Effect of Methotrexate and Vitamin A on NOR Expression in the Lining Epithelium of Gastric Mucosa in Male Wistar Rats Reghunathan Deepthinath1, Bhat K Ramachandra2, Bairy K Lakshminarayan3, Murlimanju BV4,,*, Prasad AM1 1Department of Anatomy, Melaka Manipal Medical College, Manipal Campus, Manipal University, Manipal, Karnataka, India 2Department of Anatomy, Kasturba Medical College, Manipal University, Manipal, Karnataka, India 3Department of Pharmacology, Kasturba Medical College, Manipal University, Manipal, Karnataka, India 4Department of Anatomy, Kasturba Medical College, Manipal University, Bejai, Mangalore, Karnataka, India *Corresponding author email id: flutemist@gmail.com
Abstract Background and objective: The histopathology and expression of nucleolar organising regions (NORs) of gastric mucosa were studied after the administration of methotrexate (MTX) and vitamin A (VA). The objective was to test whether the argyrophylic nucleolar organiser region (AgNOR) count increases or decreases with different doses of MTX. The literatures on AgNORs suggest that there may be a possible association between AgNOR counts and disease transformation. MTX is one of the commonest drugs used in several diseases. It is interesting to hypothesise the numbers, shape and the distribution of AgNORs after the administration of MTX. Methods: Male Wistar rats aged 4 months, maintained in our institution, were used in the present study. The rats were divided into following groups, group I - control (saline treated), group II - animals treated with 8 mg of MTX, group III - animals treated with 10 mg of MTX, group IV - animals treated with 12 mg of MTX, group V - animals treated with VA (5,000 IU), group VI - animals treated with VA (5,000 IU) and MTX (12 mg). Results: It was observed that MTX treatment showed abrupted or incomplete surface epithelium in the gastric mucosa. The gastric pits were short and epithelium showed lightly stained nuclei and cytoplasm. When MTX was given along with VA, the VA minimised the severity of damage caused by MTX. The frequency of large-size NOR was reduced after treatment with MTX. However, the medium and small-size NOR count were increased. When treated with MTX and VA, NOR frequency increased and reached almost the level of the control group. Conclusion: The present study observed that large-sized NOR decreases numerically while medium and small sized NORs increases with the administration of MTX. We believe that the NOR fusion was affected by the MTX drug. Higher the dose of MTX, lesser the NOR count and lesser the irregularity of NORs. However, VA co-administration reduces the damage produced by MTX. We believe that findings of the present study might be useful in understanding the long-term effect of MTX and its adverse effects. Top Keywords Methotrexate, NOR, AgNOR, Histopathology, Vitamin A. Top |
Introduction The nucleolar organising regions (NORs) are loops of ribosomal DNA present in the nuclei of cells, which are involved in the synthesis of rRNA.1 They are known as argyrophylic nucleolar organiser regions (AgNORs) because of their strong affinity to silver.2,3 NORs are concerned with proliferative activity of cells and are useful to study the nuclear variations in normal and abnormal conditions. [4] Methotrexate (MTX) is an analogue of folic acid and acts as a folic acid antagonist. It inhibits the enzyme dihydrofolate reductase, which is necessary for the cell division. It is an antineoplastic, antiproliferative and antimetabolite used in the treatment of certain neoplastic diseases, severe psoriasis and adult rheumatoid arthritis.5,6 It has been reported that vitamin A (VA) protects the small intestine from MTX-induced damage. |
AgNOR staining is a simple and economical technique for investigating the proliferative activity. Estimating the AgNOR count in endoscopically biopsied specimens at the margin of invasive gastric carcinoma is useful for assessing the nodal metastasis and clinical prognosis. These preoperative estimates might aid in tailoring the operative procedure and administering the adjuvant therapy.7 The quantification of AgNORs can be used as marker for proliferative activity, which might help in the diagnosis of cancer.8 |
In the present study, the histopathology and NOR expression of gastric mucosa were studied after administering the MTX, VA and combination of both. Objective of the present study was to test whether the AgNOR count increases or decreases with different doses of MTX. The literature on AgNORs suggests that there may be a possible association between AgNOR count and disease transformation. It is well know that MTX is one of the commonest drugs used in several diseases. Therefore, it is interesting to hypothesise the number, shape and the distribution of AgNORs after the administration of MTX. VA was used to check if any improvement in the MTX-induced damage to the gastric mucosa and the NOR quantity. |
