In vitro and in vivo effects of flubendiamide and copper on cyto-genotoxicity, oxidative stress and spleen histology of rats and its modulation by resveratrol, catechin, curcumin and α-tocopherol

Abstract

Background: Living organisms are frequently exposed to more than one xenobiotic at a time either by ingestion of contaminated food/fodder or due to house-hold practices, occupational hazards or through environment. These xenobiotics interact individually or in combination with biological systems and act as carcinogen or produce other toxic effects including reproductive and degenerative diseases. Present study was aimed to investigate the cyto-genotoxic effects of flubendiamide and copper and ameliorative potential of certain natural phyotconstituent antioxidants.

Method: In vitro cytogenotoxic effects were evaluated by employing battery of assays including Propidium iodide staining, Tunel assay, Micronuclei, DNA fragmentation and Comet assay on isolated splenocytes and their prevention by resveratrol (5 and 10 μM), catechin (10 and 20 μM), curcumin (5 and 10 μM) and α-tocopherol (5, 10 and 20 μM). In vivo study was also undertaken daily oral administration of flubendiamide (200 mg/kg) or copper (33 mg/kg) and both these in combination, and also all these concurrently with of α-tocopherol to Wistar rats for 90 days.

Results: Flubendiamide and copper produced concentration-dependent cytotoxic effects on splenocytes and at median lethal concentrations, flubendiamide (40 μM) and copper (40 μM) respectively produced 71 and 81% nonviable cells, higher number of Tunel+ve apoptotic cells, 7.86 and 9.16% micronucleus and 22.90 and 29.59 comets/100 cells and DNA fragmentation. In vivo study revealed significant (P < 0.05) increase in level of lipid peroxidation (LPO) and decrease in glutathione peroxidase (GPx), glutathione-S-transferase (GST) and superoxide dismutase (SOD) activities in groups exposed to flubendiamide or copper alone or both these in combination. Histopathological examination of rat spleens revealed depletion of lymphoid tissue, separation of splenocytes and rarification in splenic parenchyma of xenobiotic(s) treated groups.

Conclusion: Flubendiamide and copper induce oxidative stress and produce cytogenotoxic effects along with histoarchitectural changes in spleen. All four tested natural antioxidants (resveratrol, catechin, curcumin and α-tocopherol) reduced flubendiamide and copper-induced cytotoxic effects in rat splenocytes. Rat splenocytes are very sensitive to flubendiamide and copper-induced cytogenotoxicity, therefore, these can be effectively employed for screening of compounds for their cytogenotoxic potential. α-tocopherol was effective in restoring alterations in oxidative stress biomarkers and preventing histoarchitectural lesions in spleen.

Keywords: Copper; Cyto-genotoxicity; Flubendiamide; Oxidative stress; Splenocytes.

Fig. 1

Representative photographs of rat splenocytes showing TUNEL + ve cells (40 X) following in vitro exposure to median lethal concentration of flubendiamide alone (40 μM) and in the presence of different concentrations of natural antioxidants-resveratrol, catechin, curcumin and α-tocopherol

Fig. 2

Representative photographs of rat splenocytes showing TUNEL + ve cells (40 X) following in vitro exposure to median lethal concentration of copper alone (40 μM) and in the presence of different concentrations of natural antioxidants-resveratrol, catechin, curcumin and α-tocopherol

Fig. 3

Representative photographs of rat splenocytes showing micronuclei formation (100 X) following in vitro exposure to median lethal concentrations of flubendiamide and copper alone (40 μM) and in the presence of dimethyl sulphoxide (DMSO) and dexamethasone

Fig. 4

In vitro effect of median lethal concentration of flubendiamide and natural antioxidants at different concentrations on DNA fragmentation pattern in rat splenocytes. RV: Resveratrol (5 and 10 μM), Cath: Catechin (10 and 20 μM), A-T: α-tocopherol (5, 10 and 20 μM), Cur: Curcumin (5 and 10 μM), Flb: Flubendiamide, Dexa: Dexamethasone,Cont: Control

Fig. 5

In vitro effect of median lethal concentration of copper and natural antioxidants at different concentrations on DNA fragmentation pattern in rat splenocytes. RV: Resveratrol (5 and 10 μM), Cath: Catechin (10 and 20 μM), A-T: α-tocopherol (5, 10 and 20 μM). Cur: Curcumin (5 and 10 μM), Cu: Copper, Dexa: Dexamethasone, Cont: Control

Fig. 6

Representative photographs of rat splenocytes showing comet formation following their in vitro exposure to median lethal concentrations of flubendiamide and copper

Fig. 7

Section of spleen of rat from control group showing healthy histoarchitecture with red (arrow head) and white pulp (arrow) in splenic parenchyma (10X H&E stain)

Fig. 8

Spleen section of copper sulphate (33 mg/kg) exposed group (IV) showing mild depletion of lymphoid tissue from white pulp (arrow) (10 X H&E stain)

Fig. 9

Spleen section of flubendiamide (200 mg/kg) exposed group (V) showing separation of splenocytes and rarefication (arrow) in splenic parenchyma (10 X H&E stain)

Fig. 10

Spleen section of flubendiamide (200 mg/kg) + copper sulphate (33 mg/kg)-exposed group (VI) showing separation of splenocytes and rarefication (arrow) in splenic parenchyma (40 X H&E stain)

Fig. 11

Spleen section of rats treated with α-tocopherol (100 mg/kg) + copper sulphate(33 mg/kg) of group (VII) showing normal histo-architecture with abundant lymphoid tissue in the white pulp (arrow) suggestive of amelioration (40 X H&E stain)

Fig. 12

Spleen section of rats treated with α-tocopherol (100 mg/kg) + flubendiamide (200 mg/kg) of group (VIII) showing normal histoarchitecture, abundant lymphoid tissue in the white pulp (arrow) suggestive of amelioration (40 X H&E stain)

Fig. 13

Spleen of rats treated with α-tocopherol (100 mg/kg) + flubendiamide (200 mg/kg) + copper sulphate (33 mg/kg) of group (IX) showing apparently healthy histoarchitecture with ample red (arrow head) and white pulp (arrow) in splenic parenchyma suggestive of amelioration (10 X H&E stain)