|Year : 2019 | Volume
| Issue : 1 | Page : 231-236
Effects of acetylsalicylic acid on rats: An in vivo experimental study in azaserine-rat model
Hasan Yildiz1, Erkan Kalipci2, Haydar Oztas3, Deniz Yildiz1
1 Department of Biology, Faculty of Science, Mustafa Kemal University, Antakya, Turkey
2 Department of Environmental Engineering, Faculty of Engineering and Architecture, Nevsehir Haci Bektas Veli University, Nevsehir, Turkey
3 Department of Biology Education, Ahmet Kelesoglu Faculty of Education, Necmettin Erbakan University, Konya, Turkey
|Date of Web Publication||13-Mar-2019|
Dr. Erkan Kalipci
Department of Environmental Engineering, Faculty of Engineering and Architecture, Nevsehir Haci Bektas Veli University, Nevsehir
Source of Support: None, Conflict of Interest: None
Aim: The effect of acetylsalicylic acid (ASA) on thiol levels was studied in a rat model of azaserine carcinogenesis.
Materials and Methods: ASA and azaserine were applied to the animals to research changes in cellular sulfhydryl (–SH) content and variations in free and protein-bound molecules containing the –SH group. Such effects in rats injected with azaserine were investigated at low (200 ppm) and high (400 ppm) concentrations of ASA over a relatively short (6 months) and a relatively long (12 months) period.
Results: Changes in the hepatic, pancreatic, and renal –SH contents were also determined.
Conclusion: Compared to the other tissues studied, the liver contained the highest levels of both free and protein-bound –SH.
Keywords: Acetylsalicylic acid, azaserine, rats, sulfhydryl content
|How to cite this article:|
Yildiz H, Kalipci E, Oztas H, Yildiz D. Effects of acetylsalicylic acid on rats: An in vivo experimental study in azaserine-rat model. J Can Res Ther 2019;15:231-6
|How to cite this URL:|
Yildiz H, Kalipci E, Oztas H, Yildiz D. Effects of acetylsalicylic acid on rats: An in vivo experimental study in azaserine-rat model. J Can Res Ther [serial online] 2019 [cited 2019 Aug 22];15:231-6. Available from: http://www.cancerjournal.net/text.asp?2019/15/1/231/237382
| > Introduction|| |
Clinically, previous epidemiological and experimental studies have shown that the development of neoplastic tissue alterations may be prevented using nonsteroidal anti-inflammatory drugs (NSAIDs).,,, There are a large number of investigations regarding the possible preventing effect of acetylsalicylic acid (ASA) on thiol levels.
In the present study, to investigate the possible inhibition effects of ASA, we used a nowadays well-known azaserine-treated rat model, first reported by Longnecker and Curphey. Neoplastic change in the pancreas was experimentally induced in this model, and the possible effects of ASA on such change were studied using biochemical techniques. In conditions such as cancer, known for an increase in the oxidative stress, antioxidant enzyme activity has been shown to be also increased due to adaptive mechanisms.
This experiment was a two-by-two comparison of animals treated by two different doses of ASA for two different durations, in animals with or without exposure to azaserine, to their controls. In addition to searching for the atypical acinar cell foci (AACF) which have been described as developing in the exocrine pancreas of rats injected with azaserine, the study determined the levels of free and protein-bound sulfhydryl (–SH) groups in the cells of the exocrine pancreas, the liver and the kidney. Certain authors have reported a low content of –SH groups in various neoplastic tissues.
Determining the free and protein-bound substances with –SH groups and comparing these values to controls may give some clues on how certain substances known as carcinogens, such as azaserine, affect –SH (thiol) levels.
| > Materials and Methods|| |
The aim of study; effect of acetylsalycilic acid (ASA) on thiol levels was studied in a rat model of azaserine cancerogenesis. A total of 144 14-day-old male albino Wistar rats weighing 14–26 g were used for the experimentation. The animals were kept in special cages in numbers not exceeding four per cage. An artificial light and dark cycles were alternated every 12 h to maintain regular biologic rhythms of experimental rats. Prior authorization had been obtained for the experiment from the Mustafa Kemal University Animal Experiments Ethical Committee on December 26, 2006 under No. 23. The experiment was conducted in compliance with the Animal Experiments Ethical Committee guideline.
