Magister Scientiae (Medical Bioscience) - MSc(MBS) / Nicotine exposure to the fetus through tobacco smoking or nicotine replacement therapy during the whole period of gestation and lactation causes diverse effects on fetal and neonatal lung development, integrity and maturation which compromise the gas exchange function of the lungs and renders this vital organ susceptible to gradual damage and different diseases in latter life. Maternal nicotine exposure during gestation and lactation results in gradual destruction of the lung parenchyma, and this leads to the combination of many small air sacs in one bigger alveoli which is a sign of emphysema. Many researchers speculated that the way in which, nicotine causes emphysema and other damage, is by inducing the formation of many reactive oxygen species (ROS), and creating an imbalance between the oxidants and the antioxidants of the body, which is termed oxidative stress. The aim of this study was to assess the effects of nicotine exposure on the lung of the fetal and neonate rat during gestation and lactation as gas exchanger, and also to see whether the supplementation of tomato juice containing lycopene, a powerful carotenoid antioxidant could protect the lungs against these effects of maternal nicotine exposure. In this study pregnant rats have been divided into 4 groups: a group which received nicotine (1mg/kg body weight/day) subcutaneously, a group that received the tomato juice only (6mg/kg body weight/day per os), a third group that received the combination of tomato juice ( 6mg /kg body weight/ day per os) and nicotine (1mg/kg body weight /day subcutaneously ) . The control group that received saline (1mg/kg body weight /day) subcutaneously and water. The injections were done during pregnancy and lactation until weaning at postnatal day 21. The results showed that maternal nicotine exposure during gestation and lactation leads to a gradual damage of the lung parenchyma and slower formation of the alveoli during the equilibrated phase of the lung growth leading to a decrease in the internal surface area required for gas exchange. Supplementation with tomato juice during gestation and lactation prevents all the adverse effects of maternal nicotine exposure on the lungs of the offspring. Since nicotine induce an increase in the oxidant levels of the mother and the fetus, my results imply that lycopene protected the lungs of the offsprings against the oxidants and thus against changes in the program that controls lung development as the animals age. This is supported by the observation that at postnatal day 84 the antioxidant. / South Africa

Philosophiae Doctor - PhD / exposed to nicotine through the blood and the milk of the mother. Nicotine is thus expected to interact with the developing fetus and the offspring of mothers who smoke or use NRT for smoking cessation, resulting in the interference with normal fetal and neonatal lung development. Maternal cigarette smoke or nicotine exposure produces adverse effects in the lungs of offspring, these include; intrauterine growth retardation, low birth weight, premature birth, reduced pulmonary function at birth, and a high occurrence of respiratory illnesses after birth. This study aimed at investigating the effects of maternal nicotine exposure during gestation and lactation on lung development in the offspring; to establish whether tomato juice can have protective effects on the fetal lung development and function in the offspring; and to determine if nicotine cases premature aging of the lungs of the offspring. It was therefore shown that maternal exposure to nicotine during gestation and lactation ad no significant effect on the growth parameters of the offspring. Maternal nicotine exposure during gestation and lactation had no effect on the growth parameters of the offspring, but resulted in compromised lung structure and function. The morphometric results demonstrated decrease in alveolar number, increase in alveolar size, and decrease in lung parenchyma of the nicotine exposed animals showing a gradual deterioration of the lung parenchyma. Structural alterations include emphysematous lesions, where the latter was accompanied by an increase in alveolar size (Lm), and a decrease in the tissue volume of the lung parenchyma. Thickening of alveolar walls was also evident and serves as an indication of remodeling of the extracellular matrix, also a characteristic of emphysema. A consequence of the gradual deterioration of the lung parenchyma is a decrease in the alveolar surface area available for gas exchange. The present study showed that the emphysematous lesions were conceivably a result of a reduced rate of cell proliferation accompanied by the increase in senescent cells numbers in the alveolar walls of the exposed offspring. The data of this study suggests that maternal nicotine exposure during gestation and lactation induces premature aging of the lungs of the offspring rendering the lungs of the offspring more susceptible to disease later in life. Since these structural changes occurred later in the life of the offspring and long after nicotine withdrawal, it is suggested that it is programmed during gestation and lactation. Smoking and NRT result in an increased load of oxidants in the mother and fetus. It also reduces the level of anti-oxidants and thereby compromising the ability of the mother to protect the fetus. It is hypothesized that this oxidant-antioxidant imbalance will program the lungs to age prematurely. The supplementation of the mother’s diet with tomato juice, rich in lycopene, other anti-oxidants such as vitamin C, as well as phytonutrients protected the lungs of the offspring against the adverse effects of maternal nicotine exposure. This supports the hypothesis mentioned above. The study further showed that the effects of grand-maternal nicotine exposure during gestation and lactation on the lungs of the F1 offspring is also transferred to the F2 offspring. This is most likely via the paternal and maternal germ line. Since tomato juice supplementation of the mother’s diet with tomato juice prevented the adverse effects of maternal nicotine exposure on the lungs of the offspring, it is conceivable that it will prevent transfer of these changes to the F2 generation. / South Africa

