Elsevier

Nutrition

Volume 22, Issues 11–12, November–December 2006, Pages 1192-1201
Nutrition

Basic Nutritional Investigation
α-Tocopherol and ascorbic acid supplementation reduced acute lung inflammatory response by cigarette smoke in mouse

https://doi.org/10.1016/j.nut.2006.08.016Get rights and content

Abstract

Objective

Short-term cigarette smoke (CS) exposure leads to acute lung inflammation through its influence over oxidants/antioxidants imbalance. Antioxidant vitamins such as ascorbic acid and α-tocopherol interact with oxidizing radicals. It is not clear if antioxidant supplementation can reduce inflammatory lung responses. Thus our aim was to analyze the effects of vitamin supplementation on the lungs of mice exposed to six cigarettes per day with histologic, cytological, and biochemical methods.

Methods

C57BL/6 mice were exposed to ambient air (control) or CS from 3, 6, 9, 12, or 15 cigarettes daily for up to 5 d. Mice alveolar macrophages and polymorphonuclear cells were counted in the bronchoalveolar lavage. Groups of CS animals received 50 mg/kg of ascorbic acid daily and/or 50 mg/kg of α-tocopherol daily as an oral supplementation (CS+C, CS+E, CS+C+E, respectively) 12 h before CS exposure. Thiobarbituric acid-reactive substances were detected and western blot to nuclear factor-κB were performed in lung extracts; metalloprotease-12 and tumor necrosis factor-α positive alveolar macrophages were quantified in the lungs processed for immunohistochemistry of the animals exposed to the smoke from six cigarettes daily for 5 d.

Results

The number of alveolar macrophages and polymorphonuclear cells in bronchoalveolar lavage (cells × 103/mL) in mice exposed to CS were increased and CS with vitamin supplementation groups presented bronchoalveolar lavage cells similar to those of control. Thiobarbituric acid-reactive substances values were reduced in vitamin supplementation groups when compared with CS and the lower value was found in the CS+C+E group. Metalloprotease-12 and tumor necrosis factor-α were more evident in CS as much as nuclear factor-κB activation when compared with control and vitamin supplementation groups.

Conclusion

Our results showed that CS induced acute lung inflammation. The inflammatory process after cigarette exposures was reduced by ascorbic acid, α-tocopherol, or more efficiently by both vitamin supplementations.

Introduction

Chronic obstructive pulmonary disease (COPD) represents a worldwide leading cause of morbidity and mortality and it is predicted to rank as the third most common cause of death by 2020 [1], [2]. Cigarette smoke (CS) is the major etiologic factor in the pathogenesis of COPD [3], [4], and thus the majority of these cases can be prevented by quitting smoking.

Cigarette smoke contains a large number of oxidants, and it has been hypothesized that many of the adverse effects of smoking may appear as a result of oxidative damage to critical biological substances [5], [6], [7]. CS exposure increases the amount of alveolar oxidants, not only because CS itself contains an expressive number of free radicals but also because it increases the number of inflammatory cells in alveoli, which spontaneously release oxidants [8]. These oxidants inactivate α1-antitrypsin and other protease inhibitors such as secretory leukoprotease inhibitor [5], [9]. Further, the recruitment of inflammatory cells increases the protease burden, thus tipping the protease-antiprotease balance further toward the protease side [10], [11], [12]. In addition, evidence suggests that oxidative stress enhancement is associated with inflammatory cell influxes to the lung followed by lipid peroxidation and by an increase in the amount of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) [13], [14]. Matrix metalloproteinases (MMPs) consist of a number of structurally related enzymes capable of digesting extracellular matrix and basement membrane components [15]. Oxidants induced by CS can directly damage components of the lung extracellular matrix such as elastin and collagen or even modify the matrix to make it more susceptible to protease attack [16]. Specific proteases derived from alveolar macrophages (AMs) and polymorphonuclear cells (PMNs) are responsible for lung injury [10], [11], [12], [17]. Extracellular matrix degradation is a complex multistep process that involves a family of zinc-dependent endopeptidases known as matrix metalloproteinases. MMPs consist of a number of structurally related enzymes capable of digesting extracellular matrix and basement membrane components [15]. MMP activity is regulated at different levels, including transcriptional control, extracellular activation of proenzymes, and active enzyme inhibition [10]. The tissue inhibitors of metalloproteinases are a family with four members identified; they form complexes with active enzymes and inhibit MMP activity [18].

