Determination Of 1, 2, 4-Trichlorobenzene-Induced DNA Damage In Mouse Peripheral Blood Lymphocytes

1,2,4 -TCB pervades in natural environment and industrial production. It causes a series of problems due to its longevity and amassment. The EPA has added it into the environmental pollutant list. There is also dispute about the genetic toxicity of the 1,2,4 - TCB. The single cell gel electrophoresis (SCGE) , or Comet Assay, is widely used to detect the DNA damage in single cells. This study exposed the peripheral blood lymphocytes of mice to 1, 2, 4 - TCB in vivo and in vitro, used SCGE to detect the DNA damage of lymphocytes to evaluate the genetic toxicity of 1,2,4 - TCB, and explored the mechanisms of genetic toxicity through catalase ( CAT) intervention.Materials and methods1. Exposure in vivo30 male Kunming mice (4 to 6 weaks old, 19 to 25g) were selected and divided into five groups at random, six mice in each. TCB was diluted in olive oil immediately before being injected intraperito-neally, repeatedly for 3 days (3 x 50,100,200mg/kg body wt. ; with 24 h interval). The genotoxic agent cyclophosphamide (3 x 150mg/ kg body wt. ; with 24 h interval) was used in positive control group. Negative control animals received olive oil intrapetitoneally repeatedly with 24 h between each injection (3 x 10 mg/kg body wt. ). Micewere killed 16 h after last injection. Using the Percoll density gradient fractionation to isolate blood lymphocytes.2. Exposure in vitroIsolated lymphocytes at 1 x 106cells/ml were treated as follow; DMSO diluted 1,2,4 -TCB (0, 10, 50, 100, 500 and 800umol/L) was applied to serum free medium 90 min at 37t; 1,4 - HQ(0, 5, 10, 25 and 50umol/L) , dissolved in distilled water, was applied to serum free medium for 90 min at 37t; H2O2 (50umol/L) was applied in H20 for 30 min at 4C.Cell viability was determined using the trypan blue exclusion assay. In all experiments, the viability of the cells was found to be greater than 85%.3. Catalase interventionIsolated lymphocytes were concurrently treated by the above exoteric exposing experiment in presence of catalase (250U/ml; 10ul/ tube).4. Single cell gel electrophoresis assay.5. Statistical analysis.All the data were input into the SSPS 10.0 database and analyzed by chi - square test and ANOVA.Results1. DNA damage induction by 1,2,4 - TCB in vivo Both comet cell percentage and mean comet length at all the doses were significantly different from the negative control, except for 50mg/kg of the 1,2,4 -TCB. They were also significantly different between high and moderate dose group.2. DNA damage induction by 1,2,4- TCB and 1,4 - HQ in vitroBoth 500 and 800umol/L group of 1,2,4 - TCB have very significant difference with the negative control group in comet cell percentage and mean comet length. Both 25 and 50umol/L group of 1,4 -HQ have very significant difference with the negative control group.3. Catalase intervention.1,2,4 - TCB, HQ and H2O2 have no significant difference with the negative control group.Conclusion1. 1,2,4 - TCB can cause DNA damage in vivo. The DNA damage become more serious as the dose of 1,2,4 - TCB increases.2. Both 1,2,4 - TCB and 1,4 - HQ can cause DNA damage in vitro. Catalase can inhibit the damage.