>Magister Scientiae - MSc / Tobacco consumption and exposure to tobacco smoke is one of the biggest contributing factors to a growing epidemic of non-communicable diseases (NCDs), primarily cancers, diabetes, cardiovascular and chronic lung diseases which account for 63% of all deaths worldwide (WHO, 2011). An increased concern is in pregnant women who smoke. They not only expose themselves to nicotine, but also their unborn child. Cigarette smoking during pregnancy is associated with many developmental and growth complications. There are critical periods within the “program” that directs normal growth and development, during which the fetus is vulnerable to the effects of external factors. During these critical periods of development the program can be changed to increase the susceptibility of the fetal organs to disease and increased risk of adverse health consequences in adulthood. Health care professionals have tried to reduce the consumption of tobacco smoke by prescribing nicotine replacement therapy (NRT) to pregnant females as an alternative to smoking, without considering the effects of nicotine on the developing embryo and the health risk that might arise after birth. It is known that nicotine induces oxidant formation with resulting oxidative effects. This induces an overload of oxidants in the fetus and a decrease in the antioxidant capacity thereof. This may interfere with normal lung development.

Magister Scientiae (Medical Bioscience) - MSc(MBS) / Cigarette smoking is one of the foremost causes of chronic obstructive pulmonary diseases such as emphysema and chronic bronchitis, and although it is the most preventable causes of death, it accounts for approximately 6 million deaths worldwide each year. Cigarette smoking during pregnancy and lactation remains one of the primary modifiable risk factors for undesirable fetal, obstetrical, and developmental outcomes. Consequently, the offspring of the smoking mother is exposed to nicotine via the blood and the milk of the mother. As a result, nicotine interacts with the developing offspring and therefore interferes with normal fetal lung development. Maternal smoking during gestation and lactation has been associated with both short and long term health risks ranging from intrauterine growth restriction to physiological abnormalities. Maternal smoking has also been strongly linked to an increased risk for pulmonary diseases and respiratory morbidity in the offspring of the smoking mother. The main objectives of this study were to determine the effects of maternal nicotine exposure during gestation and lactation on the alveolar wall composition during lung development in the offspring; if maternal nicotine exposure during gestation and lactation induces premature cellular senescence in the lungs of the offspring; to clarify the role of pulmonary fibroblasts in premature senescence; and to establish whether tomato juice supplementation will prevent premature aging in the lungs of rats that were exposed to nicotine via the placenta and mother’s milk. From the data generated in this study it was evident that maternal nicotine exposure during gestation and lactation compromises the gas exchange function of the lungs of the F1 offspring. This was prevented by supplementing the mother’s diet with tomato juice which is then received by the offspring via the placenta and mother’s milk. This is conceivably achieved by maintaining the oxidant-anti-oxidant ratio of the mother and of the developing fetus and neonate, thereby averting premature senescence caused by nicotine exposure. Moreover, the present study also demonstrates that a decrease in fibroblast density is associated with emphysematous-like lesions in the lungs of the nicotine exposed F1 progeny. Since pulmonary fibroblasts are chief contributors to the extracellular matrix of the lungs, involved in alveolar multiplication and regeneration; premature aging or cessation of the metabolically active fibroblasts largely contributes to diminished lung structure and function.

