|Year : 2010 | Volume
| Issue : 1 | Page : 17-21
Failure to detect the anti-mutagenic effect of insulin in experimental type-2 diabetic rats
Syed Imam Rabbani1, Kshama Devi1, Salma Khanam2
1 Department of Pharmacology, Al-Ameen College of Pharmacy, Opp. Lalbagh Main Gate, Hosur Road, Bangalore, India
2 Department of Pharmacognosy, Al-Ameen College of Pharmacy, Opp. Lalbagh Main Gate, Hosur Road, Bangalore, India
|Date of Web Publication||20-Sep-2010|
Department of Pharmacology, Al-Ameen College of Pharmacy, Hosur Road, Near Lalbagh Main Gate, Bangalore - 560 027
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: The oxidative stress is known to cause mutation-related disorders in diabetic patients. Materials and Methods: The present study was designed to investigate the anti-mutagenic effect of insulin in nicotinamide (NA: 230 mg/kg, i.p.) and streptozotocin (STZ: 65 mg/kg, i.p.) induced nuclear defects. Bone marrow micronucleus (MN) test and caudal epididymal sperm abnormalities were detected to find the somatic and germinal cell mutations, respectively. The antioxidant status was determined by estimating serum lipid peroxidation, catalase, superoxide dismutase and glutathione peroxidase levels. Results: The experimental type diabetes significantly (P < 0.001) reduced the antioxidant status and enhanced MN frequency and sperm defects compared to control animals. Although administration of insulin (1, 3, 5 and 7 IU/kg, s.c. for 4 weeks) significantly (P < 0.001) reduced hyperglycemia, it did not alter the antioxidant status, and somatic and germinal cell defects in diabetic rats. Conclusion: The results suggest that insulin did not have protective effect against the genotoxicity induced by NA-STZ diabetes.
Keywords: Insulin, micronucleus, oxidative stress, sperm defects
|How to cite this article:|
Rabbani SI, Devi K, Khanam S. Failure to detect the anti-mutagenic effect of insulin in experimental type-2 diabetic rats. J Pharm Negative Results 2010;1:17-21
|How to cite this URL:|
Rabbani SI, Devi K, Khanam S. Failure to detect the anti-mutagenic effect of insulin in experimental type-2 diabetic rats. J Pharm Negative Results [serial online] 2010 [cited 2020 Feb 28];1:17-21. Available from: http://www.pnrjournal.com/text.asp?2010/1/1/17/68870
| Introduction|| |
The occurrence of type-2 diabetes mellitus (T2DM) is associated with elevated level of oxidative stress, which results from hyperglycemia through glycoxidation and sorbitol system activation and from limitation of the hexose monophosphate shunt, leading to a decrease in glutathione synthesis.  Earlier studies suggested a strong relationship between hyperglycemia, oxidative stress and DNA damage. This can be evident from the presence of higher levels of DNA damaged products such as 8-hydroxy-2-deoxy guanosine (8-OHdG) in the blood of diabetic patients.  Increased nuclear damage in diabetes is reported to enhance mutations in both somatic and germinal cells. The somatic cell nuclear damages can cause diseases such as cancer, heart ailments, neurological defects and aging, while germinal cell defect can result in infertility and inheritable disorders in newborns. ,
Among the various mechanisms suggested for the anti-mutagenesis, the antioxidant property of the compound is reported to play an important role. Antioxidant exerts multiple effects to overcome the oxidative stress mediated damage on the nucleus, such as scavenging the free radical, increasing the host antioxidant enzyme levels and repairing the damaged nucleus. 