Dosages and implementation of the azaserine rat model
The azaserine rat model was used, developed by Longnecker and Curphey 1975, to study experimental pancreatic neoplasia and recently used by several authors (Khoo et al., 1991; Yıldız et al., 2008; 2013; Kalıpcı et al., 2012; Yener et al., 2013).,,, The rats received intraperitoneal injections of azaserine, at a dose of 30 mg/kg body weight, weekly for three weeks. ASA was used at two different doses (200 ppm and 400 ppm) over two different treatment durations (6 and 12 months).
In addition to the control groups for 6 months (Con6) and 12 months (Con12), the experimental groups were as follows: azaserine 6 months (Aza6), azaserine 12 months (Aza12), azaserine with ASA 200 ppm over 6 months (AzaAsp206), azaserine with ASA 400 ppm over 6 months (AzaAsp406), azaserine with ASA 200 ppm over 12 months (AzaAsp212), azaserine with ASA 400 ppm over 6 months (AzaAsp412), ASA 200 ppm over 6 months (As206), ASA 400 ppm over 6 months (As406), ASA 200 ppm over 12 months (As212), and ASA 400 ppm over 6 months (As412), making up 12 groups in all. The animals were distributed in different cages by group. The control group and azaserine-only-initiated groups (i.e., Con6, Con12, Aza6, and Aza12) received standard pellet rat feed and water ad libitum. The ASA groups were given standard rat food and water ad libitum, with the addition in their feed of ASA at a concentration of 200 ppm (AzaAsp206, AzaAsp212, Asp206, and ASP 212) or 400 ppm (AzaAsp406, AzaAsp412, Asp406, and Asp412).
Preparation of tissue homogenates
The tissue samples of each individual animal were taken out of the −60°C freezer; portions of about 100 mg were cut out and thawed at room temperature. These portions were placed in glass test tubes, and 2000 μL Tris–HCl at pH 8.2 was added and the samples were homogenized in a homogenizer. To avoid any heat damage to the –SH groups, all these operations were performed on ice.
Measurement of free and protein-bound sulfhydryl groups
The assay procedure described by Sedlak and Lindsay was used to determine the concentration of free and protein-bound –SH groups in separate samples from the liver, pancreas, and kidney tissues. The protein-bound amount was calculated by subtracting the concentration of free –SH from the total –SH value.
The Student–Newman–Keuls multiple comparison analysis of variance method was used as the comparative test, on a ProStat Version 5.04 for Windows statistical package. A P value threshold of 0.05 was accepted as statistically significant.
| > Results|| |
A total of 144 rats were used in this study, distributed in 12 distinct groups according to their receiving 200 or 400 ppm of ASA in their feed, or none, over 6 months or 12 months, and with or without azaserine pretreatment. AACF developed in the exocrine pancreas of rats in all groups pretreated with azaserine (Aza6, AzaAsp206, AzaAsp406, Aza12, AzaAsp212, and AzaAsp412); none were found in the control group or those receiving ASA only (Con6, Asp206, Asp406, Con12, Asp212, and Asp412). Free and protein-bound –SH levels in the liver, kidney, and pancreas tissues were determined.
Hepatic free –SH levels were higher in controls (Con6 and Con12) than the azaserine groups (Aza6 and Aza12) for their respective treatment durations. Among the animals treated or observed for 6 months, the –SH concentrations were significantly lower in the group receiving azaserine pretreatment only, compared to all others, that is controls (Con6), azaserine and ASA (AsaAsp206 and AzaAsp406), and ASA only (Asp206 and Asp406) [Figure 1]. A difference was also observed in the 12-month study duration, with AzaAsp412, Asp212, and Asp412 having significantly higher free –SH concentrations than Az12, while a statistical significance could not be detected for the AzaAsp212). Free –SH levels were higher in the 12-month group (Az12) than in the shorter one (Az6), but no statistical significance could be detected [Figure 1]. Protein-bound hepatic –SH concentrations were higher in the 6-month controls (Con6) than in the corresponding 12-month group (Con12) [Figure 2]. They were significantly higher in ASA-treated groups (AzaAsp206, AzaAsp406, Asp206, and Asp406) than in the azaserine-only group in the 6-month treatment. Similar differences were found comparing all groups within the 12-month treatment, that is, protein-bound hepatic –SH levels were higher in all ASA-treated groups and in controls compared to azaserine only groups (Aza12). Protein-bound –SH concentrations were higher in the animals pretreated with azaserine and observed for 12 months compared to their 6-month counterparts (40.42 ± 2.47; 32.95 ± 7.25). The group with the highest –SH concentration was AzaAsp212 (69.51 ± 2.81) [Figure 2].