Alveolar fluid clearance is driven by vectorial Na+ transport and promotes postnatal lung adaptation. The effect of insulin on alveolar epithelial Na+ transport was studied in isolated alveolar cells from 18–19-day gestational age rat fetuses. Equivalent short-circuit currents (ISC) were measured in Ussing chambers and different kinase inhibitors were used to determine the pathway of insulin stimulation. In Western Blot measurements the activation of mediators stimulated by insulin was analyzed. The ISC showed a fast dose-dependent increase by insulin, which could be attributed to an increased ENaC (epithelial Na+ channel) activity in experiments with permeabilized apical or basolateral membrane. 5-(N-Ethyl-N-isopropyl)amiloride inhibition of ISC was not affected, however, benzamil-sensitive ISC was increased in insulin-stimulated monolayers. The application of LY-294002 and Akti1/2 both completely blocked the stimulating effect of insulin on ISC. PP242 partly blocked the effect of insulin, whereas Rapamycin evoked no inhibition. Western Blot measurements revealed an increased phosphorylation of AKT after insulin stimulation. SGK1 activity was also increased by insulin as shown by Western Blot of pNDRG1. However, in Ussing chamber measurements, GSK650394, an inhibitor of SGK1 did not prevent the increase in ISC induced by insulin. The application of IGF-1 mimicked the effect of insulin and increased the ENaC activity. In addition, an increased autophosphorylation of the IGF-1R/IR was observed after insulin stimulation. We conclude that insulin rapidly increases epithelial Na+ transport by enhancing the activity of endogenous ENaC through activation of PI3K/AKT in alveolar cells.

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Tumorigenesis is often caused by the dysregulation of developmental pathways that are activated during repair, a process that recapitulates development. The Hedgehog (Hh) pathway is a signalling pathway essential for cell patterning and identity during embryogenesis. Activation of Hh signalling has been reported in small cell lung cancer progression, but the role of the Hh receptor, Patched1 (Ptch1), remains poorly understood. Therefore, it is imperative that we understand how Ptch1 is involved in development and tissue repair in order to understand its roles in cancer. This project aimed to study the role of Ptch1 during the branching process of lung development and in the regeneration of airway epithelial cells. A conditional knockout approach was utilised to excise Ptch1 by crossing Ptch1 conditional mice with Dermo1-Cre mice (Dermo1Cre+/-;Ptch1lox/lox), thereby activating the Hh pathway in the mesenchyme, independent of ligand. Dermo1Cre+/-;Ptch1lox/lox embryos died at E12.0 and showed secondary lung branching arrest leading to lobe formation defects. Expression of Ptch1, Gli1 and Foxf1 were shown to be upregulated in both proximal and distal lung mesenchyme, indicating inappropriate pathway activation and disruption of the Hh gradient. Fgf10 expression was spatially reduced in Dermo1Cre+/-;Ptch1lox/lox lungs and the addition of Fgf10 to these lungs in culture showed partial restoration of branching, thus Hh signalling was shown to regulate branching via Fgf10. Due to the patterning defect associated with our in vivo model, we took an in vitro approach to delete Ptch1 in lung explants cultures. This also showed reduced branching and validated that mesenchymal proliferation was enhanced after Ptch1 deletion, consistent with the previously reported role of Hh signalling in mesenchymal cell survival. Small cell lung cancer originates in the proximal lung and has been linked to aberrant repair processes. Therefore, Hh signalling in proximal airway repair was investigated. Ptch1 expressing cells were detected in the bronchial epithelium and stroma during homeostasis. But these cells were not detected following polidocanol-induced injury in the murine nasal septum and lung. However during naphthalene-induced repair, Ptch1 expressing cells were detected in the regenerating bronchial epithelium, suggesting that Hh dependent progenitors respond specifically to naphthalene-induced damage and perhaps are pulmonary neuroendocrine or variant Clara cells. Therefore, this project has provided insight into how Ptch1 patterns lung branching and lobe specification during development and also highlights the importance of Ptch1 in pulmonary epithelial regeneration.

Within this review, sex-specific differences in alveolar epithelial functions are discussed with special focus on preterm infants and the respiratory disorders associated with premature birth. First, a short overview about fetal lung development, the challenges the lung faces during perinatal lung transition to air breathing and respiratory distress in preterm infants is given. Next, clinical observations concerning sex-specific differences in pulmonary morbidity of human preterm infants are noted. The second part discusses potential sex-specific causes of pulmonary complications, including pulmonary steroid receptors and local lung steroid metabolism. With regard to pulmonary steroid metabolism, it is important to highlight which steroidogenic enzymes are expressed at which stage during fetal lung development. Thereafter, we review the knowledge concerning sex-specific aspects of lung growth and maturation. Special focus is given to alveolar epithelial Na+ transport as a driver of perinatal lung transition and the sex differences that were noted in this process.