Biochemical composition of a smokeless tobacco product (NASWAR) used in Pakistan

1 Introduction

Smokeless tobacco (ST) use is a growing public health concern with an estimated 360 million users across the globe (The Tobacco Atlas, 2022) of which, more than 90 % live in the South Asian region. Despite being associated with multiple disease conditions (Boffetta and Straif, 2009; Critchley and Unal, 2003), the use of ST products is gradually on the rise in South Asian countries (Chugh et al., 2023). This rise in consumption can be attributed primarily to a lack or poor implementation of ST-specific tobacco control laws and legislation (Azeem et al., 2022). An important contributor to these legislative deficiencies is the poor understanding of the risks associated with the use of these ST products. Compared to smoking tobacco such as cigarettes, there is insufficient scientific evidence on the toxicity of ST products commonly consumed in developing countries. As a result, these products are considered safe or less harmful (Wackowski et al., 2014) by the general public. The situation is further complicated by the considerable variation in ST products in terms of varieties, their composition, and associated health risks among different countries (Ahmad et al., 2020a). Nonetheless, scientific evidence from across the globe suggests that ST use is a prime risk factor for oral, pharyngeal, and esophageal cancers, coronary heart diseases, and poor birth outcomes and contributes to 0.65 million deaths each year (Siddiqi et al., 2020).

ST use is also common in Pakistan with an estimated 8 % of the population using different ST products, the commonest being Paan, Gutkha, and Naswar (Naz et al., 2018). Naswar use is mostly common among the ethnic Pashtun population of Khyber Pakhtunkhwa (KP) province, wherein around 15 % of the general population uses naswar (Khan et al., 2017). In Peshawar, the capital city of KP, around 60 % of the total tobacco users consume naswar (Ahmad et al., 2020a). Naswar is prepared in local cottage industries by mixing sundried tobacco leaves with slaked lime, ash, and flavoring agents (Zakiullah et al., 2012) and packed and sold in small poly-ethylene bags (Sajid and Bano, 2016). Naswar usage for a long time has commonly been linked with oral cancer with pooled estimates suggesting that habitual naswar users are at 10 times higher risk of developing oral cancer than non-users (Suliankatchi et al., 2019). Moreover, a variety of oral (leukoplakia, oral submucous fibrosis, caries, periodontal diseases, teeth staining, and halitosis) (Asthana et al., 2019; John et al., 2021) and systemic (cardiovascular and other) diseases are also reported in habitual naswar users (Khan et al., 2017; Sinha et al., 2016).

Although naswar use is common in KP, scientific studies exploring the composition of products are scarce. To date, the limited available evidence suggests the presence of toxic constituents (nicotine, heavy metals, tobacco-specific nitrosamines) (Ahmad et al., 2020b; Zakiullah et al., 2012) and bacterial contamination in naswar brands in KP (Habib et al., 2023). Storage of naswar may also expose it to fungal contamination (Saleem et al., 2018), and become a potential source of mycotoxins and aflatoxins with known carcinogenic potential in humans (Ahmed et al., 2023). However, efforts to identify aflatoxin presence in naswar have not been carried out as yet.

The scientific studies assessing the composition of naswar were conducted at least a decade ago (Zakiullah et al., 2012) and are not representative of the advances in preparation methods and product composition owing to the greater demand and popularity in the recent past (Ahmad et al., 2020a). Moreover, the composition of naswar products varies among different areas and regions within Pakistan. Additionally, there are brand differences in terms of the amount of tobacco, ash, and slaked lime as well as the type and nature of additives used in the products. It is, therefore, imperative to carry out the compositional analysis of the naswar products available in the market at the moment, to assess the risks associated with their toxicity. The current study aims to comprehensively assess the composition (organic and inorganic constituents), toxic and heavy metals, and microbial toxins (aflatoxins) contamination in the most commonly consumed naswar brands in the KP province of Pakistan.

2 Materials and methods

3 Results

4 Discussion

The current study, thoroughly assessed the biochemical composition of naswar brands commonly used in KP, Pakistan. The overall composition of the samples does not vary considerably across the different regions of the KP province. All the samples possess an alkaline pH ranging from 8.1 to 8.9. These results are in concordance with the findings of a recent research study reporting the basic pH of the ST products commonly consumed in India (Sharma et al., 2023). The higher and alkaline pH of naswar and other ST products is mainly due to the addition of slaked lime and ammonium bicarbonate. The alkaline pH of ST products accelerates the release of unprotonated nicotine and enhances its absorption through the oral mucosa thus increasing the risk of nicotine dependency in users (Pickworth et al., 2014). The average ash contents of the test samples were low (range: 9.6–31.4 %). Since naswar in the KP region is mostly used in moist form, the ash contents are generally low compared to other ST products (Sharma et al., 2023).

