http://informahealthcare.com/iht ISSN: 0895-8378 (print), 1091-7691 (electronic) Inhal Toxicol, Early Online: 1–6 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/08958378.2014.970787
RESEARCH ARTICLE
Genotoxicity of waterpipe smoke in buccal cells and peripheral blood leukocytes as determined by comet assay
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Hadba Jar-Allah Al-Amrah1*, Osama Abdullah Aboznada1, Mohammad Zubair Alam2, M-Zaki Mustafa ElAssouli2, Mohammad Ibrahim Mujallid1, and Sufian Mohamad ElAssouli1,2 1
Biological Sciences Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia and 2King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia Abstract
Keywords
Context: Waterpipe smoke causes DNA damage in peripheral blood leukocytes and in buccal cells of smokers. Objective: To determine the exposure effect of waterpipe smoke on buccal cells and peripheral blood leukocytes in regard to DNA damage using comet assay. Materials and methods: The waterpipe smoke condensates were analyzed by gas chromatography-mass spectrometry (GC-MS). The study was performed on 20 waterpipe smokers. To perform comet assay on bucaal cells of smokers, 10 ml of cell suspension was mixed with 85 ml of pre-warmed 1% low melting agarose, applied to comet slide and electrophoresed. To analyze the effect of smoke condensate in vitro, 1 ml of peripheral blood was mixed with 10 ml of smoke condensate and subjected for comet assay. Results: The GC-MS analysis revealed the presence of 2,3-dihydro-3,5-dihydroxy-6-methyl-4Hpyran-4on, nicotine, hydroxymethyl furancarboxaldehyde and 3-ethoxy-4-hydroxybenzaldehyde in the smoke condensates. Waterpipe smoking caused DNA damage in vivo in buccal cells of smokers. The tail moment and tail length in buccal cells of smokers were 186 ± 26 and 456 ± 71, respectively, which are higher than control. The jurak and moassel smoke condensates were found to cause DNA damage in peripheral blood leukocytes. The moassel smoke condensate was more damaging. Discussion: There is wide misconception that waterpipe smoking is not as harmful as cigarette smoking. This study demonstrated that waterpipe smoke induced DNA damage in exposed cells. Conclusion: Waterpipe smokes cause DNA damage in buccal cells. The smoke condensate of both jurak and moassel caused comet formation suggesting DNA damage in peripheral blood leukocytes.
Comet assay, DNA damage, genotoxicity, jurak, moassel, smoking, waterpipe
Introduction Tobacco smoking is the major reason of preventable cause of morbidity and mortality throughout the world. Every year more than a million smokers die prematurely out of their habit, wreaking havoc on the welfare of families and communities worldwide. Currently, developing countries are bearing most of the burden of tobacco epidemic and it is expected to continue and worsen unless an effective and comprehensive response is visible (Maziak, 2013; WHO, 2011). Cigarette smoking has been widely investigated for its genotoxicity; however, there are only few studies carried out to assess the toxicity associated with waterpipe smoking (Alsatari et al., 2012; El-Setouhy et al., 2008; Rammah et al., 2012). Waterpipe is a device widely used in Middle East
*Present address: College of Medical Applied Sciences, King Khalid University, Abha 61413, Saudi Arabia Address for correspondence: Prof. Sufian Mohamad ElAssouli. E-mail: [email protected]
History Received 28 June 2014 Revised 8 September 2014 Accepted 22 September 2014 Published online 30 October 2014
including Saudi Arabia and North African countries to smoke moassel and jurak (Al-Dawood, 2000). Moassel is a mixture of crude tobacco fermented with molasses (El-Hakim & Uthman, 1999). Different fruit flavors are added to it like apple and strawberry. In a survey on coffee shops in Cairo, Egypt, it was found the most popular form of waterpipe smoking (WHO, 2009). One head of flavored moassel smoke contains nearly 30% of nicotine, which is equivalent to the nicotine present in 20 cigarettes and thus results in 20% more plasma nicotine level (Hadidi & Mohammed, 2004). The jurak is a dark-colored paste, imported from India, is produced when a mixture of tobacco, pulpy fruit such as banana and molasses are all cooked together. In contrast to moassel, jurak contains 15% of tobacco. The jurak smoke has a pleasant, fruity odor (Zahran et al., 1982). There are three distinct types of waterpipes. The Gouza is the oldest form of waterpipe. It has a small water container (about 200–500 ml) that is made of metal (coconut shell was formerly used). The Bouri has a water container made of brass (about 200– 500 ml). The shisha is a larger (about 1000–2000 ml) and
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more decorated form of waterpipe, usually with a water container made of glass. However, it is common to see shisha water containers made of ceramics, rock-crystal or metal, including silver (WHO, 2009). The most famous one is the shisha. Other names, such as narghile, hubble-bubble and hookah, are not used in Saudi Arabia. They all share the structure of a small container half filled with water, which acts as a filter for the smoke drawn by suction from a funnelshaped tobacco holder. The tobacco is usually burned by smoldering charcoal placed on top of it. We used the term waterpipe to cover all these different names for such smoking devices. Waterpipe smoking is a type of tobacco smoking. Tobacco smoke is known to contain more than 50 identified carcinogens such as polycyclic aromatic hydrocarbons, N-nitrosamines and heavy metals (Rogers, 2008). There are several studies that linked waterpipe smoking to lung, oral, nasopharyngeal, esophageal, gastric and bladder cancer but still much work remains to be done to establish and characterize the dose– response relationships between waterpipe smoking and cancer risks (Akl et al., 2010; Feng et al., 2009; Koul et al., 2011; Rastam et al., 2010). A waterpipe smoking session may expose the smoker to more smoke over a longer period of time compared with smoking a cigarette. Typically, a cigarette smoker takes 8–12, 40–75 ml puffs over about 5–7 min and inhale 0.5–0.6 l of smoke. In contrast, waterpipe smoking sessions normally last 20–80 min, during which the smoker may take 50–200 puffs, which range from about 0.15 to 1.0 l each. Consequently, the waterpipe smoker inhale as much smoke during one session as a cigarette smoker would inhale consuming 100 or more cigarettes (Kandela, 2000). Water of the waterpipe does absorb a small amount of the nicotine, yet, as the WHO advisory states, it is likely that the reduced concentration of nicotine in the waterpipe smoke may result in smokers inhaling higher amounts of smoke and thus exposing themselves to higher levels of cancer-causing chemicals and hazardous gases (Kandela, 2000). The waterpipe smokers are at a heightened risk of developing cancer, heart disease, respiratory disease and problems with pregnancy because of the fact that the nicotine, heavy metals and carbon monoxide remain present in waterpipe even after it passes through the water (Knishkowy & Amitai, 2005; Maziak et al., 2004). It has been shown that waterpipe smoke contains 802 mg of tar, compared to 22.3 mg for cigarettes. Waterpipe smoke also contains 145 mg of carbon monoxide compared to 17.3 mg for cigarettes, pointing that there are about 36 times the tar and 8 times the carbon monoxide present in waterpipe than cigarettes (Yadav & Thakur, 2000). Another study showed that waterpipe is just as dangerous for a person’s health as cigarettes (El-Hakim & Uthman, 1999). There is common misconception that smoking a waterpipe is less harmful alternative to cigarettes, and most people who would not ever consider smoking a cigarette, smoke waterpipe regularly. This delusion has drawn more people to use waterpipe who are unaware of the toxicity associated with this kind of smoking pattern (Nuwayhid et al., 1998). Therefore, there is need to educate the people on the misconceptions and adverse health effects of waterpipe smoking, and more researches are needed to support this educational initiative.
