Corresponding Author: Abderrahim Nemmar
United Arab Emirates University, College of Medicine and Health Sciences, Department of Physiology, P.O. Box 17666, Al Ain (United Arab Emirates)
Tel. +971‐37137533, Fax +971 3 7671966, E-Mail firstname.lastname@example.org; email@example.com
Comparative Study on the Chronic Vascular Responses Induced by Regular Versus Occasional Waterpipe Smoke Inhalation in Mice
Naserddine Hamadia Sumaya Beegamb Nur Elena Zaabab Ozaz Elzakib Badreldin H. Alic Abderrahim Nemmarb,d
aDepartment of Life and Environmental Sciences, College of Natural and Health Sciences, Zayed University, Abu Dhabi, United Arab Emirates, bDepartment of Physiology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates, cDepartment of Pharmacology and Clinical Pharmacy, Sultan Qaboos University, Muscat, Al-Khod, Oman, dZayed Center for Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates
Hookah also known as waterpipe, narghile, argileh, shisha, hubble-bubble, goza, borry, qaylan, chica, and mada’a is a form of traditional smoking made of tobacco pipe with a long stretchy tube that draws the smoke through water contained in a bowel . This practice is ceased to be a middle-eastern culture where we can find it around bars in college campuses in the western hemisphere such as the United States and Europe . The popularity of WPS is rising among trendy youth, university students, and even high-school-aged children [3, 4]. There is an erroneous belief among WPS smokers that WPS is less harmful compared with cigarette smoking (CS) , and this is based on the misconception that the filtration occurs when the smoke passes through the water will diminish the levels of tar, nicotine, and other toxins . A meta-analysis conducted by Neergaard et al. suggested that daily WPS generates nicotine equivalent to daily smoking of 10 cigarettes. Furthermore, occasional WPS is comparable to smoking two cigarettes during 24-hr . There are increasing epidemiological evidences suggesting the potential of WPS of becoming a major public health problem in most Arab countries .
According to the 2015 WHO recommendation and several other reports, WPS probably is addictive as in other forms of tobacco and its consumption may lead to the same detrimental effects caused by cigarette [1, 9-11]. In support of the latter, it has been reported that WPS contains ample quantities of toxicants including nicotine, tar, carbon monoxide, polycyclic aromatic hydrocarbons, nitrosamines, volatile aldehydes, phenols and heavy metals, catechol and hydroquinone [9, 10]. Clinical and experimental studies including our own, demonstrated that WPS causes various diseases and is detrimental to various systems comparable to cigarette smoke, including cancer [10, 12], reproductive system injuries , adverse cardiopulmonary effects  and metabolic syndrome development which is considered a major risk factor for developing thrombosis [15, 16]. Furthermore, it leads to low birth weight [17, 18] and increased neonatal death rate .
It has been reported that occasional or regular WPS users are susceptible to be regular cigarette smokers, signifying the fact that WPS may be a potential leading way for CS smoking . Most of the research on smoking focused on regular smoking. However, recent population data indicate that 22%–33% of US adult smokers are intermittent smokers who do not smoke every day [21, 22]. Moreover, the number of non-daily smokers is anticipated to rise due to the high cost of tobacco and restriction rules in public areas [23, 24]. The classification of occasional smokers is based on the amount and frequency of smoking. The latter includes four categories, (1) smoking two days in the past week, (2) smoking at least 100 cigarettes in their lifetime, but not smoking daily and having smoked in the last 12 months, (3) smoking between 1 and 10 cigarettes per day in the last 30 days, or (4) smoking at least once a week but not every day [25, 26].
Accumulating evidences suggest that light and non-daily cigarette smoking are associated with increased morbidity including a similar risk of cardiovascular diseases as heavier smokers, different types of cancer, respiratory diseases and reproductive health problems [27-31]. Hence, as far as we are aware, there is a scarcity of data on the vascular and systemic effects of occasional WPS and in order to address that, we aimed to investigate the mechanisms underlying the effects of six months exposure to either Occ-WPS (30 minutes/day, 1 day/week) or Reg-WPS (30 minutes/day, 5 days/week) on thrombogenicity, platelet aggregation, endothelial integrity, inflammation, and oxidative stress.
Materials and Methods
Animals and WPS exposure
BALB/c mice of both genders aged 6–8 weeks, weighing 20–25 g (Taconic Farms Inc., Germantown, NY, USA) were housed in the local central animal facility of the College of Medicine and Health Sciences and maintained in controlled light cycle (12-h light:12-h dark cycle), humidity of 60% and, controlled-temperature (22 ± 1°C). Animals had free access to water and food ad libitum.