The administration of insulin is considered to be the best option to control the hyperglycemia if the oral hypoglycemic agents fail to attain the desired glycemic control. Insulin therapy is reported to benefit the diabetic patients in reducing the complications associated with hyperglycemia.  Administration of insulin to overweight pregnant mothers has been found to decrease the chances of gestation diabetes.  Besides, insulin treatment is reported to prevent the peripheral nerve damage in diabetic condition.  Reports also indicate that insulin therapy enhances the antioxidant status in the diabetic rats.  However, studies relating to the anti-hyperglycemic and antioxidant activities of insulin with anti-mutagenic effect are not found in the literature. Hence, the present research is aimed evaluate the anti-mutagenic activity of insulin in diabetic Wistar rats, using bone marrow micronucleus (MN) and sperm abnormality tests.
| Materials and Methods|| |
Insulin (regular) available in the market was used in the study. The stains and other reagents/chemicals used were of analytical grade and procured from Himedia Labs (P) Ltd., Mumbai, India.
Eight-week-old healthy, laboratory in-bred, male Wistar rats, weighing 180 ± 10 g, were maintained under standard laboratory conditions such as temperature 20 ± 2 o C, 12 hour light/dark cycle and provided water and pellet food (Amruth Biotech Ltd., Maharashtra, India) ad libitum. The experiments were conducted after obtaining prior approval from the Institutional Animal Ethics Committee.
Induction of type-2 diabetes
Experimental T2DM was developed in adult rats by administering streptozotocin (STZ) and nicotinamide (NA).  The animals received intraperitoneally 230 mg/kg of NA (SD Fine-Chem Ltd., Mumbai, India) dissolved in saline, 15 min before an intraperitoneal administration of 65 mg/kg of STZ (Sigma Aldrich, St. Louis, Missouri, USA) dissolved in 0.1 M citrated buffer (pH 4.5) immediately before use. Blood glucose level was estimated in a drop of blood collected from tail vein after 2 days and the animals with glucose level 180 ± 8 mg/dl were only selected for the study.
Dosage, treatment and sampling
The animals were divided mainly into six groups, i.e., control, diabetic and treatment, consisting of eight rats in each group. Four doses of insulin, viz., 1, 3, 5 and 7 IU/kg were selected as per the previous reports and also to attain different levels of anti-hyperglycemic effect. , The doses were adjusted using sterile water for injection and were administered subcutaneously everyday for 4 weeks after the induction of diabetes. In the case of control and diabetic groups, animals received saline (0.5 ml/kg) daily throughout the treatment period.
Bone marrow micronucleus test
The modified method of Schmid was followed to perform the bone marrow MN test.  The animals, after the respective treatments, were sacrificed by cervical dislocation under light anesthesia (diethyl ether, 2 ml/kg, open drop method). Animals were cut open to excise femur and tibia. Bone marrow MN slides were prepared by using the modified method of Schmid. Marrow suspension from femur and tibia bones prepared in 5% bovine serum albumin (BSA) was centrifuged at 1000 rpm for 8 min and the pellet was resuspended in a required quantity of BSA. A drop of this suspension was taken on a clean glass slide and smear was prepared on glass slide and air-dried. The slides were fixed in absolute methanol, stained with May-Grunwald-Giemsa and micronuclei (MN) were identified as two forms of RBCs (i.e., polychromatic erythrocytes as PCEs and normochromatic erythrocytes as NCEs). About 2000 PCEs and corresponding NCEs were scanned for the presence of MN using 100Χ oil immersion objective. The P/N ratio was calculated by dividing total number of PCEs with corresponding NCEs per animal. 
Sperm morphology and sperm count assay
The procedure described by Wyrobek and Bruce  was followed to study the sperm shape abnormality in cauda epididymis of the rats. One thousand sperms per animal were screened to find the different types of abnormality in one of the cauda epididymis. Six types of abnormalities such as amorphous, hookless, banana shape, fused, double headed and double tailed were evaluated and finally represented as percentage total abnormality. 
The caudal sperm count test was performed as per the method of D'Souza (2004). The spermatozoa count was obtained by counting the number of sperm cells in the four WBC chambers using a Neubauer's slide. 
In vivo antioxidant activity
Blood samples were collected from the retro-orbital plexus under light ether anesthesia. The serum was separated by centrifugation (1000 rpm for 2 min) and immediately analyzed to determine the antioxidant enzyme activity.