On a global scale, the chemical composition of ST products varies widely between different types and also within different brands of the same product (Kaur et al., 2019). In the current study, naswar samples also exhibit considerable compositional ingredients that are considered hazardous for human consumption. For example, nicotine, a well-known alkaloid in tobacco that is linked to addiction and health risks including cardiovascular diseases and cancer (Mishra et al., 2015) was the most common chemical constituent in naswar samples. Similarly, cotinine and norcotinine, the biomarkers for nicotine exposure in humans (Benowitz et al., 2009) can absorb quickly through the oral or respiratory mucosa, enter the bloodstream, and can even cross the blood–brain barrier (Shoji et al., 2023). Octadecenioc acid (stearic acid), with proven DNA damage and tumorigenic potential (Alhazmi et al., 2019) was also present in some naswar samples. Similarly, polycyclic aromatic hydrocarbons such as naphthalene which is reported in three naswar brands are a respiratory toxicant as well as a possible human carcinogen (Traboulsi et al., 2020). We have also found nitrogenous compounds in naswar samples. The accumulation of Tobacco-Specific Nitrosamines (TSNAs) in tobacco products is influenced by factors like nicotine conversion and nitrate content. Nitrates, which are chemically unstable, can react with tobacco alkaloids, either directly or through microbial digestion to form TSNAs (Habib, U. et al., 2023).

ST products that are commonly consumed in Southeast Asian populations contain additional flavoring agents with varying concentration ranges (Lisko et al., 2014). In this study, the identification of menthol in at least three naswar brands confirms the local manufacturer’s intentional addition of this flavoring agent into the product. The addition of menthol increases the attraction to new users by making the product more palatable and acts as a reinforcer (Ahijevych and Garrett, 2010). Another interesting finding of this study was the identification of ethylamphetamine carbamate in naswar samples S12. Ethylamphetamine carbamate is an amphetamine derivative and is classified as a drug with addiction and abuse potential. If ingested, it may induce hallucinations, cardiac arrhythmias, blurred vision, and a strong feeling of intoxication (Zanda and Fattore, 2017). The presence of amphetamine in these naswar samples raises important questions. It could indicate adulteration of other plant products containing amphetamines with similar properties. Alternatively, it is plausible that amphetamine, in its chemical form, was directly added to these naswar products given its ready availability in the market (Alhazmi et al., 2019). This important finding warrants further investigations to better understand the sources and health implications of such adulterations.

In addition to the organic constituents, we have also reported twelve different elements, five of which are carcinogenic in humans. These findings are also in concordance with previous reports indicating carcinogenic elements in ST products (Sami et al., 2021). Previously, several studies have identified toxic heavy metals and other hazardous elements in ST products across the world (Brima, 2016; Guezguez et al., 2021; Shetty and Hegde, 2021). It is important to note the metallic composition of individual samples varied widely among the samples reflecting differences in the amount and composition of the ingredients and also the processing, wrapping, and storage of the product. Our study, for the first time, also reported the presence of aflatoxins in naswar products. Previously, AFB1 and AFG1 aflatoxins have been reported in the ST products (Qi et al., 2017). Chronic toxicity of aflatoxins can lead to liver cell carcinoma in humans, stunted growth, weakened immunity, and cirrhosis in unhealthy individuals (Ahmed et al., 2023). While we were able to identify aflatoxins in naswar samples, the source of contamination whether it is because of direct contamination of tobacco leaves, use of additives, pH, or storage conditions is yet to be known.

Although the current study is the first ever and most comprehensive analysis of the composition of the most famous naswar brands from all the administrative regions of KP, it has some limitations. First, naswar samples used in the study were selected based on popularity, and not all brands were selected thus greatly limiting the generalizability of the study findings. Second, we could not assess the impact of storage and shelf-life variations on naswar composition. Third, the sample size per region may vary considerably but we could only test two samples per region.

5 Conclusion

The present study identified several organic (85), metallic (12) and aflatoxins (4) constituents in naswar samples collected from all administrative regions of KP, Pakistan. Naswar also possess highly addictive potential due to alkaline pH (8.61 ± 0.271) and high nicotine contents. The addictive potential coupled with the presence of carcinogenic compounds renders naswar a very dangerous product for human consumption. Given that naswar is manufactured by the cottage industry in Pakistan, steps must be taken to look into regulating their manufacture and sale.

Funding

The study received funding from the Office of Research, Innovation and Commercialization (ORIC), Khyber Medical University, Peshawar, Pakistan, and Pakistan Council of Scientific and Industrial Research, Peshawar, Pakistan.