The goal of the present study is to determine whether selected pesticides are capable of inducing double strand breaks (DSB) in an in vitro model and the recombinational pathway ensuing this damage.

Pesticides constitute a heterogeneous group of chemicals, specifically synthesized to control plagues, weeds, and unwanted organisms of all kinds. The authorized organic pesticides include several chemical groups or families 

Pesticides started being used with DDT and other organochlorides, followed by organophosphates, carbamates, pyrethroids, and herbicides like paraquat and 2,4-D. These compounds are of wide spectrum and show varying degrees of toxicity, not only for target species, but to others, unintendedly, including humans.

In agriculture, pesticides are typically applied in mixtures and exposure to these compounds has been associated with chronic adverse health effects including neurological, reproductive, respiratory, metabolic and developmental problems and cancer.  Particularly infant and childhood leukemia in relation to which they have been described as promoters since many of them do not induce genetic damage.

Genotoxic effects, however, have been documented in ecotoxicological and epidemiological studies, in animal models, as well as in vitro. Biomarkers identified include sister chromatid exchanges, chromosomal aberrations, micronuclei, and DNA breaks observed in the comet assay. 

Four serial dilutions of each pesticide or metabolite, as well for etoposide, were tested. Treatments were done in duplicate as follows: 250 microliters of whole blood were placed in 2.25 ml of RPMI-1640 and treated with the corresponding compound and concentration for 1.5 h at 37°C, after which 3 ml of 0.075 M KCl was added and incubation was continued for 30 min.

Lymphocytes were then fixed with minor modifications; briefly, cells were recovered by centrifugation at 250g for 10 min at room temperature; the supernatant was removed and formaldehyde was added at a final concentration of 4%.

Ten min later, 1 ml PBS with 0.12% Triton X-100 was added, and an incubation of 30 min was allowed at room temperature; thereafter the samples were washed with 1 ml cold PBS supplemented with 4% fetal bovine serum (FBS) and centrifuged for 8 min at  300g at 0°C.

Supernatant was discarded and 1 ml of cold 50% methanol in PBS was added. The samples were left at −20°C all night. Tubes were then centrifuged at 300g for 8 min at 0°C, the supernatant was discarded, 3 ml of cold methanol was added, and samples were kept at −20°C until analysis.

All Eight pesticides were tested for their ability to produce DSB in nonproliferating lymphocytes and to evaluate whether the classical recombinational mechanisms of DNA repair would come into action.

Two of them, paraoxon and glyphosate, were found to produce both DSB and the phosphorylation of Ku80, indicating that nonhomologous end joining recombinational DNA repair system would be activated. The type of gamma-H2AX foci observed was comparable to that induced by etoposide at similar concentrations. 

The results are of importance since these effects occurred at low concentrations in the micromolar range, in acute treatments to the cells. Effects over longer exposures in actual environmental settings are expected to produce cumulative damage if repeated events of recombination take place over time.