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 (22–1,800mg/kg) was administered by oral gavage to C57BL/6J mice at gestational day 7.75, targeting the critical period for HPE. Gene–environment interactions were investigated using Shh+/− 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 67mg/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 33mg/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

Polychlorinated Biphenyls (PCBs): Risk Factors for Autism Spectrum Disorder?

“…The utility of animal models in this context is elegantly illustrated in studies of mice harboring a single allele mutation in the sonic hedgehog (Shh) gene, which is typically silent. Relative to wild-type controls, Shh mutant mice were found to exhibit enhanced sensitivity to the teratogenic effects of the pesticide synergist piperonyl butoxide [188]. Similarly, point mutations in RYR, which are silent under normal physiologic conditions, confer susceptibility to malignant hyperthermia (MH) triggered by exposure to halogenated anesthetics [74].…”

Multifactorial Genetic and Environmental Hedgehog Pathway Disruption Sensitizes Embryos to Alcohol‐Induced Craniofacial Defects

“…Embryos were exposed from 6 to 24 hpf. This period of exposure was chosen to include the period of susceptibility for alcohol‐ and PBO‐induced HPE‐associated midfacial defects described in mice (Everson et al, 2019; Kietzman et al, 2014). At the end of the dosing period (24 hpf), the drug and embryo medium were removed and replaced with new embryo media.…”

“…It inhibits an insect’s cytochrome P450 detoxification enzymes, thereby augmenting the pesticidal activities of the active ingredient(s) ( e.g., pyrethroid pesticides). While PBO use is widespread, the potent developmental hazard of PBO was only recognized recently with the discovery that PBO can block Hh signaling (Everson et al, 2019; Horton et al, 2011; Wang et al, 2012).…”

Gene-environment interactions: aligning birth defects research with complex etiology

“…In mice, loss of both Shh alleles results in severe HPE, whereas heterozygous mice are indistinguishable from wild-type littermates. However, studies have shown that these 'silent' mutations dramatically exacerbate the teratogenicity of environmental chemicals, including ethanol and piperonyl butoxide (Everson et al, 2019;Kietzman et al, 2014). Similar experiments have shown additive or synergistic interactions between additional gene-environment pairs including Cdon and ethanol (Hong and Krauss, 2012), Gli2 and ethanol (Kietzman et al, 2014), and Gli2 and the synthetic smoothened antagonist vismodegib (Heyne et al, 2016).…”

“…Following the discovery of cyclopamine, numerous small molecules have been identified as pathway inhibitors acting through the same smoothenedtargeted mechanism (Pietrobono and Stecca, 2018;Rimkus et al, 2016). Such small molecules include diverse environmental chemicals, such as phytocannabinoids (Khaliullina et al, 2015) and a widely used pesticide synergist, piperonyl butoxide (Everson et al, 2019). Alcohol (ethanol), a known human teratogen, can also be added to this mix and has been suggested to act on multiple factors upstream of and within the Shh signaling cascade to disrupt development of the face and brain (Sulik, 2005).…”