|Figure 1: Liver free –sulfhydryl levels following 6 and 12 months period P < 0.05. *Significantly different from the control group. **Significantly different from the azaserine group|
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|Figure 2: Liver protein bound –sulfhydryl levels following 6 and 12 month periods P < 0.05. *Significantly different from the control group. **Significantly different from the azaserine group|
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In the renal tissue, there was a significantly higher level of free –SH levels in controls (Con6) compared to the corresponding azaserine-only animals (0.73 ± 0.03; 0.65 ± 0.02). Free –SH levels were significantly higher at 6 months in the azaserine and ASA groups (AzaAsp206 and AzaAsp406) than azaserine only (0.81 ± 0.03 vs. 0.74 ± 0.04; 0.65 ± 0.02). It is clear that there was no detectable significant difference between the ASA-only groups (Asp206 and Asp406), nonpretreated with azaserine, and the azaserine (Aza6) group at 6 months. The 12-month control (Con12) and azaserine (Aza12) free –SH values were not statistically significantly different. No significant differences in these values could be evidenced at 12 months between the azaserine–ASA groups (AzaAsp212 and AzaAsp412) and the azaserine only groups (Aza12), even though they appeared to be higher for the 12-month treatment groups receiving ASA. Only Asp212 and Asp412 groups –SH values were noticeably lower than all other 6-month or 12-month groups (0.50 ± 0.007; 0.51 ± 0.01) [Figure 3]. (Asp212 and Asp412); their –SH values were noticeably lower than all other 6-month or 12-month groups (0.50 ± 0.007; 0.51 ± 0.01) [Figure 3].
|Figure 3: Kidney free –sulfhydryl levels following 6 and 12 month period P < 0.05. *Significantly different from the control group. **Significantly different from the azaserine group|
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No difference was elicited between the controls and the azaserine-only groups at their respective observation duration (Con6 and Con12 to Aza6 and Aza12). Protein-bound –SH group values were significantly higher in the azaserine and ASA group (AzaAsp412) versus control (Con12) and azaserine only (Aza12) [Figure 4].
|Figure 4: Kidney protein-bound –SH levels levels following 6 and 12 month period P < 0.05. *Significantly different from the control group. **Significantly different from the azaserine group|
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Free –SH content comparisons among groups for the pancreas tissue homogenates failed to show statistically significant differences. The free –SH level was highest in the 6-month and 12-month values after azaserine and 200 ppm ASA (AzaAsp206 = 0.576 and AzaAsp12 = 0.574). Free –SH subjectively appeared to be lower in the 12-month azaserine-only group (Aza12: 0.51) compared to both controls (Con12: 0.55) and azaserine and ASA 200 ppm (AzaAsp212: 0.57), but a statistical significance was not detected [Figure 5].
|Figure 5: Pancreatic free –SH levels levels following 6 and 12 month period P < 0.05. *Significantly different from the control group. **Significantly different from the azaserine group|
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When comparing protein-bound pancreatic –SH levels for the 6-month groups, only AzaAsp406 and Asp406 were different than other with a statistical significance. As for the 12-month groups, AzaAsp212 had levels lower than Aza12 and Con12 [Figure 6]. The findings of all the permutations of the treatment are shown in [Table 1] and [Table 2].
|Figure 6: Pancreatic protein-bound –SH levels following 6 and 12 month period P < 0.05. *Significantly different from the control group. **Significantly different from the azaserine group|
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|Table 1: Liver, kidney, and pancreatic free sulfhydryl levels following 6- and 12-month time period (P <0.05)|
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|Table 2: Liver, kidney, and pancreatic protein-bound -SH following 6- and 12-month time periods (P <0.05)|
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The highest concentrations of free –SH were measured in the liver. A comparison among the experimental groups shows that the free –SH levels are higher in the kidney when compared to the pancreas, except for Asp212. The highest level of protein-bound –SH was in the liver, with the exception of Aza6; the lowest concentration was in the pancreatic tissue.
