In vivo toxicity evaluation of two polyoxotungstates with potential antidiabetic activity using Wistar rats as a model system

Abstract

In this study, the in vivo hypoglycemic effect of a donut-shaped polyanion salt (NH₄)₁₄[Na@P₅W₃₀O₁₁₀]·31H₂O {NaP₅W₃₀} and its Ag-containing derivative K₁₄[Ag@P₅W₃₀O₁₁₀]·22H₂O·6KCl {AgP₅W₃₀}, as well as their hepatotoxicity and nephrotoxicity, was evaluated. In the screening hypoglycemic study, Wistar albino rats with streptozotocin induced diabetes were treated intraperitoneally with three single doses (5, 10, and 20 mg per kg per b.w.) of both investigated polyoxotungstates. The blood glucose levels, measured before and after 2, 4 and 6 h polyoxotungstate application, showed that both studied compounds induced the most pronounced and time dependent glucose lowering effects at the doses of 20 mg kg^-1. Thus, daily doses of 20 mg kg^-1 were administered to Wistar albino rats orally for 14 days in further toxicity examinations. The serum glucose concentration and biochemical parameters of kidney and liver function, as well as a histopathological analysis of kidney and liver tissues were evaluated 14 days after the polyoxotungstate administration. Both investigated compounds did not induce statistically significant alterations of the serum glucose and uric acid concentrations, as well as some of the liver function markers (serum alanine and aspartate aminotransferases, and alkaline phosphatase activities). However, the significant decrease in serum total protein and albumin concentrations and the increase in biochemical parameters of renal function - serum urea (up to 63.1%) and creatinine concentrations (up to 23.3%) were observed for both polyoxotungstates. In addition, the detected biochemical changes were in accordance with kidney and liver histhopathological analysis. Accordingly, the hepatotoxic and nephrotoxic effects of these potential antidiabetic polyoxotungstates could be considered as mild.

Fig. 1. The effect of {NaP₅W₃₀} on the blood glucose level in STZ-diabetic rats. The investigated compound was administered intraperitoneally (t = 0 h) in three different single doses (5, 10, and 20 mg per kg per b.w.). (A) The blood glucose level was determined 2, 4, and 6 hours after the POM application. The values are expressed as mean ± S.E.M. The repeated measures ANOVA test was used to determine the statistical significance. The blood glucose levels after 2 h, 4 h, and 6 h of {NaP₅W₃₀} administration were significantly reduced (P < 0.05) in respect to the corresponding pretreatment diabetic control values for all administrated doses (5, 10, and 20 mg kg⁻¹). (B) The blood glucose level (expressed as % of diabetic control) at three {NaP₅W₃₀} doses 6 hours after the treatment.

Fig. 2. The effect of {AgP₅W₃₀} on the blood glucose level in STZ-diabetic rats. The investigated polyoxotungstate was administered intraperitoneally (t = 0 h) in three different single doses (5, 10, and 20 mg per kg per b.w.). The blood glucose level determined 2, 4, and 6 hours after the POM application. The values are expressed as mean ± S.E.M. The repeated measures ANOVA test was used to determine the statistical significance of difference among groups.

Fig. 3. The effects of {NaP₅W₃₀} (20 mg per kg per day per os) and {AgP₅W₃₀} (20 mg per kg per day per os) on serum glucose concentrations. In Control group, the rats received saline (0.9% NaCl) per os for 14 days. Blood samples were taken after 14 days POM (or saline) application. The results are presented as median (range) values. One Way ANOWA followed by Tukey's Multiple Range Test was used to determine the statistical significance of difference among groups.

Fig. 4. The effects of {NaP₅W₃₀} (20 mg per kg per day per os) and {AgP₅W₃₀} (20 mg per kg per day per os) on renal function parameters (A, B and C: serum urea, creatinine and uric acid concentration, respectively). In Control group, the rats received saline (0.9% NaCl) per os for 14 days. Blood samples were taken after 14 days POM (or saline) application. The results are presented as median (range) values. One Way ANOVA followed by Tukey's Multiple Range Test was used to determine the statistical significance of difference among groups.*P < 0.05; ***P < 0.001.

Fig. 5. The effects of {NaP₅W₃₀} (20 mg per kg per day per os) and {AgP₅W₃₀} (20 mg per kg per day per os) on liver function parameters (A, B, C, D and E: AST, ALT and ALP activities, and total protein and albumin concentrations in serum, respectively). In Control group, the rats received saline (0.9% NaCl) per os for 14 days. Blood samples were taken after 14 days POM (or saline) application. The results are presented as median (range) values. One Way ANOVA followed by Tukey's Multiple Range Test was used to determine the statistical significance of difference among groups. *P < 0.05; ***P < 0.001.

Fig. 6. Photomicrographs of HE-stained rat kidney sections. Tissues were stained with haematoxylin and eosin, and image was captured under light microscope with ×5 (a–c) and ×40 (d–f) magnifications; (a and d) = tissues from control rats treated with saline; (b and e) = tissues from rats treated with 20 mg per kg per day {NaP₅W₃₀}; (c and f) are tissues from 20 mg per kg per day {AgP₅W₃₀}-treated rats. No difference between the kidney tissues from the treated and control rats.

Fig. 7. TEM micrographs presenting the ultrastructure of tubules in examined kidneys in untreated rat (a and d), in rat treated with 20 mg per kg per day {NaP₅W₃₀} (b and e) and in rat treated with 20 mg per kg per day {AgP₅W₃₀} (c and f). Low power magnification (×2800) of tubules: unaltered tubules with prominent basal labyrinth in control rat (a), tubules with disorganized basal labyrinth and condensation of mitochondrial matrix in {NaP₅W₃₀} (b) and {AgP₅W₃₀} (c) treated rats. High power magnification (×14 000) of: the cytoplasm of epithelial cell in tubules with unaltered mitochondria in kidney of control rat (d), cytoplasm of tubular epithelial cell in kidney tubules with electron dense mitochondria (arrow) in {NaP₅W₃₀} treated animal (e), and cytoplasm of epithelial cells of kidney tubules with distorted mitochondria (arrow) in {AgP₅W₃₀} treated animal (f).

Fig. 8. Photomicrographs of HE-stained rat liver sections. Tissues were stained with haematoxylin and eosin, and image was captured under light microscope with ×5 (a–c) and ×40 (d–f) magnifications; (a and d) = tissues from control rats treated with saline; (b and e) = tissues from rats treated with 20 mg per kg per day {NaP₅W₃₀} showing: discrete fields of focal nescrosis between lobules (arrows); (c and f) are tissues from 20 mg per kg per day {AgP₅W₃₀}-treated rats showing: focal necrosis in periportal regions (arrows).

Fig. 9. TEM micrographs presenting the ultrastructure of liver (×3500) in untreated rat (a), in rat treated with 20 mg per kg per day {NaP₅W₃₀} (b), and in rat treated with 20 mg per kg per day {AgP₅W₃₀} (c). Ultrastructural features of unaltered hepatocytes in control rat (a). Liver tissue with field of necrosis accompanied with extravasated erythrocytes in rat treated with {NaP₅W₃₀} (b). Small fields of coagulation necrosis in {AgP₅W₃₀} treated rat (c).