Substantial Toxic Effect of Water-Pipe Smoking on the Early Stage of Embryonic Development

Substantial Toxic Effect of Water-Pipe Smoking on the Early Stage of Embryonic Development Abstract Background Water-pipe smoking (WPS) is the most widespread tobacco use in the Middle-East, and is rapidly spreading globally. Smoke from WP contains most of the compounds present in cigarette smoke, although in different proportions. WPS is associated with the risk of several human diseases; however, its impact on the early stage of normal development has not been investigated yet. Thus, in this investigation, we assess the effect of WPS on the embryo at the early stage of development. Methods Chicken embryos at 3 days of incubations were used in this study. Meanwhile, we explored the outcome of WPS on angiogenesis using the chorioallantoic membrane (CAM) of the chicken embryos. Finally, quantitative real-time polymerase chain reaction was used to study the regulation of some key control genes of cell proliferation, apoptosis, and migration. Results Our data reveal that WPS inhibits angiogenesis of the CAM and in embryos in comparison with their matched controls; in addition, WPS-exposed embryos show slight reduction in their sizes. We also noted that around 80% of WPS-exposed embryos die before 10 days of incubation. More significantly, WPS induces upregulations of BCL-2, Caspase-8, ATF-3, INHIB-A, and Cadherin 6 genes, which are important key regulators of cell apoptosis, proliferation, and migration. Conclusion Our data reveal, for the first time, that WPS has very toxic effects during the early stage of embryogenesis. Thus, we believe that further studies are required to elucidate the pathogenic effect of WPS on human health especially on the embryo at the early stage of its development. Implications This investigation addresses an important gap on the outcome of WPS during the early stage of embryogenesis. Data of this study point out that WPS can have a very toxic effect on the embryo at this stage. Additionally, results from this report display for the first time that WPS can damage normal angiogenesis of the embryo thus provoking a significant number of embryonic death. Moreover, this study reveals that this effect can occur via the deregulation of several genes related to cell apoptosis, proliferation, and migration. Introduction Tobacco smoking is the major cause of preventable morbidity and mortality worldwide and accounts for 6 million deaths each year (World Health Organization). Tobacco smoking can be used in different formulas, ranging from cigarette, cigar smoking and e-cigarettes in addition to water-pipe. The incidence of tobacco consumption has alarmingly increased among both males and females especially in developing countries.1 Smoking has been causally linked to numerous health consequences including cancers, stroke, coronary heart disease, pneumonia, respiratory effects, diabetes mellitus, rheumatoid arthritis, impaired immune functioning, and inflammation.2 On the other hand, it has been well-documented that secondhand smoke can cause the same effects as direct smoking.3–5 Thus, environmental secondhand smoke is estimated to cause yearly more than 600,000 premature deaths worldwide.6 Water-pipe smoking (WPS) is the most widespread tobacco use in the Middle-East region, and its prevalence around the globe is rapidly increasing. It has been largely used to smoke tobacco flavored with flowers, spices, and fruit with some regional and cultural differences.7,8 Thus, the introduction of flavored tobacco, social acceptability, café and restaurant culture, social interaction, and lack of Shisha specific policy and regulations are important factors for shisha prevalence globally.8 This can be partly explained by the common misconception that WPS is less harmful than cigarettes, particularly since several studies have shown a gap in knowledge regarding the harmful effects of WPS.8,9 Nevertheless, recent studies show clearly the harmful effects of WPS on human health, which include chronic bronchitis, emphysema, coronary artery disease, lung, gastric and esophageal cancers, periodontal disease, obstetrical complications, osteoporosis, and mental health problems, along with significant decrease in lung function and exercise capacity.9 Herein, it is important to highlight that WPS has the same types of toxicants as cigarette smoke, including high levels of nicotine, heavy metals, particulate matter, and various carcinogens.9–11 In addition, the charcoal used to heat the tobacco can also raise health risks by producing high levels of pollutants, such as carbon monoxide, metals, and cancer-causing chemicals.10 Moreover, secondhand smoke from WPS can also cause serious risk of respiratory diseases in addition to other health disorders in exposed nonsmokers.12,13 As we mentioned above, cigarette smoking exerts multiple adverse effects but abundant evidence, mostly in adults, suggests that oxidative stress and free radical damage is a major pathophysiological factor. In addition, smoking during embryogenesis is known to contribute to numerous poor birth outcomes, such as low birth weight and preterm birth, as well as life-long health and developmental problems.14–16 Thus, several molecular studies have analyzed the genotoxic effects of tobacco smoke exposure in fetal tissues. These investigations revealed that cigarette smoking can induce DNA damage, chromosomal instability and disruption of angiogenesis in addition to gene deregulations associated with the extracellular matrix, apoptosis, angiogenesis, response to stress, as well as cell proliferation and migration.15–17 In this context, it is important to highlight that the chicken embryo model was largely used to study embryo-toxicity of cigarette smoking as well as other toxic substances and new materials.14,15,18,19 Thus, it is clear that smoking in different forms can cause a major effect on normal embryonic development. However, the impact of WPS during embryogenesis has not been investigated yet. Thus, in this investigation, we examined, for the first time, the outcome of WPS on the early stage of embryonic development using chicken embryo as a model. Our data show clearly that WPS can induce damage to the normal development of the embryo via the deregulation of several key