Three Generations, One Future: A Systematic Analysis on Nicotine's Effect across generations in C. elegans.
Taki, Faten Ahmad
Tobacco smoking is a worldwide epidemic that is responsible for diseases and death rates that surpass those attributed to a combination of other causes (e.g. cancer, HIV, accidents). A major mediator of tobacco-smoke related negative consequences is nicotine. Nicotine is an addictive poison that entraps users in a vicious cycle of constant drug seeking and reinforcement. Despite the public health policies and laws enforced to decrease the habitual smoking, it is still prevalent, especially among adolescents. According to WHO, 40% of children and up to 60% of teenagers are passively and actively exposed to tobacco smoke. Early life stages are more vulnerable and sensitive to environmental and life experienced stresses. At that stage, stresses can have enduring effects that not only persist until adulthood, but are also inherited to the subsequent generations. With respect to nicotine, a wealth of studies have investigated the dose and time-dependent effects of this chemical on multiple systems including cell lines and model organisms. However, the transgenerational effect of nicotine exposed during post-embryonic stages has not been reported. On the molecular level, an increasing number of popular findings that show the involvements of certain microRNAs in physiological processes have expanded to include response to nicotine. Nevertheless, a systematic profiling of microRNA expression levels is yet to be determined. In our study, we employed C. elegans as our model to investigate the transgenerational effect of nicotine exposure limited to the post-embryonic larval stages of the parent F0 generation. Two concentrations (20[mu]M and 20mM) were chosen based on previous studies. We investigated the effect of nicotine on the behavior of L4 C. elegans (N2) across three generations (F0, F1, and F2). Here we report that nicotine altered the sinusoidal locomotion, body bends, and forward and backward speeds across three generations. Such represented an enduring and heritable addiction initiated by parental post-embryonic nicotine exposure. In addition our qRT-PCR results showed that direct nicotine exposure throughout the larval stages (30 hours), altered the systematic miRNA expression profiles in L4 C. elegans in a dose-dependent manner. Through target prediction analyses coupled with background research, fos-1 was predicted to be a key mediator of the addiction-like behavior in C. elegans larvae. Conclusively, our results offer novel insights on the sensitivity of early developmental stages to nicotine exposure. The behavioral transgenerational effect as well as the parental altered miRNA profiles will set the basis for future miRNA transgenerational analyses coupled with target and pathway validation. With this in mind, the need for suitable reference genes for normalization and reliable interpretations is necessary. We dedicated our last objective to identify reference gene candidates to serve this purpose. Based on results from five statistical approaches (geNorm, NormFinder, BestKeeper, dCt method, and RefFinder), we report that the expression levels of tba-1 and cdc-42 were the most stable among all of sixteen compiled genes. Taken together, our work is preliminary for a new research direction concerned with nicotine that would help support public health policies and awareness campaigns to further stress on the risks and dangers of tobacco addiction.
Taki, Faten Ahmad. (January 2013). Three Generations, One Future: A Systematic Analysis on Nicotine's Effect across generations in C. elegans. (Master's Thesis, East Carolina University). Retrieved from the Scholarship. (http://hdl.handle.net/10342/4186.)
Taki, Faten Ahmad. Three Generations, One Future: A Systematic Analysis on Nicotine's Effect across generations in C. elegans.. Master's Thesis. East Carolina University, January 2013. The Scholarship. http://hdl.handle.net/10342/4186. August 21, 2018.
Taki, Faten Ahmad, “Three Generations, One Future: A Systematic Analysis on Nicotine's Effect across generations in C. elegans.” (Master's Thesis., East Carolina University, January 2013).
Taki, Faten Ahmad. Three Generations, One Future: A Systematic Analysis on Nicotine's Effect across generations in C. elegans. [Master's Thesis]. Greenville, NC: East Carolina University; January 2013.
East Carolina University