Vitamins are important for lung development, being involved in several reactions regarding alveoli growth and regeneration [19] and lung protection [20]. Antioxidant vitamins such as ascorbic acid (vitamin C) and α-tocopherol (vitamin E) have been described as potent substances due to their interaction with oxidizing radicals [21]. Vitamin C is known to scavenge aqueous reactive oxygen species by rapid electron transfer and thus inhibits lipid peroxidation and reduces the level of oxidized vitamin E [22], [23] and vitamin E terminates the chain reaction of lipid peroxidation in membranes and lipoproteins [23], [24]. Daily oral administration of 10 mg/kg of all-trans retinoic acid in feed induced an inhibition in the growth of squamous cell carcinoma [25]. Moreover, 5 consecutive days of vitamin C or E (100 mg/kg daily) pretreatment completely prevented the formation of DNA single-strand breaks induced by CS in the lung, stomach, and liver of mice [26]. These vitamins also have beneficial effects over other mechanisms such as infectious diseases [27], and doses of 20, 30, and 50 mg/kg of food of tocopherol were equally effective in preventing aspirin-induced gastric lesions in rats [28], although it is not clear if vitamin C or E supplementations decrease lung inflammation induced by CS. After setting up an acute model of CS-induced inflammation in mice [29], our aim in this study was to analyze the effects of vitamin C and/or E supplementations on AM and PMN influxes. The following procedures were performed: Western blot to determine levels of nuclear factor-KB (NFKB), immunohistochemistry to quantify MMP-12 and TNF-α and biochemical analysis for lipid peroxidation, with the purpose of understanding the mechanisms of lung inflammation induced in this model.

Section snippets

CS exposure

A three-step experiment was undertaken to analyze the effect of antioxidant vitamins E and C over the inflammatory cells influx into pulmonary parenchyma. All procedures were carried out in accordance with conventional guidelines for experimentation with animals and the local committee approved the experimental protocols.

Results

Figure 1 shows the effects of cigarette doses in BAL 24 h after the last smoke exposure (n = 5 each for group). The number of AMs increased significantly according to cigarette doses of 6cig, 9cig, and 15cig (P < 0.05) and was 3.4 times more significant with 12cig (P < 0.001) when compared with the control group. The number of PMNs increased only in mice exposed to 6cig (P < 0.01) compared with the control group.

Figure 2 shows the effects of 6cig exposure from 1 to 5 d (n = 5 each for group).

Discussion

Cigarette smoke has been implicated as the major risk factor for the development of COPD [7], [12]. The adverse action of the CS is due to the presence of a large variety of compounds such as nicotine, benzo(a)pyrene, oxidants, and free radicals that could initiate, promote, and/or amplify oxidative damage [34], [35]. CS is the major determinant of diseases related to oxidative stress and the individual variations in the incidence and extension of COPD are numerous [36]. Specific reasons such

Conclusion

Alveolar macrophages, NFκB, MMP-12, and TNF-α significantly increase in response to CS exposure. The antioxidant nutrients vitamins C and E prevent this response to some degree. The inflammatory process after cigarette exposures was reduced by ascorbic acid, α-tocopherol, or more efficiently by both vitamin supplementations.

References (51)

  • S.H. Kim et al.

    Influence of smoking on markers of oxidative stress and serum mineral concentrations in teenage girls in Korea

    Nutrition

    (2003)
  • K. Tarwadi et al.

