced by methotrexate in rats. Histological and immunohistochemical study

Accepted Manuscript α-Lipoic acid ameliorates oral mucositis and oxidative stress induced by methotrexate in rats. Histological and immunohistochemical study

Amal A.M. Ahmed, Manar A.A. Selim, Norhan M El-Sayed PII: DOI: Reference:

S0024-3205(17)30001-2 doi: 10.1016/j.lfs.2017.01.001 LFS 15119

To appear in:

Life Sciences

Received date: Revised date: Accepted date:

12 November 2016 31 December 2016 2 January 2017

Please cite this article as: Amal A.M. Ahmed, Manar A.A. Selim, Norhan M El-Sayed , α-Lipoic acid ameliorates oral mucositis and oxidative stress induced by methotrexate in rats. Histological and immunohistochemical study. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Lfs(2017), doi: 10.1016/j.lfs.2017.01.001

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ACCEPTED MANUSCRIPT α-lipoic acid ameliorates oral mucositis and oxidative stress induced by methotrexate in rats. Histological and immunohistochemical study. Amal A.M. Ahmeda, Manar A.A.Selimb, Norhan M El-Sayedc.

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Department of Cytology & Histology, Faculty of Veterinary Medicine, Suez Canal

University, Ismailia, Egypt. c

Department of Oral biology, Faculty of Dentistry, Suez Canal University, Ismailia, Egypt.

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Department of Pharmacology & Toxicology, Faculty of Pharmacy, Suez Canal University,

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Ismailia 41522, Egypt.

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Correspondence at:

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Norhan M El-Sayed, PhD

Department of Pharmacology & Toxicology, Faculty of Pharmacy, Suez Canal University,

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Ismailia 41522, Egypt e-mail: [email protected]

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Tel: 002-01227222915

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Fax: 002-064-3230741

ACCEPTED MANUSCRIPT Abstract Aim Oral mucositis is a common adverse effect of Methotrexate (MTX) that may limit its clinical use. Oxidative stress and apoptosis have been proposed to mediate MTX toxicity. The current study was conducted to assess the conceivable protective effect of α-lipoic acid (LA) against

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MTX induced toxicity on both buccal and lingual mucosae.

into three groups; control, MTX-treated group

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Thirty male wister rats were allocated

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Main Methods

subjected to single intraperitoneal injection of MTX (20 mg/kg, i.p.) and LA- treated group

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treated with daily intraperitoneal injection of LA (10 mg/kg, i.p.) for 5 weeks before MTX injection (20 mg/kg, i.p.). Rats were then sacrificed under anesthesia then their buccal and

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lingual mucosae were dissected out and processed for biochemical and histopathological studies. Biomarkers of oxidative stress and integrity of nuclear DNA (nDNA) were estimated.

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Immunostaining was used to determine Bax and PCNA localization.

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Key findings

MTX-treated rats showed increased levels of MDA and fragmentation of DNA in addition to

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reduction of GSH levels and activities of catalase and SOD. Histological examination of MTX-treated rats demonstrated degenerative changes that involved the surface epithelium

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and lamina propria of their buccal and lingual mucosae. Immunohistochemical results of MTX-treated rats revealed strongly positive Bax and weakly positive PCNA staining reactivity of the nuclei of the basal and parabasal cells of the surface epithelium. However, LA significantly attenuated MTX-evoked alterations in the previous-stated parameters highlighting its antioxidant and anti-apoptotic potential. Significance

ACCEPTED MANUSCRIPT LA may be suggested to be a prospective candidate to ameliorate MTX-induced oral mucositis.

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Key words: Oral mucositis, Methotrexate, Lipoic acid, Oxidative stress, Apoptosis, Rat

ACCEPTED MANUSCRIPT 1. Introduction Oral mucositis is a common painful, inflammatory and debilitating adverse effect of cancer therapy. 50% of cancer patients under standard doses of chemotherapy are affected and the percentage goes up to 80 % in patients who received high doses of anticancer treatment [1]. The mucosa of the lips, tongue, mouth, floor of mouth and soft palate are easily affected by antineoplastic agents such as Methotrexate (MTX) than keratinized hard tissues [2].

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Generally, MTX as an inhibitor of folic acid reductase can induce oral mucositis through inhibition of DNA synthesis and consequently leads to reduction in the renewal capabilities

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of the basal epithelium and inhibition of mucosal cell proliferation [3]. Collectively these events lead to mucosal atrophy, collagen collapses, and ultimate ulceration. Normally the oral

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mucosa is characterized by higher rate of replication which makes it highly susceptible to cytotoxic agents like MTX.

