Indian Journal of Research in Homeopathy

ORIGINAL ARTICLE
Year
: 2020  |  Volume : 8  |  Issue : 2  |  Page : 91--99

Assessment of health status during exposure to cigarette smoke using blood chemistry, lung tissue histology and myocardial metabolism


Chibuzor Stella Ukonu, FO Awobajo, AA Adejare, CN Anigbogu 
 Department of Physiology, Faculty of Basic Medical Sciences, College of Medicine, University of Lagos, Lagos, Nigeria

Correspondence Address:
Ms. Chibuzor Stella Ukonu
Department of Physiology, Faculty of Basic Medical Sciences, College of Medicine, University of Lagos, Lago
Nigeria

Abstract

Background and Objectives: Cardiorespiratory function is critical to well-being. The effect of cigarette smoke exposure (CSE) as a pollutant on human health is of great interest and requires an adequate investigation in order to reduce the burden of its complications. This study was designed to investigate the effect of cigarette smoke on cardiovascular function and airway muscle functional anatomy using an experimental setup. Materials and Methods: Twenty adult male guinea pigs were grouped into four different groups and exposed to different concentrations of cigarette smoke. On the 12th week of exposure, blood pressure parameters were determined with the aid of a pressure transducer connected to a PowerLab data acquisition syste m. Vascular reactivity was also measured in relation to norepinephrine (NE), acetylcholine (ACh), and sodium nitroprusside (SNP). Myocardial oxygen demand (MOD) and baroreceptor reflex sensitivity were equally evaluated. Animals were sacrificed through cervical dislocation, and the lung and trachea were har vested for histological studies. Results: Cigarette smoke significantly decreased blood pressure and heart rate (HR), baroreceptor sensitivity, and MOD (P < 0.05). Furthermore, cigarette smoke significantly increased vascular reactivity to NE by increasing mean arterial blood pressure (MABP) and HR (P < 0.05) but reduced vascular reactivity to graded doses of ACh and SNP by decreasing MABP (P > 0.05). Red blood cell and platelet concentration as well as sodium and calcium level in the blood were all increased at high dose of exposure. Conclusion: CSE resulted in alteration in histological architecture of the lungs and trachea. CSE decreased blood pressure, baroreflex sensitivity, MOD, and vascular reactivity response of MABP to ACh and SNP and increased HR and vascular reactivity response to NE. Furthermore, it also negatively altered the normal architecture of the lung and trachea.



How to cite this article:
Ukonu CS, Awobajo F O, Adejare A A, Anigbogu C N. Assessment of health status during exposure to cigarette smoke using blood chemistry, lung tissue histology and myocardial metabolism.Niger J Exp Clin Biosci 2020;8:91-99


How to cite this URL:
Ukonu CS, Awobajo F O, Adejare A A, Anigbogu C N. Assessment of health status during exposure to cigarette smoke using blood chemistry, lung tissue histology and myocardial metabolism. Niger J Exp Clin Biosci [serial online] 2020 [cited 2021 Apr 17 ];8:91-99
Available from: https://www.njecbonline.org/text.asp?2020/8/2/91/309164


Full Text



 Introduction



The cardiovascular and respiratory systems ensure the adequate supply of oxygenated blood to all body tissues. Tobacco smoking and its deleterious effect on human health is a public health challenge worldwide with huge financial burden on the government and significant reduction in life expectancy among active and passive smokers.[1],[2] Cigarette smoking practiced for recreational purposes or for pleasure sensation is a common activity all over the world, especially in developing countries because of the prevailing socioeconomic challenges.[3] Smoking has been linked to the incidence of cancer and tumor growth[4] with both acute and chronic adverse effects on the respiratory, hematological, and cardiovascular system.[5],[6] According to report, exposure to cigarette smoke increases the risk of developing cardiovascular diseases, hypertension, inflammation, stroke, clotting, and respiratory disorders.[7] Evidence suggests that exposure to cigarette smoke even at low level will have marked increased risk of cardiovascular dysfunction.[6] There is really no safe level of exposure to tobacco smoke;[8] thus, several key scientific findings have revealed that different levels of exposure to cigarette affect the cardiovascular and airway muscle function.[5],[8] This study intends to shed more light on the possible physiological mechanism behind the effect of exposure to cigarette smoke on respiratory airway functions and the cardiovascular system in experimental model.