Top Methods Animals Male Wistar rats aged 4 months, maintained in our institution, were used in the present study. Animals were maintained under controlled conditions of light, temperature and humidity at an air conditioned animal house. The average weights of animal were 200 g. The study protocol has been approved by Animal Ethical Committee of our institution. The rats were divided into following groups, group I - control (saline treated), group II - animals treated with 8 mg of MTX, group III - animals treated with 10 mg of MTX, group IV - animals treated with 12 mg of MTX, group V - animals treated with VA (5,000 IU), group VI - animals treated with VA (5,000 IU) and MTX (12 mg). Animals were injected intraperitoneally with different doses of MTX on alternative days for 1 week and sacrificed on the seventh day by using ether. The animal groups which were injected with VA and MTX+VA were also sacrificed by using ether. Tissue Processing, Perfusion and Staining The animals were deeply anaesthetised with ether and fixed on a dissection board. The chest cavities were opened to expose the heart. About 15 ml of 0.9% saline was perfused through the left ventricle at the rate of 1 ml/min. It was followed by perfusion of 10% formalin (about 250 ml) at the same flow rate. The stomach was removed in all the rats and kept in 10% formalin for 48 h (post fixation). Paraffin blocks were made in an embedding bath; sections of 3–5 μm thickness were cut from the blocks using the rotary microtome. Sections were mounted on the air-dried gelatinised slides. The tissues were de-paraffinised in xylene, then transferred to water through descending grades of alcohol (absolute, 90, 70 and 50% alcohol). The sections were washed in tap water and stained with Erlich's haemotoxylin for 5 min. After this, the sections were again washed in tap water. Then stained with 1% of eosin for 30 s and dehydrated with ascending grades of alcohol, cleared in xylene and mounted in Canada balsam. Finally, the tissues were observed under microscope for assessing the histological state of the tissue. Studying the NOR Preparation of AgNO3 solution To prepare 50% of silver nitrate solution, 5 g of silver nitrate powder was dissolved in 10 ml of distilled water and filtered through Whatman filter paper in a dark room. The solution was stored in a dark bottle at 4°C and used within 1 week. Preparation of gelatin One gram of powdered gelatin was dissolved in 49 ml of distilled water and 1 ml of formic acid. To dissolve faster, the mixture was kept in hot water bath at 60°C for 10 min. The solution was stored in a dark bottle at 4°C and used within 1 week. Silver nitrate staining The rapid staining and de-staining method developed in our lab9 were performed throughout the present study. Four drops of silver nitrate solution and two drops of gelatin solution were added on to the slide. The solutions were gently mixed and spread over the slide. The slides were gently warmed and passed to and fro over the Bunsen's burner for 15–20 s (till the slides turn into golden yellow). During the staining process, precautions were taken to avoid over heating of the slides. The slides were rinsed in distilled water and air dried. Over stained slides were flooded with either 50% hydrogen peroxide for 1 s or 25% hydrogen peroxide for 15 s. The process was monitored under the microscope and precautions were taken that the cellular architecture, composition and staining ability were not altered. The dissolved silver was washed from the sections under running water. Microscopic Observation and NOR Count Standard protocols were followed for recording the number and quantification of the size and shape of AgNOR dots.10 Areas with minimal cell overlap and no artefact were demarcated for counting. The weak and dark stained slides were not evaluated. The NORs appeared as black dots within the orange coloured nuclear background. Dots were defined as discrete homogenous silver precipitates with well-defined edges. Overlapped dots with well-defined edges were counted as greater than one when these appeared while viewing through different focal planes. Dots lying in a group with indistinguishable boundaries were treated as one. Dots outside the nucleolus were not considered. Observations were completed within a week of staining and the counting was performed, using oil immersion at x400. NOR Size and Shape Size of the NOR was measured using ocular micrometre (calibrated with stage micrometre). These groups were classified into three groups (Figure 1) on the basis of their diameter (small ≤1 μm, medium 