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Clinically, mucositis can be classified into four categories according to World Health Organization (WHO); grade 0, no mucosal lesion; grade 1 (mild), erythematous area; grade 2

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(moderate), mucosal erythema or ulceration, allowing intake of solid food; grade 3 (severe), ulcerated area, only liquids allowed; and grade 4 (life threatening), mucositis requiring enteral or parenteral nutrition [4]. Histologically, the disease involves four successive phases:

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initiation of an inflammatory/vascular phase, upregulation, signalling and amplification

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phase, an ulcerative/microbiological phase, and finally healing phase. The initial phase is usually acute and is characterised by release of inflammatory cytokines such as interleukin1 from the epithelium and the connective tissues. The epithelial phase is typically observed

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about four to five days after administration of cytotoxic agents including MTX as that interferes with DNA synthesis of the oral mucosa epithelium. Consequently this results in

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reduction of epithelial renewal and appearance of ulcerative lesions. The third phase is signaling and can be complicated by microbiological infection thus it is the most symptomatic phase. This ulcerative phase occurs as a result of collapses of mucosal barriers concomitantly with neutropenia. Lastly the healing phase which involves the formation of pseudo membrane to coat the ulcer [5]. α-Lipoic acid (LA) is a powerful endogenous antioxidant and important cofactor in mitochondrial dehydrogenase reactions. LA is a disulfide compound that acts as a coenzyme in pyruvate dehydrogenase and α-ketoglutarate dehydrogenase mitochondrial reactions, thus it plays a fundamental role in mitochondrial energy metabolism [6]. It is soluble in both lipid

ACCEPTED MANUSCRIPT and water environments. Being lipid soluble, LA is vastly effective at combating free radicals, including lipid peroxides that can be found in cellular membranes. Its water solubility allows gaining free access to the cytosol, where it efficiently scavenges reactive oxygen species at their mitochondrial source. In addition to its antioxidant potential, LA could regenerate endogenous non-enzymatic antioxidants including reduced glutathione (GSH) and vitamin C which in turn can recycle vitamin E and coenzyme Q10 [7]. This study

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for oral mucositis induced by a high single toxic dose of MTX.

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was designed to investigate the effectiveness of LA (10mg/kg/day) as a preventive treatment

ACCEPTED MANUSCRIPT 2.Materials and methods 2.1 Animals Thirty adult male wister rats weighting 80-120 g were used in the present study; they were obtained from the National Centre of Research (Cairo, Egypt). Rats were housed in stainless steel cages in a normal light – dark cycle at around 25 °C. They were fed standard laboratory feed and had free access to water. Body weights of rats were recorded every week. All

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animals received professional care in accordance with the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health. This study was carried

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out in accordance with the Guidelines of the Animal Care and Use Committee at Faculty of

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veterinary medicine, Suez Canal University (approval no. 2016089).

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2.2 Drugs and chemicals

Methotexate was purchased as vial (ACDIMA International, Shanxi Powerdone Pharmaceutical Co.Ltd) dissolved in 0.9 % saline. Lipoic acid was obtained in form of

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thiotacid® vial (Amriya Pharmaceutical Industries, Cairo, Egypt) and was dissolved in 0.9 %

2.3 Experimental design

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saline.

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Thirty rats were randomly allocated into three groups (10 rats each), Group I: untreated rats (normal control) receiving saline (0.9 % NaCl); Group II: rats receiving single dose of MTX (20 mg/kg/i.p); Group III: rats treated with LA (10mg /kg/day/i.p) for 5 weeks before the

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administration of a single dose of MTX (20 mg/kg/i.p). Four days after MTX treatment, all rats were sacrificed by cervical dislocation under ketamine anesthesia (80 mg/kg, i.p.). A

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longitudinal incision on rats’ buccal mucosae of both sides of their mouths was made and then the buccal mucosae from both sides were removed in a spindle-like fragment. In addition, lingual mucosae were dissected out and both are processed for biochemical and histopathological studies. 2.4 Determination of oxidative stress markers For estimation of different oxidative stress parameters, part of each of buccal mucosa and tongue was ice-cooled, homogenized in 10% phosphate buffer (pH 7.4) using a Teflon homogenizer (Glass Col homogenizer system, Vernon hills, USA), and then centrifuged at 3000×g for 15 min at 4 °C. The supernatant was assayed for malondialdehyde (MDA), GSH

