1 SUBMITTED 21 FEB 22 1 REVISIONS REQ. 10 MAY & 1 JUN 22; REVISIONS RECD. 12 MAY & 12 JUN 22 2 ACCEPTED 15 JUN 22 3 ONLINE-FIRST: JUNE 2022 4 DOI: https://doi.org/10.18295/squmj.6.2022.043 5 6 Cytotoxic and Genotoxic Effects of Waterpipe on Oral Health Status 7 A systematic review and meta-analysis 8 *Ricardo Grillo,1 Mehdi Khemiss,2 Yuri Slusarenko da Silva3 9 10 1Department of Oral and Maxillofacial Surgery, Faculdade São Leopoldo Mandic, 11 Campinas, Brazil; 2Department of Dental Medicine, Fattouma Bourguiba University 12 Hospital, University of Monastir, Monastir, Tunisia; 3Department of Oral & 13 Maxillofacial Surgery, UniFG University Center, Guanambi, Brazil. 14 *Corresponding Author’s e-mail: doutorgrillo@uol.com.br 15 16 Abstract 17 A worldwide increase in waterpipe consumption can be observed. The present 18 systematic review aims to assess cytotoxic and genotoxic impacts on oral health related 19 to waterpipe smoking. We searched MEDLINE, Cochrane Library, and Dimensions 20 evaluating if waterpipe smokers (P) have any cytotoxic or genotoxic effects on oral cells 21 (I) compared to non-smokers (C) regarding mouth neoplasms (O). PRISMA guidelines 22 were adopted for the current systematic review. Review Manager was utilized for 23 statistical analysis (p < 0.05). A risk of bias and summary were performed to assess the 24 grade of the 20 included articles. With some of the articles included, a forest plot was 25 created in different levels. Waterpipe smoking is harmful to oral health, causing 26 cytotoxic and genotoxic effects on oral cells with a Risk Difference of 0.16. It causes a 27 series of detrimental cellular and genetic modifications such as acanthosis, epithelial 28 dysplasia, and hyperparakeratosis. Changes in DNA methylation and p53 expression 29 were assessed among others. In addition, waterpipe has a bunch of carcinogenic 30 compounds. Even with few publications on the subject, articles are very devastating in 31 confirming the carcinogenicity of waterpipe smoking. Waterpipe smoke is cytotoxic and 32 2 genotoxic. Due to the release of many organic compounds, it increases the incidence of 33 oral cancer. 34 Keywords: Mouth Neoplasms; Oral Health; Smoking Water Pipes; Tobacco Use; 35 Toxicity Measure. 36 37 Introduction 38 Tobacco may be smoked in different ways. Waterpipe is one form of tobacco use that 39 has been gaining popularity during the last decades. A systematic review conducted in 40 2018 showed that waterpipe use prevalence was alarmingly high in the Eastern 41 Mediterranean and European regions, especially among youth.1 42 43 The waterpipe smoke contains a wide range of carcinogens such as naphthylamines, 44 tobacco-specific nitrosamines, polycyclic aromatic hydrocarbons, primary aromatic 45 amines, and carbon monoxide carbonyls like formaldehyde, acetaldehyde, or acrolein 2. 46 Waterpipe use has been associated with DNA damage and cell death, and these 47 genotoxicity and cytotoxicity are involved in oral carcinogenesis 3. Laboratory-based 48 investigations have shown various genomic and transcriptomic alterations previously 49 categorized in various cancers 4. In fact, Walters et al. 5 observed changes in DNA 50 methylation at 727 locations in the genome. DNA methylation may predispose the cells 51 to cancer by activating specific genes and repressing others 6. It also plays a significant 52 role in metastasis 7. In addition, nuclear changes in the oral mucosa cells of waterpipe 53 smokers (WS) were reported 8. These changes occur in the early stages of cancer and 54 may be used as biomarkers to screen oral dysplastic and malignant lesions 9. 55 56 However, the contribution of waterpipe use to the development of oral cancer is not 57 well-established 2. Furthermore, the few available studies on this topic were not focused 58 explicitly on oral cancers 10. Systematic reviews determine whether scientific evidence 59 is consistent and can be generalized across populations 11. Therefore, this study aimed to 60 systematically review the scientific literature regarding the cytotoxic or genotoxic 61 effects of waterpipe smoking on oral mucosal cells. 