Abstract
This study aimed to evaluate the extrapulmonary effects of exposure to cigarette smoke (CS) through the analysis of blood components and histopathological examinations of the trachea and diaphragm muscle (DM) in C57BL/6 mice. Thirty-six animals were exposed to six cigarettes per day for 5 days. The mice were divided into a control group (CG) and groups exposed to CS for 1 (CS1D), 2 (CS2D), 3 (CS3D), 4 (CS4D), and 5 (CS5D) days. The trachea, DM, and blood were collected for morphometric and biochemical analyses. In comparison with the CG, CS4D and CS5D mice showed an increased influx of inflammatory cells into the DM and trachea. Increased glycogen deposits in the tracheal tissue of CS3D mice were observed, compared with that in CG, CS1D, and CS2D mice. In the blood serum, the number of inflammatory cells and the concentration of cholesterol increased in CS1D mice, compared with the CG. Alanine aminotransferase (ALT) levels were elevated in CS5D mice, compared with those in CS3D and CS4D mice. Aspartate aminotransferase (AST) levels were elevated in CS3D and CS5D mice, compared with those in the CG. Urea levels were significantly increased in CS5D mice, compared with CS1D mice. Our results showed extrapulmonary effects of short-term exposure to CS in adult mice.
Introduction
Tobacco use is the main isolated cause of illness and death in the world, including lung cancer, pulmonary emphysema, chronic bronchitis, and heart disease (Dalrymple et al., 2015). Tobacco currently kills about 6 million people each year and this number could increase to more than 10 million by the year 2030 (Petyaev, 2016).
Cigarette smoke (CS) contains approximately 6000 chemical components (Dalrymple et al., 2015). Direct and indirect exposure to CS leads to an excessive production of proteases that activate an inflammatory cascade, culminating in a systemic response to tissue damage, inflammation induction, and redox imbalance (Cantin and Richter, 2012). Exposure to CS leads to an increase in the number of macrophages, neutrophils, and lymphocytes in the lung. Macrophages secrete pro-inflammatory substances such as cytokines and metalloproteinases that induce local inflammation (Barnes et al., 2003; Bosken et al., 1992), leading to an increase in the number of leukocytes in the blood, particularly neutrophils. The main consequences of CS inhalation are related to damage the respiratory tract, which is the primary risk factor for the development of chronic obstructive pulmonary disease (COPD), a disease characterized by a chronic inflammatory process that results in lung injury (Biswas and Rahman, 2009; Domej et al., 2014; Wouters et al., 2002). CS is the major etiological factor in the pathogenesis of COPD, which is characterized by irreversible airway changes and is associated with a chronic inflammatory response of the lungs that is mediated by inflammatory proteins including cytokines, chemokines, adhesion molecules, and inflammatory enzymes (Barnes, 2008; Wang et al., 2014). COPD consists of four forms of anatomical lesions: emphysema, remodeling of the lower airways, vascular remodeling associated with pulmonary hypertension, and metaplasia/hypertrophy of the mucosal glands of the bronchi associated with hypersecretion of mucus (Zhou et al., 2013).
The trachea is the main route of air conduction. It is lined with pseudostratified columnar epithelium and goblet cells and represents one of the lines of defense of the respiratory tract, since it possesses mucus-secreting cells that act against inhaled external agents (Elliott et al., 2007). Ciliary beat produces movements that move mucus in the cephalic direction to ensure clearance of the airways. However, exposure to CS causes hyperplasia of mucus-producing cells and a decrease in the ciliated area of the epithelium (Simet et al., 2010). Another essential organ for ventilation is the diaphragm muscle (DM), which is a skeletal striated muscle that separates the chest and abdominal cavities and can inflate and deflate the lungs, resulting in inspiratory and expiratory movements. Compounds in CS can cause hyperresponsiveness of the airways, leading to inflammation and impaired function of the respiratory system (Merrell and Kardon, 2013; Shimada et al., 2012).
The pulmonary effects of inhaling CS are well established, as recently demonstrated by our group (Campos et al., 2013). However, the effects of CS on other organs have been described to induce and/or worsen various heart, liver, and kidney diseases that are related to inflammatory processes (Bazzano et al., 2003; Kruger et al., 2015; Orth and Ritz, 2002).
We previously demonstrated the effects of CS in the lungs of mice exposed to CS for 5 days (Campos et al., 2013). In the present study, we aimed to evaluate the extrapulmonary effects of exposure to CS through the analysis of blood components and histopathological examinations of the trachea and DM in C57BL/6 mice.
Materials and methods
Animals
Male, 8-week-old C57BL/6 mice were housed under controlled conditions in standard laboratory cages (Laboratory of Experimental Nutrition, Department of Food—School of Nutrition, Federal University of Ouro Preto). During all experiments, animals were maintained at ambient temperature (21 ± 2°C) under a diurnal light cycle in a vivarium with food and drinking water supplied ad libitum. All in vivo experimental protocols were approved by the Institutional Ethics Committee (#2010/38).