ACCEPTED MANUSCRIPT in addition to the activities of endogenous antioxidant enzymes like catalase and superoxide dismutase (SOD) using colorimetric kits from Biodiagnostic (Giza, Egypt) according to the manufacturer’s protocol. MDA was measured based on its reaction with thiobarbituric acid (TBA) in acidic medium at temperature of 95 °C for 30 min to form a pink colored product. The absorbance of the reaction product was measured spectrophotometrically at 534 nm [8, 9]. Tissue reduced GSH contents were assessed according to the manufacturer’s instruction(

nitrobenzoic acid)

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Biodiagnostic, Giza, Egypt) using a method based on the reduction of 5,5` dithiobis (2to produce a yellow compound. The absorbance was measured

colorimetrically at 412 nm [10]. SOD activity was determined spectrophotometrically

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according to Nishikimi et al. [11] method based on the capabilityof the enzyme to inhibit the

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phenazine methosulphate-mediated reduction of nitroblue tetrazolium dye and then the absorbance was read at 560 nm. Moreover, the activity of catalase was estimated depending

enzymes were expressed as u/mg protein.

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2. 5 DNA laddering assay

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on its reaction with H2O2 as previously described [12, 13]. All the activities of antioxidant

Among the characteristics of apoptosis is the cleavage of double-stranded DNA in the linker

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region between nucleosomes via endogenous endonucleases and the generation of mono- and oligonucleosomes of 180 bp or multiples. To assess endonuclease-dependent ladder-like

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DNA fragmentation by gel electrophoresis, genomic DNA was extracted from both buccal mucosa and tongue using Wizard® Genomic DNA Purification kit (Promega Corporation, Madison, WI, USA) according to the manufacturer's instructions. All of the extracted

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genomic DNA samples were then loaded onto agarose gel (15 μg/lane) and subjected to constant voltage mode electrophoresis (in a large submarine at 4 V/cm, for 4 h) on a 1.5%

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agarose gel containing 0.5 μg/ml ethidium bromide. Gels were visualized by G:Box Gel Documentation system (Syngene, USA). 2.6 Histopathology and immunohistochemistry. Buccal and lingual mucosae specimens were immediately fixed in 10% neutral buffered formalin, dehydrated in ascending grades of ethyl alcohol, cleared in xylol, embedded in three changes of paraffin wax and sectioned at 4-6 µm thick sections. Microscopic slides were subjected to routine histological technique and stained with hematoxylin and eosin (H&E) [14]. Sections were examined by a blinded investigator without knowledge of any other data on the experimental groups.

ACCEPTED MANUSCRIPT For immunohistochemical analysis of Bax and PCNA, buccal and lingual sections (5-μm) from each rat was deparaffinized and incubated in citrate buffer (pH = 6) in a microwave oven for antigen retrieval. Activity of endogenous peroxidases was quenched by applying 0.3 % H2O2 to the sections. A Vectastain rabbit blocking reagent was used to prevent nonspecific binding. Polyclonal rabbit anti-Bax (Cat#ab7977, Abcam, Cambridge Science Park, Cambridge, UK) was used as primary antibodies. Mouse monoclonal anti-PCNA (Cat #

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MS-862-P, Thermo Scientific, CA, USA) was used as primary antibodies. Antibody bindings were visualized by using avidin-biotin complex (ABC kit, Vector laboratories).

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After overnight incubation, secondary antibodies were then incubated with each section for 5 min. After washing ABC reagent was used then incubated with the

diaminobenzidine

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peroxidase enzyme substrate for 3 min. Sections were counterstained with hematoxylin to enhance the nuclear staining. For negative controls, adjacent sections were processed with the

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same steps with the exception of the primary antibodies. All procedures were done according to Vectastain Elite ABC Kit (Rabbit IgG, catalogue number PK-601, Vector Laboratories,

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California, USA). Images were obtained from buccal mucosa and the dorsal surface of the tongue by means of a digital camera (Olympus Dp25, Japan). For detection of % area of apoptotic and proliferation cells immunostained with Bax and PCNA respectively, images of

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selected parts of buccal and lingual mucosae were analyzed using the ImageJ software

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developed by the National Institute of Health (Bethesda, Maryland, USA). 2.7 Morphometric analysis

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For histological analysis of epithelial thickness of both buccal and lingual epithelium, five histological sections were selected for measurement at 10x magnification. At random, five

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fields from each section were evaluated. Measurement of epithelial thickness was performed by using imageJ program. 2.8 Statistical analysis Data were collected and expressed as mean ± S.E.M. For statistical analysis, one-way ANOVA, followed by Bonferroni's test for multiple comparisons was applied. Data analysis was performed employing the statistical package for social sciences, version 21 (SPSS Software, SPSS Inc., Chicago, USA). The level of significance was set at P values <0.05.