62 63 3 Materials and Methods 64 A systematic review and meta-analysis were conducted according to the Preferred 65 Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines 12. 66 This study protocol was registered in the PROSPERO database. 67 68 Cytotoxic and genotoxic original studies were considered in this systematic review. In 69 addition, inclusion criteria were considered: a) inclusion of waterpipe usual users; b) 70 any cytotoxic or genotoxic effects; c) comparison to group control. 71 72 The first hit was conducted online by two independent reviewers (RG and MK) in 73 MEDLINE (via PubMed), Cochrane Library, Health Virtual Library (BVS) and 74 Dimensions from inception until Dec 12th of 2021. Terms selected in the primary 75 articles selected to justify this review were combined with Boolean operators (OR / 76 AND), answering the acronym PICO (population, intervention, control, and outcome). 77 The following strategy was used: ((((hookah) OR (shisha) OR (waterpipe) OR 78 (“waterpipe”) OR (narghile)))) AND (((oral) OR (oral health) OR (dental) OR 79 (buccal))) NOT (systematic review). PECO acronym to be answered was: Do waterpipe 80 smokers (P) have any cytotoxic or genotoxic effects on oral cells (I) compared to non-81 smokers (C) regarding mouth neoplasms (O)? 82 83 Exclusion criteria were: 1. clinical changes, and 2. radiographic modifications, 3. 84 Studies performed out of head, face and neck region, and 4. Animal studies. 85 Comparative studies but with no conclusion specific to waterpipe toxicity were 86 excluded. Studies that met the inclusion criteria or those with doubtful information 87 either in the title or abstract were selected for full-text assessment in this review’s 88 second round. 89 90 Two different reviewers (RG and MK) independently extracted the following data from 91 the included studies for analysis: year of study, demographic data, cytotoxic and/or 92 genotoxic effect, waterpipe, and control group sizes. Any discrepancies were resolved 93 by consensus. In the case of persistence, arbitration was performed by a third author 94 (Y.S.S.). Alteration such as micronuclei, pyknosis, karyorrhexis and karyolysis were 95 discussed. 96 4 To assess the studies’ quality, the risk of bias was assessed according to the Quality 97 Assessment Tool for Diagnosis Accuracy Studies (QUADAS-2) 13. The results were 98 used in Review Manager Software 5.4 (Review Manager (RevMan) [Computer 99 program]. Version 5.4. Copenhagen: The Nordic Cochrane Centre, The Cochrane 100 Collaboration, 2014). Results were considered statistically significant with a 95% 101 confidence interval. 102 103 The QUADAS-2 Tool was assessed through risk of bias and risk of applicability across 104 studies to evaluate the following questions: (1) Patient selection: description of patient 105 selection and inclusion; (2) Index text: description of the index test, its conduction and 106 interpretation; (3) Reference standard: description of the reference standard, its 107 conduction and interpretation; (4) Flow and timing of ach included article: description 108 of the patient who did not receive the index test or reference standard and who were 109 excluded. 110 111 Results 112 The first bibliographic search redeemed 346 records from databases. BVS have returned 113 no result. Duplicates were removed, remaining 181 articles. After screen reading and 114 excluding paper unrelated to our search, 38 remained. Reports from the same 115 authors/co-authors or same study center were excluded 14–19 as such reviews, comments, 116 letters, hypotheses, and expert opinions 3,20–26. Two exclusive microbiological studies 117 were removed 27,28, same to animal studies 29,30. One study was not found 31. Manual 118 search has retrieved no additional paper. The searched records distribution and the 119 number of studies finally selected are shown in the flow diagram (Fig. 1). 120 121 The full text of all data sets viewed from the first round was independently checked for 122 the same reviewers’eligibility. A total of 20 articles were included in this review. 123 Studies included authors, year of publication, demographic data, cytotoxic or genotoxic 124 evaluation, and the number of patients in waterpipe and control groups are in Table 1. 