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Since studies on health effects of waterpipe are lacking, we planned to examine the genotoxic effects of waterpipe smoking and its condensate on buccal cells and in peripheral blood leukocytes. Genotoxicity was evaluated using the single cell gel electrophoresis (SCGE) also known as comet assay.
Materials and methods Collection of buccal cells This study was performed on 20 waterpipe smokers recruited from different places such as coffee shops and resting areas in Jeddah, Saudi Arabia. Selection criteria: waterpipe smokers were those who use only waterpipe (jurak or moassel) to smoke daily but no cigarette. All subjects were apparently healthy adult males aged between 28 and 65 years, mean age was 37.55 years and were not alcoholics or on medication. All the subjects belonged to middle class families. The frequencies of smoking of subjects ranged from 1 to 4 times a day. Buccal cells from 20 matched apparently healthy nonsmokers were used as control. This study was approved by the Institutional Review Boards of the Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia, prior to the start of the study. All subjects were informed about the study, and written consents were obtained from all of them before taking samples. The time chosen for the sampling of buccal cells was late evening when the subjects usually smoked with waterpipe using jurak or moassel. The only stipulation was that the subject must have smoked at least jurak or moassel for a minimum of 15 min. The samples (buccal cells) were collected at around 11:00 PM by scraping the buccal mucosa with a wooden spatula. The buccal cells were collected in a tube containing 1 ml of Minimal Essential Media (Gibco, Cleveland, TN), wrapped in aluminum foil to protect them from light, stored in refrigerator at 4 C and processed at around 9:00 AM the next day. In this way, the buccal cells were stored for nearly 10 h. Collection of human peripheral blood leukocytes Human peripheral blood leukocytes were collected from non smokers’ healthy adult males who were not taking any drug or medicine and alcohol. The samples were collected in EDTAcontaining vacutainers. Collection of waterpipe smoke condensate To collect waterpipe smoke condensate of jurak and moassel, the hose of waterpipe was connected to a pump fitted with pre-weighed glass fiber filter. The glass fiber filter was replaced every five minutes. Condensate of waterpipe smoke were extracted from these glass fibers using ethanol in a preweighed beaker, then evaporated to dryness at room temperature, and the beaker was weighed again. Finally, the jurak and moassel smoke condensate were dissolved in water so that the concentration was 44.6 mg/ml and 12.8 mg/ml, respectively. Gas chromatography-mass spectrometry analyses of the smoke condensate The smoke condensate was analyzed for organic components by gas chromatography-mass spectrometry (GC-MS). For the
Waterpipe genotoxicity
DOI: 10.3109/08958378.2014.970787
GC-MS analysis, the condensate was dissolved in dichloromethane and injected into the GC-MS machine DB1707/ DB5 carried out at Wessling, Germany. The following program was used: 2 ml injection volume, initial temperature: 50 C, maximum temperature: 350 C, initial time: 3.20 min and equilibration time: 0.50 min.
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SCGE on buccal cells The tubes containing buccal cells were centrifuged at 200 g for 10 min. The supernatant was decanted, and the cell pellet was washed with 500 ml phosphate buffer saline (PBS) and centrifuged. The buccal cells suspension was counted in hemocytometer; 10 ml of this suspension was mixed with 85 ml of pre-warmed (40 C) low melting agarose 1% (w/v). Cells in low melting agarose were applied to a Trevigen comet slideÔ (Gaithersburg, MD) and incubated at room temperature until the gel layer solidified, and then it was layered with 50 ml trypsin solution (0.25% trypsin, 1 mM EDTA in Hanks balanced salt solution) and incubated for 30 min at 37 C. Then slides were washed with PBS. Cell lysis was achieved by treating slides with proteinase-K (1 mg/ml) for 60 min following which the comet slides were left in alkaline solution for 20 min at room temperature in the dark. The gel on slide was subjected to electrophoresis for 20 min (0.9 V/cm) at pH 9.1 in electrophoresis buffer. At the completion of electrophoresis, the slides were rinsed by dipping several times in distilled water. Fixation and precipitation was carried out by immersing the slides in 70% ethanol for 5 min and then air dried. The gels were stained with ethidium bromide (50 mg/ml) and visualized by fluorescence microscope (40). Soon after the preparation of slides, they were analyzed for comet parameters using LAI Comet analysis system, HCSA version 2.3.4 (Loats Associates, Westminster, MD). Slides were then stored at room temperature in a slide box containing silica beads to avoid loss of humidity for less than a week so that if necessary arise they can be analyzed again. Earlier, we carried out electrophoresis at pH 13, but at this high pH, the buccal cells were completely disintegrated. Similar findings were also observed by other investigators who showed that buccal cells sustained massive damage and disintegrated at high pH, but lower pH were extremely resistant to lysis, an essential step in the comet assay. Therefore, successful lysis was achieved by using 0.25% trypsin for 30 min followed by proteinase K (1 mg/ml) treatment for 60 min. When this procedure was performed on cells pre-embedded in agarose on a microscope slide, followed by electrophoresis in 0.01 M NaOH, 1 mM EDTA, pH 9.1, 18 min at 12 V, a satisfactory comet image was obtained (Ostling & Johanson, 1984; Szeto et al., 2005). Genotoxicity of smoke condensate as determined by comet assay using human peripheral blood leukocytes The smoke condensate (10 ml) extracted above was added with 1 ml of peripheral blood in the tube; 10 ml of this mixture was mixed with 85 ml of pre-warmed (40 C) 1% (w/v) low melting agarose for 60 min. Cells in low melting agarose were applied to a Trevigen comet slideÔ and incubated at room temperature until the gel layer solidified, then the slides were
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immersed for 20 min in freshly prepared cold lysing solution (2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Tris, pH 10, 1% sodium sarcosinate), 1% Triton X-100 and 10% DMSO were added just before use for a minimum of 60 min at 4 C. The slides were then laid in horizontal electrophoresis apparatus filled with freshly prepared alkaline solution, pH 13, following which electrophoresis was performed at 1.0 V/cm for 20 min. At the completion of electrophoresis, the slides were rinsed by dipping several times in distilled water. Fixation and precipitation was carried out by immersing the slides in 70% ethanol for 5 min and then air dried. The gels were stained with ethidium bromide (50 mg/ml) and visualized by fluorescence microscope (40) (Elassouli et al., 2007; Singh et al., 1988). Soon after the preparation of slides, they were analyzed for comet parameters using LAI Comet analysis system, HCSA version 2.3.4 (Loats Associates).
