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

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.

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.

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 (22-1,800mg/kg22−1,800mg/kg<math><mrow><mn>22</mn><mo>-</mo><mn>1,800</mn><mspace></mspace><mi>mg</mi><mo>/</mo><mi>kg</mi></mrow></math>) was administered by oral gavage to C57BL/6JC57BL/6J<math><mrow><mi>C</mi><mn>57</mn><mtext>BL</mtext><mo>/</mo><mn>6</mn><mi>J</mi></mrow></math> mice at gestational day 7.75, targeting the critical period for HPE. Gene-environment interactions were investigated using Shh+/-Shh+/−<math><mrow><mi>S</mi><mi>h</mi><msup><mi>h</mi><mrow><mo>+</mo><mo>/</mo><mo>-</mo></mrow></msup></mrow></math> mice, which model human HPE-associated genetic mutations.

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 67mg/kg67mg/kg<math><mrow><mn>67</mn><mspace></mspace><mi>mg</mi><mo>/</mo><mi>kg</mi></mrow></math>. 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 33mg/kg33mg/kg<math><mrow><mn>33</mn><mspace></mspace><mi>mg</mi><mo>/</mo><mi>kg</mi></mrow></math>.