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3. Results 3.1. Effect of LA treatment on occurrence of diarrhoea MTX administration led to the occurrence of diarrhoea and reduction of food intake in all rats. However, only 2 out of 10 rats treated with LA exhibited signs of diarrhoea.

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3.2. Biomarkers of oxidative stress: Table (1) points out the disturbances in some biochemical markers of oxidative stress such as MDA, GSH contents, catalase and SOD activities, in both buccal mucosa and tongue of

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normal and MTX-treated rats. To examine the effect of MTX-evoked oxidative stress, first

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MDA levels were measured. MDA is a well-recognized biomarker of oxidative damage to lipid contents in cell membrane. In rats acutely intoxicated with MTX (20 mg/kg/i.p), the

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MDA levels in both buccal mucosa and tongue significantly increased. Administration of LA (10 mg/kg, i.p) for 5 weeks prior to MTX exposure diminished MTX-induced rise in MDA contents in both oral mucosa and tongue tissues. LA demolished MDA contents to nearly

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normal values. These results obviously designated that treatment of LA blocked MTXinduced lipid peroxidation in both buccal mucosa and tongue tissues. In addition, rats acutely

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exposed to MTX (20 mg/kg/i.p) showed a significant fall in reduced GSH levels compared to control group. Treatment with LA (10 mg/kg/i.p) for 5 weeks before MTX injection reversed

normal levels (Table 1).

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the decline in the reduced GSH levels in both buccal mucosa and tongue tissues to approach

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The present study showed that activities of catalase and SOD in both buccal mucosa and tongue were significantly attenuated in MTX-treated rats compared to normal control.

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However, a marked rise in catalase and SOD activities were showed upon administration of LA in MTX-treated rats. Interestingly, LA is capable to recover SOD and catalase activities to similar levels of the control (Table 1). Thus, treatment with LA can protect both buccal mucosa and tongue tissues from the toxic effect of MTX by combating free radicals evidenced by ameliorating the changes in biomarkers of oxidative stress. 3.3. Fragmentation of nuclear DNA Figure 1 depicts the qualitative alterations in the integrity of the genomic DNA extracted from the buccal mucosa (lane 1, 2 and 3) and tongue tissues (lane 4, 5 and 6) of different study groups. Results from agarose gel electrophoresis show that nDNA isolated from

ACCEPTED MANUSCRIPT untreated control rats (lane 1 and 4), LA (lane3 and 6) exhibited total ladder and smear negativity. However, MTX exposure resulted in a characteristic DNA ladder pattern with mixed smearing in both buccal mucosa and tongue as a marker of apoptosis. In addition, a dramatic oligonucleosome-length degradation of genomic DNA was observed (lane 2 and 5, Figure 1). These results demonstrated that LA treatment (10mg/kg, i.p) abolished the ladder pattern of genomic DNA cleavage in both buccal mucosa and tongue of MTX-treated rats.

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Therefore, it apparently provided evidence to its protective effect against MTX-induced apoptosis.

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3.4. Histological evalutation:

Histological examination of the buccal mucosa of control rats showed normal histological

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features of surface epithelium, underlying lamina propria and submucosa (Fig.2 A1 and A2).The epithelium was formed of keratinized stratified squamous epithelium characterized

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by foldings towards the underlying lamina propria, forming regular, broad, few and short epithelial ridges (Fig.2 A1). Epithelium of control rats was formed of four layers of normal

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structure, basal cell layer, prickle cell layer, granular cell layer and keratinous layer. Lamina propria of control rats showed regular arrangement of collagen fibres, fibroblasts and small sized blood vessels. Submucosa was formed of densely packed collagen fibres and fat cells

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(Fig. 2 A1&A2). MTX-treated rats showed significant increase in the buccal epithelial

