Micronucleus and comet assay in the peripheral blood of juvenile rat: Establishment of assay feasibility, time of sampling and the induction of DNA damage

Abstract

Micronucleus (MN) and comet assay can successfully detect the genetic damage in two different cell population of peripheral blood i.e. erythrocytes and lymphocytes. The present study was aimed to investigate the kinetics of MN formation as well as to identify the time of maximum MN induction in the peripheral blood erythrocytes in juvenile rats and thereafter to examine its relationship with the observed DNA damage in the lymphocytes as determined by comet assay. The rat peripheral blood micronucleus (PBMN) assay is generally not preferred owing to the selective elimination of micronucleated cells from the circulation by spleen. However, inefficient splenic removal of micronucleated cells in the juvenile Sprague–Dawley (SD, 26 days) rats conferred advantage to be a suitable model for PBMN assay. The kinetics of MN formation and DNA damage in the peripheral blood were determined with cyclophosphamide (50 mg/kg), chlorambucil (30 mg/kg), methotrexate (20 mg/kg), cisplatin (5 mg/kg) and paclitaxel (0.5 and 1 mg/kg). All the tested chemicals with different mechanisms of action have induced time-dependant changes in the MN frequency in the peripheral blood erythrocytes. Comet assay in the peripheral blood lymphocytes also revealed similar pattern of rise and fall in the DNA damage. MN frequency and the different comet assay parameters exhibited significant positive correlation with all the tested chemicals and in both of the assays the peak was observed in between 36 and 48 h post-treatment. Results of the present study clearly demonstrates that MN frequency in the peripheral blood erythrocytes exhibits positive correlation with the DNA damage in the peripheral blood lymphocytes as evident from different comet assay parameters. Further, the study highlights the detection of DNA damage in two different cell population and establishes the complementary nature of these two bioassays for the genotoxicity testing by combining a conventional technique to the smarter one.