After one week of acclimatization to the experimental conditions, the mice were indiscriminately separated into 3 groups, air (control), Occ-WPS and Reg-WPS. The WPS exposure protocol has been performed according to previously described methods [32, 33]. Mice were placed in soft restraints and connected to the exposure tower. Using a nose-only exposure system connected to a waterpipe device, the animals were exposed to either air or WPS by their noses (inExpose System, SCIREQ, Canada). Animals were exposed to mainstream WPS generated by commercially available apple-flavored tobacco. For each daily session, 10 grams of tobacco were placed into the WPS head. At the end of WPS exposure session, the remaining tobacco was discarded.
Control mice were exposed to air only. The duration of the session was 30 min/day. Regarding Reg-WPS, mice were exposed to WPS, 5 days/wk for 6 months [32, 33] and for Occ-WPS group, mice were exposed to WPS, 1 day/wk for 6 months. The WPS exposure procedure was monitored by a computerized system. A computer-monitored puff was produced every 1 min (consisting of a 2 s puff time of WPS after that a 58 s of fresh air). Twenty-four hours following the last exposure session, various vascular endpoints were assessed. The total number of animals used in the present study was 83. They were distributed as described below.
Induction of thrombosis in pial arterioles and venules of mouse photochemically
In vivo pial arteriolar and venular thrombogenesis was assessed in separate set of mice at the end of the 6 months exposure period to either Occ-WPS (n=6) or Reg-WPS (n=7) or air (n=7), according to a previously described technique [32, 33]. Briefly, the trachea was intubated after the induction of anesthesia with urethane (1 mg/g body wt ip), and a 2-Fr venous catheter (Portex, Hythe, UK) was inserted in the right jugular vein for the administration of fluorescein (Sigma- Aldrich). Thereafter, a craniotomy was first performed on the left side, using a microdrill, and the dura was stripped open. Only untraumatized preparations were used, and those showing trauma to either the microvessels or underlying brain tissue were discarded. Animals were then placed on the stage of a fluorescence microscope (Olympus, Melville, NY) attached to a camera and DVD recorder. A heating mat was placed under the mice, and body temperature was raised to 37°C, as monitored by a rectal thermoprobe connected to a temperature reader (Physitemp Instruments). The cranial preparation was moistened continuously with artificial cerebrospinal fluid of the following composition (in mM): 23 NaHCO3, 5 KCl, 3 NaH2PO4, 4 MgSO4, 124 NaCl, 2.5 CaCl, and 10 glucose (pH 7.3–7.4). A field containing arterioles and venules of 15–20 µm in diameter was chosen. Such a field was taped before and during the photochemical insult. The photochemical insult was carried out by injecting fluorescein (0.1 ml/mouse of 5% solution) via the jugular vein, which was allowed to circulate for 30–40 s. The cranial preparation was then exposed to stabilized mercury light. This combination produces endothelium injury of the arterioles and venules. This, in turn, causes platelets to adhere at the site of endothelial damage and then aggregate. The platelet aggregates and thrombus formation grow in size until complete arteriolar or venular occlusion. The time from the photochemical injury until full vascular occlusion (time to flow stop) in arterioles and venules was measured in seconds. At the end of the experiments, animals were euthanized by an overdose of urethane.
Prothrombin time (PT) and activated partial thromboplastin time (PTT) measurement in plasma in vitro
In a separate set of mice, at the end of the 6 months exposure period to either Occ-WPS (n=6) or Reg-WPS (n=6) or air (n=7), animals were anesthetized, and the blood was withdrawn from the inferior vena cava and placed in citrate solution (3.2%) (ratio of the blood to anticoagulant: 9:1) for PT and aPTT measurement according to a previously described technique [32, 33]. The PT was measured on freshly collected platelet-poor plasma with human relipidated recombinant thromboplastin (Recombiplastin, Instrumentation Laboratory, Orangeburg, NY) in combination with a Merlin coagulometer (MC 1 VET, Merlin). The aPTT was measured with automated aPTT reagent from bioMerieux (Durham, NC) using a Merlin coagulometer (MC 1 VET, Merlin). Normal plasma used as the reference for both PT and aPTT was prepared by pooling equal portions of platelet-poor plasmas from the blood of seven untreated mice.
Platelet aggregation in mouse whole blood
The platelet aggregation assay in whole blood obtained from a separate set of mice exposed to Occ-WPS (n=7) or Reg-WPS (n=7) or air (n=6) for 6 months was performed as described before [32, 33]. After anesthesia, the blood was withdrawn from the vena cava and placed in citrate (3.2%). Aliquots of 100 µl were added to the wells of a Merlin coagulometer (MC 1 VET, Merlin, Lemgo, Germany). After incubation with ADP (1 µM) for 3 min at 37.2°C, Blood samples were stirred for 3min. At the end of this period, 25µl samples were removed and fixed on ice in 225 ml cellFix (Becton Dickinson, Franklin Lakes, NJ). ADP induction of platelet aggregation is reflected by a decrease in counted single platelets in the blood obtained from Occ-WPS or Reg-WPS or air-exposed mice.