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thickness in compared to control and lipoic-treated rats (Fig. 2 D). The epithelial ridges became longer and broader (Fig. 2B1). In addition, the basal cells lacked their normal architecture; the epithelial-connective tissue interface showed disintegration of the basement

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membrane at certain areas with loss of basal cells adhesion and invasion of some basal cells towards the subepithelial-connective tissue (Fig. 2 B2).Vacuoles of different sizes were observed in the Malpighian layer (Fig. 2 B2). The prickle cell layer showed swelling of their

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cells. The granular cell layer increased in thickness and showed apparent mild increase of basophilic keratohyaline granules in their cytoplasm and the keratinous layer showed increase in thickness (hyperkeratosis) and had an irregular outer surface. Few inflammatory cells were detected in the lamina propria of mucosae of MTX-treated rats. Buccal mucosa of LA-treated rats presented improvement in their histological structures; the surface epithelium showed decrease in the extent of swelling of its cells, the epithelial ridges kept their normal characteristic pattern with regular, broad, few and short epithelial ridges (Fig. 2C1 & C2). Absence of vacoulation revealed in the Malpighian layer (Fig. 2 C2). Furthermore, LA-

ACCEPTED MANUSCRIPT treated rats showed decrease in the degree of disintegration and dissociation of the collagen fibres (Fig. 2C2). Microscopic examination of the dorsal surface of tongue of control rats showed normal histological features of surface epithelium (Fig. 3 A1). It had different types of papillae including filiform, fungiform papillae and circumvallate papillae. Basal cell layer was organized with euchromatic nuclei (Fig. 3 A2). However, MTX-treated rats showed

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degenerative changes of the surface epithelium. Filiform papillae were atrophic, their number and height were apparently decreased (Figure 3B1). The prickle cell layer showed swelling

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of their cells (Fig. 3B2). The granular cell layer appeared thickened and showed mild increase of basophilic keratohyaline granules in their cytoplasm and the keratinous layer showed

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hyperkeratosis. Underlying collagen fibers of MTX-treated rats appeared dissociated; associated with high infiltration inflammatory cells in compared to control rats (Fig. 3 B2).

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Interestingly, examination of the epithelium of LA-treated rats displayed obvious improvement in its histological structure and restore its integrity. Apparent increase in the

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number and height of the filiform papillae was observed (Fig. 3 C1). Hyperplasia of the Malpighian layer without disintegration of the basement membrane was recorded (Fig. 3 C2). MTX-treated rats showed significant increase in the lingual epithelial thickness in compared

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3.5. Immunohistochemistry.

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to control and LA-treated rats (Fig. 3 D).

Examination of buccal and lingual mucosae immunostained with Bax monoclonal antibody,

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control rats exhibited weakly positive staining reactivity of cells of the surface epithelium and weak immunostaining of underlying lamina propria (Fig. 4A and 4D). In contrast, strongly positive staining reactivity of the cells of the prickle and granular cell layers of the epithelium

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of MTX-treated rats and strong immunostaining of underlying lamina propria (Fig. 4B and 4E). Epithelium of LA-treated rats displayed weakly positive staining reactivity and weak immunostaining of underlying lamina propria (Fig. 4C and 4F). Mean % area of apoptotic cells immunostained with Bax in both buccal and lingual epithelium was significant; MTXtreated rats showed an increase in the mean % area of apoptotic cells in compared to control and LA-treated rats (Fig. 4 H&I). Examination of buccal mucosae immunostained with PCNA displayed that the basal and parabasal cells of epithelium had strongly positive immunoreactivity (Fig. 5 A) in control rats, weakly positive reaction in MTX rats (Fig. 5 B) and moderately to strongly positive

ACCEPTED MANUSCRIPT reaction in LA group (Fig. 5 C).Immunostaining of dorsal surface of the lingual epithelium with PCNA revealed that; basal cell layers had strongly positive reaction of control group (Figure 5 D), weakly positive in MTX group (Fig. 5 E) and strongly positive PCNA staining reactivity in LA group (Figure 5 F). Mean % area of proliferation cells immunostained with PCNA in both buccal and lingual epithelium was significant; MTX-treated rats showed decrease in the mean % area of proliferative cells in compared to control and LA-treated rats

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(Fig. 5 H&I).

