Effects Of Neonicotinoid Insecticides Imidacloprid Exposure On Reproductive And Genetic Toxicity

Objective:Neonicotinoid insecticides are the kind of importance of insecticides after Carbamate,Organophosphates and Pyrethriods insecticides.Imidacloprid（IMI）is the earliest and largest neonicotinoid insecticides on the market.Currently,imidacloprid is commonly detected in fruits and vegetables,and some samples showed residues above the maximal residue limit.However,few data report the human exposure to imidacloprid.Some studies suggest that IMI may be an environmental endocrine disruptor,but no research on IMI’s effect on human and its mechanisms is available.The present study is intended to detect urinary IMI and 6-chloronicotinic acid（6-ClNA,which is a common metabolite of the neonicotinoids）concentrations among pregnant women in a birth cohort,and to analyze the potential association of neonicotinoid insecticides exposure with reproductive effects and genetic damage.TK6cells and female ICR mice experiments were used to explore the potential mechanisms.Methods:The population study was based on a birth cohort has been established in Laizhou Wan（Bay）,Shandong province.The present study extracted information from maternal questionnaire and medical records and selected bio-samples in the following explorations:（1）296 maternal urinary IMI and 6-ClNA concentrations were detected with HPLC-MS/MS.（2）Binary logistic regression and multiple linear regression were used to analyze the association between maternal IMI and 6-ClNA levels and their menstruation history,childbearing history,and condition of present pregnancy extracted from questionnaire and medical records.（3）Enzyme-linked immunosorbent assay was applied to detect the concentrations of 8-hydroxy-2’-deoxyguanosine（8-OHdG）in maternal urine.Multiple linear regression was used to analyze the association between maternal IMI and 6-ClNA levels and 8-OHdG concentrations.In the experimental study,to explore the reproductive effects and its mechanisms of IMI,a total of 48 seven-week old female ICR mice were randomly divided into four groups:control group,low dose group（6 mg/kg/d）,middle dose group（12 mg/kg/d）,high dose group（24 mg/kg/d）.Mice exposed to IMI by gavage for 30continuous days.Vaginal smears were taken in order to determine the estrous cycle of female mice.24 hours after the last exposure,the ovary,uterus,brain,liver,kidney and spleen were collected to calculate the organ coefficients.The levels of follicle-stimulating hormone（FSH）,luteinizing hormone（LH）,progesterone（P）and estradiol（E₂）in mice serum were detected by enzyme-linked immunosorbent assay.The ovarian and uterine tissues histopathology was estimated under light microscope based on HE staining,the apoptosis of granulosa cells in the ovary was detected using the TUNEL assay.In order to explore the genetic toxicity and its mechanisms of IMI,8-OHdG levels in serum were measured by ELISA,in vivo ICR mice liver cells micronucleus tests and comet assays,and in vitro TK gene mutation experiments were used.Results:（1）IMI was detectable in urine samples in 98.3%of the pregnant women,and the median was 0.07μg/L（not adjusted for creatinine）and 0.15μg/g（creatinine-adjusted）.The detection rate of 6-ClNA in urine samples was in 98.0%,and the median was 5.03μg/L（not adjusted for creatinine）and 9.58μg/g（creatinine-adjusted）.（2）Maternal 6-ClNA levels were associated with decreased menstrual period and lower risks of menorrhalgia.（3）Maternal IMI levels were associated with increased 8-OHdG concentrations in urine.（4）In female ICR mice,luteal cells of the ovarian tissues were significantly increased in middle and high dose groups,and uterine tissues were oedematous in the high dose group.A small quantity of granulosa cells apoptosis in the ovary was found by TUNEL.（5）In TK gene mutation assay,IMI significantly increased TK mutations with a dose-effect relationship in 0.1 1,5 and 10μg/mL（P<0.05）.Conclusion:（1）Pregnant women in the study area were widely exposed to IMI.Maternal neonicotinoids metabolite 6-ClNA levels are likely to be related to menstrual period and menorrhalgia rather than childbearing history and condition of present pregnancy.Maternal IMI exposure is likely to be associated with oxidative genetic damage.（2）IMI exposure can damage the ovarian and uterine tissues of female mice.（3）IMI has certain mutagenicity to TK6 cells.