125 126 Three studies were in vitro 4,32,33, and biological samples were obtained from patients in 127 17 studies 8,31,33-37,39-48. In addition, the levels of pro-inflammatory cytokines, the 128 receptor activator of nuclear factor-κB (RANKL), and osteoprotegerin were evaluated 129 34–36. 130 5 131 Six studies investigated the genotoxic effect of waterpipe smoke 4,32,33,37–39. A comet 132 assay was performed in one study 33. Cell line-based models were used to understand 133 the mechanisms of action of waterpipe smoke on oral cells 4,32. Immortalized non-134 transformed normal human oral keratinocytes (OKF6/TERT1) chronically (eight 135 months) exposed to waterpipe smoke were developed by Patil et al. 4. When phenotypic 136 alterations were studied, they revealed genomic anomalies in OKF6/TERT1-waterpipe 137 cells, with some overexpressed and some downregulated genes. In the other study that 138 developed a cell line-based model, two human normal oral epithelial were treated with 139 100g/L of waterpipe smoke solution for two days 32. When examined, both cells became 140 more elongated and showed decreased cell-cell contact compared to untreated ones. 141 This epithelial-mesenchymal transition was accompanied by the deregulation of a set of 142 genes related to oncogenesis 32. 143 144 On the other hand, eight studies evaluated nuclear changes in cytology samples from the 145 buccal mucosa of patients. Some pathological assessments were performed, including 146 micronuclei (DNA aggregates separate from the primary nucleus), karyorrhexis (nuclear 147 fragmentation), karyolysis (complete dissolution of nuclear components), pyknosis 148 (shrinkage or condensation of a cell), acanthosis (benign abnormal thickening of the 149 stratum spinosum), hyperparakeratosis (abnormal keratinization of the epidermal 150 stratum coreum), and epithelial dysplasia (architectural and cytological epithelial 151 changes). 152 153 The mean of micronuclei, cell nucleus perimeter, and area was contrasting in the WS 154 group compared to NS one 8,38–42. In addition, the mean percentages of karyorrhexis, 155 karyolysis, and pyknosis had substantial changes 43–45. Other histopathologic changes 156 like acanthosis, hyperparakeratosis, and epithelial dysplasia were associated with 157 waterpipe use. An increased oral cancer incidence were related to different types of 158 tobacco use 46,47. 159 160 Waterpipe smoke was associated with changes in DNA methylation 37. In fact, about 161 64% of global DNA methylation was detected in DNA samples isolated from WS 162 compared to NS. In addition, promoter methylation of the MLH1 gene was observed in 163 the oral epithelium of the WS group 37. 164 6 The tumor suppressor protein p53 mutations were also associated with waterpipe use 165 48,49. This alteration could lead to apoptosis, suppression of the cell cycle, senescence, 166 differentiation, and DNA repair 48. 167 168 A meta-analysis was carried out with RevMan 5.4. A forest plot created only with 169 RevMan was possible with different levels of variation. This happened because articles 170 use different cells to assess cytotoxicity and genotoxicity in different ways (Fig.2). Of 171 the 20 articles included, nine rated genotoxicity and 11 rated cytotoxicity. The evidence 172 on literature is that waterpipe smoke causes several cytotoxic and genotoxic effects on 173 oral cells with a risk difference (RD) of 0.16 (95% CI 0.09-0.23, P < 0.00001). 174 175 The graph for the risk of bias (Fig. 3) was created with RevMan 5.4 using the 176 QUADAS-2 protocol. The high quality of the items can be seen in this picture. Articles 177 come from all over the world, mainly from the Middle East (13), three multicentre 178 studies, two from Africa and one each from Europe and South America. This can be 179 explained by the higher and more frequent consumption of waterpipe in the Middle 180 East. 181 182 Discussion 183 Although waterpipe use is a world-spread epidemic, several included studies are 184 possible to note a colossal concern in Middle East countries where waterpipe is smoke 185 is very usual 32,40,48. 