ACCEPTED MANUSCRIPT 4. Discussion Oral mucositis is a common side effect of MTX that may limit its use in the oncology setting. The present work demonstrated that rats that received MTX showed an increase in the thickness of epithelium of buccal mucosa, swelling of the prickle cells, hyperkeratosis in addition to inflammatory cell infiltration in the lamina propria. Collectively these histopathological changes suggest that exposure to MTX can evoke an initial During this phase, both buccal mucosa and

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inflammatory/vascular phase of mucositis.

tongue release free radicals and various inflammatory mediators to induce oxidative stress.

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Moreover, it has been demonstrated that increase in the oxidative stress was implicated in an inflammatory response in rat mucosa [15]. This was suggested to be resulted from the

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oxidative stress-induced activation of proinflammatory cytokine NF- kappaB [15]. The observed increases in MDA levels and decrease of GSH besides the reduction the activities of

MTX in both the buccal and lingual mucosae.

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antioxidant enzymes (SOD and catalase) are proofs of oxidative stress status induced by

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MTX can evoke extensive tissue damage mediated by oxidative stress. Previous studies demonstrated that MTX induces oxidative stress in several tissues evidenced by increasing

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MDA levels and decreasing SOD activities [16-19]. MTX was found to diminish activities of the cytosolic nicotinamide adenosine diphosphate (NADP)–dependent dehydrogenases and

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NADP malic enzymes and that can decrease the cellular content of nicotinamide adenosine diphosphate hydrogen (NADPH) [20]. Normally, NADPH is utilized by GSH reductase to maintain adequate levels of active form of GSH. Therefore, the significant decline in GSH

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content demonstrated in the present study in both buccal and lingual mucosae of rats treated by MTX could result in impairment of endogenous antioxidant defence system, thus exposing

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the cells to free radicals. Generation of free radicals results in the opening of the mitochondrial permeability transition pore with concomitant discharge of cytochrome c from its mitochondrial store to cytoplasm leading to induction of apoptotic pathway. The present findings of DNA laddering assay showed a significant DNA fragmentation in buccal mucosa and tongue tissue after administration of MTX. MTX is a dihydrofolic acid analogue that couples to the dihydrofolic acid reductase enzyme to inhibit the synthesis of tetrahydrofolate, which is essential for DNA replication [21]. In addition MTX antagonizes purine and pyrimidine synthesis resulted in DNA defects, leading to cell cycle arrest and hence apoptosis. Increased free radicals may trigger morphological and structural changes of the

ACCEPTED MANUSCRIPT nucleus causing denaturation and damage of DNA integrity, which participate in the induction of apoptosis. This is confirmed by the immunohistochemical results of the buccal and lingual mucosae of rats which received MTX and incubated with Bax monoclonal antibody showed strongly positive staining reactivity of the cells of the prickle and granular cell layers of the epithelium. This suggests that cell loss in the buccal and lingual mucosae of MTX-induced

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mucositis may be mediated by apoptosis (programmed cell death). The current findings depict that the intrinsic pathway through regulation of the bcl-2 family of proteins, was

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activated by MTX and this is consistent with observed up-regulation of the pro-apoptotic gene bax. This is in agreement with Koppelmann et al (2012) who demonstrated that MTX-

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induced apoptosis in the small intestine of rat was mediated by intrinsic pathway [22]. It has been showed that MTX treatment resulted in increase in p53 expression and in turn leads to

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apoptosis in female rats [23].

MTX can directly inhibit DNA replication and mucosal cell proliferation as evidenced by

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weakly positive PCNA staining reactivity of the nuclei of the basal and parabasal cells of the surface epithelium of the buccal and lingual mucosae of MTX-treated rats. This lead to

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decline in the renewal potential of the basal epithelium, subsequently this result in epithelial atrophy as shown in the filiform papillae of the tongue. Generally, the oral mucosa is

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characterised by high rate of replication which make it highly susceptible to cytotoxicity of MTX.

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Fascinatingly, LA was revealed to be a promising candidate in ameliorating oral mucositis induced by MTX. LA improved the histological structures of buccal and lingual mucosae as

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it reduced the extent of swelling of the epithelial cells and degree of disintegration and dissociation of the collagen fibres of the lamina propria. Increased contents of reduced GSH and the activities of SOD and catalase upon LA administration suggest that it can mediate its protection via modulation of endogenous antioxidant defence system. The antioxidant capacity of LA neutralises the generated free radicals due to MTX evoked oral mucositis. Being water- and fat- soluble, LA is very effective in combating free radicals including MDA at cell membrane and scavenging free radicals formation at their mitochondrial source. LA is reduced intracellularly to dihydrolipoic acid (DHLA), which is even more powerful as an antioxidant [6]. The LA/DHLA redox couple can scavenge a variety of free oxygen radicals including superoxide and peroxyl radicals. In addition, LA and/or DHLA can chelate a

