The insecticides permethrin and chlorpyrifos show limited genotoxicity and no leukemogenic potential

Epidemiological and clinical studies have revealed that maternal exposure to pesticides-insecticides during pregnancy is associated with an increased risk of infant or childhood acute leukemia. The household insecticides permethrin and chlorpyrifos, which are members of the pyrethroid and organophosphate families of pesticides, respectively, have been associated with both the induction of MLL rearrangements (MLLr) and the development of infant acute leukemia. Despite the epidemiological association between insecticides and increased risk of leukemia, whether such insecticides act as topo isomerase II DNA-damaging poisons remains unknown and studies testing the biological plausibility of such an association are lacking. Here, we assessed the genotoxicity, induction of MLLr and leukemogenic potential of permethrin and chlorpyrifos by taking advantage of cutting-edge in vitro and in vivo models using prenatal, neonatal and adult hematopoietic stem and progenitor cells (HSPC). Our data suggest that the insecticides tested show no cytotoxicity, limited genotoxicity and no leukemogenic potential in human and murine HSPC in vitro and in vivo. We initially assessed whether acute exposure to etoposide (1 μM), permethrin or chlorpyrifos (10 μM or 50 μM) induces MLL breaks in undifferentiated human embryonic stem cells (hESC) and CD34 HSPC derived from hESC, cord blood or adult peripheral blood (Figure 1A). Acute exposure (24 h) to either permethrin or chlorpyrifos consistently induced MLL breaks in 3–7% of embryonic, neonatal and adult CD34 cells in a doseindependent manner (Figure 1B, C). Embryonic and somatic CD34 cells were sensitive to the three treatments (Figure 1C). Of note, whereas embryonic and neonatal CD34 cells were slightly more sensitive to etoposide than to insecticides (Figure 1C), no differences were found for adult CD34 cells, suggesting that the potential genotoxicity of the insecticides may have a more relevant etiological impact in adult MLLr acute leukemia. Chronic exposure to low doses of etoposide has been reported to induce apoptosis, MLL breaks and major chromosomal abnormalities in hESC. To test whether this occurred after exposure to insecticides, we treated hESC for 24 h with 10 μM of either permethrin or chlorpyrifos followed by a daily “booster” dose (2 μM) for 40 days (Figure 1D). After 5 days of recovery (without treatments), MLLr breaks and gross genomic abnormalities were assayed by interphase fluorescence in situ hybridization (iFISH), G-banding and comparative genomic hybridization (Figure 1E-G). In contrast to the frequency of MLL breaks (3–7%) observed after acute exposure (Figure 1C), MLL breaks were scarcely detectable upon chronic exposure to permethrin or chlorpyrifos, suggesting a legitimate repair of the DNA damage/double strand breaks (DSB) at the MLL locus (Figure 1E). Likewise, karyotyping and comparative genomic hybridization analysis revealed no numerical or structural chromosomal alterations (Figure 1F) or DNA gains or losses after chronic exposure (Figure 1G). It has been previously suggested that the chromosomal topology and chromatin structure resulting from early apoptosis may represent the underlying substrate for MLL chromosomal translocations to occur. Here, in contrast to etoposide, which did cause significant cell death at doses as low as 1 μM, neither permethrin nor chlorpyrifos induced cell death/apoptosis at doses 50-fold higher (Online Supplementary Figure S1A), supporting the concept that the absence of high-grade DNA fragmentation may represent a chromatin physical impediment for MLL DSB to fuse in-frame with a partner gene and encode an oncogenic fusion protein. Inverse polymerase chain reaction assays confirmed the absence of in-frame MLL fusions (data not shown). Overall, these data indicate that chronic exposure to insecticides neither enriches for MLL breaks nor generates MLL fusion oncogenes or gross genomic instability. To test the bona fide ability of permethrin and chlorpyrifos to function as topoisomerase II poisons, and their ability to generate DNA-DSB we performed an in vivo complex of enzyme assays to analyze covalent genomic DNA/topoisomerase II complexes in live cells. Treatment of hESC with permethrin and chlorpyrifos at 10–500 μM induced only minimal poisoning of the topoisomerase II isoforms (a and b), whereas as little as 1 μM etoposide induced significant poisoning of both isoforms (Online Supplementary Figure S1B). Similarly, monitoring of g-H2AX by western blotting (Online Supplementary Figure S1C) or fluorescent activated cell sorting (FACS) analysis (Online Supplementary Figure S1D-F) revealed that the pesticides were unable to induce DSB in hESC, neonatal or adult CD34 cells at relatively high concentrations, whereas etoposide potently induced DSB that were slightly repaired to some extent over time in somatic CD34 cells but not in hESC (Online Supplementary Figure S1F). Overall, the data indicate that permethrin and chlorpyrifos are not topoisomerase II poisons and do not generate DNA-DSB in embryonic or somatic CD34 cells. We next sought to determine whether prolonged in vivo exposure of human CD34 HSPC to pesticides induces MLL breaks or initiates leukemia (Figure 2A). Immunodeficient (NSG) mice xenotransplanted with cord blood-derived human CD34 HSPC were exposed for 12 weeks to permethrin or chlorpyrifos in the drinking water to mimic the environmental exposure in humans. Our target concentration for pesticide exposure was 10 mg/kg/day, and 5 mg/kg/day for etoposide. As the actual concentration to which mice are exposed will depend on their weight and their consumption of drinking water over the experimental period, we measured both parameters weekly. Results revealed an actual intake of permethrin, chlorpyrifos and etoposide only 10–20% lower than the theoretical concentrations (8.5, 9 and 4.2 mg/kg/day, respectively) (Online Supplementary Figure S2A-C). Importantly, the permethrin and chlorpyrifos metabolites 3-BPA and TCPy were readily detected by gas chromatography-mass spectrometry in serum and urine as soon as 48 h after pesticide exposure, confirming consistent exposure through administration of drinking water (Online Supplementary Figure S2D). iFISH analysis at sacrifice revealed a small but significant increase in the frequency of bone marrow-engrafted human CD45 cells harboring MLL breaks in etoposidetreated mice, but not in permethrinor chlorpyrifostreated mice (Figure 2B). FACS analysis at sacrifice revealed similar levels of human graft and normal multilineage (immature, myeloid and B-cell lymphoid) engraftment in the bone marriow and peripheral blood of etoposide-, permethrinand chlorpyrifos-treated mice (Figure 2C, Online Supplementary Figure S2E), with no evidence of splenomegaly (Figure 2D). These findings indicate that chronic in vivo exposure to the indicated Letters to the Editor