 Materials and Methods



Experimental animals

Twenty male guinea pigs with weight ranging between 220 g and 470 g were procured from the Animal House, College of Medicine of the University of Lagos, and were allowed to acclimatize for 2 weeks under standard housing condition (ventilated room with 12/12-h light/dark cycle at 24°C ± 2°C). The guinea pigs were fed ad libitum with standard animal chow (animal care complete ration) and water, supplemented with Tridax procumbens.

Experimental design

Animal grouping and exposure to cigarette smoke

The guinea pigs were randomly divided into three groups. Group one (GR-1) served as the control (unexposed to cigarette smoke). GR-2 was exposed to two sticks of cigarette smoke daily (one stick in the morning and one stick in the afternoon) for 12 weeks. GR-3 was exposed to four sticks of cigarette smoke daily (two sticks in the morning and two sticks in the afternoon) for 12 weeks.[9] Group 4 was exposed to four sticks of cigarette smoke daily (two sticks in the morning and two sticks in the afternoon) for 4 weeks, followed by withdrawal to room air for another 8 weeks. The smoking chamber method used was according to that of Awobajo et al.[10] model and each bout of exposure lasted for 15 min. All procedures were carried out in accordance with the guidelines of the Animal Ethics Committee of the College of Medicine, the University of Lagos, with clearance number: CM/HREC/12/16/093 and in accordance with the guidelines of the National Academy of Science's Guide for the Care and Use of Laboratory Animals (2011).

The Animals were kept in cigarette smoke chamber containing graded concentration of cigarette smoke according to Awobajo et al.[10] Exposure to cigarette smoke lasted for 12 weeks in the case of Groups 2, 3, and 4, while the withdrawal group was exposed for 4 weeks, followed by 8 weeks of withdrawal to normal laboratory room environment. The body weight of the animals was measured weekly before the exposure to cigarette smoke (onset of the experiment) and after exposure using an electronic weighing scale (Camry, model: EK3250). Percentage change in weight was calculated as:

[INLINE:1]

Determination of blood pressure parameters, myocardial oxygen demand, and baroreflex sensitivity

Animals were anesthetized on the final day with urethane-chloralose (25% [w/v] - 1% [w/v], 3.5 mL/kg body weight) and the carotid artery cannulated and connected to a pressure transducer connected to a computerized data acquisition system with LabChart-7 pro software (PowerLab-4/24T, model MLT844/P; AD Instruments). The heart rate (HR) (beats/min) was determined by counting the number of arterial pulses that were displayed on the recorder over a period of 60 s. The pulse pressure was determined by subtracting the diastolic blood pressure (DBP) from the systolic blood pressure (SBP) (systolic pressure – diastolic pressure) (mmHg). The mean arterial blood pressure (MABP) was also determined from the formula: diastolic pressure +1/3 (systolic pressure – diastolic pressure) (mmHg). All these data were equally obtained from the auto-calculation from the PowerLab system.

Myocardial oxygen demand (MOD) (rate pressure product) was calculated from the product of SBP and HR (SBP × HR) (mmHg × beats/min). The baroreflex sensitivity (BRS) was equally determined. Both common carotid arteries were isolated in the neck and looped around with loose ligatures after the cannulation. A bilateral carotid occlusion was carried out by occluding both carotid arteries for 45 s with clip. Blood pressure and HR changes were determined.[11] BRS was calculated as change in HR per unit change in MABP (ΔHR/ΔMAP) (beats/min/mmHg).[12] Vascular reactivity was determined by recording the blood pressure and HR after injecting norepinephrine (NE), acetylcholine (ACh), and sodium nitroprusside (SNP) through the right cannulated femoral vein.

Hematological parameters

About 1 ml of blood samples was collected by cardiac puncture into EDTA tubes and used for a full blood count using an auto blood analyzer (BC-5300 Autohematology analyzer, Mindray). The remaining blood was drained into sterile plain sample bottles tubes, allowed to clot for 20 min, and then centrifuged at 7000 rpm for 15 min using an ultracentrifuge, and the serum was collected and kept in Eppendorf tubes at -20°C until used for the electrolyte concentration analysis. The following electrolytes were measured: Na+, K+, Ca2+, and Cl- using the blood analyzer (Nova 16 electrolyte/chemistry analyzer, manufactured by Nova Biomedical, USA).