1–3 μm and large >3 μm). On the basis of shape, NORs were classified into regular (with round or oval, well-defined margin) and irregular (with irregular serrated margin). In each category, the size of dots was documented. Statistical Analysis Data on the mean NOR count; size-wise distribution of dots was tested using one-way analysis of variance. Since the test of homogeneity of variance showed high significance between different groups, data were reanalysed using square root transformation. For each dose and distribution, mean NOR count, standard deviation, F-ratio and P-value were computed. Parameters Studied The histopathological study of MTX and dose-wise expression of NORs in the stomach was considered in the present study. The data were collected and analysed on the basis of following parameters in the gastric mucosa: histopathological study of MTX-treated rats, histopathological study of VA-treated rats, histopathological study of MTX- and VA-treated rats, NOR study of MTXtreated rats, NOR study of VA-treated rats and NOR study of MTX and VA-treated rats. Top Results Histopathology of Gastric Mucosa In the present study, it was observed that MTX treatment showed abrupted or incomplete surface epithelium in the gastric mucosa when compared to the control sample (Figure 2a). The gastric pits were short and the epithelium showed light stained nuclei and cytoplasm (Figure 2b). When MTX was given along with VA, the VA minimised the severity of damage caused by MTX (Figure 2c). Expression of NORs The data showed overall decrease in the total NOR count (numerical) from the control to 12 mg dose MTX (Table 1). Large-sized NORs showed a statistically significant (P<0.05) numerical decrease from control to 12 mg dose MTX. A statistically significant difference was found between the control and 10 mg dose of MTX (P<0.05). However, no significant difference (P>0.05) was found between 10 and 12 mg doses of MTX. There was no significant difference (P>0.05) found between the control and 8 mg doses of MTX. In medium-sized NORs, the value indicated that there was a numerically high count in 12 mg dose MTX. But it was not significant statistically (P<0.05). It showed a statistically high significance between 10 and 12 mg doses of MTX (P<0.05). However, no statistical difference (P>0.05) found between control vs 8 and 10 mg doses of MTX. Whereas in the small-sized NORs, the values indicated that there was a numerically high count in 10 mg dose MTX (P<0.05). A statistically high significant difference (P<0.05) was found between control vs 8 mg dose and 12 mg dose MTX as well as between 10 and 12 mg doses of MTX. The regular shaped NORs showed a numerical increase (P<0.05) from control to 12 mg dose MTX. Data showed a statistically high difference (P<0.05) between control and drug-treated groups as well as between 8 and 12 mg doses of MTX. However, no statistical significance (P>0.05) observed between 10 and 12 mg doses of MTX. Irregular shaped NORs showed a numerical decrease (P<0.05) from control to 12 mg dose MTX. Data showed a statistically high difference (P<0.05) between control and drug-treated groups as well as between 8 and 12 mg doses of MTX. However, no statistical significance (P>0.05) observed between 10 and 12 mg doses of MTX. When VA was given along with MTX the NOR expression was reversed. The count was significantly increased (P<0.05) in the VA+MTX treated group, when compared to 12 mg dose MTX-treated group. The count was almost close to that of the control group (P<0.05). Top Discussion MTX is an antimetabolite drug widely used in the cancer chemotherapy; however, gastrointestinal toxicity is one of its serious side effects. The antimitotic effect of MTX is known to cause malabsorption syndrome. However, the mechanism of toxicity has not been completely clarified. Alterations to mucosal surfaces of the gastrointestinal tract and diarrhoea are well-documented side effects of MTX in humans, attributable to MTX acting on the rapidly dividing mucosal cells.11,12 MTX injury is exacerbated by malnutrition and consumption of an elemental diet,13−17 but alleviated by co-administration of VA,18,19 suggesting that diet plays an important role in this mode of injury. Moreover, being a high affinity inhibitor of dihydrofolate reductase, MTX indirectly affects the synthesis of thymidylate, suppressing the DNA synthesis.12 Additionally, it was demonstrated that the cytosolic Nicotinamide Adenine DinucleotidePhosphate (NADP)-dependent dehydrogenases and NADP malic enzyme were inhibited by MTX, suggesting that the drug could decrease the availability of NADPH in the cells.20 Under normal conditions, NADPH is used by glutathione (GSH) reductase to maintain the reduced state of cell GSH, which is known