Nicotine-responsive genes in cultured embryonic mouse lung buds: interaction of nicotine and superoxide dismutase

Nicotine exposure during prenatal development may be a cause of the abnormal lung function seen in infants born to smoking women. Previously we used an organ culture system to demonstrate that nicotine directly affects branching morphogenesis and gene expression in embryonic mouse lung buds. Here we attempt to identify genes potentially involved in the nicotine response and explore the relationship between gene expression changes and stimulation of branching. DNA microarray technology, analyzed by DChip software, and semi-quantitative RT-PCR were applied to RNA samples from embryonic lung buds grown in presence or absence of nicotine. Four genes, BAX, calcyclin, osteopontin and Cu-Zn superoxide dismutase (SOD1), identified by the microarray as showing changes in mRNA level with nicotine treatment were investigated in detail. RT-PCR showed that nicotine exposure resulted in significant decreases in mRNA levels for BAX, calcyclin and osteopontin, but nicotine did not affect the mRNA level of SOD1. Nicotine-induced changes in BAX, calcyclin and osteopontin mRNAs showed a general correlation with stimulation of branching, implying a common mechanism for effects of nicotine on branching and on gene expression. BAX, calcyclin and osteopontin mRNA levels were found to be developmentally regulated, but only the effect of nicotine on BAX mRNA was parallel to the developmental change in vivo, suggesting that nicotine action cannot be explained simply as a stimulation of the embryonic lung's developmental program. Addition of exogenous SOD to the culture medium resulted in increased branching similar to that caused by nicotine, but, unexpectedly, branching was not increased relative to control when nicotine and SOD were co-administered, suggesting interfering mechanisms of action of the two agents. Exogenous SOD was found to alter mRNA levels of BAX, calcyclin and osteopontin in a pattern that differed from that seen in response to nicotine. Gene expression changes seen with co-administration of nicotine and SOD yielded further evidence of interaction between these agents. In conclusion, three putative nicotine-responsive genes were identified whose expression was also influenced by developmental stage and by exogenously added SOD. A common mechanism likely underlies nicotine's effects on both branching and gene expression in this system. Our evidence also suggests that nicotine and SOD stimulate branching by distinct but interacting mechanisms.