regulator genes of cell proliferation, apoptosis, and migration which are important events in embryogenesis. Methods Smoking Machine Protocol and WPS Preparation A standard smoking protocol (Aleppo/Beirut Method) was used as described in refs. 20,21, which consists of a total of 171 puffs of 0.53 l volume, a puff duration of 2.6 seconds, and an inter puff interval of 17 seconds. The water-pipe was prepared by padding the head with 10 g of brand tobacco mixture known as “Two Apples”, covering it with aluminum foil and perforating the foil to allow air passage. A charcoal, “Three Kings” brand quick-light briquette, was ignited and placed on top of the head at the beginning of the smoking session. Water in the water bowl was changed at the beginning of every smoking session. The condensate (smoking) was collected using regular laboratory filter paper. Filters were dried and weighted before and after collecting smoke and drying. Afterwards, smoked-filters were solved in PBS or RPMI medium (Qiagen, Toronto, ON, Canada) with final concertation of 20 mg/mL of smoking particles; then PBS and RPMI solutions were filtered using 0.45 μm (Costar, Concord, ON, Canada). This study was approved by QU-IBC Committee of Qatar University number 15/16–17. Embryos and In Ovo WPS Treatment At day 3 White Leghorn chicken embryos were exposed to two puffs (1.6 L WPS/0.5 m2) for 5 minutes or treated by 25 μg suspension of WPS in PBS per embryo. The total number of WPS-exposed and control embryos were 78 and 27, respectively. The treatment and exposure methods were described in refs. 19,20. Subsequently, embryos were sacrificed at days 7–10 of incubation and autopsied; small pieces of brain and heart tissues were removed for RNA extraction and quantitative real time-PCR (qPCR) analysis. Macroscopy and Microscopy Analysis Stereomicroscope was used to examine WPS-exposed or treated embryos in comparison with their matched controls. In our analysis, we focused on blood vessel development on the CAM, as well as in the embryo, particularly brain and liver tissues as previously described in refs. 19,22. RNA Isolation and Quantitative Real-Time PCR (qRT-PCR) Analysis Total RNAs from brain and liver tissues were isolated using RNA extraction kit (QIAGEN Canada Inc, ON, Canada) according to the manufacturer’s protocol. One microgram of total RNA from each sample was used to synthesize cDNA, using Superscript II reverse transcriptase (Invitrogen, Waltham, MA, USA). Afterwards, cDNAs were used as templates for qRT-PCR detection using the SensiFAST SYBR & Fluorescein kit (FroggaBio Scientific solutions, Toronto, ON, Canada). The sequence of primers used to amplify B-Cell lymphoma-2 (BCL-2), Caspase-8, Activation transcription factor-3 (ATF-3), Inhibin β-A (INHIB-A) and Cadherin 6 genes and glyceraldehyde 3 phosphate dehydrogenase genes, are described in Table 1. The amplification reactions were carried out with the Applied Biosystems 7500 Fast Real-Time PCR tool (Applied Biosystems, Waltham, MA, USA). The comparative ΔΔCt method was used for relative quantification of the amount of mRNA in each sample once they were normalized to glyceraldehyde 3 phosphate dehydrogenase transcript levels. Genes’ expressions were calculated relative to unexposed embryo tissues messenger RNA (mRNA) levels, which were used as control. SD was expressed as % of Ct values of three independent experiments. Table 1. Primer Sets for BCL-2, CASPAS-8, ATF-3, INHB-A, and Cadherin-6 type-2 Used for Real-Time PCR Amplification Gene names  Primers  BCL-2 (B-cell/lymphoma-2)  5′-GGATGCCTTTGTGGAATTGT-3′    5′-GTCCAAGATAAGCGCCAAGA-3′  CASPAS-8  5′-TCCTCTTGGGCATGACTACC-3′  5′-TGTCAATCTTGCTGCTCACC-3′  ATF-3 (Activating transcription factor-3)  5′-AAAAGCGAAGAAGGGAAAGG-3′  5′-ATACAGGTGGGCCTGTGAAG-3′  INHIB-A (Inhibin β-A)  5′-GCCACCAAGAAACTCCATGT-3′  5′-GCAACGTTTTCTTGGGTGTT-3′  CDH6-2 (Cadherin 6, type 2)  5′-TGCTTCTGGTTGCTGTTTTG-3′  5′-GCAGCTTGCCAACATACTGA-3′  GAPDH (Glyceraldehyde-3-phosphate dehydrogenase)  5′-CCTCTCTGGCAAAGTCCAAG-3′  5′-CATCTGCCCATTTGATGTTG-3′  Gene names  Primers  BCL-2 (B-cell/lymphoma-2)  5′-GGATGCCTTTGTGGAATTGT-3′    5′-GTCCAAGATAAGCGCCAAGA-3′  CASPAS-8  5′-TCCTCTTGGGCATGACTACC-3′  5′-TGTCAATCTTGCTGCTCACC-3′  ATF-3 (Activating transcription factor-3)  5′-AAAAGCGAAGAAGGGAAAGG-3′  5′-ATACAGGTGGGCCTGTGAAG-3′  INHIB-A (Inhibin β-A)  5′-GCCACCAAGAAACTCCATGT-3′  5′-GCAACGTTTTCTTGGGTGTT-3′  CDH6-2 (Cadherin 6, type 2)  5′-TGCTTCTGGTTGCTGTTTTG-3′  5′-GCAGCTTGCCAACATACTGA-3′  GAPDH (Glyceraldehyde-3-phosphate dehydrogenase)  5′-CCTCTCTGGCAAAGTCCAAG-3′  5′-CATCTGCCCATTTGATGTTG-3′  View Large Statistical and Survival Analysis and Angiogenesis Quantification The difference between the number of deaths of WPS-exposed embryos and their matched controls was compared using 2 × 2 Chi square table; meanwhile, survival curves for the two groups were estimated using the Kaplan–Meier Estimator and compared using log-rank test. In parallel, angiogenesis was quantified using the AngioTool program version 0.6a as described by Zudaire et al.23 Three main measurements were compared: vessels area, number of junctions, and total vessels length. Statistical significance was calculated for each of the three measurements. SPSS 64-bit version 23 was applied to carry out the previous tests where probabilities less than .05 were considered statistically significant. Results In order to study the effect of WPS on the embryo at the early stage of development, we examined the outcome of WPS exposure on the chicken embryo at the third day of incubation. Seventy-eight embryos were exposed to WPS, as described in the method section; in parallel, 27 embryos were used as controls and exposed to lab air or buffer solution. While examining the embryos daily, we found that approximately 50% of the embryos died 1 day after the treatment, compared to the control groups in which less than 10% of the embryos died (Table 2); this effect on the control groups could be associated with the manipulation, as reported in our previous studies.19,22 Four days after treatment, around 80% of WPS-exposed embryos died in comparison with their matched controls (Table 2). Thus, WPS decreases significantly the survival probability of the exposed embryos in comparison with their matched controls (Figure 1). Survived embryos were autopsied