Results Analysis of jurak and moassel smoke condensate by GC-MS Analysis of jurak and moassel smoke condensate by GC-MS revealed the presence of many organic compounds. Number of compounds detected in moassel smoke condensate was higher than the jurak smoke condensate. Both smoke condensates were found to contain 5-hydroxymethyl-5furancarboxaldehyde and 3-ethoxy-4-hydroxybenzaldehyde, which are known for their genotoxic and carcinogenic properties (Table 1). The polycyclic aromatic hydrocarbons like benzo[a]anthracene, chrysene, benzo[b]fluoranthene,
Table 1. Compounds screened for their presence in jurak and moassel smoke condensates using GC-MS.
Compound name 1,2,3-Propantriol 2,3-Dihydro-3,5-dihydroxy-6-methy l-4H-pyran-4on 5-Hydroxymethyl-5-furancarboxaldehyde Nicotine 3-Ethoxy-4-hydroxybenzaldehyde Dihydro methyl jasmonate 1,2-Propandiol Vanillin Naphthalene Acenaphthylene Acenaphthene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzo[a]anthracene Chrysene Benzo[b]fluoranthene Benzo[k]fluoranthene Benzo[a]pyrene Dibenz[ah]anthracene Benzo[ghi]perylene
Jurak smoke condensate (mg/kg)
Moassel smoke condensate (mg/kg)
0.820 2.1
0.83 0.56
1.389 1.57 0.46 0.759 – – – – – – – – – – – – – – – – –
2.18 2.16 2.12 2.16 5.278 0.926 – – – – – – – – – – – – – – –
– Not detected. Detection limit of the GC-MS machine was 1 mg.
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Table 2. Comparison of comet assay parameters in buccal cells between waterpipe smokers and non smokers.
Comet assay parameters
Buccal cells of water pipe smokers (n ¼ 20)
Buccal cells of non-smokers (n ¼ 20)
% variation from buccal cells of non smokers
Tail moment Tail length % tail DNA Fragmented DNA
186 ± 26 456 ± 71 97 ± 19 32 ± 3.3
0.05 ± 0.001 9 ± 1.3 1.12 ± 0.02 3.4 ± 0.03
371.9 4966.0 8560.0 841.0
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Values are mean ± SD; from each subject, 50 buccal cells were subjected randomly for comet analysis.
benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a,h]anthracene, anthracene, fluoranthene and pyrene were not detected in either of jurak and moassel smoke condensate. Comet assay on buccal cells of waterpipe smokers The results of comet assay on buccal cells taken from smokers soon after their smoking session are listed in Table 2. Since there was not much difference in the results of buccal cells from jurak and moassel smokers, we have combined their values in the table. For comet analysis, 50 buccal cells were selected randomly from each of 20 smokers and non-smokers, and the mean values and their standard deviations are reported. The tail moment in buccal cells of smokers was found to be 186 ± 26, which is 371.9% higher than the tail moment in buccal cells of non-smokers. The other comet parameters like tail length, % tail DNA and fragmented DNA were 456 ± 71, 97.0 ± 19 and 32.0 ± 3.3, respectively, in buccal cells of smokers, whereas in control group (non smokers), the values of tail length, % tail DNA and fragmented DNA were extremely low. Genotoxicity of jurak and moassel smoke condensate using comet assay on human peripheral blood leukocytes The comet result on the human peripheral blood leukocytes from non smokers and without any exposure to jurak or moassel smoke condensate (control) were found to have mean tail moment 0.01, fragmented DNA 2.8, percent tail DNA 0.31% and tail length as 2.0. These comet results suggest no damage in DNA of control cells (Table 3). When peripheral blood leukocytes were exposed to Jurak or moassel smoke condensate with 10 ml of sample for 10 min caused comet formation in 40% of cells (data not shown). The comets were observed in 88% of cells when incubation temperature was increased to 60 min. Among randomly selected 50 cells for comet analysis on peripheral blood leukocytes from non smokers treated for 60 min with jurak smoke condensate, the mean tail moment was found 12.61 ± 7.41 (minimum 0.69, maximum 28.93), % tail DNA was found in the range from 4.78 to 39.64 (mean 22.36 ± 8.87) and the range of tail length observed between 39 and 294 with mean tail length of 160.74 ± 47.66 (Table 3). Moassel smoke condensate was also found causing comet formation when it was exposed to peripheral blood leukocytes taken from non smokers. Among 50 cells selected randomly for comet analysis, the tail moment
Table 3. Comparison of comet assay parameters in human peripheral blood leukocytes between jurak and moassel smoke condensate.
Comet assay parameters
Jurak smoke condensate
Moassel smoke condensate
Tail moment 12.61 ± 7.41 21.86 ± 13.33 Tail length 160.74 ± 47.66 213.10 ± 75.22 % tail DNA 22.36 ± 8.87 29.03 ± 9.77 Fragmented DNA 5.09 ± 1.41 5.23 ± 1.43
% variation from moassel smoke condensate 73.35 32.5 29.83 2.75
Negative control (peripheral blood leukocytes from non smokers and without any treatment) values: tail moment ¼ 0.01; tail length ¼ 2.0; % tail DNA ¼ 0.31; and fragmented DNA ¼ 2.78.
was found in the range from 3.27 to 49.97 (mean 21.86 ± 13.33). The mean % tail DNA of 50 cells having comet was found to be 29.03 ± 9.77 (minimum 12.79, maximum 46.81) and the mean tail length was 213.10 ± 75.22. These values suggest significant damage in DNA of treated cells (Table 3). On comparing the effect of jurak and moassel smoke condensate on peripheral blood leukocytes, it was observed that moassel smoke condensate was generally more damaging than jurak smoke condensate. Mean of tail moment, % tail DNA and tail length of moassel smoke condensate treated peripheral blood leukocytes were significantly higher than the jurak smoke condensate-treated cells. However, no significant difference was observed between jurak and moassel smoke condensate with regard to fragmented DNA.
