Application of cytogenetic endpoints and comet assay on human lymphocytes treated with atorvastatin in vitro

Abstract

This study investigated the genotoxic potential of atorvastatin on human lymphocytes using comet assay, structural chromosome aberrations (CA) and sister-chromatid exchange (SCE) analysis. Lymphocyte cultures were treated with a single drug at a concentration of 30.21 ng/mL. For comet assay, cells exposed to atorvastatin for 24 h, 48 h and 72 h were embedded in agarose slides, lysed with alkaline lysis solution and exposed to an electric field. DNA migrated within the agarose and formed comets whose length depends on the amount of DNA damage. For analysis of structural CA, cells were grown on medium for 48 h and for SCE analysis for 72 h. Structural CA did not induce significant damage to the genome, although a higher CA frequency was observed in cells treated with atorvastatin for 3 h, 20 h and 48 h than in control samples. Results of the SCE analysis did show statistically significant differences in the mean SCE number between atorvastatin-exposed and control human lymphocytes and between different exposure times. Comet assay also showed increased DNA damage caused in atorvastatin-exposed human lymphocytes than in corresponding control cells for exposure times of 24 h, 48 h and 72 h for the tail length and for 72 h for the tail moment. Results obtained in this study point to the significance of biological indicators providing information on the primary genome damage after long-term exposure, which can help to establish drug therapeutic concentrations that do not put patients with high blood cholesterol to a greater treatment-related risk.

Keywords:

• Chromosome aberrations
• sister-chromatid exchange
• comet assay
• peripheral blood human lymphocytes
• atorvastatin

Acknowledgments

The authors gratefully acknowledge the cooperation of all the volunteers who participated in this study. We wish to thanks BSc. Snježana Ramic for excellent technical assistance and BSc. Martina Durinec for statistical advice. This investigation was supported by the Croatian Ministry of Science, Education and Sports (grant No. 0022-0222148-2125).

Notes

*Incidence of CA was evaluated by analyzing 200 metaphases.

^a Statistically significant increase compared to corresponding control (P < 0.05, ANOVA).

^b Statistically significant increase compared to sample treated with atorvastatin in time period of 3 hours (P < 0.05, ANOVA).

^a Statistically significant increase in compare to corresponding control (P < 0.05, ANOVA).