Protective effects of tumor necrosis factor alpha inhibitors on methotrexate-induced pancreatic toxicity

Background. Methotrexate (MTX), a folate antagonist, is commonly used in the treatment of many different types of cancer and inflammatory diseases, including pancreatic cancer, although its side effects on the pancreas have not yet been researched. The mechanism of MTX-induced toxicity is not well known, and it has been reported in high-dose toxicity studies that the pancreas is sensitive to toxic effects.


Introduction
Methotrexate (MTX) was discovered in 1948 by Farber as an effective treatment for acute leukemia and is an antimetabolite, antifolate, and chemotherapeutic drug. 1,2he effect of DNA synthesis in the "S" phase by inhibition and the affinity of dihydrofolic acid to inhibit the dihydrofolate reductase enzyme appear with the use of MTX.Thus, there is a drop in tetrahydrofolic acid levels and purine synthesis is inhibited. 3,42][3][4][5] Nevertheless, its use is associated with side effects such as fibrosis affecting the liver, lung, and other organs. 1,2,5Acute intense MTX side effects are dose-dependent.Bone marrow suppression, gastrointestinal upsets or ulcers, and liver fibrosis are well-known acute or common side effects of MTX therapy.[6][7][8][9][10][11] Methotrexate has been used in the treatment of pancreatic cancer, but there has been no research into its side effects on the pancreas, an organ which is relatively sensitive to various toxic substances and drugs.][7][8][9][10][11] However, the development of tolerance to MTX has been reported in patients over time.The tolerance mechanism of the metabolites in MTX is not known. 1,2,4,5,11Physicians increase the dosage of MTX to counter the development of tolerance.Accordingly, the risk of MTX toxicity increases.It was found in recent studies that an MTX overdose caused oxidative stress, leading to the release of proinflammatory cytokines and further tissue damage. 5,6,11umor necrosis factor alpha (TNF-α) plays a key role in the mechanism of immune-mediated inflammatory diseases, such as RA, ankylosing spondylitis, Crohn's disease, ulcerative colitis, psoriasis, and psoriatic arthritis.Inflammation induces the overproduction of inflammatory cytokines, such as TNF-α, interleukin: IL-1β, IL-6, IL-15, and IL-18, while TNF-α has been shown to increase the number of pancreatic cancer cells. 12It has been reported in the literature that TNF-α cytokines modulate the autocrine growth regulatory pathways in pancreatic cancer cells. 13Antagonists of TNF-α, such as adalimumab (ADA), infliximab, etanercept (ETN), golimumab, and certolizumab pegol, have been widely used in the treatment of RA.Inhibition of TNF results in the down-regulation of abnormal and progressive inflammatory processes, resulting in the prevention of target organ damage. 12,131][12][13] The combination of MTX and ADA has not been studied for its ability to counter toxicity in the pancreas.][16] The aim of this study was to determine whether TNF-α inhibitors have a preventive or toxicity-enhancing effect on MTX-induced pancreatic toxicity.

Animal testing
Thirty male albino Sprague Dawley ® rats, aged 3 months and weighing 250-300 g, were procured from the Animal Care and Research Unit for this study (Recep Tayyip Erdogan University, Rize, Turkey).The animals were kept at a constant temperature (21 ±3°C) and according to a constant photoperiod and temperature regime (12-h light/dark cycle).
During the experimental period, all of the subjects were given fed ad libitum pellets containing 21% crude protein (purina) and given drinking water daily.The animals were selected according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals, prepared by the National Academy of Sciences and published by the National Institutes of Health.Recep Tayyip Erdogan University Animal Care and Research Unit (Rize, Turkey).
This study was approved by the Institutional Animal Ethical Committee Recep Tayyip Erdogan University Animal Care and Research Unit (Rize, Turkey).
The subjects were randomly allocated to 3 groups of 8 rats each (n = 8), with similar biological and physiological characteristics: Group 1: control group (healthy), Group 2: MTX group and Group 3: MTX + ADA group.
Isotonic saline solution, mixed with an equal volume of MTX, was administered by ip.injection to Group 1.
A single-dose injection of MTX (Emthexate-s ® , 50-mg ampule), at a dose of 20 mg/kg, was given by ip.injection to Groups 2 and 3, and a single dose of 5 mg/kg ADA (REMICADE ® ) was administered ip. to Group 3.
All the rats were sacrificed under anesthesia with ketamine hydrochloride 50 mg/kg, intramuscularly (Ketalar, Parke-Davis, Istanbul, Turkey) 5 days after receiving the MTX injection.

