Small molecule screening identified three compounds with demonstrable human exposure as Hh signaling inhibitors, which appear to act downstream of Ptc1 and upstream of Gli1 with variably weaker potency than the index Hh inhibitor cyclopamine. The screening approach and execution appears to have been independently validated by a recent report examining the effect of exogenous 17-β-estradiol exposure on chondrocranial development in the zebrafish (Fushimi et al., 2009
). The authors demonstrated that exposure above 5 μM caused craniofacial dysmorphia grossly mimicking that of both shh-
mutant and cyclopamine-exposed zebrafish (Wada et al., 2005
). Exposure to 20 μM 17-β-estradiol caused a reduction in ptc1
expression while not affecting the expression of shh.
That these effects were not reversed by an estrogen receptor antagonist suggests an independence from nuclear estrogen receptor signaling and, along with the data presented herein, argues that 17-β-estradiol acts as a direct Hh pathway antagonist.
Each of the three compounds identified here has recognized routes for human exposure but none has been linked to human birth defects consistent with Hh signaling inhibition. One animal study investigating the teratogenic potential of tolnaftate, a synthetic antifungal agent, was negative (Noguchi et al., 1966
). Treatment of pregnant mice and rats by subcutaneous injection of 1,000 mg/kg/d tolnaftate from E8.5 to 13.5 (where morning of vaginal plug = E0), did not cause a significant incidence of fetal abnormalities. No report of tolnaftate teratogenicity in humans has been published.
Ipriflavone is a synthetic isoflavone of worldwide use as a supplement for its touted anabolic and bone density building properties. Several studies have examined in vivo
pharmacokinetics of ipriflavone in animal models as well as humans. In rats, the bioavailability of ipriflavone was approximately 24% (Kim et al., 2002
). In healthy human volunteers, ipriflavone exhibited a half-life of 10-12 hrs (Ma et al., 1997
). To date, no studies examining the teratogenic potential of ipriflavone in humans or animal models have been published.
17-β-estradiol, the most significant human estrogen, plays critical roles in sexual development, reproduction, and other biological effects. Serum levels fluctuate during the normal menstrual cycle, generally remaining below 25 nM. Circulating levels rise during pregnancy but generally remain under 100 nM, significantly lower than our observed in vitro EC50 concentration for Hh signaling inhibition of 2.75 μM. Exogenous 17-β-estradiol is also administered for several clinical purposes including hormonal supplementation, birth control, and prevention of osteoporosis; however, levels achieved by exogenous administration are generally within the physiologic range.
We find it unlikely, under normal circumstances, that humans are exposed to inhibitory concentrations of any one of these compounds in isolation. Indeed, epidemiological studies would likely have identified an environmental chemical with high potency and teratogenic potential. However, this does not exclude a role of these compounds or other yet to be identified environmental Hh inhibitors as contributors to etiologically-complex birth defects like CLP and HPE. The small molecule libraries sampled here represent only a small fraction of the total number of compounds with relevant human exposure and it is likely that other, more potent inhibitors exist in the environment. Moreover, Hh signaling antagonists can produce additive inhibitory effects (Lipinski et al., 2007
A particularly noteworthy aspect of HPE etiopathogenesis in relation to this line of study is the presence of heterozygous mutations in several Hh pathway genes in both affected probands as well as clinically normal obligate carriers (Nanni et al., 1999
; Roessler et al. 2003
). This variable phenotypic penetrance suggests an interaction of multiple genetic lesions or single lesions and environmental or chemical insults. That three of 4,240 evaluated compounds exhibited specific Hh pathway inhibitory activity, demonstrates the potential for Hh pathway perturbation as a result of environmental exposure. These findings justify additional, more comprehensive searches for Hh inhibitors, as well as studies examining the interaction of such chemical insults in the context of identified genetic predispositions.