Serum lipid peroxidation
The procedure described by Ohkawa et al. was followed to estimate the lipid peroxidation (LPO). The principle depends on the reaction between thiobarbituric acid with malondialdehyde, a secondary product of lipid peroxidation, at pH 4. The reddish pink color developed was estimated at 532 nm, which indicates the extent of peroxidation. The extent of lipid peroxidation is expressed as nmol/mg protein. 
The estimation of catalase (CAT; EC 188.8.131.52) activity was done by determining the decomposition of H 2 O 2 at 240 nm in an assay mixture containing phosphate buffer (0.25 M, pH 7). One international unit of catalase utilized is that amount which catalyzes the decomposition of 1 mM H 2 O 2 per minute at 37°C and expressed in terms of unit/mg protein. 
The principle for measuring the superoxide dismutase (SOD; EC 184.108.40.206) depends on detecting the O2− generated during auto-oxidation of hydroxylamine. During the oxidation, nitroblue tetrazolium (NBT) was reduced and nitrite was produced in the presence of ethylenediaminetetraacetic acid (EDTA), which could be detected colorimetrically at 560 nm. The concentration of SOD is expressed as units/mg protein. 
Glutathione peroxidase (GPx; EC 220.127.116.11) activity was assayed based on the modified method of Paglia and Valentine, described by Heath and Tappel.  A 100 μl sample of the serum was incubated for 5 min at 37°C with stock solution (0.12 mM NADPH and 1 unit of glutathione reductase prepared in the Tris buffer) in a final volume of 1.65 ml. Then, 50 μl of cumene hydroperoxide (1 mg/ml) was added to start the reaction, and the absorbance at 340 nm was monitored for the rate of disappearance of NADPH and the GPx value is represented as μg of glutathione consumed/min/mg protein.
Blood glucose estimation
A drop of blood was collected from the tail vein and applied to the test zone of the glucose strip for immediate measurement of the fasting glycemia (mg/dl) using the Ascensia ENTRUST glucometer (Bayer healthcare Ltd., Mumbai, India).
The statistical analyses were done by one-way analyses of variance (ANOVA) followed by multiple comparisons by Bonferroni test for bone marrow MN test,  while data on epididymal sperm shape abnormalities and sperm count were analyzed employing a nonparametric test, the Mann-Whitney U test.  The antioxidant data were analyzed by one way ANOVA. P < 0.05 was considered to indicate significant difference.
| Results|| |
The bone marrow MN test indicated that experimental type-2 diabetes after the administration of NA-STZ, significantly (P < 0.001) increased the MN frequency in PCEs and NCEs, and decreased the P/N ratio compared to the control animals. Insulin (regular) was tested at 1, 3, 5 and 7 IU/kg and the results indicated that none of the doses significantly prevented the nuclear damage in the erythrocytes compared to the diabetic animals [Table 1].
The sperm abnormality test revealed that NA-STZ treated diabetic rats showed significant (P < 0.001) increase in the sperm shape abnormality and reduced the sperm count and weight of testis compared to the control animals. Administration of insulin produced no significant alteration in the sperm shape and sperm count defects in the diabetic rats. The treatment also did not increase the diminished weight of testis in diabetic condition [Table 2].
|Table 1 : Effect of insulin on the frequency of bone marrow MN in NA-STZ-induced diabetic rats|
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|Table 2 : Effect of insulin on the sperm morphology and sperm count in NA-STZ-induced diabetic rats|
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The serum antioxidant status indicated that NA-STZ significantly (P < 0.001) increased the LPO and decreased the CAT, SOD and GPx compared to the diabetic animals. However, none of the tested doses of insulin reversed significantly the diminished antioxidant profile in the diabetic animals [Table 3]. The blood glucose estimation indicated that insulin exhibited a dose-dependent reduction in the NA-STZ-induced hyperglycemia. The highest tested dose of insulin (7 IU) reversed the elevated blood glucose to the normal level in the diabetic rats [Figure 1].