Histological preparation
Pancreatic tissue samples were fixed in 10% formalin.After fixation, the specimens were dehydrated in an ascending series of alcohol, cleared in xylene, and embedded in paraffin.The paraffin blocks were cut 4-5 µm thick, using a microtome (Leica ® RM2125 RTS), and the sections were stained with hematoxylin and eosin (Applichem GmbH, Darmstadt, Germany), according to the guidelines that govern conventional light microscopes.

Immunohistochemical preparation
Paraffin blocks of the pancreatic samples were cut into 1-2 µm semi-thin sections.The sections obtained for immunohistochemical staining were left in xylene for 10 min, twice, and after being passed through an alcohol series (70-99%) for 5 min, were kept in a 3% H 2 O 2 solution for 10 min.After washing with phosphate buffered saline solution (PBS), they were heated 4 times in a citrate buffer for 5-10 min at 800 W of power, and allowed to stand for 20 min in a secondary blocking agent.Each preparation was allowed to stand for 60-75 min in various dilutions (1/50-1/200) of anti-laminin antibody staining (Polyclonal Guinea Pig Anti-Insulin ® [clinical application for IS002], and Polyclonal Rabbit Anti-Human Somtastatin ® [clinical application for A0566], both Dako Denmark A/S, Denmark).The 3,3'-diaminobenzidine (DAB) solution was used as a chromogenic dye, and stained with Mayer's hematoxylin for contrast dye.Phosphate buffered saline solution was used for the negative controls instead of primary antibodies.The preparation was closed off with a suitable agent and photographed.According to the blind scoring evaluation performed by 2 histologists, the positivity was divided into the following 4 categories according to the percentage value: mild (+), moderate (++), severe (+++), and very severe (++++).

Pathological assessment of tissue
Parts of the tissue were fixed and embedded in paraffin for histological analysis.Blinded pathological scoring of the pancreas, by an experienced pathologist from Recep Tayyip Erdoğan University, Rize, Turkey, was performed with respect to edema, bleeding, inflammatory cell infiltration, and necrosis, according to Schmidt's report (Table 1). 14

Statistical analysis
Statistical analysis was performed using SPSS ® v. 18 (IBM; Chicago, USA).Schmidt's score was calculated. 14The data was expressed as mean ±standard error of the mean (SEM).Statistical analysis was performed using one-way analysis of variance, followed by Tukey's test for each paired experiment value.A p-value of p < 0.05 was considered to be significant.The sections obtained by immunohistochemical staining were evaluated and graded by 2 blinded histologists and the positivity was expressed in terms of percentage and divided into 4 grade categories.Data was expressed as median ±standard deviation (SD).The obtained values were statistically compared with the Kruskal-Wallis test (p < 0.05) to each other.

Histological results
The morphologic structure of the tissues in the control group was observed to have a normal histology (Fig. 1A, 1B).
The histopathologic features of the Group 2 samples were atypical islets of Langerhans, mild edema, necrosis, and inflammatory infiltration.Necrotic cells were observed in the islets of Langerhans and acinar cells.Necrotic acinar cells, with a pyknotic nucleus in the center, partial karyorrhexis, and a bright border due to cytoplasmatic shrinkage and cell degeneration, were observed.Connective tissue trabeculae were noted to increase considerably in volume with leukocytic infiltration (Fig. 1C-1E).
Complete destruction of islet of Langerhans cells, due to the toxicity of MTX, appeared in the Group II rats compared to Group 1 (Fig. 1A-1D).A decrease in necrotic cells was also observed in islets of Langerhans and acinar cells (Fig. 1F) after the administration of the ADA/MTX combination therapy.

Stereological results
All the study rats were scored by both investigators using Schmidt's histopathological scoring method.There was no more than a 0.5-point deviation in their discernment.Schmidt's histopathological scoring steps, shown in Table 2, were applied.

Statistical results
Significantly higher mean edema, necrotic cell, and inflammatory scores, compared to those obtained for Group 1, were recorded for Group 2 (p < 0.05).Significantly decreased edema, necrotic cell, and inflammatory scores were recorded for Group 3, when compared to Group 2 (p < 0.05) (Table 2).