ACCEPTED MANUSCRIPT number of transitional metals like Cu2+, Zn2+and regenerate endogenous antioxidants like Vitamin C and Vitamin E. LA can raise intracellular levels of GSH by being a transcriptional inducer to genes controlling GSH synthesis via Nrf2/ARE signalling pathway [24] besides to its capability to raise cysteine uptake [25]. In the present study, LA treatment prevented the laddering pattern of DNA in both buccal mucosa and tongue in addition to preservation of endogenous antioxidant levels. This is consistent with the immunohistochemical results of the

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buccal and lingual mucosae of rats treated with lipoic acid for 5 weeks and incubated with bax monoclonal antibody showed weakly positive staining reactivity of the cells of the prickle and granular cell layers of the epithelium. The underlying lamina propria revealed

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weakly positive staining reactivity of bax in the fibroblasts and blood vessels. Indeed, it has

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been showed that LA up-regulates the anti-apoptotic protein Bcl-2 in endothelial cells of male rat [26]. Together this postulates that LA increases the survival of the buccal and lingual

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mucosae by regulation of intrinisic pathway via downregulation of pro-apoptotic bax. Moreover, the immunohistochemical results of the buccal and lingual mucosae of rats which

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treated with LA for 5 weeks prior to MTX revealed strongly positive PCNA staining reactivity of the nuclei of the basal and parabasal cells of the surface epithelium indicating increase in the rate of turnover and cell renewal . Previous study by Dadhania et al (2010)

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have depicted the protective effect of LA against MTX-induced intestinal toxicity in rat

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through overcome oxidative stress, enterocyte DNA fragmentation and reducing TUNEL positive cells and p53 expression [27]. Furthermore, LA was found to lessen oxidative stress in several models of neurodegenerative diseases [28], diabetic neuropathy [29] and acute

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renal injury[30]. LA augments the function of endogenous antioxidants such as vitamin E and vitamin C. The synergy of antioxidants reduces the oxidative stress in the body, preventing

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and reversing tissue damage and initiating cell repair to help reduce tissue inflammation and promote healing of damaged tissues. Conclusion

To conclude, LA is a promising agent that can lessen the incidence and severity of oral mucositis following MTX therapy. LA can accelerate mucosal healing and modify the course of the biologic process of oral mucositis. Using LA in oncology settings and in patients with rheumatoid arthritis promises to substantially reduce MTX-related oral mucositis and improve patient quality of life.

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Conflict of interest The authors declared that there are no conflicts of interest. Funding sources

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This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. References

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[29] N. Papanas and Ziegler D, Efficacy of alpha-lipoic acid in diabetic neuropathy, Expert Opin. Pharmacother. 15 (2014) 2721-2731 [30] J .Zhang and P.A.McCullough, Lipoic Acid in the Prevention of Acute Kidney Injury, Nephron. 134 (2016)133-140

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ACCEPTED MANUSCRIPT Table 1 Effects of lipoic acid (10 mg/kg, i.p) on some biochemical indicators of oxidative stress, namely MDA, GSH, catalase and SOD activity, in both buccal mucosa and tongue of methotrexate-treated rats. Tongue MTX

normal control 2.92+0.24

10.36+0.66a

MTX + LA 4.33+0.31b

114.6+9.16

61.17+3.29 a

98.48+5.3 b

104.5+4.67

54.19+9.34

81.46+5.26 b

94.05+6.87

46.17+3.14 a

84.91+4.75 b

91.01+3.73

42.6+6.99

82.13+4.51 b

62.7+3.85

28.7+3.19 a

56.81+3.29 b

26.31+3.09

50.74+3.27 b

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8.82+0.25a

MTX + LA 3.820+0.23b

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MDA (nmol/ mg protein) GSH (μmol/mg protein) catalase (U/mg Protein) SOD (U/mg protein)

normal control 3.04+0.19

buccal mucosa MTX

59.47+3.21

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Values are expressed as means ± S.E.M. n=10 for each experimental group.

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Superscript letters indicate a significant difference at P≤0.05 using one-way ANOVA followed by the Bonferroni's test for multiple comparisons. a, indicates significant differences from control rats. b, indicates significant differences from MTX-treated rats.