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Genotoxicity assessment of antidiabetic formulation (ADPHF6) in human lymphocytes by single cell gel electrophoresis (comet assay) - an in vitro study

Devanand Shanmugasundaram1, Kumar Perumal2, Changam S. Sasikumar1*, Sanjay M. Cherian3 and Kotturathu M. Cherian4
  • 1 Frontier Mediville (Affiliated to University of Madras), Department of Cellular & Molecular Biochemsitry, India
  • 2 Sri Ramachandra University, Biotechnology, India
  • 3 Frontier Lifeline Hospital, Cardio Thoracic Surgery, India
  • 4 Frontier Lifeline Hospital, Cardio Thoracic Surgery, India

Levels of Reactive Oxygen Species (ROS) molecules during aerobic metabolism are often regulated by unique endogenous antioxidant system. During hyperglycaemic condition, accumulation of excess fatty acids & glucose in adipose tissue (Wright Jr E., 2006) results in increased levels of ROS. When ROS molecules overwhelms the cells antioxidant defence system, it ends up in cellular oxidative stress; which in turn is reported to cause oxidative DNA damage & intervene damage to macromolecules & cellular membranes (Ahmad et al., 2013). Our novel anti-hyperglycaemic polyherbal formulation (ADPHF6) had already illustrated significant inhibitory activity against α-amylase & α-glucosidase enzymes and also scavenging free radicals (in vitro models). The present study demonstrates the protective effect of formulation against H2O2 induced DNA damage in human lymphocytes by Single Cell Gel Electrophoresis (SCGE) assay. Experimental procedures were approved by Institutional Human Ethics Committee of Frontier Lifeline Hospital, Chennai, India (FLL/IEC/02/2014). Peripheral human lymphocytes were isolated (Duthie et.al, 2002) and subjected for Cell viability by Trypan blue exclusion method. The alkaline SCGE assay was carried out to determine the level of DNA damage in ADPHF6 treated cells with minor modifications from Singh et al., 1988. Frosted microscopic slides were pre-coated with 1% NMA followed by 1% LMA and incubated for 15 min at 15-20o C. 100 μL of freshly prepared cell suspension (2 x 104 cells) was mixed with 0.5% LMA & casted on microscopic slide. The cells were immersed in lysing solution for 2 hours at 4O C and washed in TBE buffer for 5 min at RT. All the slides were treated with fresh alkaline solution for 20 minutes for expression of alkali-labile damage. Electrophoresis was performed at 24 V for 20 min at RT. Slides were washed in neutralizing buffer for 5 min at RT. All the groups were stained with Acridine Orange (20µg/ml) & Propidium Iodide (20µg/ml) and visualized under microscope (Nikon Eclipse 80i). Reaction mixture of Lymphocytes (suspended in RPMI-1640) with H2O2 (50 µM) served as Positive Control (PC); Lymphocytes in RPMI-1640 served as Vehicle control; Lymphocytes with H2O2 (50 µM) & Quercetin (50 mM) served as Standard Control. Lymphocytes treated with ADPHF6 aqueous extract (50 µg/ml) & H2O2 (50 µM) in various concentrations served as Test group. In untreated group (Vehicle control), % of tail DNA found to be null. Cells treated with 50 µM H2O2 (Positive control) showed maximum % of tail DNA (>90%) compared to Standard control group (< 30%). ADPHF6 treated cells (50 µg/ml) with 50 µM H2O2 showed significant decrease in % of tail DNA (< 20%). Though mammalian cells are equipped with impressive range of antioxidant enzymes & molecules, but in oxidative stress conditions these agents fails to normalize the redox status of cell. Various scientific investigations in recent times have stated that natural based antioxidants proved to be an alternative therapy for ROS mediated effects. To support this hypothesis, findings from our present study based on SCGE assay clearly suggests that, our anti-hyperglycaemic polyherbal formulation possesses strong antioxidant property in scavenging Free Radical molecules which restore the redox homeostasis in cells. Further studies are required to confirm the potential role of ADPHF6 against Type II Diabetes Mellitus involving in vivo models.

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Acknowledgements

We would like to thank Prof.T.S.Lokeswari, Head of Department, Department of Biotechnology, Faculty of Biomedical Sciences & Technology, Sri Ramachandra University, India for utilizing instrumental facility. Authors also thank Dr.Solomon F.D.Paul, Head of Department, Department of Human Genetics, Faculty of Biomedical Sciences & Technology, Sri Ramachandra University, India for utilising Fluorescent Microscope facility.

References

1. Wright, Jr. E., Scism Bacon, J. L., and Glass, L. C., (2006). Oxidative stress in type 2 diabetes: the role of fasting and postprandial glycaemia. Int. J. Clin. Pract. 60, 308–314.
doi: 10.1111/j.1368-5031.2006.00825.x
2. Ahmad et al., (2013). In vitro antioxidant potential of dicliptera roxburghiana. BMC Complementary and Alternative Medicine. 13, 140. doi:10.1186/1472-6882-13-140
3. Duthie, S. J., Pirie, L., Jenkinson, A. M., and Narayanan, S., (2002). Cryopreserved versus freshly isolated lymphocytes in human biomonitoring: endogenous and induced DNA damage, antioxidant status and repair capability. Mutagenesis. 17, 211-214. doi: 10.1093/mutage/17.3.211.
4. Singh N.P., McCoy, M. T., Tice, R. R., and Schneider E. L., (1988). A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 175, 184-191. doi: 10.1016/0014-4827(88)90265-0

Keywords: Comet Assay, Single cell gel electrophoresis, DNA Damage, Polyherbal formulation, Lymphocytes

Conference: ICAW 2015 - 11th International Comet Assay Workshop, Antwerpen, Belgium, 1 Sep - 4 Sep, 2015.

Presentation Type: Poster Discussion

Topic: Clinical applications of the comet assay

Citation: Shanmugasundaram D, Perumal K, Sasikumar CS, Cherian SM and Cherian KM (2015). Genotoxicity assessment of antidiabetic formulation (ADPHF6) in human lymphocytes by single cell gel electrophoresis (comet assay) - an in vitro study. Front. Genet. Conference Abstract: ICAW 2015 - 11th International Comet Assay Workshop. doi: 10.3389/conf.fgene.2015.01.00069

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Received: 03 May 2015; Published Online: 23 Jun 2015.

* Correspondence: Dr. Changam S Sasikumar, Frontier Mediville (Affiliated to University of Madras), Department of Cellular & Molecular Biochemsitry, Chennai, Tamilnadu, 600101, India, sheelsasic@yahoo.co.in