GC-MS Analysis and Cytotoxicity Evaluation of Shammah ( Smokeless Tobacco ) Samples of Jazan Region of Saudi Arabia as Promoter of Cancer Cell Proliferation

Substance Abuse Research Centre, Jazan University, P.O. Box 114, Jazan 45142, Saudi Arabia Department of Pharmaceutical Chemistry, College of Pharmacy, Jazan University, P.O. Box 114, Jazan 45142, Saudi Arabia Department of Pharmacology and Toxicology, Faculty of Pharmacy, Umm Al-Qura University, Makkah 21955, Saudi Arabia Medicinal and Aromatic Plants Research Institute, National Center for Research, P.O. Box: 2424, Khartoum 11111, Sudan Department of Pharmacognosy, College of Pharmacy, Jazan University, P.O. Box 114, Jazan 45142, Saudi Arabia


Introduction
Smokeless tobacco (ST) is used by over 300 million people worldwide, mostly from South Asian and Middle Eastern countries [1].In Saudi Arabia, the use of ST is more prevalent in the southern region (Jazan Province) due to its proximity to Yemen, where its use and trade is legal.Shammah is a locally manufactured form of smokeless tobacco.It is an addictive substance, used by placing in the oral cavity, and saliva produced is ingested thereafter.Generally, shammah is produced mainly from the tobacco leaves, including N. tabacum, N. rustica, N. glauca, or N. nepalensis [2,3].e tobacco leaves are further mixed with other materials such as carbonate of lime, ash, black pepper, volatile oils, and other coloring and flavoring agents to enhance its properties [4].A number of harmful substances, including at least 28 potential carcinogens, have been identified in shammah; among these substances, nitrosamines are reported to be the most harmful [5].
Smoked tobacco have been reported to increase the risk of developing at least 14 types of cancer and is responsible for about 87% of deaths from lung cancer and 25-30% of general cancer deaths [6].On the contrary, the chewed ST has been responsible for about 400,000 oral cancer cases so far, which represents around 4% of all the cancer types [6].In addition to that, the use of smokeless tobacco is also associated with a wide range of adverse health issues, including cardiovascular diseases, weight loss, increased blood urea, and creatinine levels which seem to cause serious degenerative alterations and lead to periportal fibrosis in liver and edematous and calcified changes in renal glomerulus [7].Aqueous extracts of ST hinder growths of some oral flora, which affect their balance, causing oxidative stress via degradation of nicotine into hydroxynicotine and cotinine-N-oxide inside the bacterium [8,9].Furthermore, disruption of sperm-head morphology, decreased total sperm count, increased oxidative stress, and high genotoxic and germ cell toxic effects have been seen in animals treated with tobacco [10].According to the Saudi Arabia cancer incidence report of 2014, the most common cancer in the Jazan region is colon cancer in males and breast cancer in females [11].
Despite the fact that nicotine itself is not carcinogenic, its derivatives such as 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone and N′-nitrosonornicotine might be responsible for the carcinogenic effect [12].Moderate and reversible toxicity along with weight loss was found in the esophagus, stomach, liver, kidneys, and lungs [13].
e presence of ammonia, benzo[a]pyrene, cadmium, nickel, nicotine, nitrates, and tobacco-specific nitrosamines increase the risk of probabilistic cancer [14].e use of tobacco is thought to cause systematic stress in humans with increased inflammation and RBC membrane damage [15].Heavy metals such as arsenic and nickel have synergistic effects with risk factors associated with oral cancer [16,17].
To the best of our knowledge, no data are available on the constituents and cytotoxicity of ST/shammah locally manufactured in Saudi Arabia.erefore, this could be the first study to explore the constituents of shammah samples from the Jazan region using GC-MS and their carcinogenic properties on the breast, ovary, and colon cancer cells, by MTT cytotoxicity assays.We assume that this study will effectively highlight the carcinogenic potential of locally used shammah and could strengthen the social and governmental efforts to minimize the use of these substances among the local inhabitants and stimulate the awareness in the society about the adverse health effects associated with the use of shammah.