at 7–8 days of incubation; we noted that WPS inhibits the angiogenesis of the chorioallantoic membrane (CAM) by approximately 30% (Figures 2 and 3) and in the embryo, especially the brain (data not shown). On the other hand, our data revealed that WPS can harm the survival of embryos since all WPS-exposed embryos are smaller in comparison with their controls at the same age, 7 and 8 days of incubation; meanwhile, it is important to highlight that 62 of 78 embryos died before 8 days (p < .001) of incubation. Table 2. Number of Chicken Embryos Used in This Investigation Embryo  Number of cases  Number of embryos died before 10 days of incubation (%)  WPS-exposed  78  62 (79.48)  Control  27  6 (22.22)  Embryo  Number of cases  Number of embryos died before 10 days of incubation (%)  WPS-exposed  78  62 (79.48)  Control  27  6 (22.22)  We note that approximately 80% of WPS-exposed embryo die 4 days after exposure. The embryos were exposed to WPS at 3 days of incubation as described in the Methods section. WPS = Water-pipe smoking. View Large Figure 1. View largeDownload slide Survival analysis of water-pipe smoking (WPS)-exposed embryos and their matched controls. It is clear that WPS significantly decreases the survival probability of the exposed embryos in comparison with their control (p < .001); this analysis was performed as illustrated in the Methods section. Figure 1. View largeDownload slide Survival analysis of water-pipe smoking (WPS)-exposed embryos and their matched controls. It is clear that WPS significantly decreases the survival probability of the exposed embryos in comparison with their control (p < .001); this analysis was performed as illustrated in the Methods section. Figure 2. View largeDownload slide Outcome of water-pipe smoking (WPS) on angiogenesis of the chicken embryo chorioallantoic membrane (CAM) at 7 days of incubation (4 days after WPS-exposure). We remark that WPS inhibits angiogenesis of the CAM in comparison with their matched control. The embryos were exposed to WPS at 3 days of incubation as described in the Methods section. Figure 2. View largeDownload slide Outcome of water-pipe smoking (WPS) on angiogenesis of the chicken embryo chorioallantoic membrane (CAM) at 7 days of incubation (4 days after WPS-exposure). We remark that WPS inhibits angiogenesis of the CAM in comparison with their matched control. The embryos were exposed to WPS at 3 days of incubation as described in the Methods section. Figure 3. View largeDownload slide Quantification of chorioallantoic membrane (CAM) angiogenesis of chicken embryos at 7 days of incubation using AngioTool program. Blood vessels area and junction numbers were compared between water-pipe smoking (WPS)-exposed CAMs and their matched controls. It is clear, from this analysis, that WPS inhibits angiogenesis of the CAM in WPS-exposed embryos in comparison with untreated ones. Embryos were exposed to WPS at 3 days of incubation as illustrated in the Methods section. Figure 3. View largeDownload slide Quantification of chorioallantoic membrane (CAM) angiogenesis of chicken embryos at 7 days of incubation using AngioTool program. Blood vessels area and junction numbers were compared between water-pipe smoking (WPS)-exposed CAMs and their matched controls. It is clear, from this analysis, that WPS inhibits angiogenesis of the CAM in WPS-exposed embryos in comparison with untreated ones. Embryos were exposed to WPS at 3 days of incubation as illustrated in the Methods section. Subsequent, and based on earlier studies related to tobacco smoking during pregnancy16,17 and our own recent investigation vis-à-vis embryonic toxicity,19 we examined the expression of BCL-2, CASPAS-8, ATF-3, INHB-A, and Cadherin-6 type-2 genes in brain and heart tissues from WPS-exposed embryos in comparison with unexposed ones (controls) by qRT-PCR; we found that BCL-2, CASPAS-8, ATF-3, INHB-A, and Cadherin-6 type-2 genes are upregulated in WPS-embryonic tissues in comparison with tissues from control embryos (Figure 4a, b). These results are consistent with the outcome of tobacco smoking studies on the embryo as well as our recent investigation related to the toxicity of nanoparticles in chicken embryos. In this regard, it is important to emphasize that these genes are essential regulators of cell proliferation, apoptosis, and cell migration,24–29 which are critical events during embryogenesis. Figure 4. View largeDownload slide (a, b) Quantitative real-time polymerase chain reaction analysis of BCL-2, Casapas-8, ATF3, INHIB-A, and CDH6 genes in brain (a) and heart (b) tissues of chicken embryos at 8 days of incubation. We note that WPS induce an upregulation of BCL-2 (1), Casapas-8 (2), ATF-3 (3), INHIB-A (4), and CDH6 (5) in six samples (embryos) in comparison with control tissues. The T test was used to compare the expression of these genes in the control tissues and water-pipe smoking (WPS)-exposed ones. The embryos were exposed to WPS at 3 days of incubation as described in the Materials and Methods section. Figure 4. View largeDownload slide (a, b) Quantitative real-time polymerase chain reaction analysis of BCL-2, Casapas-8, ATF3, INHIB-A, and CDH6 genes in brain (a) and heart (b) tissues of chicken embryos at 8 days of incubation. We note that WPS induce an upregulation of BCL-2 (1), Casapas-8 (2), ATF-3 (3), INHIB-A (4), and CDH6 (5) in six samples (embryos) in comparison with control tissues. The T test was used to compare the expression of these genes in the control tissues and water-pipe smoking (WPS)-exposed ones. The embryos were exposed to WPS at 3 days of incubation as described in the Materials and Methods section. Discussion In this investigation, we explored for the first time the outcome of WPS during embryogenesis. Indeed, the effect of WPS is not sufficiently studied in conjunction with human health; however, the few existing data indicate clearly that WPS can be harmful to animals and human cells.30,31 For example, in lungs, the toxic effects of WPS may be associated with the robust and unusual inflammatory response that includes early onset of fibrosis.32 Nevertheless, the effect of WPS on the embryo has not been explored yet, especially at the early stage of development. We reasoned that using the chicken embryo model would be a good approach to determine the