Di(2‐ethylhexyl)phthalate Induces Hepatic Tumorigenesis through a Peroxisome Proliferator‐activated Receptor α‐independent Pathway: Yuki Ito, et al. Department of Occupational and Environmental Health, Nagoya University Graduate School of Medicine—Di(2ethylhexyl)phthalate (DEHP), a commonly used industrial plasticizer, causes liver tumorigenesis presumably via activation of peroxisome proliferator‐activated receptor alpha (PPARα). The mechanism of DEHP tumorigenesis has not been fully elucidated, and to clarify whether DEHP tumorigenesis is induced via PPARα, we compared DEHP‐induced tumorigenesis in wild‐type and Pparα‐null mice. Mice of each genotype were divided into three groups, and treated for 22 months with diets containing 0, 0.01 or 0.05% DEHP. Surprisingly, the incidence of liver tumors was higher in Pparα‐null mice exposed to 0.05% DEHP (25.8%) than in similarly exposed wild‐type mice (10.0%). These results suggest the existence of pathways for DEHP‐induced hepatic tumorigenesis that are independent of PPARα. The levels of 8‐OHdG increased dose‐dependently in mice of both genotypes, but the degree of increase was higher in Pparα‐null than in wild‐type mice. NFκB levels also significantly increased in a dose‐dependent manner in Pparα‐null mice. The protooncogene c‐jun‐mRNA was induced, and c‐fos‐mRNA tended to be induced only in Pparα‐null mice fed a 0.05% DEHP‐containing diet. These results suggest that increases in oxidative stress induced by DEHP exposure may lead to the induction of inflammation and/or the expression of protooncogenes, resulting in a high incidence of tumorigenesis in Pparα‐null mice.

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 plasticizer di(2-ethylhexyl) phthalate (DEHP) has been widely used in the manufacture of polyvinyl chloride-containing products such as medical and consumer goods. Humans can easily be exposed to it because DEHP is ubiquitous in the environment. Recent research on the adverse effects of DEHP has focused on reproductive and developmental toxicity in rodents and/or humans. DEHP is a representative of the peroxisome proliferators. Therefore, peroxisome proliferator-activated receptor alpha (PPARα)-dependent pathways are the expected mode of action of several kinds of DEHP-induced toxicities. In this review, we summarize DEHP kinetics and its mechanisms of carcinogenicity and reproductive and developmental toxicity in relation to PPARα. Additionally, we give an overview of the impacts of science policy on exposure sources.