as an important cytosolic antioxidant, protective against reactive oxygen species (ROS). Thus, the significant reduction in GSH levels promoted by MTX leads to a reduction of effectiveness of the antioxidant enzyme defence system, sensitising the cells to ROS.21 MTX binds tightly to the dihydrofolate reductase, the enzyme which catalyses the NADPHdependent reduction of dihydrofolate to tetrahydrofolate, which is utilised as a one carbon carrier. This is required for the synthesis of purine and pyramidine, including thymidilate synthesis. This is essential for DNA synthesis and cell division.12 On the other hand, it has been suggested that VA may be involved in the regulation of DNA and RNA synthesis.22,23 |
Basically, cell cycle is the ‘programme’ for cell growth and division. There are four broad phases of the cell cycle: G1 (and G0), S, G2 and M. The G1 (gap 1) phase is characterised by gene expression and protein synthesis. This is the only part of cell cycle regulated primarily by extra-cellular stimuli (like mitogens and adhesion). When MTX is given, a small proportion of the cells are observed to be passed into the G2 phase, where synthesis of cellular components necessary for mitotic cell division takes place. The majority of them are blocked at the S phase where replication of the DNA genome is observed to take place.24 This phase enables the cells to grow and produce all the necessary proteins for the DNA synthesis. Cancer cells escape from the controls on cell division and the action mechanism of anti-neoplastic drug is based on the stopping of uncontrolled cells at any one stage of the cell cycle.25 To further understand the relationship between the cell cycle and quantity of AgNOR proteins, it is necessary to determine the phases of cell cycle during which expression of AgNOR varies and to know the most variable proteins in each phase.26 The higher value indicates that the major parts of the cells are in the S-G2 phase and correlatively few are in the G1 phase and this characterises a rapid cell cycle.26 However, in the present study, the count was decreased from the control to higher dose of MTX. This indicates that major part of the cells is in the G1 phase and correlatively few cells are in the S-G2 phase. |
NORs stained with colloidal silver techniques, evidence sites of active rRNA transcription. It has been proven that AgNOR undergo a rise in number and variations in size and shape in conditions, which traditionally involve enhanced cell proliferation and rRNA transcription. AgNOR have been described as a marker of malignant transformation in multiple entities.27 AgNOR proteins are a set of argyrophilic nucleolar proteins that accumulate in highly proliferating cells, whereas their expression is very low in non-proliferating cells. Some of these proteins remain associated with the NORs during mitosis. In situ, the expression of AgNOR proteins is measured globally by quantification of the level of silver staining using the morphometry and image analysis. |
In the present study, the total NORs irrespective of their shape and size followed a progressively decreasing pattern, which correlated with the dose of MTX. An apparent decrease in the total NOR count in the present study could be due to inactive cell proliferation because of the blockage in the S phase of the cell cycle, decrease in cell ploidy, resulting in a real decrease in AgNORbearing chromosomes or decrease in the transcriptional activity. It has been generally found that quantity of silver-stained NOR proteins is strictly related to the cell proliferation.28 VA has reversed the activity of MTX. The irregularities of NORs refer to aggregation of more than one NOR in a site, thus reflecting the greater transcriptional rate. In the present study, the total numbers of irregular dots were less in high dose of MTX than the control group and the total number of regular NORs were less in control than in high-dose MTX-treated group. This indicates that the transcriptional rate was less in high doses of MTX than in the control group. Less transcriptional rate reduced the total NOR count, which in turn reduced the overlapping or aggregation of NORs in a single cell. When VA was given after the high dose of MTX, it reversed the activity of MTX. |
