Biological Monitoring Method for Urinary Neonicotinoid Insecticides Using LC‐MS/MS and Its Application to Japanese Adults: Jun UEYAMA, et al. Department of Pathophysiological Laboratory Sciences, Field of Radiological and Medical Laboratory Sciences, Nagoya University Graduate School of Medicine—Objectives: Agricultural use of neonicotinoid (NEO) insecticides has been increasing in recent years, but their biological monitoring methods have been scarcely reported. In this study, we developed and validated a rapid and sensitive method for quantifying urinary NEO concentrations using high‐performance liquid chromatography‐tandem mass spectrometry (LC‐MS/MS).Methods: After phosphate‐induced acidification of a urine sample, urinary NEOs were trapped by a solid‐phase extraction column and eluted with methanol for acetamiprid, imidacloprid, thiacloprid, thiamethoxam, clothianidin and dinotefuran and with an acetonitrile and methanol solution (1:1, v/v) containing 5% NH₃ for nitenpyram. Aseparation analysis was performed by LC‐MS/MS within 10 minutes for the sample. This method was applied to first morning urine obtained from 52 Japanese (40.9 ± 10.5 years old, mean ± standard deviation) without occupational NEO exposure.Results: The linear dynamic ranges and their limit of quantification (LOQ, signal to noise ratio=10) levels were 0.3−20 or 50 μg/l (r=0.998−0.999) and 0.05−0.36 μg/l, respectively. The absolute recovery was 64−95%, and the intra‐ and inter‐day precisions were less than 16.4% (relative standard deviation, %RSD). This method was successfully applied for analysis of NEOs in human urine samples obtained from 52 adults. The frequencies of individuals who showed more than LOD levels was above 90% for imidacloprid, thiamethoxam, clothianidin and dinotefuran, more than 50% for acetamiprid and thiacloprid and 29% for nitenpyram.Conclusions: These results indicated that our new method could be applied to biological monitoring of NEO exposure even at environmental exposure levels in Japanese adults without occupational spraying histories.

Permethrin, a popular synthetic pyrethroid insecticide used to control noxious insects in agriculture, forestry, households, horticulture, and public health throughout the world, poses risks of environmental exposure. Here we evaluate the reproductive toxicity of cis-permethrin in adult male ICR mice that were orally administered cis-permethrin (0, 35, or 70 mg/kg·d) for 6 wk. Caudal epididymal sperm count and sperm motility in the treated groups were statistically reduced in a dose-dependent manner. Testicular testosterone production and plasma testosterone concentration were significantly and dose-dependently decreased with an increase in LH, and a significant regression was observed between testosterone levels and cis-permethrin residues in individual mice testes after exposure. However, no significant changes were observed in body weight, reproductive organ absolute and relative weights, sperm morphology, and plasma FSH concentration after cis-permethrin treatment. Moreover, cis-permethrin exposure significantly diminished the testicular mitochondrial mRNA expression levels of peripheral benzodiazepine receptor (PBR), steroidogenic acute regulatory protein (StAR), and cytochrome P450 side-chain cleavage (P450scc) and enzyme and protein expression levels of StAR and P450scc. At the electron microscopic level, mitochondrial membrane damage was found in Leydig cells of the exposed mouse testis. Our results suggest that the insecticide permethrin may cause mitochondrial membrane impairment in Leydig cells and disrupt testosterone biosynthesis by diminishing the delivery of cholesterol into the mitochondria and decreasing the conversion of cholesterol to pregnenolone in the cells, thus reducing subsequent testosterone production.

The effect of the redox state of human serum albumin (HSA) on the antioxidant properties of the entire body has been a focus of recent research. The usefulness of HSA redox state as a biomarker for reducing oxidative stress has been investigated in clinical settings; however, evidence for its significance as a health index in non-clinical settings is yet to be established. This study aimed to examine the associations between HSA redox state and the atherosclerotic indices of carotid intima-media thickness (IMT) and plaque formation in a rural Japanese population. We conducted a cross-sectional study as part of a health check-up program in the rural area of Hokkaido, Japan, at the end of August 2013. A total of 281 residents (124 men and 157 women) were included in the final analysis. Lifestyle-related data were obtained through a self-reported questionnaire, and ultrasound examinations were performed to measure IMT and determine plaque formation. The high-performance liquid chromatography postcolumn bromocresol green method was used to separate HSA into human nonmercaptalbumin and human mercaptalbumin (HMA). We found a significant negative relationship between the fraction of HMA [f(HMA)] and IMT (standardized β = − 0.132, p = 0.03). Moreover, f(HMA) was significantly associated with plaque formation (p < 0.01) with an odds ratio of 0.89 (95% confidence interval, 0.81–0.97) for every 10% increment in f(HMA). We found that the HSA redox state, as determined by f(HMA), was associated with atherosclerotic indices in Japanese subjects. These results suggest that the HSA redox state indicates the risk of developing atherosclerosis.