CS exposure protocol
The animals were exposed to six commercial full-flavor filtered Virginia cigarettes (10 mg of tar, 0.9 mg of nicotine, and 10 mg of carbon monoxide), three times per day (morning, afternoon, and evening) for a total of 5 days as described previously (Pires et al., 2011). The groups exposed to CS for 1, 2, 3, 4, and 5 days were called CS1D, CS2D, CS3D, CS4D, and CS5D, respectively (n = 6 per group). Briefly, each group of mice was placed in an inhalation chamber (40 cm long, 30 cm wide, and 25 cm high), inside an exhaustion chapel. A cigarette was coupled to a plastic 60-mL syringe so that puffs could be captured and subsequently expelled into the exposure chamber. One liter of smoke from one cigarette was aspirated with this syringe (20 “puffs” of 50 mL), and each puff was immediately injected into the chamber. All animals in each group were maintained in these CS-exposed conditions (3%) for 6 min, after which the cover of the chamber was removed and the exhaust fan of the chapel was turned on, evacuating the smoke within 1 min. Two cigarettes per inhalation period were used. Mice in the control group (CG; n = 6) were subjected to the same procedures but were exposed to ambient air. Control and exposed animals were euthanized by cervical dislocation 24 h after the last treatment (Campos et al., 2013, 2014).
Histology
The trachea and DM were removed and immersed in a fixative solution for 48 h. The material was then processed as follows: tap water bath for 30 min, 70% and 90% alcohol baths for 1 h each, two baths in 100% ethanol for 1 h each, and embedding in paraffin (Demircan et al., 2016). Serial 5-μm sagittal sections were obtained from the trachea and DM, then stained with hematoxylin and eosin for histological analyses and with periodic acid-Schiff (PAS) to quantify mucus production (Lima et al., 2015).
Hemogram and biochemical analyses of blood serum
Two aliquots of blood were collected from each animal in polypropylene tubes containing, or not, 15 μL of anticoagulant. One aliquot was transferred to the Pilot Clinical Analysis Laboratory (LAPAC-OP) to perform a blood count and a white blood cell count. The other aliquot was centrifuged at 10,000 r/min for 15 min, and the supernatant was removed for the measurement of cholesterol, glucose, urea, creatinine, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) by the enzymatic colorimetric method using a commercial kit (Bioclin®, Quibasa, Belo Horizonte, Brazil) (Soares et al., 2016). Parameters including the white cell count, hemoglobin level, red blood cell count, and hematocrit were assessed using a CM200 random access clinical analyzer (Wiener Lab, Rosario, Argentina).
Morphometric analyses
Morphometric analyses were performed at the Multiuser Laboratory of the Research Center for Biological Sciences of the Federal University of Ouro Preto. Twenty images obtained from histological slides of the trachea and DM were digitized using a DM5000b optical microscope and a CM300 digital microcamera (Leica, Wetzlar, Germany). The images were scanned with a 40× objective. We used a representative image at 40× magnification with a 100-μm ruler to calibrate a ruler in pixels derived from the ImageJ software program (NIH, Bethesda, Maryland, USA), such that 434 pixels equaled 100 μm, and measured five areas of each captured image (Campos et al., 2014).
Statistical analysis
Data with a normal distribution were assessed using univariate analysis of variance followed by the Tukey post hoc test. Data are expressed as mean ± standard error of the mean. Differences were considered significant at p < 0.05. All analyses were performed with GraphPad Prism version 5.00 (GraphPad Software; San Diego, California, USA) for Windows 7.
Results
Histological and morphometric analyses of DM and trachea
Morphometric analyses of the DM and trachea in our experimental model showed an increased influx of inflammatory cells in CS4D and CS5D mice, compared with that in the CG. Staining with PAS showed an increase in glycogen deposition in the tracheal tissue of CS3D mice, compared with that in CG, CS1D, and CS2D mice (Figure 1). However, there was a decrease in the thickness of the tracheal epithelium in CS4D and CS5D mice, compared with that in CG, CS1D, CS2D, and CS3D mice (Table 1).
Photomicrographs of histological sections (5 μm) of trachea and diaphragm muscle tissues collected from mice in the control group (CG) and cigarette smoke (CS)-exposed groups. (a to c) Group exposed to ambient air; (d to f) group exposed to CS for 1 day; (g to i) group exposed to CS for 2 days; (j to l) group exposed to CS for 3 days; (m to o) group exposed to CS for 4 days; (p to r) group exposed to CS for 5 days. PAS staining–positive cells are indicated by thin arrows. Sections shown in (a), (b), (d), (e), (g), (h), (j), (k), (m), and (n) were stained by hematoxylin and eosin and visualized at ×440 magnification. Sections shown in (c), (f), (i), (l), (o), and (r) were stained by periodic acid-Schiff and visualized at ×440 magnification. PAS: periodic acid-Schiff.