A B C
Increased edema, acinar edema, and necrotic cells were observed in islets of Langerhans and acinar cells in Group 2, compared to those found in Group 1 (p < 0.05) (Table 2) in our study on MTX-induced pancreatic toxicity.It was also found that atypical islets of Langerhans and connective tissue trabeculae were observed to increase considerably in volume.A reduction in the amount of insulin and somatostatin was also found.An accumulation of mast cells in the connective tissue was observed.3][24] Methotrexate is used for the treatment of pancreatic cancer, but has been shown to destroy the beta cells of the pancreas, primarily through oxidative stress. 1,2,5,24In support of the data in the literature, we determined that in our study the number of insulin and somatostatin-positive cells significantly decreased in Group 2, when compared to that in Group 1 (Fig. 2B, 3B).
It has been proposed in previous studies that MTX increases lipid peroxidation and oxidative stress, leading to an increase in reactive oxygen species (ROS). 5,24It has also been reported that ROS play a key role in decreasing immune system defense. 5,24,25In addition, intense proinflammatory cytokine release, due to excessive ROS formation, has been reported during MTX treatment for cancer and autoimmune diseases. 5,6,24,25Accumulation of ROS leads to neutrophil infiltration and proinflammatory cytokine release, which triggers apoptosis. 5,6,24,25][26] A decrease in islets of Langerhans insulin and somatostatin-immunopositive cells was shown in this study following the administration of MTX (Fig. 2B, 3B).In the statistical evaluations performed, significant differences were observed between the control and MTX groups in terms of anti-insulin and anti-somatostatin immunopositivity according to the Kruskal-Wallis test (p < 0.05).b In the statistical evaluations performed, significant differences were observed between the control and MTX + ADA groups in terms of anti-insulin and anti-somatostatin immunopositivity according to the Kruskal-Wallis test (p < 0.05).c In the statistical evaluations performed, significant differences were observed between the MTX and MTX + ADA groups in terms of anti-insulin and anti-somatostatin immunopositivity according to the Kruskal-Wallis test (p < 0.05).MTX -methotrexate; ADA − adalimumab.

Discussion
The side effects of MTX on the pancreas have not been researched.However, high-dosage substances were reported to have a toxic effect on the pancreas.[18][19][20][21][22][23]

A B C
It has been suggested that TNF-α plays a key role in the mechanism of immune-mediated inflammation in recent studies.Inhibition of TNF results in the downregulation of abnormal and progressive inflammatory processes, resulting in target organ damage. 4,5,10,11,19,27t has been experimentally proven that TNF inhibitors prohibit organ tissue damage by suppressing the formation of TNF-α, proinflammatory cytokines, and nitric oxide. 5,26,27 decrease in edema, acinar cell edema, and necrotic cells was observed in Group 3 (p < 0.05) (Table 2) after the administration of ADA.An increase in islets of Langerhans insulin and somatostatin-positive cells was also demonstrated following the application of ADA treatment to Group 3 (Fig. 2C, 3C).
As a result, ADA was shown to have a protective effect against MTX-induced pancreatic injury.It was shown in this study that ADA reduced inflammatory levels and provided better protection to the insulin and somatostatin cells than that provided by MTX.This was a pilot study.Thus, further studies are needed in this regard to confirm our findings.

5 5 Fig. 2 .
Fig. 2. Histopathologic examination of liver tissue stained by anti-insulin IHC staining method by light microscopy A − control group, insulin positive area; B − MTX group, insulin negative area; C − MTX + ADA group, insulin positive area.

Fig. 3 .
Fig. 3. Histopathologic examination of liver tissue stained by anti-somatostatin IHC staining method by light microscopy: A − control group, somatostatin positive area; B − MTX group, somatostatin negative area; C − MTX + ADA group, somatostatin positive area.

Table 2 .
Statistical results bMeans in the same column by the same superscript letter are not statistically significantly different under the Tukey test (α = 0.05).Results are mean ±standard error of the mean.a vs control Group p < 0.05; b vs MTX Group p < 0.05.MTX -methotrexate; ADA − adalimumab.

Table 3 .
Anti-insulin and anti-somatostatin shown in the pancreas tissues by the method of immunoperoxidase in immunohistochemical staining