Materials and Methods
2.1.Shammah Material.Shammah powder was collected from different producers in Jazan, Abu Arish, Sabiya and Ahad al Masarihah cities of the Jazan region.A total of eight shammah samples were collected, which include Arishi shammah from Jazan city (S1), Arishi shammah from Sabiya (S2), Arishi shammah from Ahad al Masarihah (S3), Adani cold shammah from Jazan city (S4), Adani cold shammah from Abu Arish (S5), Adani cold shammah from Sabiya (S6), special shammah from Sabiya (S7), and Khadrah shammah from Ahad al Masarihah (S8).ese shammah varieties also differ in their color as the Arishi shammah is yellow in color and is also called as yellow shammah, whereas other types are brown, white, black, and grey shammah depending upon the additives used as the coloring agent.As discussed earlier, shammah is manufactured locally by mixing powdered tobacco leaves with several other constituents that are added externally to increase its acceptability and potency.ese ingredients include several known substances such as lime, ash, black pepper, volatile oils, coloring agents, and flavoring agents and several unknown substances that are added by the manufacturer to impart unique properties to the product.Many of these substances are harmful to human health and can cause cancer if exposed for long time.

Preparation of Different Extracts.
Each of the eight shammah samples (0.2 g) was extracted and homogenated at 5000 R/sec with different polar (water and ethanol) and nonpolar solvents (chloroform and petroleum ether).After 24 h, the extracts were filtered, concentrated by distillation, and dried under reduced pressure to obtain a dark-colored mass.e residues were stored at 4 °C in the dark for GC-MS analysis.e crude ethanolic extract of shammah samples was prepared for MTT cytotoxic investigations according to method described by Harborne [18].In brief, 50 g of shammah powder was macerated in 300 mL of 80% (v/v) aqueous ethanol for 72 h at room temperature with shaking.
e supernatant was filtered through 0.45 μm filter paper and dried.

GC-MS Analysis of the Extracts.
GC-MS analyses were carried out on a Shimadzu Gas Chromatograph instrument fitted with a capillary column TR-5MS (30 m × 0.25 mm) and film thickness 0.25 μm. e carrier gas He was used at a flow rate of 1.2 mL/min.e initial temperature was 70 °C, which was increased at a rate of 15 °C per min to 290 °C and held for 30 min.e chromatograph was coupled to Shimadzu QP2010 Ultra MS detector 70 eV.

Identification of Constituents.
Constituents were identified by GC-MS by comparing their retention indices with those of authentic standards available in the laboratory or with the retention indices, which were in close agreement with reference.e fragmentation patterns of mass spectra were compared with those stored in the spectrometer database using the NIST08 and Wiley 9 built-in libraries.

Cell Culture.
ree cancer cell lines MCF7 (human breast adenocarcinoma), A2780 (human ovary adenocarcinoma), and HT29 (human colon adenocarcinoma) and one normal cell line MRC5 (normal human fetal lung fibroblast) were used in this study.All cells were obtained from the American Type Culture Collection (ATCC).e three cancer cells were subcultured in RPMI-1640 media (10% FBS), while MRC5 was maintained in the Eagles minimum essential medium (EMEM, 10% FBS), all incubated at 37 °C, 5% CO 2 , and 100% relative humidity.e cytotoxicity of the eight shammah samples was evaluated by the MTT assay as per the method described in the literature [19].e three cell lines and one normal fibroblasts were separately cultured in 96 well (3 × 10 3 /well) and incubated at 37 °C overnight.Final compound concentrations 0, 6.25, 12.5, 25, 50, and 100 μg/ mL (DMSO 0.1%; n � 3) were used in the study.Plates were incubated for 72 h, followed by addition of MTT to each well.Plates were further incubated for 3 h, the supernatant were aspirated, and DMSO was added to each well.Absorbance was read on multiplate reader (Bio-Rad PR4100).Optical density of the purple formazan A 550 is proportional to the number of viable cells.Compound concentration causing 50% inhibition (IC 50 ) compared to control cell growth (100%) was determined.GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego, California, USA was used for analysis.

