Narghile water-pipe smoking has been getting popularity in different countries (Chaaya et al., 2004). It’s being offered as a form of pleasure in different cafes and restaurants. It is even more common among teenagers and assumed to be relatively safer than cigarette smoke. Bad impact of water-pipe smoke on our respiratory tract is largely underestimated among common people (Shafagoj et al., 2002). According to a report published by WHO (world health organization) it is suggested not be an alternative of cigarette (Maziak et al., 2004). In case of narghile, tobacco is heated by a combusted carbon source and smoke containing different flavors is passed through water therefore more toxic and cancer causing chemicals contact the respiratory tract during inhalation as compared to cigarette (Shihadeh ., 2003;). Knowing these facts we aim to analyze the DNA of narghile smokers by using highly efficient DNA sequencing technologies and comparing it with the non-smokers DNA. We hypothesize that the chemicals present in the smoke of narghile can cause lesions in the DNA of respiratory tract cells which can raise the chances of cancer. By doing this analysis we will be able to address the effects of narghile smoke on cells of our respiratory tract.
1. PROJECT SUMMARY
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DNA sample from airway epithelial cell of 10 individuals having previous exposure of Narghile water-pipe smoking will be taken and genome sequencing will be done by Illumina genome analyzer platform II. Sequencing data will be compared with nonsmoker’s genome. By using alignment tool Burrows-Wheeler Aligner (BWA) and SHORE analysis we will be able to get mutational spectrum induced by narghile smoke in human respiratory epithelial layer. Frequencies of SBS and Indels will be calculated by using statistical analysis. Data will be presented to highlight the effects of Narghile water-pipe smoking on our health including risk of cancer.
HYPOTHESIS OF RESEARCH
Narghile water-pipe smoke can induce genome wide mutation in the respiratory epithelial layer cells, which can increase the chances of respiratory tract associated cancer and other linked diseases.
Narghile water-pipe smoking is hazardous to health because it contains several toxic compounds which can damage the cells of our respiratory tract. It is commonly used as an alternative of smoking and considered to be less harmful than cigarette smoke. Average session for Narghile lasts 40 minutes which means 0.15 to 0.5 liters of smoke is being inhaled during that session (Alan et al., 2004; Djordjeviv et al., 2000). Although tobacco free mixtures of different flavors and molasses are also available (chaouachi 2007), but their impact on our health are needed to be defined. Here we hypothesize that the narghile smoke can induce genome wide mutations in the respiratory tract epithelial cells which can lead to various dangerous diseases including pulmonary cancer and other kinds of respiratory tract cancer. Secondly the genome wide comparison between cigarette smoker and narghile water-pipe smoker will answer to many question about the health impact of narghile smoke.
Project is based on genomic analysis to determine genome wide mutation caused by hazardous chemicals present in narghile water-pipe smoke. Lung cancer can be a consequence of mutations caused by cigarette smoke (Lee et al.,2010) therefore we are interested to analyze the mutational spectrum of narghile smokers to get some idea about its impact on human health. Water pipe tobacco smoking is regarded as a threat to human health (WHO, 2005). It has been reported that oral kertenocytes can be effected by narghile smoke which damage the respiratory tract (Rastam et al., 2010). By using recent advancements in next generation sequencing technologies we will be able to point out the genome level lesion caused by various dangerous chemicals present in narghile smoke.
Narghile water smoke consists of flavored tobacco which is slowly burnt along with molasses and caramelized sugar which is then passed along the water and inhaled with much longer puffs (Alan et al., 2004). By comparing the genome of narghile smoker and non-smoker control using Illumina genome analyzer II including next generation alignment software, the frequencies of various mutations including single base substitutions, small and large insertion and deletions can be calculated. By comparing it with reference genome and previously reported data about mutational spectrum induced by cigarette smoke, the underestimation about risk and severity of narghile smoke can be eliminated. Secondly risk of pulmonary associated cancers can be elucidated by computing the frequencies and positions of mutations in the coding and regulatory regions of genome.
4A.BACKGROUND OF THE RESEARCH PROBLEMS TO BE ADDRESSED
As in case of Narghile water-pipe, smoke is passed through water and some of nicotine released from the burnt tobacco gets absorbed in the water but still reasonable amount of nicotine is present which can harm. As compared to cigarette in case water-pipe smoke also contains fumes of burnt sugar as well as very high amount of puff has been inhaled (Maziak W. et al., 2004 ; Shafagoj et al., 2002). Several studies at genome and transcriptome level has been conducted while the literature Narghile water-pipe smoking health risks at genetic level is still limited.