Histological analyses of the trachea and DM from the CG and CS groups. a
DM: diaphragm muscle; CS: cigarette smoke; CG: control group; CS1D: group exposed for 1 day; CS2D: group exposed for 2 days; CS3D: group exposed for 3 days; CS4D: group exposed for 4 days; CS5D: group exposed for 5 days; ANOVA: analysis of variance; PAS: periodic acid-Schiff; SEM: standard error of the mean.
aValues with a common superscript letter do not differ. Values without a common superscript letter indicate statistical differences. Data were expressed as mean ± SEM and were analyzed by one-way ANOVA followed by the Tukey post hoc test (p < 0.05).
Analysis of inflammatory cells, hemoglobin, red cells, and blood hematocrit
There was an increase in the number of inflammatory cells in the blood of CS1D animals, compared with that of CG animals. We observed no statistically significant differences in the other parameters analyzed (Table 2).
Hemogram of blood serum from the CG and CS groups.a
CS: cigarette smoke; CG: control group; CS1D: group exposed for 1 day; CS2D: group exposed for 2 days; CS3D: group exposed for 3 days; CS4D: group exposed for 4 days; CS5D: group exposed for 5 days; ANOVA: analysis of variance; SEM: standard error of the mean.
aValues with a common superscript letter do not differ. Values without a common superscript letter indicate statistical differences. Data were expressed as the mean ± SEM and were analyzed by one-way ANOVA followed by the Tukey post hoc test (p < 0.05).
Biochemical analysis of blood serum
Exposure to CS caused changes in biochemical parameters related to liver and renal disorders. There was an increase in the cholesterol levels in CS3D, CS4D, and CS5D mice, compared with that in the CG. ALT levels were higher in CS5D mice than in CS3D and CS4D mice, while AST levels showed an increase in CS2D and CS5D mice, compared with those in the CG. There was also a significant increase in urea content in CS5D mice, compared with that in CS1D mice (Table 3).
Biochemical analysis of blood serum from the CG and CS groups.a
CS: cigarette smoke; CG: control group; CS1D: group exposed for 1 day; CS2D: group exposed for 2 days; CS3D: group exposed for 3 days; CS4D: group exposed for 4 days; CS5D: group exposed for 5 days; ANOVA: analysis of variance; SEM: standard error of the mean.
aValues with a common superscript letter do not differ. Values without a common superscript letter indicate statistical differences. Data were expressed as mean ± SEM and were analyzed by one-way ANOVA followed by the Tukey post hoc test (p < 0.05).
Discussion
The long-term effects of exposure to CS, mainly in the lungs, are well established; however, studies on the toxic effects of CS after short-term exposure and in other organs remain scarce (Csabai et al., 2016; Dong et al., 2016; Kruger et al., 2015). Pinho-Ribeiro et al. (2017) demonstrated the effects of atorvastatin and simvastatin on mouse lung repair after long-term exposure to CS. A few other studies have analyzed the effects of CS after short-term exposure. Campos et al. (2013) reported the effects of CS in the lungs of mice exposed to CS for 5 days. Pena et al. (2016) also demonstrated the effects of CS in the lungs of mice exposed to CS. In the airways, a primary defense mechanism is performed by goblet cells that produce the mucus present on the epithelium. This is crucial for the physiological function of the mucociliary mechanism that is responsible for expelling inhaled exogenous particles into the upper airway, so that they can be released. For this process to occur correctly, it is necessary that the physical and chemical characteristics of the epithelium are kept intact. We observed an overproduction of mucus, characterized by glycogen storage, in the tracheal epithelium of animals after the third day of exposure to CS.
CS contains numerous toxic particles and compromises the functioning of various organs. The respiratory system is the main target of exposure to these particles, since it is the first contact route with the external environment through inhalation. Oxidants present in CS can severely damage lung and airway tissues by stimulating alveolar macrophages and endothelial cells to produce reactive oxygen species and cytokines that attract neutrophils and other inflammatory cells to the site of injury, triggering an inflammatory response (Campos et al., 2014; Li et al., 2012). High levels of reactive oxygen species induce an inflammatory cascade by activating pro-inflammatory transcription factors such as nuclear factor-κB (Rahman, 2012). Previous work by our group has shown an increase of mucus production in the trachea of rats exposed to other toxic substances such as formaldehyde (Lima et al., 2015). Besides increasing the production of epithelial mucus, CS can cause morphological changes that affect functionality (Knowles and Boucher, 2002; Lima et al., 2015). Our results showed injury and a thinner tracheal epithelium in mice exposed to CS. According to Randell et al., the existence of a pseudostratified columnar epithelium is necessary to ensure the proper functionality of the trachea (Randell, 2006; Seifart and Plagens, 2007). Furthermore, it was observed that the pseudostratified epithelium of the trachea suffered metaplasia when exposed to smoke, becoming squamous (Randell, 2006).