Measurement of tissue factor, tissue plasminogen activator (tPA), P-selectin and E- selectin concentrations in the plasma
The concentrations of tissue factor, P-selectin and E-selectin in the plasma obtained from mice exposed to Occ-WPS (n=8) or Reg-WPS (n=8) or air (n=8) were measured by enzyme-linked immunosorbent (ELISA) assays using commercially available kits obtained from R&D systems (Duo Set, Minneapolis, MN, USA). Regarding tPA, the ELISA kit was obtained from Molecular Innovations (Novi, Michigan, USA).
Erythrocyte and platelet count, hemoglobin concentration and hematocrit were assessed in the same mice reported above. The animals were anesthetized intraperitoneally with pentobarbital sodium (45 mg/kg), and blood was then drawn from the inferior vena cava in EDTA (4%). A sample was used for platelet and red blood cell counts and hematocrit determination using an ABX VET ABC Hematology Analyzer with a mouse card (ABX Diagnostics, Montpellier, France). The remaining blood was centrifuged for 15 min at 4 °C at 900g, and the plasma samples obtained were stored at -80°C until further analysis.
Measurement of lactate dehydrogenase (LDH) activity in the plasma
The LDH activity was measured using commercial kits (Sigma Chemical, St. Louis, MO, USA) which determine the conversion of lactate to pyruvate in the presence of LDH with an equivalent lessening of NAD in the plasma of the mice reported above. The formation of NADH from the reaction can show a difference when measured in absorbance at 340 nm.
Measurement of TNFα, IL-1β, 8-isoprostane and total antioxidant capacity levels in the plasma
The proinflammatory and oxidative stress markers were assessed in the plasma of the mice reported above. The concentration of the pro-inflammatory cytokines, tumor necrosis factor-α (TNFα) and interleukin-1β (IL-1β) were measured by ELISA assays using commercially available kits obtained from R&D systems (Duo Set, Minneapolis, MN, USA). Protein content in each sample was measured by Bradford’s method, as described earlier [32, 33].
The levels of 8-isoprostane and total antioxidants were quantified according to the manufacturer’s instructions provided in the commercially available assay kits obtained from Cayman Chemicals (Michigan, USA).
All graphs were produced using GraphPad Prism Version 7 for Windows software (GraphPad Software Inc, San Diego, CA, United States). Data were expressed as means ± SEM. To assess whether the measured parameters were normally distributed, the Shapiro-Wilk normality test was used. Normally distributed data were tested by using one-way analysis of variance ANOVA followed by Holm-Sidak’s multiple comparisons test. P<0.05 was considered significant.
Photochemically induced thrombosis in pial arterioles and venules of mouse in vivo
Fig. 1 shows the effect of Occ-WPS or Reg-WPS exposure on photochemically induced thrombosis in both pial arterioles and venules of mice. The exposure to either Occ-WPS or Reg-WPS showed a prothrombotic tendency in both pial arterioles and venules. Compared with air-exposed mice, the occlusion time in pial arterioles was significantly shortened following exposure to either Occ-WPS (P<0.00001) or Reg-WPS (P<0.00001) (Fig. 1A). Similarly, in pial venules, Occ-WPS (P<0.00001) or Reg-WPS (P<0.00001) caused a marked reduction of the thrombotic occlusion time. Moreover, in the pial venules there was a statistical significance between Occ-WPS and Reg-WPS groups (P<0.00001) (Fig. 1B).
PT and aPTT
Fig. 2 illustrates the impact of 6 months exposure to either Occ-WPS or Reg-WPS on PT and aPTT in mouse plasma. The shortening of PT and aPTT is indicative of hypercoagulability tendency in the blood. The exposure to Occ-WPS or Reg-WPS induced a statistically significant shortening of PT (P<0.00001, Fig. 2A) and aPTT (P<0.00001, Fig. 2B) compared with plasma obtained from air-exposed mice.
Platelet numbers and platelet aggregation in vitro
As shown in Fig. 3A, Occ-WPS exposure did not affect platelet number in the blood compared with air exposed group. However, Reg-WPS exposure induced significant increase in the platelet numbers (P=0.03). In addition, there was a statistically significant increase in the number of platelets in Reg-WPS group compared with Occ-WPS one (P=0.04). We found that whole blood obtained from mice exposed to either Occ-WPS or Reg-WPS and incubated in vitro with ADP displayed a significant platelet aggregation (P=0.005-P=0.00001) compared with blood collected from mice exposed to air. Moreover, there was a statistical significance between Occ-WPS and Reg-WPS groups (P=0.005) (Fig. 3B).
