Developmental Toxicity Assessment of Piperonyl Butoxide Exposure Targeting Sonic Hedgehog Signaling and Forebrain and Face Morphogenesis in the Mouse: An in Vitro and in Vivo Study

Abstract

Background: Piperonyl butoxide (PBO) is a pesticide synergist used in residential, commercial, and agricultural settings. PBO was recently found to inhibit Sonic hedgehog (Shh) signaling, a key developmental regulatory pathway. Disruption of Shh signaling is linked to birth defects, including holoprosencephaly (HPE), a malformation of the forebrain and face thought to result from complex gene-environment interactions.

Objectives: The impact of PBO on Shh signaling in vitro and forebrain and face development in vivo was examined.

Methods: The influence of PBO on Shh pathway transduction was assayed in mouse and human cell lines. To examine its teratogenic potential, a single dose of PBO ($22-\mathrm{1,800}\mathrm{mg}/\mathrm{kg}$) was administered by oral gavage to $C57\text{BL}/6J$ mice at gestational day 7.75, targeting the critical period for HPE. Gene-environment interactions were investigated using $Sh{h}^{+/-}$ mice, which model human HPE-associated genetic mutations.

Results: PBO attenuated Shh signaling in vitro through a mechanism similar to that of the known teratogen cyclopamine. In utero PBO exposure caused characteristic HPE facial dysmorphology including dose-dependent midface hypoplasia and hypotelorism, with a lowest observable effect level of $67\mathrm{mg}/\mathrm{kg}$. Median forebrain deficiency characteristic of HPE was observed in severely affected animals, whereas all effective doses disrupted development of Shh-dependent transient forebrain structures that generate cortical interneurons. Normally silent heterozygous Shh null mutations exacerbated PBO teratogenicity at all doses tested, including $33\mathrm{mg}/\mathrm{kg}$.

Discussion: These findings demonstrate that prenatal PBO exposure can cause overt forebrain and face malformations or neurodevelopmental disruptions with subtle or no craniofacial dysmorphology in mice. By targeting Shh signaling as a sensitive mechanism of action and examining gene-environment interactions, this study defined a lowest observable effect level for PBO developmental toxicity in mice more than 30-fold lower than previously recognized. Human exposure to PBO and its potential contribution to etiologically complex birth defects should be rigorously examined. https://doi.org/10.1289/EHP5260.