|Table 3 : Effect of insulin on the serum antioxidant status and glucose level in NA-STZ-induced diabetic rats|
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|Figure 1 : Effect of insulin on blood glucose level in NA-STZ induced diabetic rats|
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| Discussion|| |
The present study indicated that NA-STZ-induced diabetes in Wistar rats significantly enhanced the nuclear defects in the erythropoietic and germinal cells, besides reducing the antioxidant status. NA-STZ-induced diabetic model in Wistar rats is commonly used since different levels of stable hyperglycemia can be produced, which are suitable for long-term studies. Further, this method of chemical diabetes is reported to mimic the clinical T2DM, especially in terms of insulin secretion in response to glucose load. The mechanism suggested for these responses is the partial protection offered by the NA against the STZ mediated oxidative damage to the pancreatic beta cells. 
The mutation testing assumes importance since it helps in identifying not only the mutagen but also the anti-mutagenic potential of the test compound. Bone marrow MN test and sperm abnormalities test are commonly employed assays to determine the mutations in somatic and germinal cells, respectively.  The MN test detects the genomic alterations arising from chromosomal damage and/or damage to the mitotic apparatus caused by clastogenic or aneurogenic agents. MN in young erythrocytes arises primarily from chromosomal fragments or lagging chromosomes that are not incorporated into daughter nucleus at the time of cell division in the erythropoietic blast cells, and the changes in the incidences of MN are considered to reflect the chromosomal damage in somatic cells.  On the other hand, estimation of epididymal sperm count, motility and morphology provides vital data on the nuclear integrity and fertilization capacity of the male germinal cells.  Higher incidences of sperm abnormality reflect the changes that the male germinal cell has undergone during its development and maturation stage in the epididymis. 
Several mechanisms have been suggested for anti-mutagenesis, such as inhibition of absorption of mutagens, complexation and deactivation of mutagens, inhibition of tumor promotion and progression and scavenging of free radicals. Antioxidant property of a compound plays an important role in the anti-mutagenesis.  Natural antioxidants like vitamins, tannins and flavonoids are known anti-mutagens against environmental and chemical mutagens. , Antidiabetic agents such as gliclazide, metformin, glimepiride and glibenclamide have also shown anti-genotoxic effect due to antioxidant property. These agents are reported to benefit the diabetic patients in reducing both hyperglycemia and also the mutagenic complications associated with enhanced oxidative stress. ,,,
In this study, following 4 weeks of insulin (regular) treatment to NA-STZ diabetic animals, although reduction in the elevated blood glucose was observed [Figure 1], the incidences of MN frequency and sperm abnormalities as well as the diminished antioxidant status was not altered [Table 1], [Table 2], [Table 3]. These observations indicated that the tested doses of insulin did not protect the bone marrow erythrocytes and spermatozoa cells against the reactive oxygen species induced lesion in diabetic condition. Earlier studies suggested that insufficient availability of test agent in the host system could be one possible reason for the lack of anti-mutagenic effect.  Sometimes, the drug might also fail to counteract the relevant cause for nuclear damage as observed in the case of vitamin C which failed to reduce the MN incidences caused by albendazole.  The rapid metabolism of a drug by the enzymes present in gut and liver could also contribute to the loss of anti-genotoxic property.  Considering these reports, it can be suggested that rapid metabolism of insulin might have caused insufficient concentration in host cell. This could have failed to elicit the antioxidant enzyme defense required for preventing the nuclear defects in erythropoietic and sperm cells. In this direction, further studies involving the longer acting insulin could be planned to find the role of sustained availability of insulin on oxidative stress and mutagenesis in diabetic condition.
| Conclusion|| |
From the present study, it can be concluded that the administration of insulin (regular) had no modulatory effect on oxidative stress, incidences of MN in erythrocytes and sperm abnormalities at tested doses and route of administration in NA-STZ-induced diabetic rats.
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[Table 1], [Table 2], [Table 3]