We found early neoplastic changes in the pancreas and liver. However, AACFs in different numbers were observed in all groups treated with azaserine in the pancreas. AACF in the pancreas shows a different appearance and it can be distinguished easily from the surrounding healthy tissue. According to previous studies performed with azaserine initiation, only a small amount of these acinar focus may lead to pancreatic carcinoma. In this study, AACFs have clear limits and they did not look like invasive structures [Figure 7].
|Figure 7: Atypical acinar cell foci observed microscopically in the pancreas in all of the treatment groups with azaserine. Atypical acinar cell foci were easily distinguished from acinar cells in the normal pancreatic parenchyma (H and E)|
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| > Discussion|| |
Our study has been determined that the rats in the control group had higher levels of –SH, either free or protein bound, than azaserine-treated animals. Rats treated with ASA have been shown a higher –SH levels than azaserine-only groups (Aza6 and Aza12), after 6 and 12 months. In the azaserine-only animals, the concentrations of both free and protein-bound –SH were lower in the hepatic tissue than for groups treated with ASA. A similar finding has been obtained for the 6-month results in the kidney tissue. As for the pancreas, there was an appearance of high levels only in the animals treated with 200 ppm of ASA, not confirmed by statistical significance.
Concentrations of SH were generally different among controls, azaserine-treated, and ASA-treated animals. Hepatic levels were increased in both the 6-month and 12-month ASA treatment groups compared to azaserine. There was an increase in renal tissue levels of free –SH in the 12-month ASA groups, while the protein-bound –SH values were increased in the 6-month ASA animals. A subjective appearance of an elevation in free –SH levels in some of the 6-month and 12-month groups and in the 6-month results of the bound –SH could not be confirmed by statistical analysis.
van Lieshout et al.studied the effect of various NSAIDs on glutathione and glutathione transferase concentrations in different gastrointestinal organs. They reported significant changes in glutathione levels associate with indomethacin in the upper, middle, and lower intestine; piroxicam produced differences in the esophageal concentrations and ASA in the middle gut. In other words, different active ingredients in the NSAID family increase glutathione levels in different sectors of the gastrointestinal tract. Similarly, ASA affected thiol group concentrations in the liver differently than in the pancreas.
Kumar et al. reported an increase in somatotropin (GSH) in the liver following administration of a cancerogen. Increased GSH levels were also found in gastrointestinal adenocarcinomas, whereas other authors found reduced levels of GSH in various neoplastic diseases. Measurements of –SH-containing compound concentrations seem to result in variable values.
Many different factors may cause a fall in the cellular glutathione levels. Vitamin C deficiency may, for example, cause a fall in the glutathione levels, as also do exercise fatigue, diabetes, cystic fibrosis, and HIV infection. Oxidative stress using chemical compounds is known to reduce glutathione levels. We observed that hepatic and renal glutathione levels were reduced, compared to the controls and the ASA-treated animals, following azaserine application, confirming earlier, similar reports.
Sato et al. found, in a study on mouse lymphoma cell cultures, that the glutathione and cysteine levels were markedly reduced in case of an insufficiency of the cysteine transport activity.
Reports from earlier experimental studies indicate that glutathione levels are different in healthy and cancerous tissues. Inci et al. found low levels of antioxidant protective enzymes in hepatic cancer cells, a result that parallels ours. They reported that the GSH level in the cancerous tissue was significantly elevated compared to the adjoining healthy tissues. Daly et al.found the glutathione S-transferase (π klas) enzyme level to be higher in the AACF. el-Sharabasy et al. reported higher glutathione levels and glutathione reductase activity in breast cancer cells compared to healthy tissue.
Differences in the levels of compounds with thiol groups were observed in this study between the experimental group and the controls.
Our study found lower levels of both free and bound –SH in the azaserine-treated rats than in the controls. Rats treated with ASA had higher free and bound –SH levels than all azaserine-treated groups (Aza6 and Aza12). One may say that this fall of free and protein-bound –SH in the rats injected with azaserine is somehow related to the neoplastic changes. Expliciting the interaction between the azaserine and the thiol groups may be useful in understanding neoplastic changes.
New studies to investigate whether such changes may be an early harbinger of neoplasia and other studies similar to ours to discover the detailed interaction of thiol groups and cancer should be warranted.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2]