influence of WPS during the normal development of the embryo. In this study, we report for the first time that WPS causes a dramatic effect on the embryo at 3 days of incubation, since ~80% of our WPS-exposed embryos die after 4 days of treatment; thus, WPS dramatically affects the survival probability of the exposed embryos in comparison with their controls. Meanwhile, we noted that WPS-exposed embryos are smaller in comparison with their matched controls. These data are consistent with several investigations related to survival rate, size, and weight of embryos and newborns exposed to cigarette smoking.14,33,34 Moreover, our study revealed that WPS can harm the chicken embryo and inhibit angiogenesis of the CAM by ~30% after 3–4 days of exposure; this was accompanied by the induction of important necrosis in different organs, such as the liver and others (data not shown), which could consequently provoke a high rate of mortality in WPS-exposed embryos. These effects were reported by several authors vis-à-vis the outcome of tobacco smoking on embryos.14–16,35 More significantly, it has been demonstrated that tobacco smoking can affect several key gene regulators of angiogenesis, cell survival, and apoptosis such as VEGFA, PGF, FLT1, HIF1A, TP53, BAX, and BCL-2.16,17,36,37 On the other hand, a recent study revealed that WPS can induce lung inflammation and oxidative stress in mice. This was accompanied with DNA damage, enlargement of alveolar spaces and ducts, and the impairment of lung functions.32 In addition, it was shown that tumor necrosis factor-α and interleukin-6 concentrations were significantly increased in the lung following the exposure to WPS in animal models.31 Data from this investigation point out that the heart of WPS-exposed animals show an increase in the expression of inducible nitric oxide synthase and cytochrome C. Meanwhile, it was noted that WPS exposure significantly increased heart DNA damage.32 Moreover, in our lab, we are presently investigating the outcome of WPS on human noninvasive oral cancer cells; our preliminary data indicate that WPS enhances the cell motility of these cancer cells, which is accompanied by a downregulation of E-cadherin (Sadek et al., in preparation). Thus, it is clear that WPS can affect the physiology of the exposed organism via the deregulation of key genes. Herein, we explored for the first time the regulation of BCL-2, CASPAS-8, ATF-3, INHB-A, and Cadherin-6 type-2 genes in brain and heart tissues of WPS-exposed embryos; this was based on earlier studies related to tobacco smoking during pregnancy16,17 and our own recent investigation vis-à-vis embryonic toxicity.19 Moreover, it is well-documented that BCL-2, CASPAS-8, and ATF-3 are important key regulators of cell death and apoptosis.25–27 Meanwhile, it was recently demonstrated that INHB-A and Cadherin-6 type-2 can regulate mesenchymal condition (EMT) event,24,28,29 which is a complex process that allows cells to migrate to ectopic sites during embryogenesis as well as cancer metastasis.37 Based on this fact, we examined the expression of BCL-2, CASPAS-8, ATF-3, INHB-A, and Cadherin-6 type-2 genes in brain and heart tissues from WPS-exposed embryos in comparison with their matched control ones; our data indicate that these genes are upregulated under the effect of WPS in our embryos which can explain the dramatic influence of WPS during embryonic lifecycle. These data are consistent with our previous work regarding the outcome of single-walled carbon nanotubes (SWCNTs) on embryogenesis, wherein we demonstrated that SWCNTs can provoke a significant toxic effect at the early stage of normal development. Our data pointed out that BCL-2, CASPAS-8, ATF-3, INHB-A, and Cadherin-6 type-2 genes are major targets of cytotoxic and genotoxic outcome of SWCNTs in the embryo.19 Additionally, these gene targets of SWCNTs were confirmed in human bronchial epithelial cell models by our group.38 Similarly, WPS can deregulate these genes in human as well as animal organs and tissues including lung.17,31 Herein, we report that WPS also provokes embryonic death at the early stage of embryogenesis, which could be driven by the inhibition of angiogenesis through the deregulation of BCL-2, CASPAS-8, ATF-3, INHB-A, and Cadherin-6 type-2, and possibly other genes involved in normal embryonic development. Conclusion In this investigation, we demonstrate for the first time that WPS has a very toxic effect on the early stages of embryogenesis in the avian model. In parallel, it was identified that BCL-2, CASPAS-8, ATF-3, INHB-A, and Cadherin-6 type-2 genes are important targets of WPS in the embryo. Therefore, this study provides clear evidence that WPS can harm the normal development of the embryo. However, further studies are required to elucidate the pathogenic effect of WPS on human health especially on the embryo at the early stage of its development. Funding This work was supported by the College of Medicine of Qatar University and grant # QUUG-CMED-CMED-15/16-2 from Qatar University. Declaration of Interests The authors have no conflicts of interest to declare. The sponsor had no role in the design and conduct of the study or in the preparation, review, or approval of the manuscript. Acknowledgments The authors would like to thank Mrs. A. Kassab and Mr. D. Octeau for their critical reading of the manuscript. The authors AAA, MYH, and KWS contributed equally to this work. References 1. Lipari RN. Trends in smokeless tobacco use and initiation: 2002 to 2012. The CBHSQ Report . Rockville, MD; 2013. 2. Rojewski AM, Baldassarri S, Cooperman NAet al.  ; Comorbidities Workgroup of the Society for Research on Nicotine and Tobacco (SRNT) Treatment Network. Exploring issues of comorbid conditions in people who smoke. Nicotine Tob Res . 2016; 18( 8): 1684– 1696. Google Scholar CrossRef Search ADS PubMed  3. Chen R, Clifford A, Lang L, Anstey KJ. Is exposure to secondhand smoke associated with cognitive parameters of children and adolescents?–a systematic literature review. Ann Epidemiol . 2013; 23( 10): 652– 661. Google Scholar CrossRef Search ADS PubMed  4. Liu R, Bohac DL, Gundel LA, Hewett MJ, Apte MG, Hammond SK. 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1462-2203
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1469-994X
D.O.I.
