Prophylactic role of Enhydra fluctuans against arsenic-induced hepatotoxicity via anti-apoptotic and antioxidant mechanisms

Abstract

Objectives: The present study was undertaken to evaluate the prophylactic effect of aqueous extract of Enhydra fluctuans (AEEF) against NaAsO₂-induced hepatotoxicity.

Methods: The cytoprotective effect of AEEF against NaAsO₂ (10 µM) toxicity was measured on isolated murine hepatocytes. The effect on lipid peroxidation, protein carbonylation, cellular redox markers and signal proteins were measured after incubating the hepatocytes with NaAsO₂ (10 µM) + AEEF (400 µg/ml). Finally, the prophylactic effect of AEEF (50 and 100 mg/kg) against NaAsO₂ (10 mg/kg) toxicity was measured by in vivo assay in experimental mice.

Results: In vitro bioassay on isolated mouse hepatocytes confirmed cytoprotective effect of AEEF. The NaAsO₂ treatment significantly (P<0.01) increased the levels of lipid peroxidation, protein carbonylation with concomitant reduction (P<0.01) of antioxidant enzymes and reduced glutathione levels in hepatocytes. In addition, NaAsO₂ significantly (P<0.05–0.01) altered the expression of intrinsic (Bad↑, Bcl-2↓, cleaved-caspase 3↑ and cleaved-caspase 9↑) and extrinsic (Fas↑, Bid↑, cleaved-caspase 8↑) transcription proteins participating in the apoptotic event. However, AEEF treatment could significantly rescue the aforementioned parameters near-normal levels. In in vivo bioassay, NaAsO₂ intoxication increased (p<0.01) bioaccumulation of As along with the abnormalities in haematological parameters and redox imbalance in the livers of experimental mice. Treatment with AEEF, however, could significantly (P<0.05–0.01) restore the hematological and redox parameters to the near-normal levels, with histological studies of livers supporting the protective role of AEEF.

Discussion: Presence of substantial quantity of ascorbic acid, phenolics and flavonoids in the extract may be responsible for overall protective effect.

Keywords:

• Apoptosis
• Arsenic toxicity
• Enhydra fluctuans
• NaAsO₂
• Oxidative stress

Acknowledgments

The financial support of the Council for Scientific and Industrial Research, New Delhi, India is gratefully acknowledged through Senior Research Fellowship to Mr Tarun K. Dua [CSIR SRF Ref. No.: 09/096(773)/2013-EMR-I dated 15 March 2013]. The authors are grateful to Jadavpur University, Kolkata, India for providing the necessary facilities for this study. The authors are thankful to Mr Tristan Glover, Research Fellow, Discovery Biology, Eskitis Institute of Drug Discovery, Griffith University, Australia for his kind assistance in the correction of language. Finally, the authors would like to thank the Editor and reviewers for their time and valuable comments to improve the quality of this manuscript.

Disclaimer statements

Contributors TKD and SD contributed equally to this paper.

Funding Council of Scientific & Industrial Research (CSIR), New Delhi, India.

Conflicts of interest None.

Ethics approval The principles of Laboratory Animals Care³⁴ and the guidelines of our institutional Animal Ethical Committee (Reg. no. 0367/01/C/CPCSEA) were followed throughout the experiment.