Genotoxicity Of Nitenpyram On Human Bone Marrow Mesenchymal Stem Cells

Nitenpyram is a neonicotinoid insecticide, which was well known to have a low toxicity for mammals, and therefore it was widely used in agriculture, aquaculture and home life. Recent toxicological studies showed that nitenpyram was toxic to non-traget organisms, inducing the potential hazards to the ecological environment, mammals and even human health. Toxic mechanism of nitenpyram was unclear, and no results were reported about the toxicity of nitenpyram on human cells. The employment of human mesenchymal stem cells (hBMSCs) could effectively improve the accuracy of the results of chemical safety assessment and the relevance with human health, and help to improve the test efficiency and determine the mechanisms of toxicity. The purpose of this study is toevaluate the genotoxicity of nitenpyram and mechanism of action based on the use of in vitro hBMSCs culture, and to identify early biomarkers of genotoxicity.Water-soluble formazan method (WST-8) was used to detect cells viability after exposure to nitenpyram at concentrations ranging from 0 to 2500 ?g/mL in hBMSCs. The results showed that cells viability and proliferation were not significant inhibition. In vitro micronucleus assay was used to detect DNA damage on hBMSCs, and Annexin-FITC assay was used to detect cells apoptosis. The results showed that micronucleus frequencies and nuclear buds frequencies significantly exceeded those of the negative control after exposute to nitenpyram on hBMSCs, increasing with enhancing dosages. Micronucleus frequencies in hBMSCs treated by nitenpyram at 125 ?g/mLand above, were significantly higher than the negative control group. The response concentrations of MN detection is below the concentrations at which apoptotic bodies were observed, thus eliminating the interference of apoptotic bodies on micronucleus count. The results suggested that nitenpyram caused chromosomal damage effects in hBMSCs, causing potential genotoxicity. Reactive oxygen content, total antioxidant capacity and the vitality of superoxide were used to detect oxidative stress in hBMSCs. The reuslts indicated that reactive oxygen content increased significantly after exposure to nitenpyram at 250 ?g/mL and above, and the total antioxidant capacity decreased, and the vitality of superoxide showed abnormal as well. In addition, the alkaline phosphatase activity and the staining of Alizarin Red were used to detect osteogenic differentiation. The results suggested that nitenpyram exposure decreased the alkaline phosphatase activity and the staining of Alizarin Red for calcified nodules in hBMSCs, and then suppressed the osteogenic differentiation of hBMSCs. Furthermore, the key genes were detected on DNA damage response genes expression pathways by real-time quantitative PCR (RT-qPCR), and the results found ATM, ATR, p53, p21, Gadd45a, H2AX and Bax genes differential expression.In summary, the present study found that nitenpyram caused genotoxic damage in hMSCs, with the regulation by ATM and p53 genes on DNA damage response genes pathway. DNA damage increased intracellular reactive oxygen species, decreased total antioxidant capacity, resulting in the cellular redox system imbalance and affecting cell osteogenic differentiation. The results suggest that the hBMSCs model is a good alternative methodto animal genotoxicity testing, and the use of early biomarkers help to predict and evaluate the potential harm of exogenous chemicals to human body.