Miller and Marty TCDD exposure during a critical window of gestation produced disruption of branching morphogenesis in the rat mammary gland, resulting in smaller glands, limited branch- ing, decreased numbers of terminal end buds, and a lack of substantial alveolar lob- ule development independent of hormonal effects (Fenton et al. 2002; Vorderstrasse et al. 2004). This effect was dependent on the time of exposure: It was observed after exposure on GD15 but not GD20, and persisted at least until postnatal day 45 (postpubertal) (Fenton et al. 2002). Retinoids are involved in embryonic kid- ney patterning and development, including branching morphogenesis, and are impor- tant in kidney development. The Ret receptor modulates ureteric bud branching morpho- genesis (Gilbert 2002). Nitrofen expo- sure resulted in hypoplastic kidneys in rats (Montedonico et al. 2007). Exposure of rats in utero to the herbi- cide atrazine has been associated with altered branching morphogenesis in the mam- mary gland, although little detail is known of the exact mechanism of action (Rayner et al. 2005). Similarly, the mammary glands of offspring exposed during GD17–GD19 displayed delay in development of mature gland structures and less epithelial branching. Importantly, the offspring of atrazine-treated dams were unable to provide adequate nutri- tional support for the F 2 offspring, resulting in decreased pup weight gain. Clearly, the impact of exposure during critical windows of development to environ- mental chemicals that disrupt branching mor- phogenesis has been demonstrated in various organ systems dependent on this process. Chemicals that inhibit this process in one organ should be investigated for similar effects on lung development. Nicotine and alveolarization. The glyco- lytic pathway is very active during the alveo- lar phase of lung development and provides energy and precursors to the lung. Maternal exposure to nicotine in the rat resulted in sustained or permanent suppression of gly- colysis and glycogenolysis in the lung tis- sue of the pup (Maritz 1986, 1988), due to reduced synthesis of phosphorylase and phosphofructokinase (the rate-limiting step in glycolysis) in nicotine-exposed animals (Kordom 2004; Kordom et al. 2002; Maritz 2004). As a result of inhibited glycolysis, Na+/ K+-ATPase is inhibited, which may result in swelling and bleb formation of alveolar type I cells (reviewed by Maritz 2008). In rats, maternal dosing with nicotine dur- ing pregnancy and lactation, at doses that did not alter fetal growth, resulted in a significant decrease in number of alveoli and increase in the alveolar volume at maturity in nicotine-ex- posed pups relative to controls (Maritz 2002; 1160 Maritz and Windvogel 2003). The increase in alveolar volume in nicotine-exposed pups was attributed to slower alveolar septal for- mation, flattening of the alveoli as they aged, and disappearance of alveolar walls leading to larger alveoli. The histopathology of nico- tine-exposed animals resembled early emphy- sema (Maritz and Windvogel 2003). Routine pathologic examination of these lungs would likely fail to demonstrate these relatively subtle changes. Such irreversible changes could result in dysfunction (emphysema) in later life. Xenobiotics and cell differentiation and repair. In the rabbit, Clara cells actively dif- ferentiate postnatally, reaching maturity at 4 weeks. Despite lower levels of CYP450 activ- ity in immature neonatal Clara cells, these cells were more susceptible to injury at lower doses of 4-ipomeanol than adult cells (Plopper et al. 1994). Smiley-Jewell et al. (2000) treated rab- bits with ipomeanol during three postnatal time periods that represent early, intermediate, and final stages of Clara cell maturation. In rabbits treated with a single dose of 4-ipo- meanol during early or middle periods of dif- ferentiation (up to 9 days of age), bronchiolar cell differentiation and repair were inhibited. The period of greatest susceptibility was during mid-differentiation at 7 days of age. On exam- ination at 6 weeks, rabbits treated at day 7 had more squamous cells, fewer ciliated cells, reduced expression of Clara cell markers, and undifferentiated cuboidal cell ultrastructure compared with those treated at adulthood. In those treated at later stages of development (21 days), repair was completed during this time period, and the bronchiolar epithelium was indistinguishable from controls at 6 weeks (Smiley-Jewell et al. 2000). Thus, the extent of repair was related to the stage of cellular differentiation at the time of injury but inde- pendent of the degree of injury (Smiley-Jewell et al. 1998, 2000). Exposure during the criti- cal window of early development dispropor- tionately inhibited differentiation and repair of bronchiolar Clara cells, resulting in persistent, possibly permanent effects. Similarly, naphthalene, another bio- activated Clara cell toxicant, has been found to induce injury at much lower doses in immature mice (Fanucchi et al. 1997). 1-Ni- tronaphthalene, an atmospherically formed nitroaromatic, more severely affects immature than mature Clara cells in both mice and rats (Fanucchi et al. 2004). These findings suggest that Clara cell injury by bioactivated chemicals in the devel- oping animal is not necessarily predicted by the level of CYP450 enzyme activity. There is no reason to believe this is restricted to mice, rats, and rabbits. Susceptibility of the neonate to injury cannot be predicted by the findings in adults. Early-life injury may be a precursor to dysfunction later in life. Widespread Environmental Chemicals That Disrupt Lung Development Ozone. Lung development occurs not only prenatally but through childhood, and lung function grows at least through adolescence. Therefore, exposure to environmental toxi- cants may affect underlying structural and functional aspects of the lung during a wide window. This may increase susceptibility to asthma as well as other diseases. An elegant series of studies conducted at University of California–Davis evaluated the impact of ozone (O3) and allergens on lung development in rhesus monkeys. These studies integrated early-life exposures through multiple windows of susceptibility to observe functional and structural changes relevant to human lung development and lung disease. Tran et al. (2004a) described conducting airway growth studies in monkeys, with airways doubling in length and increasing by 33% in diame- ter between 1 and 6 months of age. Postnatal exposure to O3, alone or combined with house dust mite antigen (HDMA), resulted in changes in bronchiolar growth patterns, inhibiting growth in diameter and promoting growth in length (Figure 3), as well as reduc- ing the number of conducting airway branches (by as many as six generations) (Fanucchi et al. 2006; Plopper et al. 2007). These changes appeared to be permanent because they per- sisted after 6 months of recovery. In monkeys, the smooth muscle bundles grow along with the conducting airways and have characteristic alterations depending on location and age (Tran et al. 2004a). Muscle bundles progress with age from a primarily perpendicular orientation relative to the long axis of the airway in early development to an orientation with a large percentage of bun- dles > 30° from perpendicular. This process is disrupted by exposure to HDMA or O3 in rhesus monkeys (Fanucchi et al. 2006; Tran et al. 2004b). The smooth muscle bundles were thickened, and their orientation rela- tive to the airway in the respiratory bronchi- oles was altered by exposure to O3 such that a much higher percentage of the bundles were < 15° perpendicular to the long axis of the bronchiole, an alteration that may increase air- way hyperreactivity (Plopper et al. 2007). The changes noted above from O3 exposure were not associated with changes in lung volume or function as measured by routine testing. The effect of the structural changes found after exposure to HDMA and O3 is consistent with alterations in airflow and resistance found in asthmatics (James and Carroll 2000; Plopper et al. 2007; Tran et al. 2004b). Epidemiologic evidence suggests that exposure to O3 is asso- ciated with asthma induction in children (McConnell et al. 2002) and reduced lung ovlume 118 | number 8 | August 2010 • Environmental Health Perspectives