186 187 Waterpipe smoke condensate reveals many organic compounds like nicotine, tar, heavy 188 metals, polycyclic aromatic hydrocarbons (naphthalene, phenanthrene, fluoranthene), 189 aldehydes (5-hydroxymethyl-5-furancarboxaldehyde, 3-ethoxy-4-190 hydroxybenzaldehyde), moreover carbon monoxide, well-known substances for their 191 genotoxic and carcinogenic properties 33,40,43. Formaldehyde was detected in waterpipe 192 five times higher than in one 2R4F cigarette 33. A 2R4F cigarette is a standard reference 193 cigarette. The tobacco industry and academic laboratories uses this reference to 194 standardize test items and inhalation toxicity research. 195 196 High values for all critical comet assay parameters (a sensitive technique for DNA 197 damage detection) in buccal cells were found, suggesting waterpipe use is composed of 198 7 DNA-damaging ingredients 32,33,37,38,44. For example, DNA methylation could reach 199 increases of 64% 37; samples with 10% methylation are considered significantly 200 methylated. 201 202 One technique to evaluate the impact of environmental factors on genetic stability is the 203 investigation of micronucleus, products of early events in human carcinogenic 204 processes, especially on the oral cavity; it is considered a biomarker of genotoxicity 41. 205 Total micronuclei (TMN) and cells with micronuclei (CMN) were significantly higher 206 among waterpipe users than never smokers and very similar to cigarette-smokers 8,41,49. 207 Furthermore, there was no association between TMN and CMN with lifetime duration 208 of use, time to first waterpipe smoke of the day, and the number of hagars per day or 209 week 8. Waterpipe use was also related to chromosomal aberrations 33 and enhanced 210 level of micronuclei 8,33,38–41. 211 212 In waterpipe smoke mixtures, mutagenic and genotoxic contaminants are present on low 213 levels, but they are challenging to be detected since a few components are in high 214 concentrations 33. In addition, genotoxicity is not related to a specific compound but a 215 set of properties and chemical interactions of the sample as a whole 33. Waterpipe use is 216 related to genomic and gene expression alterations, both RNA 4,32 and DNA 33,37,44. 217 218 Waterpipe use increased the risk of histopathologic changes, including acanthosis, 219 epithelial dysplasia, hyperparakeratosis, and the development of abnormal rete ridges 46. 220 Acanthosis and epithelial dysplasia in WS were similar to cigarette-smokers (CS) 46. 221 Cytomorphometric quantitative analysis showed higher values for waterpipe users than 222 in NS, including nuclear and cell perimeter, cytoplasm size, cell area, nuclear-223 cytoplasmic ratio, and relation big diameter of nucleus/small diameter of nucleus ratio 224 42,45 besides induction of heterochromatinization of cell nuclei, a situation caused by 225 different stress factors 42,44. There is an increase in many multinucleated cells, pyknosis, 226 karyorrhexis, karyolysis than in NS 38,39,43–45 and slightly higher than in CS 38,43. Higher 227 incidence of vacuolization of cytoplasm concerning NS and even CS 45. Malignant and 228 pre-malignant lesions have a nuclear-cytoplasmic ratio increase. 229 230 Pro-inflammatory cytokine levels (Interleukin-1β, Interleukin-6, Interleukin-3, and 231 Tumor Necrosis Factor-α) were statistically higher among waterpipe-users when 232 8 compared to NS and similar to another kind of tobacco users 32,34–36. Cell necrosis and 233 apoptosis have a higher relation to carcinomas. 234 235 Protein p53 expression has a relation to regulation of apoptosis and genomic stability, a 236 crucial role in tumor suppression, named “guardian of the genome”. WS have a 237 significantly higher p53 mutation than non-smokers 48,49 in samples with malignant, pre-238 malignant, or even normal oral epithelium. This correlation is similar to CS group 49. In 239 addition, the repair index of oral mucosa cells of WS is significantly lower than in NS 240 39. It must be said that the cytotoxic effects of waterpipe are more correlated to time 241 exposure than cigarette smoking 50. 