According to the World Health Organization (WHO), cigarette smoking is 22.2% prevalent worldwide and Pakistan has 34% prevalence (1). Cigarette smoke (CS) adversely affect every vital organ of the body by altering several key cellular pathways and has become one of the leading cause of early death (2).

Cigarette smoking is a combustion process that leads to the aerosolization of thousands of toxic chemicals including carbon monoxide, hydrogen cyanide, and polycyclic aromatic hydrocarbons (3). Many of the components in CS chemically react with oxygen to generate free radicals and inhibit protective antioxidants (4). Through the combustion of noxious chemicals and generation of harmful reactive oxygen species (ROS), CS induces widespread tissue damage in a manner that mimics biological aging (5). Many in vitro and in vivo studies have elucidated mechanisms involved in cigarette smoke–induced inflammation, DNA damage, and autophagy, and the subsequent cell fates, including cell death, cellular senescence, and transformation mentioning key cause of over production of ROS (6, 7).

Physiologically, ROS molecules are formed by the partial reduction of molecular oxygen (O₂) and it reacts with other molecules and play a role in cell development, proliferation, differentiation, oxygen sensing and immunity through reversible oxidative modifications of macromolecules; but when present in excess, they can cause cellular oxidative damage (8). CS induces overproduction of ROS molecules which leads to oxidation of macro molecules and cause their conversion into peroxynitrite, hydroquinone and hydrogen peroxide which in turn cause genomic instability, oxidative DNA damage, shortening of telomere length, protein oxidation and lipid oxidation in lung epithelial and other supporting cells (9).

Prior studies have shown that tobacco smoking induces premature cellular senescence in lung epithelial cells and basal progenitor cells (10, 11). CS directly and via over production of ROS induces early cellular aging in lung tissues and nasal epithelial cells (12). Radical- and oxidant-mediated modification of proteins, nucleic acids, lipids, sugars, and consequent damage of cells play crucial roles in the genesis of a large number of age-related diseases (13).

The first effected organs from smoking are oral and nasal cavities. There are multiple studies available of impact of ROS due to CS on nasal cavity, bronchial and pulmonary tissues but limited data is available on its impact on oral cavity in smokers. This study was designed to see the impact of CS on ROS production and cellular aging in oral mucosal cells of smokers in comparison with non-smokers. We also evaluated the differential ROS and cellular aging between young and old smokers.

This study demonstrated a significant difference of ROS and CYR61 gene expression between smokers and nonsmokers. Levels of ROS were remarkably higher in smokers, and they exhibited overexpression of CYR61 gene as compared to non-smokers. Smoking cigarettes is an unhealthy practice that impacts human health. Huge amounts of ROS quickly react with numerous molecules within the cell, which affects the cellular physiological processes which in turn increases the risk of age-related diseases like diabetes, cardiovascular disorders, and cancers (14). However, there is still lack of research on how each component in CS can affect multiple human organs, tissues or cells (15).

CS impacts multiple tissues and cells, but limited data is available on oral mucosal cells. A study by Samanta et al. demonstrate that CS exposure produces oxidative damage, not only in lung tissue but also in muscle tissue, having an additional effect on respiratory muscle (16). Another study reveals higher impact of Cigarette Smoke Exposure on Organotypic Bronchial Epithelial Tissue Cultures with production of high ROS (16). Our study also reports the high amount of ROS in smokers and as compared to non-smokers in oral mucosal cells. Over production of ROS causes radical and oxidant-mediated modification micro and macro molecules of the cell which leads to alter the cell physiology including DNA damage, cancers and cell aging (17).

Many studies have shown transcriptome of smokers has altered gene expression and that these alterations are reproducible in different series of smokers (18). Many of those gene families are age-related genes. CS is correlated with up-regulation of the expression of CYR61 gene and DNA damage in buccal epithelial cells of smokers (19). Hyung-Geun et al. reported epithelial cell death and tissue loss in response to prolonged smoking both in vivo and in vitro (20). Another study revealed CYR61 gene was overexpressed in smokers compared to non- smokers, which was also associated with adipogenesis and inflammation in bronchial epithelial cells (21).

Of note, we compared ROS and cellular aging differences between young and old smokers. Levels of ROS were significantly higher in young smokers compared to old smokers. To the best of our knowledge, this is the first study which has reported the comparison between two smoker groups of different ages for ROS and cellular aging.

Currently the genetic aspects are unable to comprehensively explain the risk and prognosis of cigarette smoking related diseases. Evidence supports the significance of epigenetic alterations due to CS in the start and progression of diseases. Our results are like previous studies which were performed in animal models and humans.

We found significantly increased ROS and cellular aging in oral mucosal cells of smokers especially in young smokers. This is an alarming situation and might be the leading cause of oral pathologies and other smoking related diseases at a young age.