Discussion Over the past two decades, the comet assay or SCGE has become one of the standard methods for assessing DNA damage (Collins, 2004). The application of this test includes genotoxicity testing (Hartmann et al., 2003; Tice et al., 2000), biomonitoring (Somorovska´ et al., 1999), molecular epidemiology, ecogenotoxicology (Dixon et al., 2002; Verschaeve & Gilles, 1995), DNA damage and repair (Green et al., 1992). The comet test was first developed and described by Ostling & Johanson (1984) for detecting damages in DNA at single cell level under neutral conditions. The test was subsequently modified and improved by several researchers (Olive et al., 1990; Singh et al., 1988). The composition of organic compounds in waterpipe smoke has been less investigated as compared to the cigarette smoke. However, there are few reports, which characterize the waterpipe smoke using GC-MS and LC-MS techniques (Schubert et al., 2012; Shihadeh & Saleh, 2005). In this study, we found several organic compounds such as naphthalene, phenanthrene, fluoranthene, 5-hydroxymethyl-5-furancarboxyaldehyde, 3-ethoxy-4-hydroxybenzaldehyde. Shihadeh & Saleh (2005) characterized the waterpipe smoke using GCMS and found phenanthrene (0.78 mg), fluoranthene (0.22 mg) and chrysene (0.112 mg) during a 171-puffs smoking session. Burning of organic matrix, such as jurak or moassel, generates a complex mixture of compounds in smoke through a variety of processes. The smoke formation occurs by pyrolysis, oxidation, decarboxylation, dehydration, condensation, distillation and sublimation. El-Aasar & El-Marzabani (1991)
Waterpipe genotoxicity
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DOI: 10.3109/08958378.2014.970787
reported 142 compounds in their study on waterpipe smoke composition. These compounds include higher alkenes, alkynes, aromatic hydrocarbons, alkaloids, phenols, phenolic ethers, esters, alcohols, cyclic and polycyclic hydrocarbons. In our study, polynuclear aromatic hydrocarbons, the major carcinogenic and co-carcinogenic agents in tobacco smoke were not detected in the jurak and moassel smoke condensate. These results are in agreement with previous reports on waterpipe smoke (El-Aasar & El-Marzabani, 1991). However, these results are in contradiction with the findings of Sepetdjian et al. (2008) and Shihadeh & Saleh (2005) who have reported the presence of polynuclear aromatic hydrocarbons in the waterpipe smoke. The highest temperature that tobacco mixture can reach in waterpipe, just under the burning charcoal, is 450 C; however, the PAH are usually formed on burning of tobacco, which exceeds this temperature (Monzer et al., 2008; Shihadeh, 2003). This may be the probable reason of not detecting the PAH in the waterpipe smoke in our study. The other reason may include the sensitivity of the GC-MS machine. The GC-MS machine used for the analysis of PAHs in this study does not detect a quantity less than 1 mg of the PAHs. Monzer et al. (2008) found that charcoal contributes to more than 95% of benzo[a]pyrene production in the waterpipe mainstream smoke. In a study to characterize the components of waterpipe smoke using LC-MS, it was found that it contains formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, etc. in varying amounts ranging from 5.71 mg to 208 mg per smoking session. Formaldehyde was detected up to 111 ± 12 mg per smoking session using waterpipe. This value is five times higher when compared to one 2R4F reference cigarette (Schubert et al., 2012). The study further observed the total carbonyls were present was in the range of 689– 253 mg per session among five waterpipe smoke samples. In this study, we have found high values for all the important comet parameters in buccal cells collected from smokers soon after their active smoking session, which suggests that waterpipe smoke contains DNA-damaging ingredients. It is difficult to compare the findings of this study with other studies since there are only few studies conducted on waterpipe smoke genotoxicity particularly using comet assay. In a study, significantly higher frequencies of micronuclei formation in exfoliated cells of the buccal mucosa were observed in waterpipe smokers compared with never smokers (El-Setouhy et al., 2008). In another study, though conducted on cigarette smoke condensate, was also found genotoxic in comet assay performed on human lymphocytes (Jianlin et al., 2009). In this study, the jurak and moassel smoke condensate were found genotoxic in comet assay on human peripheral blood leukocytes. Previous studies also found waterpipe smoke as genotoxic in other genotoxicity assays. In a genotoxicity study on waterpipe smoke using chromosomal aberration, it was found that waterpipe smoke significantly increased the frequency of chromosomal aberrations. Furthermore, the chromosomal aberrations induced by waterpipe smoke were higher than that induced by cigarette smoke (Alsatari et al., 2012). Many studies have shown a strong correlation between inductions of chromosomal aberrations and the risk of cancer (Norppa et al., 2006; Ray et al., 2001).
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The genetic risk in hookah smoke (a type of waterpipe commonly used in India) has been assessed by Yadav & Thakur (2000). The investigators selected mitotic index, chromosomal aberrations, sister chromatid exchanges and satellite associations for genotoxicity analysis. The researchers found all parameters of genotoxicity showing a significant increase in the smoker group compared with control group (Yadav & Thakur, 2000). In another study, waterpipe use has been shown to enhance the level of micronuclei in buccal mucosa cells of smokers (Boulos et al., 2009). Khabour et al. (2011) reported significantly higher frequencies of sister chromatid exchanges in waterpipe smokers compared with cigarette smokers. In our study on peripheral blood leukocytes using comet assay, we found a significant increase in tail moment of jurak and moassel smoke condensate treated cells compared with control cells. On comparing the results of jurak and moassel smoke condensate, we observed that moassel smoke condensate was more genotoxic than jurak smoke condensate. These results may be explained by the fact that moassel contains more tobacco (30%) than jurak, which contains 15% of tobacco. In complex mixtures such as waterpipe smoke, the mutagenic and genotoxic contaminants usually present at such a low levels, which is difficult to be detected (Courty et al., 2004; Umbuzeiro et al., 2001). The identification of specific chemical substances with genotoxic activity in complex mixture is difficult because few compounds are present at high concentrations. Moreover, most of the times genotoxic activity cannot be attributed to specific compounds in the mixture but rather to the set of properties and chemical interactions of the sample as a whole (Hartnik et al., 2007).
Acknowledgements Authors are also thankful to King Fahd Medical Research Center for providing research facilities to conduct experimental work.
Declaration of interest This research work was generously funded by King Abdulaziz City for Science and Technology by grant No.A-S-11-0788.