10.1093/ntr/ntx135
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Abstract

Abstract Background Water-pipe smoking (WPS) is the most widespread tobacco use in the Middle-East, and is rapidly spreading globally. Smoke from WP contains most of the compounds present in cigarette smoke, although in different proportions. WPS is associated with the risk of several human diseases; however, its impact on the early stage of normal development has not been investigated yet. Thus, in this investigation, we assess the effect of WPS on the embryo at the early stage of development. Methods Chicken embryos at 3 days of incubations were used in this study. Meanwhile, we explored the outcome of WPS on angiogenesis using the chorioallantoic membrane (CAM) of the chicken embryos. Finally, quantitative real-time polymerase chain reaction was used to study the regulation of some key control genes of cell proliferation, apoptosis, and migration. Results Our data reveal that WPS inhibits angiogenesis of the CAM and in embryos in comparison with their matched controls; in addition, WPS-exposed embryos show slight reduction in their sizes. We also noted that around 80% of WPS-exposed embryos die before 10 days of incubation. More significantly, WPS induces upregulations of BCL-2, Caspase-8, ATF-3, INHIB-A, and Cadherin 6 genes, which are important key regulators of cell apoptosis, proliferation, and migration. Conclusion Our data reveal, for the first time, that WPS has very toxic effects during the early stage of embryogenesis. Thus, we believe that further studies are required to elucidate the pathogenic effect of WPS on human health especially on the embryo at the early stage of its development. Implications This investigation addresses an important gap on the outcome of WPS during the early stage of embryogenesis. Data of this study point out that WPS can have a very toxic effect on the embryo at this stage. Additionally, results from this report display for the first time that WPS can damage normal angiogenesis of the embryo thus provoking a significant number of embryonic death. Moreover, this study reveals that this effect can occur via the deregulation of several genes related to cell apoptosis, proliferation, and migration. Introduction Tobacco smoking is the major cause of preventable morbidity and mortality worldwide and accounts for 6 million deaths each year (World Health Organization). Tobacco smoking can be used in different formulas, ranging from cigarette, cigar smoking and e-cigarettes in addition to water-pipe. The incidence of tobacco consumption has alarmingly increased among both males and females especially in developing countries.1 Smoking has been causally linked to numerous health consequences including cancers, stroke, coronary heart disease, pneumonia, respiratory effects, diabetes mellitus, rheumatoid arthritis, impaired immune functioning, and inflammation.2 On the other hand, it has been well-documented that secondhand smoke can cause the same effects as direct smoking.3–5 Thus, environmental secondhand smoke is estimated to cause yearly more than 600,000 premature deaths worldwide.6 Water-pipe smoking (WPS) is the most widespread tobacco use in the Middle-East region, and its prevalence around the globe is rapidly increasing. It has been largely used to smoke tobacco flavored with flowers, spices, and fruit with some regional and cultural differences.7,8 Thus, the introduction of flavored tobacco, social acceptability, café and restaurant culture, social interaction, and lack of Shisha specific policy and regulations are important factors for shisha prevalence globally.8 This can be partly explained by the common misconception that WPS is less harmful than cigarettes, particularly since several studies have shown a gap in knowledge regarding the harmful effects of WPS.8,9 Nevertheless, recent studies show clearly the harmful effects of WPS on human health, which include chronic bronchitis, emphysema, coronary artery disease, lung, gastric and esophageal cancers, periodontal disease, obstetrical complications, osteoporosis, and mental health problems, along with significant decrease in lung function and exercise capacity.9 Herein, it is important to highlight that WPS has the same types of toxicants as cigarette smoke, including high levels of nicotine, heavy metals, particulate matter, and various carcinogens.9–11 In addition, the charcoal used to heat the tobacco can also raise health risks by producing high levels of pollutants, such as carbon monoxide, metals, and cancer-causing chemicals.10 Moreover, secondhand smoke from WPS can also cause serious risk of respiratory diseases in addition to other health disorders in exposed nonsmokers.12,13 As we mentioned above, cigarette smoking exerts multiple adverse effects but abundant evidence, mostly in adults, suggests that oxidative stress and free radical damage is a major pathophysiological factor. In addition, smoking during embryogenesis is known to contribute to numerous poor birth outcomes, such as low birth weight and preterm birth, as well as life-long health and developmental problems.14–16 Thus, several molecular studies have analyzed the genotoxic effects of tobacco smoke exposure in fetal tissues. These investigations revealed that cigarette smoking can induce DNA damage, chromosomal instability and disruption of angiogenesis in addition to gene deregulations associated with the extracellular matrix, apoptosis, angiogenesis, response to stress, as well as cell proliferation and migration.15–17 In this context, it is important to highlight that the chicken embryo model was largely used to study embryo-toxicity of cigarette smoking as well as other toxic substances and new materials.14,15,18,19 Thus, it is clear that smoking in different forms can cause a major effect on normal embryonic development. However, the impact of WPS during embryogenesis has not been investigated yet. Thus, in this investigation, we examined, for the first time, the outcome of WPS on the early stage of embryonic development using chicken embryo as a