MTX is known to cause a number of adverse effects. The most reported adverse effects include ulcerative stomatitis, leucopenia, nausea and abdominal distress. Severe haematological gastrointestinal toxicity, intestinal pneumonitis, liver damage, renal failure, intellectual deficit and wide spectrum of neurotoxic reactions were also reported.29 Other adverse effects are malaise, undue fatigue, chills and fever, dizziness and decreased resistance to infections. In the alimentary system, it shows gingivitis, pharyngitis, stomatitis, anorexia, nausea, vomiting, diarrhoea, gastrointestinal tract ulceration and bleeding. Administration of VA decreases the MTXinduced damage to the small intestine. This protective effect of VA may have clinical applications in cancer chemotherapy.30 The administration of VA before high-dose MTX may protect against drug-induced D-xylose malabsorption in children with cancer.31 Some studies have shown that biochemical modulation used to prevent MTXinduced malabsorption by VA co-administration will be of great use in MTX cancer chemotherapy.32 The oral administration of MTX to rats, inhibits DNA synthesis in crypt cells. VA co-administration protects the salvage pathway of pyrimidine synthesis and de novo purine synthesis in crypt cells.19 It has been shown histologically, biochemically and physiologically that VA protects the small intestine from the damage, which is induced by MTX.18 |
VA protects the gastric mucosal damage produced by MTX administration. In the present study, NOR frequency was reduced when treated with MTX. In contrast, when treated with MTX and VA, NOR frequency increased and reached almost the level of the control group. This is in accordance to the clinical reference of VA for the prevention and minimisation of diarrhoea in MTX treatment.33 Madhyasta et al.34 reported that VA protects against the MTX-induced genetic damage. It was also reported that both VA and leucovorin can protect against the genetic damage, which is induced by MTX.35 The present study confirms that VA minimises the genotoxic and cytotoxic effect of MTX. In the other study,36 the authors found that MTX treatment showed disruption of normal hepatic plates, vacuolation of the hepatocytes, nuclear variability and enlarged blood vessels. In that study36 also VA protected the damages produced by the MTX administration. |
We believe that NOR quantification is a handy and potentially useful biomarker, in the cancer management. However, the limitation is its role in assessing the efficacy of antitumor therapy, as compared to that of histological examination, serological markers or imaging modalities, is less clear. |
Top Conclusion It is evident from the present study that MTX decreased the total count of AgNORs. A decrease in the AgNOR count was due to the inactive proliferation of cells (anti-proliferative activity of MTX). The inactive cell proliferation was because of the blockage in the ‘S’ phase of the cell cycle by MTX. MTX decreased the cell ploidy, resulting in a real decrease in AgNOR-bearing chromosome. Here, MTX reduced the transcriptional activity of the cells. This was another reason for the reduction of AgNOR count. The irregularity of NORs refers to the aggregation of more than one NOR in a site, thus reflecting greater transcriptional rate. However, MTX reduced the transcriptional activity and decreased AgNOR count in high-dose group compared to the control group. Higher the dose of MTX, lesser the NOR count and lesser the irregularity of NORs. However, VA co-administration reduced the damage produced by MTX. We believe that the findings of the present study may be useful in understanding the long-term effect of MTX and to overcome or minimise its adverse effects. The present study observed that large-sized NOR decrease numerically, while medium- and small-sized NORs increase with the administration of MTX. We believe that the NOR fusion was affected by the MTX drug. |
Top Figures | Figure 1:: Showing the NORs in stomach (A: large sized; B: medium sized; C: small sized)
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| | Figure 2:: Showing the histopathology (eosin and haemotoxylin stain) of the gastric mucosa (a) Control rat; (b) 12 mg methotrexatetreated rat; (c) vitamin A- and 12 mg methotrexate-treated rat.
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Table | Table 1:: Expression of NORs in VA- and MTX-treated rat's stomach (mean±SD from 6 animals/group)
| | Experimental group | Total | Large | Medium | Small | Regular | Irregular | | Control | 101.5±2.62 | 28.33±1.69 | 40.16±3.38 | 31.5±4.46 | 52.5±2.43 | 47.5±2.43 | | 8 mg MTX | 89.16±2.54 | 26.83±1.57 | 36.66±2.21 | 39.83±3.02 | 56.5±2.06 | 43.5±2.06 | | 10 mg MTX | 75±2.23 | 25.33±1.24 | 32.5±2.14 | 42.16±2.79 | 58.66±1.97 | 41.33±1.97 | | 12 mg MTX | 51±2.16 | 23.66±1.24 | 41.5±1.97 | 34.83±2.19 | 62.5±1.7 | 37.5±1.7 | | VA | 101.83±2.67 | 29.33±1.1 | 41.16±3.38 | 29.5±3.45 | 53.66±1.69 | 46.33±1.69 | | VA+MTX | 99.66±1.49 | 28.66±1.24 | 40±2.76 | 31.33±2.49 | 53.16±2.73 | 46.83±2.73 |
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| Abbreviations: MTX, methotrexate; NOR, nucleolar organising region; VA, vitamin A. | |
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