Investigating the impact of tobacco particulate matter and selected components on monoamine oxidase activity, protein expression, and gene expression in brain SH-SY5Y cells : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Health Sciences at Massey University, Wellington, New Zealand
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Date
2023
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Massey University
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Abstract
Smoking addiction is one of the most widely discussed topics today due to a large number of smokers and the millions of lives it claims every year. Though a large population makes effort to quit smoking, the quit attempts mostly end in relapse, indicating the complex nature of smoking addiction. Nicotine is believed to be the major component responsible for the addiction, however, nicotine replacement therapy (NRT) has not proved to be a completely satisfying approach to smoking cessation. The low efficacy of NRT, as well as much research related to smoking addiction, suggest the role of non-nicotinic components in smoking addiction. It is hypothesised that monoamine oxidase inhibitors (MAOIs) present in tobacco smoke play a role in smoking addiction by prolonging nicotine’s reinforcing effect. Based on this hypothesis, Tobacco Research Group, Wellington has identified six candidate MAOIs in cigarette smoke. This PhD project aims to investigate the effect of nicotine, tobacco particulate matter (TPM) and the candidate MAOIs on MAO activity, MAO protein levels, MAO genes expression and global gene expression.
A human neuroblast SH-SY5Y cell line was exposed to different regimens, which included ethanol (control), nicotine, TPM and the cocktail of candidate MAOIs for a period of 1, 3, 5 and 7 days. A modified kynuramine assay was performed after SH-SY5Y cells were exposed to the different treatments to determine the effect of the exposure treatments on MAO activity and to identify the optimum period of exposure that would result in maximum MAO inhibition. Exposure for a period of 3 days was chosen as an optimum period of time for exposure and for expression and whole genome experiments. Similarly, a MTT assay was performed to determine if the exposure treatment had any cytotoxic effect. Change in MAO protein and MAO gene expression after exposure to the different treatments for the optimum period of time were then determined using Western blot and qPCR, respectively. Finally, the effect of exposure treatments for an optimum period of time on global gene expression was determined using RNA sequencing (RNAseq) technology.
It was observed that nicotine did not have any significant MAO inhibitory effect compared to the control in any of the treatments examined. TPM and MAOIs caused significant inhibition of total MAO activity when exposed for 1 and 3 days. However, no significant inhibition was seen in the exposure for 5 and 7 days. Change in MAO A and MAO B gene and protein expression levels after the exposure treatment for 3 days was not observed. Nevertheless, several genes were found to have differential expression after exposure to the treatments for 3 days. Many of these differentially expressed genes were linked with diseases and conditions related to smoking and addiction. The results suggest that the candidate MAO inhibitors identified by the Tobacco Research Group, Wellington could be the primary contributors of the MAO inhibitory property observed in cigarette smoke. This data could also possibly answer the major question regarding the component responsible for MAO inhibition by cigarette smoke in smokers. Further research is required to fully elucidate and understand the mechanisms behind the MAO inhibition from the MAOIs, and a better understanding of these mechanisms may provide a framework for the development of novel smoking cessation therapies.
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Figure 7 is reproduced with permission.
Keywords
Tobacco smoke, Composition, Particulate matter, Monoamine oxidase, Inhibitors, Gene expression