model. Our data show clearly that WPS can induce damage to the normal development of the embryo via the deregulation of several key regulator genes of cell proliferation, apoptosis, and migration which are important events in embryogenesis. Methods Smoking Machine Protocol and WPS Preparation A standard smoking protocol (Aleppo/Beirut Method) was used as described in refs. 20,21, which consists of a total of 171 puffs of 0.53 l volume, a puff duration of 2.6 seconds, and an inter puff interval of 17 seconds. The water-pipe was prepared by padding the head with 10 g of brand tobacco mixture known as “Two Apples”, covering it with aluminum foil and perforating the foil to allow air passage. A charcoal, “Three Kings” brand quick-light briquette, was ignited and placed on top of the head at the beginning of the smoking session. Water in the water bowl was changed at the beginning of every smoking session. The condensate (smoking) was collected using regular laboratory filter paper. Filters were dried and weighted before and after collecting smoke and drying. Afterwards, smoked-filters were solved in PBS or RPMI medium (Qiagen, Toronto, ON, Canada) with final concertation of 20 mg/mL of smoking particles; then PBS and RPMI solutions were filtered using 0.45 μm (Costar, Concord, ON, Canada). This study was approved by QU-IBC Committee of Qatar University number 15/16–17. Embryos and In Ovo WPS Treatment At day 3 White Leghorn chicken embryos were exposed to two puffs (1.6 L WPS/0.5 m2) for 5 minutes or treated by 25 μg suspension of WPS in PBS per embryo. The total number of WPS-exposed and control embryos were 78 and 27, respectively. The treatment and exposure methods were described in refs. 19,20. Subsequently, embryos were sacrificed at days 7–10 of incubation and autopsied; small pieces of brain and heart tissues were removed for RNA extraction and quantitative real time-PCR (qPCR) analysis. Macroscopy and Microscopy Analysis Stereomicroscope was used to examine WPS-exposed or treated embryos in comparison with their matched controls. In our analysis, we focused on blood vessel development on the CAM, as well as in the embryo, particularly brain and liver tissues as previously described in refs. 19,22. RNA Isolation and Quantitative Real-Time PCR (qRT-PCR) Analysis Total RNAs from brain and liver tissues were isolated using RNA extraction kit (QIAGEN Canada Inc, ON, Canada) according to the manufacturer’s protocol. One microgram of total RNA from each sample was used to synthesize cDNA, using Superscript II reverse transcriptase (Invitrogen, Waltham, MA, USA). Afterwards, cDNAs were used as templates for qRT-PCR detection using the SensiFAST SYBR & Fluorescein kit (FroggaBio Scientific solutions, Toronto, ON, Canada). The sequence of primers used to amplify B-Cell lymphoma-2 (BCL-2), Caspase-8, Activation transcription factor-3 (ATF-3), Inhibin β-A (INHIB-A) and Cadherin 6 genes and glyceraldehyde 3 phosphate dehydrogenase genes, are described in Table 1. The amplification reactions were carried out with the Applied Biosystems 7500 Fast Real-Time PCR tool (Applied Biosystems, Waltham, MA, USA). The comparative ΔΔCt method was used for relative quantification of the amount of mRNA in each sample once they were normalized to glyceraldehyde 3 phosphate dehydrogenase transcript levels. Genes’ expressions were calculated relative to unexposed embryo tissues messenger RNA (mRNA) levels, which were used as control. SD was expressed as % of Ct values of three independent experiments. Table 1. Primer Sets for BCL-2, CASPAS-8, ATF-3, INHB-A, and Cadherin-6 type-2 Used for Real-Time PCR Amplification Gene names  Primers  BCL-2 (B-cell/lymphoma-2)  5′-GGATGCCTTTGTGGAATTGT-3′    5′-GTCCAAGATAAGCGCCAAGA-3′  CASPAS-8  5′-TCCTCTTGGGCATGACTACC-3′  5′-TGTCAATCTTGCTGCTCACC-3′  ATF-3 (Activating transcription factor-3)  5′-AAAAGCGAAGAAGGGAAAGG-3′  5′-ATACAGGTGGGCCTGTGAAG-3′  INHIB-A (Inhibin β-A)  5′-GCCACCAAGAAACTCCATGT-3′  5′-GCAACGTTTTCTTGGGTGTT-3′  CDH6-2 (Cadherin 6, type 2)  5′-TGCTTCTGGTTGCTGTTTTG-3′  5′-GCAGCTTGCCAACATACTGA-3′  GAPDH (Glyceraldehyde-3-phosphate dehydrogenase)  5′-CCTCTCTGGCAAAGTCCAAG-3′  5′-CATCTGCCCATTTGATGTTG-3′  Gene names  Primers  BCL-2 (B-cell/lymphoma-2)  5′-GGATGCCTTTGTGGAATTGT-3′    5′-GTCCAAGATAAGCGCCAAGA-3′  CASPAS-8  5′-TCCTCTTGGGCATGACTACC-3′  5′-TGTCAATCTTGCTGCTCACC-3′  ATF-3 (Activating transcription factor-3)  5′-AAAAGCGAAGAAGGGAAAGG-3′  5′-ATACAGGTGGGCCTGTGAAG-3′  INHIB-A (Inhibin β-A)  5′-GCCACCAAGAAACTCCATGT-3′  5′-GCAACGTTTTCTTGGGTGTT-3′  CDH6-2 (Cadherin 6, type 2)  5′-TGCTTCTGGTTGCTGTTTTG-3′  5′-GCAGCTTGCCAACATACTGA-3′  GAPDH (Glyceraldehyde-3-phosphate dehydrogenase)  5′-CCTCTCTGGCAAAGTCCAAG-3′  5′-CATCTGCCCATTTGATGTTG-3′  View Large Statistical and Survival Analysis and Angiogenesis Quantification The difference between the number of deaths of WPS-exposed embryos and their matched controls was compared using 2 × 2 Chi square table; meanwhile, survival curves for the two groups were estimated using the Kaplan–Meier Estimator and compared using log-rank test. In parallel, angiogenesis was quantified using the AngioTool program version 0.6a as described by Zudaire et al.23 Three main measurements were compared: vessels area, number of junctions, and total vessels length. Statistical significance was calculated for each of the three measurements. SPSS 64-bit version 23 was applied to carry out the previous tests where probabilities less than .05 were considered statistically significant. Results In order to study the effect of WPS on the embryo at the early stage of development, we examined the outcome of WPS exposure on the chicken embryo at the third day of incubation. Seventy-eight embryos were exposed to WPS, as described in the method section; in parallel, 27 embryos were used as controls and exposed to lab air or buffer solution. While examining the embryos daily, we found that approximately 50% of the embryos died 1 day after the treatment, compared to the control groups in which less than 10% of the embryos died (Table 2); this effect on the control groups could be associated