Quantification of the tissue distribution and accumulation of the neonicotinoid pesticide clothianidin and its metabolites in maternal and fetal mice.

Neonicotinoids (NNs) are commonly used pesticides that have a selective agonistic action on insect nicotinic acetylcholine receptors. Recent evidence has shown that NNs have adverse effects in the next generation of mammals, but it remains unclear how NNs transferred from dams to fetuses are distributed and accumulated in fetal tissues. Here, we aimed to clarify the tissue distribution and accumulation properties of the NN clothianidin (CLO) and its 6 metabolites in 7 tissues and blood in both dams and fetuses of mice administered CLO for a single day or for 9 consecutive days. The results showed that the total concentrations of CLO-related compounds in the brain and kidney were higher in fetuses than in dams, whereas in the liver, heart, and blood they were lower in fetuses. The multi-day administration increased the total levels in heart and blood only in the fetuses of the single administration group. In addition, dimethyl metabolites of CLO showed fetus/dam ratios >1 in some tissues, suggesting that fetuses have higher accumulation property and are thus at higher risks of exposure to CLO-related compounds than dams. These findings revealed differences in the tissue-specific distribution patterns of CLO and its metabolites between dams and fetuses, providing new insights into the assessment of the developmental toxicity of NNs.

Sex-specific behavioral effects of acute exposure to the neonicotinoid clothianidin in mice.

Although neonicotinoids are among the major classes of pesticides that affect mammalian nervous systems, little is known about sex differences in their effects. This study aimed to examine whether the neurobehavioral effects of a neonicotinoid, clothianidin (CLO), differed between sexes. Male and female C57BL/6N mice were orally administered CLO (5 or 50 mg/kg) at or below the chronic no-observed-adverse-effect-level (NOAEL) and subjected to behavioral tests of emotional and learning functions. Changes in neuroactivity in several brain regions and the concentrations of CLO and its metabolites in blood and urine were measured. Acute CLO exposure caused sex-related behavioral effects; decreases in locomotor activities and elevation of anxiety-like behaviors were more apparent in males than in females. In addition, male-specific impairment of short- and long-term learning memory by CLO exposure was observed in both the novel recognition test and the Barnes maze test. Male-dominant increases in the number of c-fos positive cells were observed in the paraventricular thalamic nucleus in the thalamus and in the dentate gyrus in the hippocampus, which are related to the stress response and learning function, respectively. The concentrations of CLO and most metabolites in blood and urine were higher in males. These results support the notion that male mice are more vulnerable than females to the neurobehavioral effects of CLO and provide novel insights into the risk assessment of neonicotinoids in mammalian neuronal function.

Aging-related changes in the sensitivity of behavioral effects of the neonicotinoid pesticide clothianidin in male mice.

Neonicotinoids, which act as agonists of the nicotinic acetylcholine receptors of insects, are widely used pesticides worldwide. Although epidemiological studies revealed that the detection amounts of neonicotinoids in urine are higher in the elderly population than other age-groups, there is no available information regarding the risks of neonicotinoids to older mammals. This study was aimed to investigate aging-related differences in the behavioral effects of the neonicotinoid pesticide clothianidin (CLO). We acutely administered a sub-NOAEL level (5 mg/kg) of CLO to adult (12-week-old) and aging (90-week-old) mice and conducted four behavioral tests focusing on the emotional behavior. In addition, we measured the concentrations of CLO and its metabolites in blood, brain and urine. There were age-related changes in most parameters in all behavioral tests, and CLO significantly decreased the locomotor activity in the open field test and elevated plus-maze test in the aging group, but not in the adult group. The concentrations of most CLO and its metabolites were significantly higher in the blood and brain and were slightly lower in the urine in the aging group compared to the adult group. These findings should contribute to our understanding of age-related differences in the adverse effects of neonicotinoids in mammals.

Establishment of an organ culture system to induce Sertoli cell differentiation from undifferentiated mouse gonads.

Organ culture systems are useful for elucidating the process of testicular differentiation from mammalian undifferentiated genetically male gonads, as they permit various experiments, including experiments involving the control of gene expression. However, without addition of testicular differentiation-related factors, it is difficult to induce the formation of testis cord from immature gonads by a time point earlier 12 tail somites (ts) that corresponding to 11.0 days post coitum (dpc). In this study, we attempted to establish an organ culture system that induces testis formation from immature gonads (around 8 ts: 10.5 dpc) just before Sry (sex-determining region of the Y chromosome) expression. A paired gonad-mesonephros complex of around 8 ts was placed in the groove of an agarose gel block and put the semi-cylindrical piece of agarose gel to maintain the gonad morphology. The gonads were cultured in the gas phase for 96 hr. As a result, testis cord-like structures appeared in many genetically male gonads. Cells expressing the Sertoli cell markers Sox9 (SRY-box 9) and Amh (anti-Müllerian hormone) were observed, while granulosa cell marker Foxl2 (forkhead box L2) was not detected. In addition, Sox9- and Amh-expressing cells were observed throughout the entire gonad in many individuals. Amh mRNA expression was also upregulated. Surprisingly, formation of a partial testicular structure was observed from more immature gonads (6 ts). These results show that our gonadal organ culture system is useful for elucidating the regulation mechanism of Sry expression in undifferentiated bipotential gonads.