GC-MS Analysis.
Using GC-MS analysis, we were able to separate and identify various constituents of shammah samples efficiently.Representative chromatograms of three shammah samples are shown in Figure 1.All components identified in shammah samples along with their retention times are summarized in Table S1, whereas the major toxic/ carcinogenic components are listed in Table 1 along with their structures.
Last sample, the Khadrah (green color) shammah procured from Ahad al Masarihah (S8) also characterized and found to possess various types of chemical constituents including alkanes, siloxanes, pyridine derivatives, and fatty acid esters.Nicotine was present in all the extracts ranging from 10.12 to 87.91%, with the maximum amount again observed in petroleum ether extract.

Cytotoxicity Assay.
e MTT assay revealed that all the eight samples promoted the growth of MCF7, A2780, and HT29 cancer cells, as all the IC 50 values (except samples no. 4 Journal of Chemistry S5 and S6 against A2780) were between 25 and 70 μg/mL.e samples S1-S5 and S7 were found to be more cytotoxic against MRC5 normal cells compared to the cytotoxicity of these samples on the three cancer cells (Table 2).On the other hand, samples S6 and S8 have exhibited less cytotoxic property against MRC5 (IC 50 48 and 54 μg/mL, respectively) compared to their effect on the three cancer cells.

Discussion
From different materials, additives, and flavors used in manufacturing shammah, the major ingredient tobacco contains at least 50 carcinogens [6].
e degree and intensity of exposure to carcinogens in terms of duration and quantity is crucial in developing cancer [20].
is may indicate the increased danger of extracted and swallowed components of smokeless tobacco over the components of smoking tobacco.Smokeless tobacco (shammah) is used by placing about 10 g into the mouth cavity, and it is placed between the gum and the lip, under the cheek or below the tongue on the floor of the mouth.It is then sucked slowly for a period of 15 minutes to few hours, which vary from 4.   e saliva generated meanwhile is swallowed, resulting in significantly high concentration of shammah components ingested.
ese alkanes can cause chemical pneumonitis and slow death [21].Tetradecane is shown to be a carcinogen and tumor promoter in a two-stage experiment of benzo[a]pyrene carcinogenicity in mice [21].It has also been found to enhance mitogenic response of murine spleen lymphocytes to the lectin phytohemagglutinin [21].However, pentadecane is not a known toxic compound but may be harmful if taken in higher quantity by inhalation, ingestion, or skin absorption.Pentadecane, when aspirated into the lungs, has acted as an asphyxiant and was incubated in vitro with rabbit heart mitochondrial model [22].On the contrary, dodecane can be irritating to mucous membranes [21].It has also been shown to be a promoter of skin carcinogenesis for ultraviolet radiation [21].Dodecane did not increase mutations when tested in the Ames Salmonella typhimurium assay with and without metabolic activation.However, it was able to enhance mutagenesis induced by methylazoxymethanol acetate at the ouabain resistance locus [21].Tetratriacontane is similar to homologous series of n-alkanes ranging from n-C12-n-C31 and was found in all samples of the liver, heart, kidneys, muscle, and adipose bovine tissues [23].Other potential carcinogenic components detected in shammah samples include fatty acid methyl 7-octadecenoate, which has been reported to promote skin tumor formation in mice [21].Components with a lower degree of toxicity include 1,2-benzenedicarboxylic acid dibutyl ester (toxic for reproduction), and hexanedioic acid, which have produced kidney and testis damage when given to the experimental animals for a longer period of time [24].
Nicotine and nicotine derivative ((1s,2s)-nicotine-Noxide) were also identified in shammah samples included in this study.Nicotine is a pale yellow to dark brown liquid present in 2-8% of the tobacco leaves dry mass.Cotinine is a predominant metabolite of nicotine, which have been studied for its antidepressant activity.1,3-Dimethylurea is a colorless crystalline powder with low toxicity and is being used for synthesis of caffeine, pharmaceuticals, and herbicides.Cis-13-docosenoamide/erucylamide is an amide of docosenoic acid that reduces mobility and awareness in rats, whereas 9-octadecenamide induces physiological sleep.