    Linkages of antioxidant, micronutrient, and socioeconomic status with the degree of oxidative stress and lens opacity in indian cataract patients

    Nutrition

    (2004)
  • C.C. Hsieh et al.

    Opposite effects of low and high dose supplementation of vitamin E on survival of MRL/lpr mice

    Nutrition

    (2005)
  • E. Sapey et al.

    COPD exacerbations2: Aetiology

    Thorax

    (2006)
  • D.E. O’Donnell et al.

    COPD exacerbations3: Pathophysiology

    Thorax

    (2006)
  • P. Mallia et al.

    Mechanisms and experimental models of chronic obstructive pulmonary disease exacerbations

    Proc Am Thorac Soc

    (2005)
  • M.D. Evans et al.

    Cigarette smoking, emphysema, and damage to alpha 1-proteinase inhibitor

    Am J Physiol

    (1994)
  • I. Rahman et al.

    Oxidative stress and redox regulation of lung inflammation in COPD

    Eur Respir J

    (2006)
  • J.R. Spurzem et al.

    Pathogenesis of COPD

    Semin Respir Crit Care Med

    (2005)
  • W. MacNee

    Pulmonary and systemic oxidant/antioxidant imbalance in chronic obstructive pulmonary disease

    Proc Am Thorac Soc

    (2005)
  • M.D. Evans et al.

    Damage to human alpha-1-proteinase inhibitor by aqueous cigarette tar extracts and the formation of methionine sulfoxide

    Chem Res Toxicol

    (1992)
  • P.J. Barnes et al.

    Chronic obstructive pulmonary disease: molecular and cellular mechanisms

    Eur Respir J

    (2003)
  • A. Churg et al.

    Macrophage metalloelastase mediates acute cigarette smoke-induced inflammation via tumor necrosis factor-alpha release

    Am J Respir Crit Care Med

    (2003)
  • W. MacNee

    Pathogenesis of chronic obstructive pulmonary disease

    Proc Am Thorac Soc

    (2005)
  • A. Churg et al.

    Tumor necrosis factor-alpha drives 70% of cigarette smoke-induced emphysema in the mouse

    Am J Respir Crit Care Med

    (2004)
  • Cited by (59)

    • Tempol reduces inflammation and oxidative damage in cigarette smoke-exposed mice by decreasing neutrophil infiltration and activating the Nrf2 pathway

      2020, Chemico-Biological Interactions
      Citation Excerpt :

      Although COPD does not occur in patients under acute exposure to cigarette smoke, the use of acutely cigarette smoke-exposed mice as a model may contribute to reveal lung injury pathways related to COPD [6,22]. The chosen mice model has been well-established for studies investigating the initial damage triggered upon cigarette smoke exposure [6,23–26], in addition to show some similarity to humans with heavy smoking behavior [27,28]. Therefore, here we examine the effects of tempol administration in mice acutely exposed to cigarette smoke focusing on inflammation, oxidative damage and the Nrf2 pathway.

    • Taurine treatment decreases inflammation and oxidative stress in lungs of adult mice exposed to cigarette smoke

      2018, Regulatory Toxicology and Pharmacology
      Citation Excerpt :

      In the BALF, there was a higher influx of total leukocytes and macrophages in CS-exposed animals. These data corroborate a study by Campos et al. (2013) wherein a temporal analysis of the cellular influx in C57BL/6 mice exposed to CS was performed, and a study by Bezerra et al. (Silva Bezerra et al., 2006), who observed an increase in macrophage numbers in the BALF of C57BL/6 mice exposed to CS. The inhalation of particles present in CS induces a local response in the lungs that is initiated by alveolar macrophages and airway epithelial cells.

    View all citing articles on Scopus

    This study was supported with a grant from the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro. Manuella Lanzetti and Wagner Alves Pimenta received academic bursaries from the Conselho Nacional de Desenvolvimento Científico e Tecnológico and UERJ.

    View full text