Histological studies

The lungs and trachea were carefully dissected out under ice and fixed in 10% formal saline and changed every 3 days for four times before histological processing. Histological slides of the tissues were prepared and stained with hematoxylin and eosin.

Data analysis

Data were analyzed using GraphPad Prism 5 (GraphPad Software, 2365 Northside Dr. Suite 560, San Diego, California 92108) and subjected to one-way analysis of variance and least significant difference post hoc multiple comparison test. Results were displayed mean ± standard error of the mean.

 Results



Effect of cigarette smoke on body weight

[Table 1] showed a progressive percentage of weekly weight gained in the control group (3.96, 11.03, 11.04, and 13.03) within the 12 weeks of the experiment. Guinea pigs exposed to low dose of cigarette smoke over 12 weeks recorded a progressive percentage weekly weight gain (0.84, 2.45, 3.96, and 6.47) with the highest weight gain at the 12th week of cigarette smoke exposure (CSE). The high-dose group also recorded a progressive percentage of weekly weight gain (1.68, 6.06, 6.52, and 10.24) within the same 12 weeks of experiment. The recovery group (high dose + withdrawal [HDW]) progressively gained weight weekly (2.24, 4.27, 5.33, and 8.46) within the experimental period.{Table 1}

Effect of cigarette smoke on blood pressure and heart rate

Exposure to low-dose cigarette smoke significantly decreased the SBP, DBP, PP, and MABP of the test groups including the reversal group when compared with the control group. The HR was also significantly decreased (P < 0.05) in the LD and the HDW groups, while a significant increase in HR was recorded in the HDW group compared with the control result.

Effect of cigarette smoke on baroreflex sensitivity

The effect of cigarette smoke on change in HR and change in MABP and BRS is seen in [Table 2]. Change in HR showed a significant increase (P < 0.05) in the test group when compared with the control. Change in MABP showed a significant increase (P < 0.05) in the high-dose and withdrawal groups when compared with the control. A significant decrease (P < 0.001) in baroreceptor reflex sensitivity was recorded in the test group when compared with the control.{Table 2}

Effect of cigarette smoke on myocardial oxygen demand (rate pressure product)

A significant decrease (P < 0.001) in MOD was noted across the test groups when compared with the control in [Table 3]. A significant decrease (P < 0.001) was also observed in the low- and high-dose group when compared with the withdrawal group.{Table 3}

Effect of cigarette smoke on vascular reactivity

[Table 4] shows the effect of cigarette smoke on vascular reactivity responses (MABP and HR) to NE, ACh, and SNP at graded concentration.{Table 4}

Effect of cigarette smoke on vascular reactivity responses to norepinephrine

A significant increase (P < 0.05) in MABP and HR was noted in the vascular responses to NE in the control and test groups when compared to their respective baseline level.

Effect of cigarette smoke on vascular reactivity responses to acetylcholine

A significant decrease (P < 0.05) in HR was noted in the test groups when compared to their baseline in response to ACh. There was a significant decrease (P < 0.05) in MABP and HR of the control group when compared to their baseline in response to graded doses (0.01 M and 0.1 M) of ACh.

Effect of cigarette smoke on vascular reactivity responses to sodium nitroprusside

A significant decrease (P < 0.05) in HR was noted in the test groups when compared to their baseline in response to SNP. There was a significant decrease (P < 0.05) in MABP and HR of the control group when compared to their baseline in response to graded doses (0.01 M and 0.1 M) of SNP.

Effect of cigarette smoke on serum electrolyte level

[Table 5] shows the effect of cigarette smoke on serum electrolyte (K+, Na+, Cl-, and iCa2+) level. A significant increase (P < 0.05) in Na + and Cl- level was observed in the high-dose test group when compared with the control. A significant increase (P < 0.01) in Ca2+ was also noted in the high-dose test group when compared with the control. A comparison between the high-dose and the withdrawal groups showed a significant decrease in the Ca2+ level of the withdrawal group.{Table 5}

Effect of cigarette smoke on hematological parameters

[Table 5] shows the effect of cigarette smoke on white blood cell (WBC), hemoglobin (HGB), red blood cell (RBC), hematocrit (HCT), and platelet (PLT) count. A significant increase (P < 0.01) in WBC count was noted in the high-dose test group when compared with the control.