with the manipulation, as reported in our previous studies.19,22 Four days after treatment, around 80% of WPS-exposed embryos died in comparison with their matched controls (Table 2). Thus, WPS decreases significantly the survival probability of the exposed embryos in comparison with their matched controls (Figure 1). Survived embryos were autopsied at 7–8 days of incubation; we noted that WPS inhibits the angiogenesis of the chorioallantoic membrane (CAM) by approximately 30% (Figures 2 and 3) and in the embryo, especially the brain (data not shown). On the other hand, our data revealed that WPS can harm the survival of embryos since all WPS-exposed embryos are smaller in comparison with their controls at the same age, 7 and 8 days of incubation; meanwhile, it is important to highlight that 62 of 78 embryos died before 8 days (p < .001) of incubation. Table 2. Number of Chicken Embryos Used in This Investigation Embryo  Number of cases  Number of embryos died before 10 days of incubation (%)  WPS-exposed  78  62 (79.48)  Control  27  6 (22.22)  Embryo  Number of cases  Number of embryos died before 10 days of incubation (%)  WPS-exposed  78  62 (79.48)  Control  27  6 (22.22)  We note that approximately 80% of WPS-exposed embryo die 4 days after exposure. The embryos were exposed to WPS at 3 days of incubation as described in the Methods section. WPS = Water-pipe smoking. View Large Figure 1. View largeDownload slide Survival analysis of water-pipe smoking (WPS)-exposed embryos and their matched controls. It is clear that WPS significantly decreases the survival probability of the exposed embryos in comparison with their control (p < .001); this analysis was performed as illustrated in the Methods section. Figure 1. View largeDownload slide Survival analysis of water-pipe smoking (WPS)-exposed embryos and their matched controls. It is clear that WPS significantly decreases the survival probability of the exposed embryos in comparison with their control (p < .001); this analysis was performed as illustrated in the Methods section. Figure 2. View largeDownload slide Outcome of water-pipe smoking (WPS) on angiogenesis of the chicken embryo chorioallantoic membrane (CAM) at 7 days of incubation (4 days after WPS-exposure). We remark that WPS inhibits angiogenesis of the CAM in comparison with their matched control. The embryos were exposed to WPS at 3 days of incubation as described in the Methods section. Figure 2. View largeDownload slide Outcome of water-pipe smoking (WPS) on angiogenesis of the chicken embryo chorioallantoic membrane (CAM) at 7 days of incubation (4 days after WPS-exposure). We remark that WPS inhibits angiogenesis of the CAM in comparison with their matched control. The embryos were exposed to WPS at 3 days of incubation as described in the Methods section. Figure 3. View largeDownload slide Quantification of chorioallantoic membrane (CAM) angiogenesis of chicken embryos at 7 days of incubation using AngioTool program. Blood vessels area and junction numbers were compared between water-pipe smoking (WPS)-exposed CAMs and their matched controls. It is clear, from this analysis, that WPS inhibits angiogenesis of the CAM in WPS-exposed embryos in comparison with untreated ones. Embryos were exposed to WPS at 3 days of incubation as illustrated in the Methods section. Figure 3. View largeDownload slide Quantification of chorioallantoic membrane (CAM) angiogenesis of chicken embryos at 7 days of incubation using AngioTool program. Blood vessels area and junction numbers were compared between water-pipe smoking (WPS)-exposed CAMs and their matched controls. It is clear, from this analysis, that WPS inhibits angiogenesis of the CAM in WPS-exposed embryos in comparison with untreated ones. Embryos were exposed to WPS at 3 days of incubation as illustrated in the Methods section. Subsequent, and based on earlier studies related to tobacco smoking during pregnancy16,17 and our own recent investigation vis-à-vis embryonic toxicity,19 we examined the expression of BCL-2, CASPAS-8, ATF-3, INHB-A, and Cadherin-6 type-2 genes in brain and heart tissues from WPS-exposed embryos in comparison with unexposed ones (controls) by qRT-PCR; we found that BCL-2, CASPAS-8, ATF-3, INHB-A, and Cadherin-6 type-2 genes are upregulated in WPS-embryonic tissues in comparison with tissues from control embryos (Figure 4a, b). These results are consistent with the outcome of tobacco smoking studies on the embryo as well as our recent investigation related to the toxicity of nanoparticles in chicken embryos. In this regard, it is important to emphasize that these genes are essential regulators of cell proliferation, apoptosis, and cell migration,24–29 which are critical events during embryogenesis. Figure 4. View largeDownload slide (a, b) Quantitative real-time polymerase chain reaction analysis of BCL-2, Casapas-8, ATF3, INHIB-A, and CDH6 genes in brain (a) and heart (b) tissues of chicken embryos at 8 days of incubation. We note that WPS induce an upregulation of BCL-2 (1), Casapas-8 (2), ATF-3 (3), INHIB-A (4), and CDH6 (5) in six samples (embryos) in comparison with control tissues. The T test was used to compare the expression of these genes in the control tissues and water-pipe smoking (WPS)-exposed ones. The embryos were exposed to WPS at 3 days of incubation as described in the Materials and Methods section. Figure 4. View largeDownload slide (a, b) Quantitative real-time polymerase chain reaction analysis of BCL-2, Casapas-8, ATF3, INHIB-A, and CDH6 genes in brain (a) and heart (b) tissues of chicken embryos at 8 days of incubation. We note that WPS induce an upregulation of BCL-2 (1), Casapas-8 (2), ATF-3 (3), INHIB-A (4), and CDH6 (5) in six samples (embryos) in comparison with control tissues. The T test was used to compare the expression of these genes in the control tissues and water-pipe smoking (WPS)-exposed ones. The embryos were exposed to WPS at 3 days of incubation as described in the Materials and Methods section. Discussion In this investigation, we explored for the first time the outcome of WPS