Assessment of the effect of pesticide formulation of terbuthylazine on TP 53 gene integrity by using comet-FISH in human lymphocytes

Abstract

Prolonged pesticide exposure is associated with genome damage and consequently with elevated risk of cancer development. We tested low concentrations of terbutylazine and its formulation as a complex mixture, which are considered safe and likely to occur in everyday and occupational exposure. These were equivalents of acceptable daily intake (ADI) and occupational exposure limit (OEL). Human lymphocytes from 3 donors were exposed for 14 days to 0.6 ng/mL (ADI), and 8.0 ng/mL (OEL) of terbutylazine and its formulation with and without the presence of metabolic activation (S9). Primary DNA damage was monitored by using alkaline comet assay and TP 53 gene integrity by hybridizing comet assay slides with TP 53 gene probe in comet-FISH. Higher concentration (OEL) of active ingredient with and without metabolic activation showed significantly migration of DNA into comet tail compared to control. Contrary, both concentrations of terbutylazine formulation, regardless of S9 induced primary DNA damage at the levels that were significantly higher compared to pure active ingrediaent. Decreased structural integrity of TP 53 gene was observed only for higher concentration tested (OEL) of pure terbutylazine with and without S9. Opposite, structural damage of TP 53 was significantly more diminished by treatment with commercially available pesticide formulation based on terbutylazine ; as a complex mixture at both tested concentrations TP 53 gene was affected by primary DNA damage at significantly higher rate compared to pure terbutylazine. Results suggest that prolonged exposure to low concentrations of terbutylazine based formulation as complex mixture of substances, may induce the level of primary DNA damage and pose a risk to structural integrity of TP 53 gene at significantly higher rate than the exposure to pure active ingredient. This work was financially supported by the Project No. 8366 Organic Pollutants in Environment - Markers and Biomarkers of Toxicity (OPENTOX), funded by the Croatian Science Foundation.