A significant increase (P < 0.05) in RBC and PLT count was also noted in the high-dose group when compared with the control.

 Discussion



Exposure to cigarette smoke by nonsmokers is a global pandemic implicated as a risk factor in the pathophysiology of cancer and cardiovascular and respiratory disorders.[6],[9] In spite of volume of work done on cigarette smoke and human health, there is still more to be understood about the possible mechanism (s) by which these adverse effects on the cardiopulmonary functions are produced. This study attempted to elucidate the underlying pathophysiology accounting for cardiovascular and respiratory disorders in passive smokers. The finding of this study revealed that CSE resulted in a significant reduction in the average and percentage weight gained from the 1st week of exposure. This finding is in line with other studies that made the use of guinea pigs (Ardite et al., 2006). Other studies using rats recorded a significant decrease in body weight (Teneka et al., 2014).[13] The reason for this variation in body weight is due to the difference in animal model, as guinea pigs are much larger in size than rats and can tolerate prolonged smoke exposure without the rapid weight loss observed in other species (Ardite et al., 2006). The decreased rate of weight gain of the guinea pigs may be related to less calorie intake or increased energy use. Nicotine from cigarette smoke has been known to increase the metabolic rate of sympathoadrenal activation and increases energy expenditure.[14] Nicotine suppresses food intake possibly by modifying levels of neuroregulatory substance known to initiate or suppress feeding.[15] In addition, withdrawal from exposure to cigarette smoke from the result led to further increment of body weight.

Furthermore, CSE significantly decreased SBP, diastolic blood pressure, pulse pressure, and HR. The finding of this study partly agreed with a previous study which showed a decrease in HR with no change in blood pressure in normotensive and hypertensive rats exposed to CSE (Teneka et al., 2014).[13] The effect of withdrawal from CSE could not be demonstrated in this study contrary to other studies that recorded gradual recovery (Teneka et al., 2014).[13] This poor recovery recorded after withdrawal could be as a result of the short period of withdrawal, as recovery of the cardiovascular system after smoke exposure is time dependent.[6]

Baroreceptors are mechanoreceptors that detect the pressure of blood flow through blood vessel and adjust total peripheral resistance and cardiac output.[16] The sensitivity of the baroreceptors was significantly decreased by CSE. This finding is in line with the study carried out by Xiao et al. (2008). Other studies reported no effect of CSE on baroreflex function.[17] This may be due to the possible depressant effect of cigarette smoke on vasomotor center and the cardiovascular system.[14] Reduced baroreflex function is also indicative of cardiovascular disease.[18] Withdrawal from CSE does not restore BRS in this study. Other studies reported that withdrawal restores baroreceptor reflex.[19] The difference between the two studies is explained by methodological factors.[17] Cigarette smoking produces acute myocardial ischemia by adversely affecting the balance of demand for myocardial oxygen and nutrients with myocardial blood supply.[6] This study indicates a significant decrease in MOD by CSE which is in line with other findings.[20],[21] Nicotine is believed to be the primary constituent of cigarette smoke responsible for its acute adverse effects on myocardial oxygen supply and demand.[20] Carbon monoxide, another constituent of cigarette smoke, has been shown to stiffen coronary blood vessel, thereby decreasing MOD.[21] The effect of cigarette smoke on vascular reactivity responses to graded doses of NE, Ach, and SNP showed a variable alteration in the MABP and HR. In this study, a graded dose of NE was shown to significantly increase MABP and HR in the test group when compared to their basal level. The increase in MABP and HR noticed in this study was not dose dependent. This increment is in line with the review by Benowitz and Gourlay.[22] The possible mechanism for this finding is that cigarette smoke potentiates the activity of epinephrine in increasing myocardial contractility as well as HR and blood pressure.[22]