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Ostling O, Johanson KJ. (1984). Microelectrophoretic study of radiationinduced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 123:291–8. Rammah M, Dandachi F, Salman R, et al. (2012). In vitro cytotoxicity and mutagenicity of mainstream waterpipe smoke and its functional consequences on alveolar type II derived cells. Toxicol Lett 211: 220–31. Rastam S, Li FM, Fouad FM, et al. (2010). Water pipe smoking and human oral cancers. Med Hypotheses 74:457–9. Ray GN, Shahid M, Husain SA. (2001). Status of chromosome breaks and gaps in breast cancer. A follow-up study. Cancer Genet Cytogenet 130:155–9. Rogers JM. (2008). Tobacco and pregnancy: overview of exposures and effects. Birth Defects Res C Embryo Today 84:1–15. Schubert J, Heinke V, Bewersdorff J, et al. (2012). Waterpipe smoking: the role of humectants in the release of toxic carbonyls. Arch Toxicol 86:1309–16. Sepetdjian E, Shihadeh A, Saliba NA. (2008). Measurement of 16 polycyclic aromatic hydrocarbons in narghile waterpipe tobacco smoke. Food Chem Toxicol 46:1582–90. Shihadeh A, Saleh R. (2005). Polycyclic aromatic hydrocarbons, carbon monoxide, ‘‘tar’’, and nicotine in the mainstream smoke aerosol of the narghile water pipe. Food Chem Toxicol 43:655–61. Shihadeh A. (2003). Investigation of mainstream smoke aerosol of the argileh water pipe. Food Chem Toxicol 41:143–52. Singh NP, McCoy MT, Tice RR, Schneider EL. (1988). A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–91. Somorovska´ M, Szabova´ E, Vodicka P, et al. (1999). Biomonitoring of genotoxic risk in workers in a rubber factory: comparison of the Comet assay with cytogenetic methods and immunology. Mutat Res 445:181–92. Szeto YT, Benzie IFF, Collins AR, et al. (2005). A buccal cell model comet assay: Development and evaluation for human biomonitoring and nutritional studies. Mutat Res-Fund Mol Mutagenesis 578: 371–81. Tice RR, Agurell E, Anderson D, et al. (2000). Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35:206–21. Umbuzeiro GA, Roubicek DA, Sanchez PS, Sato MI. (2001). The Salmonella mutagenicity assay in a surface water quality monitoring program based on a 20-year survey. Mutat Res 491:119–26. Verschaeve L, Gilles J. (1995). Single cell gel electrophoresis assay in the earthworm for the detection of genotoxic compounds in soils. Bull Environ Contam Toxicol 54:112–19. WHO. (2009). Report on the global tobacco epidemic, 2009: implementing smoke-free environments. Geneva: World Health Organization. WHO. (2011). Report on the global tobacco epidemic, 2011. Geneva: World Health Organization. Yadav JS, Thakur S. (2000). Genetic risk assessment in hookah smokers. Cytobios 101:101–13. Zahran F, Yousef AA, Baig MH. (1982). A study of carboxyhaemoglobin levels of cigarette and sheesha smokers in Saudi Arabia. Am J Public Health 72:722–4.
RESEARCH ARTICLE
Genotoxicity of waterpipe smoke in buccal cells and peripheral blood leukocytes as determined by comet assay
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Hadba Jar-Allah Al-Amrah1*, Osama Abdullah Aboznada1, Mohammad Zubair Alam2, M-Zaki Mustafa ElAssouli2, Mohammad Ibrahim Mujallid1, and Sufian Mohamad ElAssouli1,2 1
Biological Sciences Department, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia and 2King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia Abstract
Keywords
Context: Waterpipe smoke causes DNA damage in peripheral blood leukocytes and in buccal cells of smokers. Objective: To determine the exposure effect of waterpipe smoke on buccal cells and peripheral blood leukocytes in regard to DNA damage using comet assay. Materials and methods: The waterpipe smoke condensates were analyzed by gas chromatography-mass spectrometry (GC-MS). The study was performed on 20 waterpipe smokers. To perform comet assay on bucaal cells of smokers, 10 ml of cell suspension was mixed with 85 ml of pre-warmed 1% low melting agarose, applied to comet slide and electrophoresed. To analyze the effect of smoke condensate in vitro, 1 ml of peripheral blood was mixed with 10 ml of smoke condensate and subjected for comet assay. Results: The GC-MS analysis revealed the presence of 2,3-dihydro-3,5-dihydroxy-6-methyl-4Hpyran-4on, nicotine, hydroxymethyl furancarboxaldehyde and 3-ethoxy-4-hydroxybenzaldehyde in the smoke condensates. Waterpipe smoking caused DNA damage in vivo in buccal cells of smokers. The tail moment and tail length in buccal cells of smokers were 186 ± 26 and 456 ± 71, respectively, which are higher than control. The jurak and moassel smoke condensates were found to cause DNA damage in peripheral blood leukocytes. The moassel smoke condensate was more damaging. Discussion: There is wide misconception that waterpipe smoking is not as harmful as cigarette smoking. This study demonstrated that waterpipe smoke induced DNA damage in exposed cells. Conclusion: Waterpipe smokes cause DNA damage in buccal cells. The smoke condensate of both jurak and moassel caused comet formation suggesting DNA damage in peripheral blood leukocytes.
Comet assay, DNA damage, genotoxicity, jurak, moassel, smoking, waterpipe
Introduction Tobacco smoking is the major reason of preventable cause of morbidity and mortality throughout the world. Every year more than a million smokers die prematurely out of their habit, wreaking havoc on the welfare of families and communities worldwide. Currently, developing countries are bearing most of the burden of tobacco epidemic and it is expected to continue and worsen unless an effective and comprehensive response is visible (Maziak, 2013; WHO, 2011). Cigarette smoking has been widely investigated for its genotoxicity; however, there are only few studies carried out to assess the toxicity associated with waterpipe smoking (Alsatari et al., 2012; El-Setouhy et al., 2008; Rammah et al., 2012). Waterpipe is a device widely used in Middle East
*Present address: College of Medical Applied Sciences, King Khalid University, Abha 61413, Saudi Arabia Address for correspondence: Prof. Sufian Mohamad ElAssouli. E-mail: [email protected]
History Received 28 June 2014 Revised 8 September 2014 Accepted 22 September 2014 Published online 30 October 2014
including Saudi Arabia and North African countries to smoke moassel and jurak (Al-Dawood, 2000). Moassel is a mixture of crude tobacco fermented with molasses (El-Hakim & Uthman, 1999). Different fruit flavors are added to it like apple and strawberry. In a survey on coffee shops in Cairo, Egypt, it was found the most popular form of waterpipe smoking (WHO, 2009). One head of flavored moassel smoke contains nearly 30% of nicotine, which is equivalent to the nicotine present in 20 cigarettes and thus results in 20% more plasma nicotine level (Hadidi & Mohammed, 2004). The jurak is a dark-colored paste, imported from India, is produced when a mixture of tobacco, pulpy fruit such as banana and molasses are all cooked together. In contrast to moassel, jurak contains 15% of tobacco. The jurak smoke has a pleasant, fruity odor (Zahran et al., 1982). There are three distinct types of waterpipes. The Gouza is the oldest form of waterpipe. It has a small water container (about 200–500 ml) that is made of metal (coconut shell was formerly used). The Bouri has a water container made of brass (about 200– 500 ml). The shisha is a larger (about 1000–2000 ml) and
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more decorated form of waterpipe, usually with a water container made of glass. However, it is common to see shisha water containers made of ceramics, rock-crystal or metal, including silver (WHO, 2009). The most famous one is the shisha. Other names, such as narghile, hubble-bubble and hookah, are not used in Saudi Arabia. They all share the structure of a small container half filled with water, which acts as a filter for the smoke drawn by suction from a funnelshaped tobacco holder. The tobacco is usually burned by smoldering charcoal placed on top of it. We used the term waterpipe to cover all these different names for such smoking devices. Waterpipe smoking is a type of tobacco smoking. Tobacco smoke is known to contain more than 50 identified carcinogens such as polycyclic aromatic hydrocarbons, N-nitrosamines and heavy metals (Rogers, 2008). There are several studies that linked waterpipe smoking to lung, oral, nasopharyngeal, esophageal, gastric and bladder cancer but still much work remains to be done to establish and characterize the dose– response relationships between waterpipe smoking and cancer risks (Akl et al., 2010; Feng et al., 2009; Koul et al., 2011; Rastam et al., 2010). A waterpipe smoking session may expose the smoker to more smoke over a longer period of time compared with smoking a cigarette. Typically, a cigarette smoker takes 8–12, 40–75 ml puffs over about 5–7 min and inhale 0.5–0.6 l of smoke. In contrast, waterpipe smoking sessions normally last 20–80 min, during which the smoker may take 50–200 puffs, which range from about 0.15 to 1.0 l each. Consequently, the waterpipe smoker inhale as much smoke during one session as a cigarette smoker would inhale consuming 100 or more cigarettes (Kandela, 2000). Water of the waterpipe does absorb a small amount of the nicotine, yet, as the WHO advisory states, it is likely that the reduced concentration of nicotine in the waterpipe smoke may result in smokers inhaling higher amounts of smoke and thus exposing themselves to higher levels of cancer-causing chemicals and hazardous gases (Kandela, 2000). The waterpipe smokers are at a heightened risk of developing cancer, heart disease, respiratory disease and problems with pregnancy because of the fact that the nicotine, heavy metals and carbon monoxide remain present in waterpipe even after it passes through the water (Knishkowy & Amitai, 2005; Maziak et al., 2004). It has been shown that waterpipe smoke contains 802 mg of tar, compared to 22.3 mg for cigarettes. Waterpipe smoke also contains 145 mg of carbon monoxide compared to 17.3 mg for cigarettes, pointing that there are about 36 times the tar and 8 times the carbon monoxide present in waterpipe than cigarettes (Yadav & Thakur, 2000). Another study showed that waterpipe is just as dangerous for a person’s health as cigarettes (El-Hakim & Uthman, 1999). There is common misconception that smoking a waterpipe is less harmful alternative to cigarettes, and most people who would not ever consider smoking a cigarette, smoke waterpipe regularly. This delusion has drawn more people to use waterpipe who are unaware of the toxicity associated with this kind of smoking pattern (Nuwayhid et al., 1998). Therefore, there is need to educate the people on the misconceptions and adverse health effects of waterpipe smoking, and more researches are needed to support this educational initiative.
