P-081 In utero exposure to diisopentyl phthalate (DiPeP) disrupts the expression of steroidogenesis-related genes and induces multinucleated gonocytes in the fetal rat testis

Abstract

Study question Can the plasticizer diisopentyl phthalate (DiPeP) disrupt testosterone biosynthesis and alter fetal rat testis histology similarly to other common toxic phthalates? Summary answer DiPeP can dose-dependently suppress mRNA levels of steroidogenic genes and induce multinucleated gonocytes in the fetal rat testis, resembling the effects of other toxic phthalates. What is known already DiPeP is an uncommon phthalate, whose metabolites have been ubiquitously detected in urinary samples from Brazilian pregnant women and children. These results, which contrast with the undetected levels of DiPeP metabolites in other human biomonitoring studies worldwide, led us to investigate DiPeP reproductive and developmental toxicity in rats. Our preliminary data indicate that DiPeP is a potent antiandrogenic phthalate, but no studies have been conducted to assess the impact of DiPeP exposures on the incidence of multinucleated gonocytes in the rat fetal testis, a common feature of the rat phthalate syndrome. Study design, size, duration Pregnant Wistar rats (n = 7-9 dams/group) were treated orally (gavage) with DiPeP 0 (control), 11, 33, and 100 mg/kg/day between gestation days 14-20. Dose levels were based on prior rat phthalate toxicity studies and canola oil was used as a vehicle. On gestation day 20, dams were euthanized and up to three male fetuses from each litter were removed for the collection of fetal testes and the assessment of the study endpoints. Participants/materials, setting, methods Fetal rat testes were used for enzyme immunoassay quantification of testosterone production following ex vivo incubation of testes in culture media, analysis of mRNA levels of steroidogenic genes and insulin-like factor 3 (Insl3) by quantitative polymerase chain reaction (qPCR), and histological assessment of multinucleated gonocytes by optical microscopy. Main results and the role of chance No signs of systemic toxicity were observed as indicated by the lack of alterations in maternal body weights and the unchanged number, viability, and weight of fetuses. There were no significant changes in the ex vivo testosterone production at any DiPeP dose. On the other hand, DiPeP induced significant reductions in the mRNA expression of key steroidogenic genes, including suppressed mRNA levels of the steroidogenic acute regulatory protein (Star) and cytochrome P450 family 11 subfamily A member 1 (Cyp11a1) at 100 mg/kg/day and of cytochrome P450 family 17 subfamily A member 1 (Cyp17a1) at 33 and 100 mg/kg/day. Gene expression of Insl3, which is essential for the process of testis descent, was reduced at the highest dose by 58% in relation to control, but this change was not significant (p = 0.07). The number of multinucleated gonocytes, corrected for the number of testicular cord sections or the total area of analyzed testicular cords was significantly increased in the groups exposed to 33 and 100 mg/kg/day. Chance is an unlikely explanation for our results considering the observed dose-dependent responses and the consistency of our findings with prior phthalate reproductive toxicity data. Limitations, reasons for caution The tested doses are higher than the expected human exposure levels and animal-to-human extrapolation should be done with caution. Wider implications of the findings DiPeP suppresses the expression of steroidogenic genes and induces multinucleated gonocytes in the fetal rat testis. These endpoints seem more sensitive than inhibition of ex vivo testosterone production. Although human exposure levels are lower than the doses tested here, DiPeP can potentially act cumulatively with chemicals that share similar mechanisms. Trial registration number not applicable