Impact of environmental chemicals on lung development CDH is associated with various genetic syn- dromes, recent understanding of its etiology has been changing. Originally it was believed to be due to malformation in a portion of the diaphragm, which allowed compression of the developing lung by abdominal contents enter- ing the chest cavity, resulting in the subsequent characteristic hypoplastic lung. More recently, evidence has supported a “dual hit” theory which postulates that the original injury occurs early in lung development before and not con- nected to aberrant development of the dia- phragm (Keijzer et al. 2000). According to this explanation, the already hypoplastic developing lung is then further inhibited as a result of the mechanical compression on the ipsilateral side resulting from herniation of the intestines into the thoracic cavity. Exposure to nitrofen (2,4-dichlorophenyl- p-nitrophenyl ether), a banned pesticide, has been used as a model for CDH in rodents. Using this model, Leinwand et al. (2002) showed that the initial event in experimentally induced CDH is the development of hypo- plastic lungs, which occurred early in devel- opment before the closure of the diaphragm and herniation. Therefore, compression was not the initial cause of hypoplasia. Hypoplasia has also been observed after nitrofen expo- sure in animals without CDH (Cilley et al. 1997). Lungs of nitrofen-exposed pups had 30% fewer terminal bronchioles than did con- trols, and they were developmentally immature (Leinwand et al. 2002). Similar observations in human infants with CDH include hypoplastic lungs, fewer alveoli, thickened alveolar walls, increased pulmonary interstitial tissue, and less airspace, as well as fewer bronchioles and vas- cular branches (Gallot et al. 2005). The list of effects induced by nitrofen on various signaling pathways related to branch- ing morphogenesis and lung development has been rapidly increasing. Wnt (wingless signal- ing proteins) growth factor signaling has been shown to have a role in regulation of prolifera- tion, differentiation, and lineage specifica- tion during embryonic development. In the lung, Wnt7 inactivation results in decreased branching and subsequent hypoplasia, as well as decreases in smooth muscle (Pongracz and Stockley 2006; Shu et al. 2002; Wang et al. 2005). Wnt signaling is an upstream regulator of bone morphogenetic protein 4 (BMP4) and FGF, both important in lung development. Wnt7-null mice die at birth from severe lung hypoplasia. In mice treated with nitrofen, GATA-6 (an upstream acti- vator of Wnt7b) Wnt7b, Wnt2, and BMP4, were down-regulated (Takayasu et al. 2007a). GATA-6, a zinc finger transcription protein, is an important regulator of distal epithelial cell differentiation, as well as proximal air- way development (Yang et al. 2002). Prenatal RA partially mitigated the actions of prenatal nitrofen exposure in nitrofen-induced CDH rats (Montedonico et al. 2006, 2008). Vitamin A–deficient diets have been linked to CDH in animal studies (Andersen 1941), and a small human epidemiologic study found lower levels of retinol (the active metabolite of vitamin A) in newborns with CDH than in controls (Major et al. 1998). Knockout mice deficient in RA nuclear recep- tors had an increased incidence of a spectrum of pulmonary agenesis, hypoplasia, and CDH (Mendelsohn et al. 1994). Nitrofen has been shown to disturb the RA signaling pathway at an early stage of lung development (Nakazawa et al. 2007a), and the incidence of CDH was dramatically reduced when RA was given along with nitrofen during pregnancy in rats (Babiuk et al. 2004). In rat lung explants, RA significantly increased the growth, number of lung buds, and lung area of nitrofen-induced hypoplastic lungs but had no effect on con- trols (Montedonico et al. 2006). One possible mechanism of nitrofen’s action on retinol may be to interfere with its cellular uptake during lung morphogenesis (Nakazawa et al. 2007b). Previous studies have suggested activity by inhibiting retinal dehydrogenase 2 (RALDH2), a key enzyme for generation of RA from retinal. Four chemicals shown to be able to precipitate development of CDH in animal models (nitro- fen, bisdiamine, 4-diphenyl carboxylic acid, and SB210661) all have been found to inhibit RALDH2 (Mey et al. 2003). One of the key processes in later ges- tation is the differentiation of a portion of alveolar type II cells to alveolar type I cells. That process was impaired in the nitrofen- induced CDH lung (Takayasu et al. 2007b). Although it appears that the immediate cause of decreased differentiation into type I cells was mechanical compression, this can be considered a secondary effect of the chemical exposure and was not observed in the absence of nitrofen exposure. hTyroid- disrupting chemicals and branch- ing morphogenesis. Thyroid hormone is important for normal lung development (Van Tuyl et al. 2004). For example, alveolar septa- tion, a largely postnatal structural manifes- tation, was impaired in hypothyroid mouse pups (Van Tuyl et al. 2004). The ratio of surfactant protein mRNA expression to that of corresponding proteins was influenced by both prenatal and postnatal thyroid hormone deficiency. A reduced ratio is indicative of an immature lung. Nitrofen is a diphenyl ether and, like related chemicals, has antithyroid activity (Brandsma et al. 1994), inhibiting triiodo- thyronine (T3) receptor binding. Little research has been published on the effect of other environmental chemicals with anti- thyroid activity on lung development. Dioxins, polychlorinated biphenyls (PCBs), and Environmental Health Perspectives • volume 118 | number 8 | August 2010 polybrominated diphenyl ethers are structurally similar to nitrofen and have known antithyroid activity. A significant increase in the incidence of “bronchitis” was noted in a Taiwanese cohort of children exposed to PCBs prenatally (Rogan et al. 1988), which may involve PCB impairment of immune function and/or lung development. In an epidemiologic study in the Netherlands, ten Tusscher et al. (2001) found prenatal/lactational exposure to dioxins to be related to a significant reduction in lung function in 41 healthy children between 7 and 12 years of age. Gestational exposure in rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) resulted in up-regulation of aryl hydrocarbon receptor (AhR) signaling in the developing lung and delayed lung development as evi- denced by decreased total lung airspace and increased septal area (Kransler et al. 2009). These hypoplastic changes in lung morphology were associated with functional differences in respiratory mechanics. The study suggests that AhR activation adversely affects lung develop- ment. Up-regulation of AhR activity results in decreases in thyroid hormone due to increased metabolism. Thus, decreased thyroid hormone may play a role in these findings. We identified no other studies that exam- ined developmental exposure to thyroid- disrupting environmental chemicals and lung function. Lung function studies are a sensi- tive, minimally invasive method of measuring impact on lung development. Measurements of lung function in experimental animal pups would provide additional useful information on adverse lung impacts in developmental tox- icity studies. In addition, more epidemiologic studies evaluating lung function in people exposed prenatally and postnatally to thyroid- hormone–disrupting chemicals are needed. Examples of disruption of branching mor- phogenesis by environmental chemicals in other organ systems. Branching morphogenesis is an essential developmental process for many organs that use common signaling pathways. One of the organs that exhibit branching morphogenesis is the salivary gland. TCDD exposure of cultured murine submandibular glands reduced epidermal growth factor sig- naling. This aryl hydrocarbon–associated effect inhibited branching morphogenesis and resulted in smaller glands with enlarged buds (Kiukkonen et al. 2006). This effect was seen in cells cultured at gestational day (GD) 13 but not later in development, indicating a spe- cific window of susceptibility. Development of the prostate in the mouse is also inhibited by exposure to TCDD during pregnancy and lactation. Exposure on GD13 to a single dose resulted in reduced organ weight related to an inhibition of development of prostatic epi- thelial buds (Ko et al. 2002). Inhibition of prostatic bud development by TCDD is not androgen dependent (Lin et al. 2004). 1159