242 243 There is no peak incidence in oral cancer on WS regarding age or gender 43,47 although 244 few papers included female samples due to oral mucosa alterations concern hormonal 245 changes, and waterpipe use is much more common in male than female 45,46. 246 247 Waterpipe and cigarette users had similar effects on oral mucosa 42,43,46, including a 248 substantial association increase with oral squamous cell carcinoma (OSCC) 249 development 37,43,47–49. The combination between waterpipe and shammah (Arabian 250 snuff) or waterpipe and cigarettes led to a higher incidence of oral cancer than just one 251 kind of tobacco use 47,49. The use of waterpipe has more unfavorable effects than 252 smoking cigarettes 38,42. Waterpipe and Shammah combined use increased the risk of 253 developing OSCC by nearly 35 times 47. Khat chewing did not show significant impact 254 on the development of oral cancer 47 . However, when associated with waterpipe use, 255 there was an increase of the risk 46. The cytotoxic effect of waterpipe smoke is more 256 correlated to time exposure than cigarette smoking 41,43,44. 257 258 In addition to more restrictive legislation and interventional policy aspects, tobacco 259 cessation programs must be a priority in some regions, consisting of education, 260 psychological therapy, and pharmacological aid 47 especially for young 40,44,48, who 261 believe waterpipe use is a safe addict. 262 263 9 Conclusions 264 Waterpipe use is genotoxic and cytotoxic with a Risk Difference of 0.16 (P< 0.05). It 265 seems to increase the incidence of oral cancer, contrary to popular belief. Furthermore, 266 its carcinogenicity is similar to cigarette smoke. 267 268 Conflict of Interest 269 No conflict to disclose 270 271 Funding 272 This research did not receive any specific grant from funding agencies in the public, 273 commercial, or not-for-profit sectors. 274 275 Authors’ Contribution 276 RG was involved in conceptualization, design, data collect and analysis and drafting the 277 manuscript. MK and YSS contributed to the design, data collect and analysis and 278 drafting the manuscript. All authors approved the final version of the manuscript. 279 280 References 281 1. Jawad M, Charide R, Waziry R, Darzi A, Ballout RA, Akl EA. The prevalence and 282 trends of waterpipe tobacco smoking: A systematic review. PLoS One. 283 2018;13(2):e0192191. 284 2. Bou Fakhreddine HM, Kanj AN, Kanj NA. The growing epidemic of water pipe 285 smoking: Health effects and future needs. Respir Med. 2014;108(9):1241–53. 286 3. Souza ACF, Galvani MG, de Souza DV, Ribeiro DA. 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Waterpipe tobacco 424 smoking: health effects, research needs and recommended actions for regulators. 425 Geneva; 2015. 426 427 14 Table 1: Included studies in chronological order 428 Author year Locality (Country) Cytotoxic/ Genotoxic waterpipe group control group Ali 46 2007 - (Yemen) Cytotoxic 11 11 El-Setouhy et al. 8 2008 Cairo (Egypt) Genotoxic 128 78 Al-Amrah et al. 33 2014 Jeddah (Saudi Arabia) Genotoxic 20 0 Seifi et al. 45 2014 Babol (Iran) Cytotoxic 40 40 Eker et al. 40 2016 Mersin (Turkey) Genotoxic 30 30 Naderi, Pasha 43 2017 Tehran (Iran) Cytotoxic 25 25 Volkova et al. 42 2017 Krakiv (Ukraine) Cytotoxic 13 38 Abduljabbar et al. 35 2018 Riyadh (Saudi Arabia) Cytotoxic 41 44 Alharbi et al. 47 2018 Jazan (Saudi Arabia) Cytotoxic 70 140 AlQahtani et al. 34 2018 Multicenter Cytotoxic 40 40 Mokeem et al. 36 2018 Riyadh (Saudi Arabia) Cytotoxic 40 38 Silveira et al. 44 2018 Cascavel (Brazil) Genotoxic 40 40 Zaid et al. 48 2018 Syria (Lebanon) Cytotoxic 52 53 Amer et al. 49 2019 Cairo (Egypt) Cytotoxic 16 16 Patil et al. 4 2019 Multicenter Genotoxic - - Prasad et al. 41 2019 Ajman (United Arab Emirates) Genotoxic 100 100 Taghibakhsh et al. 39 2019 Tehran (Iran) Cytotoxic 36 36 López-Ozuna et al. 32 2020 Multicenter Genotoxic - - Rajabi-Moghaddam et al. 38 2020 Birjand (Iran) Genotoxic 30 30 Sabi et al. 37 2020 Irbid (Jordan) Genotoxic 150 150 429 15 430 Figure 1: Flow diagram of included articles. 431 432 16 433 Figure 2: Forest plot generated through RevMan 5.4 434 17 435 436 Figure 3: Risk of bias graph generated through RevMan 5.4 437 438