Dipentyl phthalate induces multinucleated germ cells and reduces testosterone in the rat fetal testis with a similar dose-response

Phthalic acid esters (phthalates) are male reproductive toxicants that exert their most potent toxicity during a window of sensitivity in fetal development. In the fetal rat, exposure to phthalates reduces testosterone biosynthesis, alters the development of seminiferous cords, and induces the formation of abnormal multinucleated germ cells (MNGs). While human and mouse fetal testes may be less sensitive to the anti-androgenic effects of phthalates than the rat, our previous work demonstrated that phthalates with medium-length side chains cause both induction of MNGs and reduction of testosterone in the rat fetal testis. In this experiment, we sought to compare the relative sensitivity of testosterone reduction and MNG induction as markers of phthalate toxicity, using dipentyl phthalate (DPeP), a known toxic phthalate, and mono-(2-ethylhexyl) tetrabromophthalate (TBPH), a suspected toxic phthalate. We also sought to develop an improved image analysis method for identification of MNGs in histological sections using a neural network approach. Timed pregnant Sprague Dawley rats were exposed to 1-100 mg/kg/d DPeP, 62.5-500 mg/kg/d TBPH, or corn oil vehicle, by daily oral gavage from gestation day 16-20. Fetal testes were isolated on GD 20 for analysis of testosterone production and quantification of MNGs in histological sections. We found that DPeP induced MNGs and decreased testosterone production with similar dose response. TBPH, conversely, did not exert any toxic effect at any dose up to 500 mg/kg/d. For image analysis, we trained a convolutional neural network with a U-Net architecture using hand-labeled images with human-identified MNGs, to identify MNGs on unlabeled images. With hand-labeled images not used in model training, we assessed the performance of the model, finding it comparable to a human. We conclude that induction of MNGs is a dose-dependent marker of DPeP exposure, with sensitivity similar to testosterone production, which may be generalizable to other phthalates. The use of automated image analysis will allow data on this histopathological endpoint to be more readily collected for analysis of phthalate toxicity. Abstract does not necessarily reflect USEPA policy.

Impact/Purpose

Some of the most commonly occurring birth defects clinically identified in humans are malformations of the male reproductive tract, including hypospadias and cryptorchidism. In utero exposure to environmental chemicals has been implicated as a potential causal factor because these effects can be recapitulated in experimental in vivo studies. Particularly, reduction of fetal testis testosterone production is induced by a range of pesticides and phthalates and is a key event in the Adverse Outcome Pathway for male reproductive effects. This research is a collaborative effort between EPA researchers and investigators at Brown University on the AOP for male reproductive effects from in utero exposure to environmental chemicals, including phthalates.

Citation

Spade, D., J. Conley, C. Lambright, S. Bell, A. Zsom, AND L. Gray. Dipentyl phthalate induces multinucleated germ cells and reduces testosterone in the rat fetal testis with a similar dose-response. Society of Toxicology 2020 Annual Meeting, Anaheim, CA, March 15 - 19, 2020.