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Since studies on health effects of waterpipe are lacking, we planned to examine the genotoxic effects of waterpipe smoking and its condensate on buccal cells and in peripheral blood leukocytes. Genotoxicity was evaluated using the single cell gel electrophoresis (SCGE) also known as comet assay.
Materials and methods Collection of buccal cells This study was performed on 20 waterpipe smokers recruited from different places such as coffee shops and resting areas in Jeddah, Saudi Arabia. Selection criteria: waterpipe smokers were those who use only waterpipe (jurak or moassel) to smoke daily but no cigarette. All subjects were apparently healthy adult males aged between 28 and 65 years, mean age was 37.55 years and were not alcoholics or on medication. All the subjects belonged to middle class families. The frequencies of smoking of subjects ranged from 1 to 4 times a day. Buccal cells from 20 matched apparently healthy nonsmokers were used as control. This study was approved by the Institutional Review Boards of the Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia, prior to the start of the study. All subjects were informed about the study, and written consents were obtained from all of them before taking samples. The time chosen for the sampling of buccal cells was late evening when the subjects usually smoked with waterpipe using jurak or moassel. The only stipulation was that the subject must have smoked at least jurak or moassel for a minimum of 15 min. The samples (buccal cells) were collected at around 11:00 PM by scraping the buccal mucosa with a wooden spatula. The buccal cells were collected in a tube containing 1 ml of Minimal Essential Media (Gibco, Cleveland, TN), wrapped in aluminum foil to protect them from light, stored in refrigerator at 4 C and processed at around 9:00 AM the next day. In this way, the buccal cells were stored for nearly 10 h. Collection of human peripheral blood leukocytes Human peripheral blood leukocytes were collected from non smokers’ healthy adult males who were not taking any drug or medicine and alcohol. The samples were collected in EDTAcontaining vacutainers. Collection of waterpipe smoke condensate To collect waterpipe smoke condensate of jurak and moassel, the hose of waterpipe was connected to a pump fitted with pre-weighed glass fiber filter. The glass fiber filter was replaced every five minutes. Condensate of waterpipe smoke were extracted from these glass fibers using ethanol in a preweighed beaker, then evaporated to dryness at room temperature, and the beaker was weighed again. Finally, the jurak and moassel smoke condensate were dissolved in water so that the concentration was 44.6 mg/ml and 12.8 mg/ml, respectively. Gas chromatography-mass spectrometry analyses of the smoke condensate The smoke condensate was analyzed for organic components by gas chromatography-mass spectrometry (GC-MS). For the
Waterpipe genotoxicity
DOI: 10.3109/08958378.2014.970787
GC-MS analysis, the condensate was dissolved in dichloromethane and injected into the GC-MS machine DB1707/ DB5 carried out at Wessling, Germany. The following program was used: 2 ml injection volume, initial temperature: 50 C, maximum temperature: 350 C, initial time: 3.20 min and equilibration time: 0.50 min.
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SCGE on buccal cells The tubes containing buccal cells were centrifuged at 200 g for 10 min. The supernatant was decanted, and the cell pellet was washed with 500 ml phosphate buffer saline (PBS) and centrifuged. The buccal cells suspension was counted in hemocytometer; 10 ml of this suspension was mixed with 85 ml of pre-warmed (40 C) low melting agarose 1% (w/v). Cells in low melting agarose were applied to a Trevigen comet slideÔ (Gaithersburg, MD) and incubated at room temperature until the gel layer solidified, and then it was layered with 50 ml trypsin solution (0.25% trypsin, 1 mM EDTA in Hanks balanced salt solution) and incubated for 30 min at 37 C. Then slides were washed with PBS. Cell lysis was achieved by treating slides with proteinase-K (1 mg/ml) for 60 min following which the comet slides were left in alkaline solution for 20 min at room temperature in the dark. The gel on slide was subjected to electrophoresis for 20 min (0.9 V/cm) at pH 9.1 in electrophoresis buffer. At the completion of electrophoresis, the slides were rinsed by dipping several times in distilled water. Fixation and precipitation was carried out by immersing the slides in 70% ethanol for 5 min and then air dried. The gels were stained with ethidium bromide (50 mg/ml) and visualized by fluorescence microscope (40). Soon after the preparation of slides, they were analyzed for comet parameters using LAI Comet analysis system, HCSA version 2.3.4 (Loats Associates, Westminster, MD). Slides were then stored at room temperature in a slide box containing silica beads to avoid loss of humidity for less than a week so that if necessary arise they can be analyzed again. Earlier, we carried out electrophoresis at pH 13, but at this high pH, the buccal cells were completely disintegrated. Similar findings were also observed by other investigators who showed that buccal cells sustained massive damage and disintegrated at high pH, but lower pH were extremely resistant to lysis, an essential step in the comet assay. Therefore, successful lysis was achieved by using 0.25% trypsin for 30 min followed by proteinase K (1 mg/ml) treatment for 60 min. When this procedure was performed on cells pre-embedded in agarose on a microscope slide, followed by electrophoresis in 0.01 M NaOH, 1 mM EDTA, pH 9.1, 18 min at 12 V, a satisfactory comet image was obtained (Ostling & Johanson, 1984; Szeto et al., 2005). Genotoxicity of smoke condensate as determined by comet assay using human peripheral blood leukocytes The smoke condensate (10 ml) extracted above was added with 1 ml of peripheral blood in the tube; 10 ml of this mixture was mixed with 85 ml of pre-warmed (40 C) 1% (w/v) low melting agarose for 60 min. Cells in low melting agarose were applied to a Trevigen comet slideÔ and incubated at room temperature until the gel layer solidified, then the slides were
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immersed for 20 min in freshly prepared cold lysing solution (2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Tris, pH 10, 1% sodium sarcosinate), 1% Triton X-100 and 10% DMSO were added just before use for a minimum of 60 min at 4 C. The slides were then laid in horizontal electrophoresis apparatus filled with freshly prepared alkaline solution, pH 13, following which electrophoresis was performed at 1.0 V/cm for 20 min. At the completion of electrophoresis, the slides were rinsed by dipping several times in distilled water. Fixation and precipitation was carried out by immersing the slides in 70% ethanol for 5 min and then air dried. The gels were stained with ethidium bromide (50 mg/ml) and visualized by fluorescence microscope (40) (Elassouli et al., 2007; Singh et al., 1988). Soon after the preparation of slides, they were analyzed for comet parameters using LAI Comet analysis system, HCSA version 2.3.4 (Loats Associates).
