Therefore, pyriproxyfen is a potent ligand for Met, mimicking the function of JH and thus preventing adult transition. Previous studies in a mouse model have indicated that pyriproxyfen is stable and safe up to 5 g/kg when administered orally and is rapidly biodegraded after administration [4]. However, the effects of large doses of pyriproxyfen on mammalian immune response are still unknown. Therefore, we explored whether large doses of pyriproxyfen affect the immune response. We aimed to determine the IgG immune response to pyriproxyfen and the widely used model antigen OVA. We also monitored other aspects

of the immune profile in response to pyriproxyfen, including Estrogen antagonist IgG subtypes such as IgG1 or IgG2a, IgE production and cytokines. The four-week-old female BALB/c mice used in this study were purchased from Kyudo (Saga, Japan) and housed in a controlled Barasertib molecular weight specific pathogen-free environment

with a 12 hr light/dark cycle (lights on from 07:00 to 19:00) and temperature and humidity controlled to 23 ± 2°C and 55 ± 5%, respectively. Feed (CE-2; Clea Japan, Tokyo, Japan) and water were provided ad libitum. All procedures related to the animals and their care were approved (Certificate No. 1104474) by the Laboratory Animal Care and Use Committee of Fukuoka University. For immunization, OVA (Sigma–Aldrich, St. Louis, MO, USA) was dissolved in PBS at a concentration of 5 μg/mL. Initially, 1.9, 5.8 and 9.7 mg of pyriproxyfen (Fig. 1) (Wako Pure Chemical Industries, Osaka, Japan) Montelukast Sodium were dissolved in 100 μL of 99% ethanol and made up to 1 mL with PBS. Subsequently, 100 μL of each pyriproxyfen solution was diluted with an equivalent volume of OVA solution to provide the desired concentrations of 3, 9 and 15 mM, respectively. The control sample was made by using PBS to create 10% ethanol and then diluting this down to 5% ethanol with OVA solution to obtain the desired concentration. Imject Alum (alum; Thermo Scientific, Rockford, IL, USA) solution was prepared by mixing

1 μL of alum (40 μg/μL) in 100 μL of OVA solution according to the manufacturer’s protocol and finally diluting to 200 μL with PBS to obtain the desired concentration of 200 μg/mL. All immunizations were performed by intraperitoneal injection in a volume of 200 μL. To evaluate OVA-specific total IgG immune responses induced by pyriproxyfen, groups of 17 mice were immunized on Weeks 0, 3 and 6 with OVA in 5% ethanol (negative control), OVA containing alum (positive control) or pyriproxyfen (15 mM). Blood samples were collected from each mouse via the tail vein at 3, 5, 7 and 8 weeks. After collection, blood samples were centrifuged at 12,000 rpm for 15 min to obtain sera. The sera were heat-inactivated at 50°C for 30 min and kept at −20°C until use. Below is a brief description of detection by ELISA of OVA-specific total IgG immune responses in sera.