e siloxane (organosilicone) compounds are probably added to the mixture as surfactants (wetting agents) or antifoaming agents.Cyclohexasiloxane and dodecamethyl are widely used as conditioning agents, emollients, in personal care products, lubricants, and defoaming and antimicrobial agents [25,26].Constituents like 3-(methylnitrosamino)-proprionitrile, nitrosamines, and nicotine have been reported to induce the production of reactive oxygen species in ST, leading eventually to fibroblast, DNA, and RNA damage with carcinogenic effects in the mouth of tobacco consumers.e metabolic activation of nitrosamine in tobacco by cytochrome P450 enzymes may lead to the formation of N-nitrosonornicotine, a major carcinogen, and micronuclei, which are an indicator of genotoxicity.ese effects further enhance DNA damage, which eventually lead to oral cancer [27].
All the shammah samples were subjected to the MTT cytotoxicity assay.e results of the MTT assay and the IC 50 values of the eight tested samples on MCF7, A2780, and HT29 cancer cells and the MRC5 noncancerous/ normal cells have been shown in Table 2. e difference of the pattern of cytotoxicity produced by the tested samples against MRC5 is evident in sample S7 (14 detected component), as it has promoted HT29 cancer cell growth and was found to be cytotoxic for MRC5 cells too (Table S1, Figure 2(a)).e sample S5 (8 detected components) has also promoted HT29 cancer cell growth; however, it was less cytotoxic for MRC5 cells (Table S1, Figure 2(b)).It has indicated that if more components are present or mixed in the shammah samples, there are more possibility of both promotion of cancer cell growth and cytotoxicity of the normal cells.Samples, S6 and S8, were less cytotoxic against MRC5, which could be attributed to the fewer components detected in the GC-MS chromatogram, compared to samples S1-S5 and S7.
Studies regarding the constituents of shammah are very scarce and more emphasis has been given to the phytochemical analysis of smokeless tobacco. is study aimed to detect the chemicals added to smokeless tobacco during the manufacture of shammah and to evaluate their overall cytotoxicity.Earlier, 28 cancer-causing chemicals have been identified in ST and is regarded as known human carcinogen [29,30]. is effect is mainly due to the presence of tobaccospecific nitrosamine (TSNA) which is a proven and potent carcinogen, in addition to polycyclic aromatic hydrocarbons (PAHs) in varying concentrations.erefore, previous studies mainly aimed to detect the concentrations of nicotine, TSNA, and PAHs in various ST samples [31,32].

Conclusion
In conclusion, various toxic and carcinogenic components have been identified by GC-MS analysis of several shammah samples collected from the Jazan region.Several uncommon constituents have also been identified owing to the fact that shammah is locally manufactured and is an admixture of various additives along with tobacco leaves.ese additives are added to enhance the organoleptic properties, taste, and mood-enhancing properties of shammah.However, these additives vary from one manufacturer to another and also among shammah samples collected from different places and may have serious adverse effects on human health.e knowledge of these constituents and their harmful effects are necessary.e cytotoxicity investigations showed that these samples have promoted the proliferation of cancer cells and killing the normal human cells within rich mixtures.Correlations are clear between the composition of these shammah samples and their carcinogenic properties.us, it is more likely that the human saliva containing digested shammah could be reason for many cancers including breast, ovary, and colon cancers.

Table 1 :
e chemical formula/structure of toxic/carcinogenic components identified in various shammah types using GC-MS.