Furthermore, graded doses of ACh and SNP were shown to significantly decrease MABP and HR in the control group when compared with their baseline level but have no significant effect on the MABP of the test groups. This finding is in accordance with previous reports both in human and animal subjects. The possible mechanism for this is the deleterious effects of cigarette smoke on endothelium-dependent vasodilation.[23],[24],[25] Cigarette smoke has been found to impair endothelium-dependent vasodilation in coronary arteries of nonsmokers almost to the same extent as seen in habitual smokers.[24] The vasodilator effect of SNP is believed to be largely mediated by nitric oxide (NO). In passive and active smokers, decreased production of endothelial NO is a mechanism by which the risk of heart disease is increased.[26] A significant increase in Cl- and iCa was observed in the high-dose group. A noticeable decrease in K+ and an increase in Na+, iCa2+, and Cl- were observed in the test group. These findings are similar to that of Markiewicz et al.,[27] whereas they differ from other reports.[28],[29],[30],[31] The decrease in K+ is probably due to the depressant effect of nicotine on electrolyte.[28]

CSE resulted in a dose-dependent significant increase in WBC count, which is in line with previous findings.[32],[33] The mechanism for CSE-induced increase in WBC count is not clear but was suggested that inflammatory stimulation of the bronchial tract induced an increase in inflammatory markers in the blood which resulted in an increase in WBC count.[32] The high WBC count probably promotes cardiovascular diseases through multiple pathologic mechanisms that mediate inflammation, plug the microvasculature, induce hypercoagulability, and promote infarct expansion.[7],[32],[34] A high dose of CSE also accounted for a significant increase in RBC. Furthermore, there was a marked increase in HGB and HCT level to CSE. These findings are in line with other research work.[32],[35] The increase in HGB, RBC, and HCT level to CSE could be a compensatory mechanism.[36] However, some researchers are of the view that CSE does not cause an increase in HGB level in all animals of the same species which could be related to tolerance potential of the individual animal.[36]

CSE-induced significant increase in PLT concentration recorded is in line with other findings[37] in experimental studies in laboratory animals. The mechanism behind the increase in PLT concentration may be due to the effect of CSE in increasing fibrinogen and thromboxane levels. The increase in fibrinogen and thromboxane which are mediators of PLT activators probably led to the increased PLT count.[38] This increased PLT level probably led to an increased risk of thrombus formation which is associated with a higher risk of heart disease.[39] Withdrawal from cigarette smoke gradually restores hematological parameters, and this is time dependent.[32]

Histological finding showed that the trachea of guinea pigs exposed to cigarette smoke had altered architecture with prominent mucosa disruption, loss of the cilia, and inclusion bodies (small blackish aggregate of smoke toxicants) in comparison to the Unexposed group as seen in [Figure 1],[Figure 2],[Figure 3],[Figure 4]. This finding is in line with those of Shraideh and Najjar[40] as well as Ziad et al.[9] The possible mechanism for the observed change in the trachea epithelium was probably due to cell degeneration.[9] The observed loss of cilia in this study may be caused by nicotine in the cigarette smoke as it has been reported to have destructive effects on microtubules through alteration of their polymerization/depolymerization. Furthermore, acetaldehyde and acrolein present in cigarette have been suspected to play a role in the damage of cilia by affecting the function and beat frequency.[41]{Figure 1}{Figure 2}{Figure 3}{Figure 4}

Next, histological findings in the lung alveoli revealed the thickening of alveolar wall tissue and inflammation in all the groups of the animals exposed to cigarette smoke.[9] This thickening has been suggested to be due to an increase in cell proliferation.[9]

With withdrawal from CSE, the tissues of trachea and lung alveoli showed a partial recovery.

Interestingly, histological picture improved in the group of animals Withdrawn from CSE as seen in [Figure 5],[Figure 6],[Figure 7],[Figure 8].{Figure 5}{Figure 6}{Figure 7}{Figure 8}

Considering the result obtained in this study, it can be concluded that exposure to cigarette smoke decreases blood pressure parameters, BRS, MOD, and vascular reactivity responses to ACh and SNP, while it increases HR, HR, MABP, and vascular reactivity response to NE. It also stimulates an increase in hematological parameters as well as Na+ and Ca2+ level, while it precipitated decreased K+ level. CSE also negatively altered the normal architecture of the lung and trachea.

 Conclusion



CSE adversely affected normal healthy status by altering the blood chemistry, pulmonary microscopic architecture, baroreflex sensitivity and myocardial oxygen demand.

Acknowledgment

We acknowledge Mr. Duncan Ota of the Department of Physiology, Faculty of Basic Medical Sciences, College of Medicine, University of Lagos, for his technical help throughout the period of this research.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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