Results Analysis of jurak and moassel smoke condensate by GC-MS Analysis of jurak and moassel smoke condensate by GC-MS revealed the presence of many organic compounds. Number of compounds detected in moassel smoke condensate was higher than the jurak smoke condensate. Both smoke condensates were found to contain 5-hydroxymethyl-5furancarboxaldehyde and 3-ethoxy-4-hydroxybenzaldehyde, which are known for their genotoxic and carcinogenic properties (Table 1). The polycyclic aromatic hydrocarbons like benzo[a]anthracene, chrysene, benzo[b]fluoranthene,
Table 1. Compounds screened for their presence in jurak and moassel smoke condensates using GC-MS.
Compound name 1,2,3-Propantriol 2,3-Dihydro-3,5-dihydroxy-6-methy l-4H-pyran-4on 5-Hydroxymethyl-5-furancarboxaldehyde Nicotine 3-Ethoxy-4-hydroxybenzaldehyde Dihydro methyl jasmonate 1,2-Propandiol Vanillin Naphthalene Acenaphthylene Acenaphthene Fluorene Phenanthrene Anthracene Fluoranthene Pyrene Benzo[a]anthracene Chrysene Benzo[b]fluoranthene Benzo[k]fluoranthene Benzo[a]pyrene Dibenz[ah]anthracene Benzo[ghi]perylene
Jurak smoke condensate (mg/kg)
Moassel smoke condensate (mg/kg)
0.820 2.1
0.83 0.56
1.389 1.57 0.46 0.759 – – – – – – – – – – – – – – – – –
2.18 2.16 2.12 2.16 5.278 0.926 – – – – – – – – – – – – – – –
– Not detected. Detection limit of the GC-MS machine was 1 mg.
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Table 2. Comparison of comet assay parameters in buccal cells between waterpipe smokers and non smokers.
Comet assay parameters
Buccal cells of water pipe smokers (n ¼ 20)
Buccal cells of non-smokers (n ¼ 20)
% variation from buccal cells of non smokers
Tail moment Tail length % tail DNA Fragmented DNA
186 ± 26 456 ± 71 97 ± 19 32 ± 3.3
0.05 ± 0.001 9 ± 1.3 1.12 ± 0.02 3.4 ± 0.03
371.9 4966.0 8560.0 841.0
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Values are mean ± SD; from each subject, 50 buccal cells were subjected randomly for comet analysis.
benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a,h]anthracene, anthracene, fluoranthene and pyrene were not detected in either of jurak and moassel smoke condensate. Comet assay on buccal cells of waterpipe smokers The results of comet assay on buccal cells taken from smokers soon after their smoking session are listed in Table 2. Since there was not much difference in the results of buccal cells from jurak and moassel smokers, we have combined their values in the table. For comet analysis, 50 buccal cells were selected randomly from each of 20 smokers and non-smokers, and the mean values and their standard deviations are reported. The tail moment in buccal cells of smokers was found to be 186 ± 26, which is 371.9% higher than the tail moment in buccal cells of non-smokers. The other comet parameters like tail length, % tail DNA and fragmented DNA were 456 ± 71, 97.0 ± 19 and 32.0 ± 3.3, respectively, in buccal cells of smokers, whereas in control group (non smokers), the values of tail length, % tail DNA and fragmented DNA were extremely low. Genotoxicity of jurak and moassel smoke condensate using comet assay on human peripheral blood leukocytes The comet result on the human peripheral blood leukocytes from non smokers and without any exposure to jurak or moassel smoke condensate (control) were found to have mean tail moment 0.01, fragmented DNA 2.8, percent tail DNA 0.31% and tail length as 2.0. These comet results suggest no damage in DNA of control cells (Table 3). When peripheral blood leukocytes were exposed to Jurak or moassel smoke condensate with 10 ml of sample for 10 min caused comet formation in 40% of cells (data not shown). The comets were observed in 88% of cells when incubation temperature was increased to 60 min. Among randomly selected 50 cells for comet analysis on peripheral blood leukocytes from non smokers treated for 60 min with jurak smoke condensate, the mean tail moment was found 12.61 ± 7.41 (minimum 0.69, maximum 28.93), % tail DNA was found in the range from 4.78 to 39.64 (mean 22.36 ± 8.87) and the range of tail length observed between 39 and 294 with mean tail length of 160.74 ± 47.66 (Table 3). Moassel smoke condensate was also found causing comet formation when it was exposed to peripheral blood leukocytes taken from non smokers. Among 50 cells selected randomly for comet analysis, the tail moment
Table 3. Comparison of comet assay parameters in human peripheral blood leukocytes between jurak and moassel smoke condensate.
Comet assay parameters
Jurak smoke condensate
Moassel smoke condensate
Tail moment 12.61 ± 7.41 21.86 ± 13.33 Tail length 160.74 ± 47.66 213.10 ± 75.22 % tail DNA 22.36 ± 8.87 29.03 ± 9.77 Fragmented DNA 5.09 ± 1.41 5.23 ± 1.43
% variation from moassel smoke condensate 73.35 32.5 29.83 2.75
Negative control (peripheral blood leukocytes from non smokers and without any treatment) values: tail moment ¼ 0.01; tail length ¼ 2.0; % tail DNA ¼ 0.31; and fragmented DNA ¼ 2.78.
was found in the range from 3.27 to 49.97 (mean 21.86 ± 13.33). The mean % tail DNA of 50 cells having comet was found to be 29.03 ± 9.77 (minimum 12.79, maximum 46.81) and the mean tail length was 213.10 ± 75.22. These values suggest significant damage in DNA of treated cells (Table 3). On comparing the effect of jurak and moassel smoke condensate on peripheral blood leukocytes, it was observed that moassel smoke condensate was generally more damaging than jurak smoke condensate. Mean of tail moment, % tail DNA and tail length of moassel smoke condensate treated peripheral blood leukocytes were significantly higher than the jurak smoke condensate-treated cells. However, no significant difference was observed between jurak and moassel smoke condensate with regard to fragmented DNA.