Quantitative elucidation of maternal-to-fetal transfer of neonicotinoid pesticide clothianidin and its metabolites in mice.

Neonicotinoids (NNs), a widely used class of systemic pesticides, are regarded as exhibiting selective toxicity in insects. However, NNs are suspected of exerting adverse effects on mammals as well, including humans. To date, only adult male animal models have been subjected to general toxicity studies of NNs; fetuses have yet to be considered in this context. Here, we focused on the NN clothianidin (CLO) for the first quantitative LC-MS/MS analysis of maternal-to-fetal transfer and residual property of once-daily (single or multiple days), orally administered CLO and its metabolites in mice. The results revealed the presence of CLO and its five metabolites at approximately the same respective blood levels in both dams and fetuses. In the dams, CLO showed a peak value 1 h after administration, after which levels rapidly decreased at 3 and 6 h. In the fetuses of each group, levels of CLO were almost the same as those observed in the corresponding dams. The present results clearly demonstrated rapid passage of CLO through the placental barrier. However, metabolite-dependent differences observed in blood pharmacokinetics and residual levels. This is the first quantitative demonstration of the presence of CLO and its metabolites in fetal mouse blood.

Combined exposure to dinotefuran and chronic mild stress counteracts the change of the emotional and monoaminergic neuronal activity induced by either exposure singly despite corticosterone elevation in mice.

Dinotefuran (DIN) belongs to the neonicotinoids (NNs), a class of globally applied pesticides originally developed to exhibit selective toxicity in insects. However, several reports have suggested that NNs also exert neurotoxic effects in mammals. We previously demonstrated neurobehavioral effects of DIN on mice under non-stressful conditions. For further toxicity assessments in the present study, we investigated the effects of DIN on mice exposed to stressful conditions. After subacutely administering a no-observed-effect-level (NOEL) dose of DIN and/or chronic unpredictable mild stress (CUMS) to mice, we conducted three behavioral tests (i.e., open field test [OFT], tail suspension test [TST] and forced swimming test [FST]). In addition, serotonin (5-HT) and tryptophan hydroxylase 2 (TPH2) of the dorsal raphe nuclei (DRN) and median raphe nuclei (MRN) and tyrosine hydroxylase (TH) of the ventral tegmental area and substantia nigra (SN) were evaluated immunohistochemically. A NOEL dose of DIN or CUMS alone increased of the total distance in OFT, decreased or increased the immobility time in TST or FST, respectively, and increased the positive intensity of 5-HT and TPH2 in the DRN/MRN, and TH in the SN. These changes were suppressed under the conditions of combined exposure to DIN and CUMS, though the blood corticosterone level was increased depending on the blood DIN values and the presence of CUMS. The present study suggests the multifaceted toxicity of the neurotoxin DIN.

Immunotoxicity evaluation by subchronic oral administration of clothianidin in Sprague-Dawley rats.

Neonicotinoid pesticides (NNs) act as agonists on nicotinic acetylcholine receptors (nAChRs) of insects, and there have been concerns about the effects of NNs on the health of mammals. Since nAChRs are expressed in immune cells, it is possible that NNs disturb the immune system. However, few reports have examined the immunotoxicity of clothianidin (CLO), a widely-used NN. Here, we report the effects of CLO on immune organs and type IV allergic reactions in ear auricles. We orally administered CLO at 0, 30 and 300 mg/kg/day (CLO-0, 30 and 300) to Sprague-Dawley rats for 28 days. The effects were evaluated by organ and body weights, histopathology, and immunohistochemistry (TCRαß, CD4, CD8, CD11b, CD68, CD103). In addition, some cecal contents were subjected to preliminary gut microbiota analysis, because microbiota contribute to host homeostasis, including the immunity. Our results showed loose stool, suppression of body weight gain, significant changes in organ weights (thymus: decreased; liver: increased) and changes of the gut microbiota in the CLO-300 group. There were no obvious histopathological changes in immune organs. Granulomas of the ear auricles were found in one rat of each of the CLO-30 and 300 groups, but CLO had no apparent effect on the thickness or immunohistochemistry in the ear auricles. We present new evidence that CLO affects the thymus and intestine, and might enhance the local inflammatory response. These findings should contribute to the appropriate evaluation of the safety of NNs in the future.