during embryogenesis. Indeed, the effect of WPS is not sufficiently studied in conjunction with human health; however, the few existing data indicate clearly that WPS can be harmful to animals and human cells.30,31 For example, in lungs, the toxic effects of WPS may be associated with the robust and unusual inflammatory response that includes early onset of fibrosis.32 Nevertheless, the effect of WPS on the embryo has not been explored yet, especially at the early stage of development. We reasoned that using the chicken embryo model would be a good approach to determine the influence of WPS during the normal development of the embryo. In this study, we report for the first time that WPS causes a dramatic effect on the embryo at 3 days of incubation, since ~80% of our WPS-exposed embryos die after 4 days of treatment; thus, WPS dramatically affects the survival probability of the exposed embryos in comparison with their controls. Meanwhile, we noted that WPS-exposed embryos are smaller in comparison with their matched controls. These data are consistent with several investigations related to survival rate, size, and weight of embryos and newborns exposed to cigarette smoking.14,33,34 Moreover, our study revealed that WPS can harm the chicken embryo and inhibit angiogenesis of the CAM by ~30% after 3–4 days of exposure; this was accompanied by the induction of important necrosis in different organs, such as the liver and others (data not shown), which could consequently provoke a high rate of mortality in WPS-exposed embryos. These effects were reported by several authors vis-à-vis the outcome of tobacco smoking on embryos.14–16,35 More significantly, it has been demonstrated that tobacco smoking can affect several key gene regulators of angiogenesis, cell survival, and apoptosis such as VEGFA, PGF, FLT1, HIF1A, TP53, BAX, and BCL-2.16,17,36,37 On the other hand, a recent study revealed that WPS can induce lung inflammation and oxidative stress in mice. This was accompanied with DNA damage, enlargement of alveolar spaces and ducts, and the impairment of lung functions.32 In addition, it was shown that tumor necrosis factor-α and interleukin-6 concentrations were significantly increased in the lung following the exposure to WPS in animal models.31 Data from this investigation point out that the heart of WPS-exposed animals show an increase in the expression of inducible nitric oxide synthase and cytochrome C. Meanwhile, it was noted that WPS exposure significantly increased heart DNA damage.32 Moreover, in our lab, we are presently investigating the outcome of WPS on human noninvasive oral cancer cells; our preliminary data indicate that WPS enhances the cell motility of these cancer cells, which is accompanied by a downregulation of E-cadherin (Sadek et al., in preparation). Thus, it is clear that WPS can affect the physiology of the exposed organism via the deregulation of key genes. Herein, we explored for the first time the regulation of BCL-2, CASPAS-8, ATF-3, INHB-A, and Cadherin-6 type-2 genes in brain and heart tissues of WPS-exposed embryos; this was based on earlier studies related to tobacco smoking during pregnancy16,17 and our own recent investigation vis-à-vis embryonic toxicity.19 Moreover, it is well-documented that BCL-2, CASPAS-8, and ATF-3 are important key regulators of cell death and apoptosis.25–27 Meanwhile, it was recently demonstrated that INHB-A and Cadherin-6 type-2 can regulate mesenchymal condition (EMT) event,24,28,29 which is a complex process that allows cells to migrate to ectopic sites during embryogenesis as well as cancer metastasis.37 Based on this fact, we examined the expression of BCL-2, CASPAS-8, ATF-3, INHB-A, and Cadherin-6 type-2 genes in brain and heart tissues from WPS-exposed embryos in comparison with their matched control ones; our data indicate that these genes are upregulated under the effect of WPS in our embryos which can explain the dramatic influence of WPS during embryonic lifecycle. These data are consistent with our previous work regarding the outcome of single-walled carbon nanotubes (SWCNTs) on embryogenesis, wherein we demonstrated that SWCNTs can provoke a significant toxic effect at the early stage of normal development. Our data pointed out that BCL-2, CASPAS-8, ATF-3, INHB-A, and Cadherin-6 type-2 genes are major targets of cytotoxic and genotoxic outcome of SWCNTs in the embryo.19 Additionally, these gene targets of SWCNTs were confirmed in human bronchial epithelial cell models by our group.38 Similarly, WPS can deregulate these genes in human as well as animal organs and tissues including lung.17,31 Herein, we report that WPS also provokes embryonic death at the early stage of embryogenesis, which could be driven by the inhibition of angiogenesis through the deregulation of BCL-2, CASPAS-8, ATF-3, INHB-A, and Cadherin-6 type-2, and possibly other genes involved in normal embryonic development. Conclusion In this investigation, we demonstrate for the first time that WPS has a very toxic effect on the early stages of embryogenesis in the avian model. In parallel, it was identified that BCL-2, CASPAS-8, ATF-3, INHB-A, and Cadherin-6 type-2 genes are important targets of WPS in the embryo. Therefore, this study provides clear evidence that WPS can harm the normal development of the embryo. However, further studies are required to elucidate the pathogenic effect of WPS on human health especially on the embryo at the early stage of its development. Funding This work was supported by the College of Medicine of Qatar University and grant # QUUG-CMED-CMED-15/16-2 from Qatar University. Declaration of Interests The authors have no conflicts of interest to declare. The sponsor had no role in the design and conduct of the study or in the preparation, review, or approval of the manuscript. Acknowledgments The authors would like to thank Mrs. A. Kassab and Mr. D. 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Journal

Nicotine and Tobacco ResearchOxford University Press

Published: Apr 1, 2018

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