Transcriptomic study of the human airway epithelial layer cells of cigarette smokers and nonsmokers was conducted by using high throughput next generation sequencing technologies which reviled 50,205 SNPs and 3008 indels in the person who were healthy but cigarette smoker while 51,299 SNPs and 3138 indels were Identified in smoker suffering from lung cancer (Sathya et al., 2015). In another study It has been observed that there are aberrant methylation pattern in the promotors of tumor repressor genes in the aerodigestive tract epithelial cells of heavy smokers. Promotors of the genes retinoic acid receptor-2 (RAR-2), CDH13 (H-cadherin), p16INK4a (p16), RASSF1A (RAS association domain family 1) were found to be aberrantly methylated. These findings can be served as a marker for lungs cancer risk (Zochbauer-Muller et al., 2003).
As we know that lung cancer is more common among cigarette smokers. Here is a study which elucidated the genetic level changes which occurs in the lung cancer tissue by doing genome analysis and comparison with normal cells. They have estimated 530 somatic single nucleotide variants in the tumorigenic cells. While 391 mutations were detected in the coding regions, overall genome wide mutation rate was predicted to be close to 17.7 per mega base (Lee et al., 2010).
In a previous study mutations in the hotspot regions of 100bp in mitochondrial DNA were analyzed which were caused by cigarette smoking. Bronchial epithelial cells from smoker (smoking history of 10 years) and non-smokers (closer relatives of smokers) were sampled and 100bp sequence was PCR amplified by Pfu. Individual point mutation frequency as low as of 10-6 was detected after comparison (Coller et al., 1998).
In another comparison of lung cancer cells and non-smokers respiratory epithelial cells, revealed 3726 point mutation and 90 short insertions and deletion in the coding region. This study suggested that there is 10 fold more mutation rate in smoker as compared to non-smokers (Govindan et al., 2012). It was found out that smoking causes the enhanced expression 32 gene while 9 of the total showed reduced expression. The data was gathered after microarray studies of smoker and non-smokers aerodigestive tract (Boyle et al., 2010).
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4B.RESEARCH PLAN/ METHODOLOGY: SCHEDULE/PHASING
Outline of research methodology is given below:
Five males and female volunteer having regular exposure of Narghile smoking from last 5-10 years will be selected for the study. Nonsmoker’s genome will be used as a control to compare the extent of mutations induced by the Narghile smoke. As smoke contacts the respiratory airway epithelial cells during smoking therefore more chances of mutations are in oral and bronchial epithelial cells. Sampling of oropharynx area and the area underneath the tongue will be done by brushing method (Sidransky 1997). Same way the sample from non-smoker will be taken.
DNA extraction from the collected samples will be done by high throughput QIAGEN DNA isolation mini kit and DNA libraries will be sequenced by using Illumina Genome Analyzer II platform. After getting sequencing data further analysis will be based on alignment of
Smoker and non-smoker’s genome to identify the variations. Burrows-Wheeler Aligner (BWA) will be used for efficient alignment of reads (Li and Durbin 2010). SHORE will be used to identify the SBS and indels (valvekenes et al., 1988). Mutation will be called if at a certain position is different from reference and present among all smoker’s genome with the exception of variation in one or two individuals.
Statistical analysis of the mutation spectrum data will done to calculate the total frequencies of different type of mutations induced in coding and non-coding regions of chromosomes. Total frequencies will be compared with previously reported mutational spectrum of cigarette smoker lungs tissue (Govindan et al., 2012) (Lee et al., 2010).
Hazards and risk of Narghile water-pipe smoking has not been studied well, while there is a pile of literature on bad impacts of cigarette smoking (WHO, 2005). Due to lack of information and misconception about narghile smoking, it is getting more common among different societies (Maziak et al., 2005). Even the marketing companies involved in the business of narghile water pipes are misleading the concept about its heath impacts and regarded as safer than cigarette (Knishkowy et al., 2005). By finding out the impact of narghile smoke on the genome of human respiratory epithelial layer cells we can eliminate the misconceptions about the impacts of narghile smoke on heath. This will also point out the chances and risks of getting cancer and other dangerous diseases by its regular exposure.