Concentrations of tissue factor, tPA, P-selectin and E-selectin in the plasma
Fig. 4 exemplifies the effect of Occ-WPS or Reg-WPS on the plasma concentrations of tissue factor (Fig. 4A), tPA (Fig. 4B), P-selectin (Fig. 4C) and E-selectin (Fig. 4D). Compared with air exposed group, exposure to Occ-WPS or Reg-WPS caused a significant increase (P<0.00001) in tissue factor concentration in the plasma and there was statistical significance between Occ-WPS and Reg-WPS groups (P<0.00001). Regarding tPA, a significant reduction was observed only in the plasma of mice exposed to Reg-WPS (P=0.006). In addition, there was a statistical significance between Occ-WPS and Reg-WPS groups (P=0.02). The plasma concentrations of the adhesion molecule P-selectin were significantly increased following Occ-WPS (P=0.004) or Reg-WPS (P=0.0001) exposure compared to air exposed mice. In addition, the concentration of E-selectin was statistically increased in both Occ-WPS (P=0.01) and Reg-WPS (P<0.00001) groups compared with air exposed mice. Moreover, there was a statistical significance in the concentration of E-selectin between Occ-WPS and Reg-WPS groups (P=0.0006).
Erythrocyte numbers, hemoglobin concentration, and hematocrit
The erythrocyte numbers and hematocrit were not affected following Occ-WPS exposure for a period of 6 months compared to air exposed group. However, we observed a significant increase in the erythrocyte numbers (P=0.0004) and hematocrit (P=0.0002) following exposure to Reg-WPS (Fig. 5A and B). Similarly, as shown in Fig. 5C, hemoglobin concentration was only significantly increased (P=0.003) in Reg-WPS (Fig. 5B) group compared with air-exposed mice. In addition, there was a statistical significance between Occ-WPS and Reg-WPS groups for erythrocyte numbers (P=0.02), hematocrit (P=0.01) and hemoglobin concentration (P=0.01).
Lactate Dehydrogenase (LDH) activity in the Plasma
Our results showed no statistical changes in LDH activity in the plasma of mice occasionally exposed to WPS compared with air exposed group. In contrast, we observed a significant increase (P=0.0001) in the activity of LDH in the plasma of mice regularly exposed to WPS for 6 months (Fig. 6). Moreover, there was a statistical significance between Occ-WPS and Reg-WPS groups (P=0.0001).
Concentrations of pro-inflammatory cytokines in the plasma
The concentrations of pro-inflammatory cytokines, TNFα and IL-1β are shown in Fig. 7. We observed that Occ-WPS exposure for a period of 6 months did not have a significant effect on the plasma concentrations of TNFα and IL-1β compared with air exposed group. On the contrary, compared with air exposed group, the concentration of TNFα (P=0.003) and IL-1β (P=0.0001) in the plasma were significantly increased following Reg-WPS exposure. Moreover, both TNFα (P=0.007) and IL-1β (P=0.0006) were significantly increased in Reg-WPS group compared with Occ-WPS group.
Levels of Oxidative Stress Markers in the Plasma
The quantification of the levels of 8-isoprostane and total antioxidants following Occ-WPS or Reg-WPS exposure is illustrated in Fig. 8. Our results showed that the concentration of 8-isoprostane, a marker of lipid peroxidation in the plasma were significantly increased following either Occ-WPS or Reg-WPS compared with air exposed group (P=0.006, Fig. 8A). Likewise, compared with air exposed group, exposure to either Occ-WPS (P=0.006) or Reg-WPS (P=0.009) induced a significant increase in total antioxidants capacity (Fig. 8B).
NH contributed to the interpretation of data, drafting and revising the manuscript. SB, NEZ and OE performed the experiments. BHA contributed to the design and the editing of the manuscript. AN designed, planned, supervised all the experiments, analyzed and interpreted the data and edited the manuscript. All authors have approved the final version of the manuscript.
This work was supported by funds of the Zayed Center for Health Sciences (grants # 12R008 and 12R072) and the College of Medicine and Health Sciences (grant # 12M022) of the United Arab Emirates University and Al-Jalila Foundation (grant # AJF201701).
Statement of Ethics
The project was reviewed and approved by the Institutional Animal Care and Use Committee of the United Arab Emirates University (Approval # ERA_2017_5625) and experiments were performed in accordance with protocols approved by the Institutional Animal Care and Research Advisory Committee.
The authors declare they have no conflict of interests.
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