The Influence of maternal nicotine exposure on selected glycolytic and cytochrome P450 enzymes in developing neonatal rat lung

Abstract

The structural and functional integrity of a developing and maturing fetal and neonatal lung is critically dependent on carbohydrate metabolism. The energy derived from carbohydrate metabolism is utilized during the processes of cell growth and development. It is reported that maternal nicotine exposure during pregnancy and lactation results in the irreversible inhibition of glycolysis, for which no mechanism is currently proposed and a significant increase in glucose turnover. The principal objectives of this thesis are (1) to investigate the isoezyme patterns and transcript levels of selected glycolytic enzymes: Hexokinase (HK), Phosphofrutokinase (PFK), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and Lactate dehydrogenase (LDH) in control developing neonatal rat lung, (2) to investigate the transcript levels of selected cytochrome (CYP) P450 enzymes: CYP1A1, CYP2A3, and CYP2B1 in control developing neonatal rat lung and (3) to determine the influence of maternal nicotine exposure during gestation and lactation on the isoenzyme patterns and transcript levels of the selected enzymes in developing neonatal rat lung, in an attempt to elucidate the mechanism of inhibition of glycolysis observed. Tissue samples were obtained from the lungs of 1, 7, 14, 21, and 49-day old pups, from both control lung tissue and lung tissue exposed to nicotine during gestation and lactation (1mg/kg body weight/day). Isoenzyme separation is achieved using polyacrylamide gel electrophoresis (PAGE) techniques and was principally based on their differences in molecular weights. The PAGE gels are densitometrically analyzed and expressed as % Density/mg protein of lung tissue. Transcript levels are analyzed with the use of dot blots followed by densitometry of the blots. The results obtained for the mRNA subunits are expressed relative to ß-actin. The final data of both isoenzyme patterns and mRNA levels are analyzed statistically using the Wilcoxon unpaired T-test, in which P<0.05 is designated as significant. Results of this study show that: (1)All three isozymes, HKI, HKII and HKIII are detectable at the mRNA level however the method used is only sensitive enough to detect HKII from postnatal day 14. HKI is the dominant isoenzyme in the lung of control as well as rats exposed to nicotine at both the transcriptional and post translational levels during gestation and lactation. Furthermore, the developmental pattern of the isoenzymes as the lung matures follows the same trend in lung tissue of control and nicotine exposed offspring. (2) The three PFK sub-types are transcribed in control neonatal lung. PFK-M mRNA levels are expressed at significantly higher levels in control developing lung than PFK-L and PFK-C. However, maternal nicotine exposure during gestation and lactation results in over-expression of PFK-M and PFK-L, but has no influence on PFK-C mRNA levels. PFK-M was affected by maternal nicotine exposure during gestation and lactation, even at postnatal day 49. This implies that the long-term effect of maternal nicotine exposure on total PFK activity can be attributed to changes in the PFK-M isoenzyme activity. (3) GAPDH mRNA is over-expressed at postnatal days 14 and 21 in the lungs of rat pups exposed to maternal nicotine, however no difference is observed between control and experimental lung tissue at postnatal day 49. The expression of GAPDH mRNA of control lung increased gradually between postnatal days 1 and 49. On the other hand, GAPDH mRNA expression in lungs of nicotine exposed rat pups show a pronounced increase in expression after postnatal day 7 and reached a maximum at postnatal day 14. (4) Expression of all the LDH isoenzymes in control developing lung, except LD-1 between day 7 and day 14, decreased. LD-4 and LD-5 (the homozygous LD-M isoenzyme), the glycolytic associated isoenzymes displayed the greatest decrease. All LDH isoenzymes as well as sub-units at the mRNA level are over-expressed from day 7 onwards in the lungs of rats exposed to nicotine during pregnancy and lactation. LD-5 (the glycolytic sub-unit) and LD-M (mRNA homotetramer isoform) were particularly affected by maternal nicotine exposure. LD-1 is the dominantly expressed isoform at the transcriptional and post-transcriptional levels, in both control and nicotine-exposed lung at any age group analyzed. (5) The human orthologs of rat CYP1A1, CYP2A3 and CYP2B1 are transcribed in lung tissue of control neonatal rats and all display a significant increase in expression from postnatal day 1 to postnatal day 49. CYP2A3 is dominantly expressed at the mRNA level in control neonatal rat lung, followed by CYP2B1 and the lowest levels shown by CYP1A1. Maternal nicotine exposure results in the induction of CYP2A3 and CYP2B1, however has no influence on CYP1A1 expression. Moreover, CYP2B1 in control lung tissue remained significantly lower than CYP2A3, however in the experimental lung there are no differences between the two by day 49. It is concluded that the inhibition of glycolysis in lungs of rats exposed to nicotine during gestation and lactation is not due to changes in the HK or PFK isoenzyme levels in the lungs of the offspring. It is, however, evident that the maturing rat lung attempts to compensate for the glycolytic inhibition by over-expressing the isoforms at the mRNA level associated with glycolytic activity and gluconeogenic activity. The over-expression of GAPDH transcript levels in nicotine-exposed lung tissue takes place only when nicotine is present, and therefore maternal nicotine exposure has no long-term effects on GAPDH mRNA expression. Inhibition of the glycolytic pathway results in the over-expression of LDH at both the transcriptional and posttranscriptional levels and it is proposed that the over-expression is in an attempt to rectify the hindrance caused by maternal nicotine exposure. CYP1A1, CYP2A3 and CYP2B1 are transcribed in neonatal rat lung and induction of CYP2A3 and 2B1 in the lungs of the offspring by maternal nicotine exposure is irreversible and thus "programmed”. Furthermore, it is proposed that if the increased transcript levels reflect increases in activity of the CYP enzymes, this may explain the increase in glucose turnover observed in the lungs of offspring exposed to nicotine during gestation and lactation.