Discussion Over the past two decades, the comet assay or SCGE has become one of the standard methods for assessing DNA damage (Collins, 2004). The application of this test includes genotoxicity testing (Hartmann et al., 2003; Tice et al., 2000), biomonitoring (Somorovska´ et al., 1999), molecular epidemiology, ecogenotoxicology (Dixon et al., 2002; Verschaeve & Gilles, 1995), DNA damage and repair (Green et al., 1992). The comet test was first developed and described by Ostling & Johanson (1984) for detecting damages in DNA at single cell level under neutral conditions. The test was subsequently modified and improved by several researchers (Olive et al., 1990; Singh et al., 1988). The composition of organic compounds in waterpipe smoke has been less investigated as compared to the cigarette smoke. However, there are few reports, which characterize the waterpipe smoke using GC-MS and LC-MS techniques (Schubert et al., 2012; Shihadeh & Saleh, 2005). In this study, we found several organic compounds such as naphthalene, phenanthrene, fluoranthene, 5-hydroxymethyl-5-furancarboxyaldehyde, 3-ethoxy-4-hydroxybenzaldehyde. Shihadeh & Saleh (2005) characterized the waterpipe smoke using GCMS and found phenanthrene (0.78 mg), fluoranthene (0.22 mg) and chrysene (0.112 mg) during a 171-puffs smoking session. Burning of organic matrix, such as jurak or moassel, generates a complex mixture of compounds in smoke through a variety of processes. The smoke formation occurs by pyrolysis, oxidation, decarboxylation, dehydration, condensation, distillation and sublimation. El-Aasar & El-Marzabani (1991)
Waterpipe genotoxicity
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DOI: 10.3109/08958378.2014.970787
reported 142 compounds in their study on waterpipe smoke composition. These compounds include higher alkenes, alkynes, aromatic hydrocarbons, alkaloids, phenols, phenolic ethers, esters, alcohols, cyclic and polycyclic hydrocarbons. In our study, polynuclear aromatic hydrocarbons, the major carcinogenic and co-carcinogenic agents in tobacco smoke were not detected in the jurak and moassel smoke condensate. These results are in agreement with previous reports on waterpipe smoke (El-Aasar & El-Marzabani, 1991). However, these results are in contradiction with the findings of Sepetdjian et al. (2008) and Shihadeh & Saleh (2005) who have reported the presence of polynuclear aromatic hydrocarbons in the waterpipe smoke. The highest temperature that tobacco mixture can reach in waterpipe, just under the burning charcoal, is 450 C; however, the PAH are usually formed on burning of tobacco, which exceeds this temperature (Monzer et al., 2008; Shihadeh, 2003). This may be the probable reason of not detecting the PAH in the waterpipe smoke in our study. The other reason may include the sensitivity of the GC-MS machine. The GC-MS machine used for the analysis of PAHs in this study does not detect a quantity less than 1 mg of the PAHs. Monzer et al. (2008) found that charcoal contributes to more than 95% of benzo[a]pyrene production in the waterpipe mainstream smoke. In a study to characterize the components of waterpipe smoke using LC-MS, it was found that it contains formaldehyde, acetaldehyde, acetone, acrolein, propionaldehyde, etc. in varying amounts ranging from 5.71 mg to 208 mg per smoking session. Formaldehyde was detected up to 111 ± 12 mg per smoking session using waterpipe. This value is five times higher when compared to one 2R4F reference cigarette (Schubert et al., 2012). The study further observed the total carbonyls were present was in the range of 689– 253 mg per session among five waterpipe smoke samples. In this study, we have found high values for all the important comet parameters in buccal cells collected from smokers soon after their active smoking session, which suggests that waterpipe smoke contains DNA-damaging ingredients. It is difficult to compare the findings of this study with other studies since there are only few studies conducted on waterpipe smoke genotoxicity particularly using comet assay. In a study, significantly higher frequencies of micronuclei formation in exfoliated cells of the buccal mucosa were observed in waterpipe smokers compared with never smokers (El-Setouhy et al., 2008). In another study, though conducted on cigarette smoke condensate, was also found genotoxic in comet assay performed on human lymphocytes (Jianlin et al., 2009). In this study, the jurak and moassel smoke condensate were found genotoxic in comet assay on human peripheral blood leukocytes. Previous studies also found waterpipe smoke as genotoxic in other genotoxicity assays. In a genotoxicity study on waterpipe smoke using chromosomal aberration, it was found that waterpipe smoke significantly increased the frequency of chromosomal aberrations. Furthermore, the chromosomal aberrations induced by waterpipe smoke were higher than that induced by cigarette smoke (Alsatari et al., 2012). Many studies have shown a strong correlation between inductions of chromosomal aberrations and the risk of cancer (Norppa et al., 2006; Ray et al., 2001).
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The genetic risk in hookah smoke (a type of waterpipe commonly used in India) has been assessed by Yadav & Thakur (2000). The investigators selected mitotic index, chromosomal aberrations, sister chromatid exchanges and satellite associations for genotoxicity analysis. The researchers found all parameters of genotoxicity showing a significant increase in the smoker group compared with control group (Yadav & Thakur, 2000). In another study, waterpipe use has been shown to enhance the level of micronuclei in buccal mucosa cells of smokers (Boulos et al., 2009). Khabour et al. (2011) reported significantly higher frequencies of sister chromatid exchanges in waterpipe smokers compared with cigarette smokers. In our study on peripheral blood leukocytes using comet assay, we found a significant increase in tail moment of jurak and moassel smoke condensate treated cells compared with control cells. On comparing the results of jurak and moassel smoke condensate, we observed that moassel smoke condensate was more genotoxic than jurak smoke condensate. These results may be explained by the fact that moassel contains more tobacco (30%) than jurak, which contains 15% of tobacco. In complex mixtures such as waterpipe smoke, the mutagenic and genotoxic contaminants usually present at such a low levels, which is difficult to be detected (Courty et al., 2004; Umbuzeiro et al., 2001). The identification of specific chemical substances with genotoxic activity in complex mixture is difficult because few compounds are present at high concentrations. Moreover, most of the times genotoxic activity cannot be attributed to specific compounds in the mixture but rather to the set of properties and chemical interactions of the sample as a whole (Hartnik et al., 2007).
Acknowledgements Authors are also thankful to King Fahd Medical Research Center for providing research facilities to conduct experimental work.
Declaration of interest This research work was generously funded by King Abdulaziz City for Science and Technology by grant No.A-S-11-0788.
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