The influx of inflammatory cells into the tracheal tissue may drive some of these physiological changes, since these cells secrete inflammatory cytokines that can alter the ciliary epithelial morphology (Simet et al., 2010). We observed that the exposure to CS led to increased inflammatory cell infiltrates after the second, fourth, and fifth days of exposure to CS. Shimada et al. (2012) demonstrated that short-term exposure to hydroquinone, a substance in CS, increased the influx of inflammatory cells into the tracheal tissue of Swiss mice. In the current study, there was an increase in the number of inflammatory cells in the DM on the third day of exposure to CS, corroborating our previous results, showing that exposure to formaldehyde for 5 days caused an inflammatory response in both trachea and DM in Fischer male rats (Kurgat et al., 2016; Lima et al., 2015).
Besides causing changes in plasma biochemical parameters related to the target organs, exposure to CS is also considered to promote systemic inflammation (Bazzano et al., 2003; El-Zayadi, 2006; Orth and Ritz, 2002). Damage caused by CS to airways causes an acute increase in the influx of inflammatory cells into the bloodstream. Circulating inflammatory cells are recruited to sites of injury where they would ordinarily remove pathogens through their phagocytic capacity (Cruvinel et al., 2010; Moon et al., 2013). Our results showed a significant increase in the serum concentration of inflammatory cells only on the first day of exposure to CS. Campos et al. (2013) reported a significant increase in the influx of inflammatory cells in bronchoalveolar lavage fluid on the second day of exposure to CS. In agreement with our current data, the previous study also showed a significantly increased infiltration of inflammatory cells in the trachea and DM on the fourth and fifth days of CS exposure (Campos et al., 2013). Several studies have shown that exposure to CS caused hepatic damage. Although CS-induced hepatic damage is not considered a direct cause of liver diseases, it is related to an increased severity of injury from other causes (Alsalhen and Abdalsalam, 2014; Azzalini et al., 2010). Azzalini et al. (2010) showed that CS could induce oxidative stress and aggravate nonalcoholic fatty liver disease in obese Zucker rats. Alsalhen and Abdalsalam (2014) showed that in men from Lebanon, cigarette smoking negatively influenced hepatic function as evaluated by measuring the plasma levels of total bilirubin, ALT, AST, alkaline phosphatase, cholesterol, triglyceride, and protein. Raised levels of these components can indicate liver injury (Jang et al., 2012; Rahmioglu et al., 2009). Our data showed that even short-term exposure to CS could alter biochemical parameters related to liver function, since the serum levels of ALT, AST, and total cholesterol were significantly increased. Our data corroborate a previous study by Nemmar et al. (2013), who identified increased levels of ALT in C57BL/6 exposed to CS for 4 days. A significant increase in the circulating liver enzyme and cholesterol levels of male smokers was also identified in a study involving smoking and nonsmoking Lebanese men aged 30–60 years.
Other authors have demonstrated that long-term exposure to CS is related to declining renal function, since it is associated with glomerular hyperfiltration and proteinuria, with a greater impact on individuals who already have kidney disease (Arany et al., 2011; Maeda et al., 2011). Arany et al. (2011) observed that in patients with acute renal ischemia, the long-term exposure to nicotine was a facilitator of disease progression. A 6-year observational study monitored over 10,000 Japanese men aged 40–55 years who did not have proteinuria or renal dysfunction at baseline and showed that smoking led to a higher incidence of proteinuria and glomerular hyperfiltration in those individuals (Maeda et al., 2011). However, studies of short-term exposure to CS remain scarce (Itoh et al., 2014; Nemmar et al., 2013). Itoh et al. (2014) showed that exposure to CS for 5 days did not cause renal changes in C57BL/6 mice. The results of our study showed a significant increase in urea levels on the fifth day of exposure to CS, suggesting renal changes even in a short-term exposure model. These data are also consistent with the study by Nemmar et al. (2013), who reported an increase in the plasma levels of urea in C57BL/6 mice exposed to CS for 4 days.
In conclusion, our results showed that short-term exposure to CS induced inflammation in the DM and trachea. In addition, the morphometry of the tracheal epithelium was affected. Our data also showed changes in serum biochemical parameters related to other organs and a systemic inflammatory response. More studies are necessary to further elucidate the effects of long-term exposure to CS.
Footnotes
Authors’ note
Thais Lourenço Martins and Keila Karine Duarte Campos contributed equally as lead authors.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.