We had previously demonstrated that cigarette smoke causes oxidation of guinea pig lung microsomal proteins [14, 15, 19]. Here we demonstrate that exposure of marginal vitamin C-deficient guinea pigs to CS causes oxidation of whole lung proteins. We have used vitamin C-depleted guinea pigs to minimize the ascorbate level in the tissues. This is because ascorbate is a potential inhibitor of CS-induced oxidative protein damage [14, 15]. If the guinea pigs were fed ascorbate-rich diet (15 mg vitamin C/animal/day), the animals failed to respond to CS [15]. We further demonstrate that oxidative modification of proteins by cigarette smoke leads to inflammation, apoptosis and cellular damage of the lung (increased air space) and that black tea can prevent such cigarette smoke-induced lung damage. Others had also shown that one major deleterious effect of smoking is oxidative damage of proteins [13, 16]. Such oxidative modifications of structural proteins in the lung, including protein carbonylation, play a significant role in the etiology and progression of several human pulmonary diseases [13, 26, 29]. Oxidative stress plays an important role not only through direct injurious effects, but by involvement in the molecular mechanisms that control lung inflammation (13). One intriguing aspect of such oxidative protein damage is that the oxidized proteins become vulnerable to degradation by endogenous proteases present in the tissues [14, 29–32]. This may be a key cause of the degradation of lung structural proteins in smokers leading to degenerative diseases like emphysema, which is marked by the loss of structural matrix of the lung and its elasticity leading to impaired transfer of oxygen and carbon dioxide into and out of the blood. We have shown that protein oxidation is followed by inflammation, as evidenced by infiltration of inflammatory cells in the septal region and macrophages inside the alveoli. It is known that during phagocytosis macrophages undergo oxidative burst, accompanied by release of proteases [33]. The proteases released from activated macrophages along with the endogenous proteases present in the tissue may be involved in degrading the cytoskeletal proteins leading to destruction of alveolar membranes and septal cells in emphysema. It is thus conceivable that if oxidation of lung proteins is prevented by antioxidants, subsequent proteolysis would be prevented, and this in turn would prevent lung damage like that observed in emphysema. Here we show that oxidation of proteins (Figure 1) and accompanied damage to the lung cells (Figure 2) are both inhibited by giving the CS-exposed guinea pigs BT as the drink. The extent of lung damage by CS exposure has been evidenced by the significant increase of the surface density (S/V) of the alveolar air space (Table 1). This represents the membrane interface of each alveolar air space per unit area. It is known that the efficiency of gas exchange (O 2 and CO 2 ) is greatly regulated by the surface density [24]. The S/V is significantly increased by giving the CS-exposed guinea pigs BT as the drink. The possibility that the loss of alveoli accompanied by increased air space in the CS-exposed guinea pigs was due to inanition and comparatively less calorie intake [34] is not tenable. This is because the CS-exposed guinea pigs consumed ≈45 ± 5 g diet/day and the guinea pigs of all other groups, namely, sham control (air-exposed), BT, and CS + BT group were pair-fed with respect to the CS group. We had shown before that the inhibitory effect of BT is apparently a synergistic effect of the antioxidant flavonols present in BT, namely, theaflavins (TF), thearubigins (TR) and catechins (CT) [19]. Based on the flavonol contents of BT, as determined before [19], the amount of flavonols consumed per guinea pig per day was approximately 5 mg TF, 90 mg TR and 30 mg CT. The BT flavonols probably act by quenching the stable oxidants, which might be long-lived radicals present in CS that are apparently responsible for oxidation of the lung proteins [14, 35, 36].

Our present data and other reports indicate that along with oxidative damage, apoptosis plays a crucial role in CS-induced lung damage [1, 4–8]. Although it is hypothesized that interaction of oxidative stress and apoptosis leads to pathophysiological conditions in emphysema, the question remains to be addressed: which one is the initial event, oxidative damage or apoptosis? It has been proposed that a vicious cycle may be established, because cells undergoing apoptosis display increased oxidative stress, which further contributes to the apoptosis [5]. The role of apoptosis in such lung damage is not mere correlative, but potentially causative [6]. Here we show that the oxidative damage is the initial event, which is followed by inflammation, apoptosis and increased air space indicating emphysematous change. The biochemical events that mark such apoptotic changes are DNA fragmentation, over-expression of Bax and activation of caspase 3. We have demonstrated that marked DNA fragmentation (increase in TUNEL positive cells) occurs in lungs of CS-exposed guinea pigs given water as the drink (Figures 4 and 5). When the CS-exposed guinea pigs are given BT instead of water, there is no observable increase in the DNA fragmentation. The percentage of TUNEL positive cells are comparable to that of sham controls (Figures 4 and 5). This indicates that BT prevents CS-induced DNA fragmentation.

Aoshiba et al. [10] reported that acute cigarette smoke exposure induces apoptosis of alveolar macrophages. However, Aoshiba et al. worked with rats and the present authors with partially vitamin C-deprived guinea pigs. Also, in the present study the authors used a relatively mild challenge while that used by Aoshiba et al. [10] was more severe, as evidenced by occurrence of some degree of alveolar bleeding. This is never observed in human smokers. Moreover, the incidence of alveolar macrophage (AM) apoptosis in CS-exposed rats obtained by Aoshiba et al. [10] was much lower (3.2 %) than observed by the present authors (≈16 %). This difference might be due to the fact that rats synthesize vitamin C [21] and vitamin C present in the respiratory tract of rats might have prevented the effect of CS inhalation on AM apoptosis.

Caspases contribute to apoptosis through disassembly of cell structures by disrupting the nuclear structure and also by cleaving several cytoskeletal proteins [30, 37]. Caspases are synthesized initially as inactive single polypeptide chains that undergo proteolytic cleavage to produce subunits having active protease activity. We have shown in this report that CS causes cleavage of procaspase 3 to active caspase 3 (17 KDa, Figure 6) in the guinea pig lung. When the CS-exposed guinea pigs were given BT as a drink, activation of caspase 3 was prevented (Figure 6).

It has already been demonstrated that phosphorylated form of p53 accumulates in the nucleus in response to DNA damage [38]. We have shown here that although the level of p53 in the guinea pig lung remains unaltered after exposure to CS, the level of phosphorylated p53 is markedly increased (Figure 7). Phosphorylation of p53 and its trnslocation in the nucleus is accompanied by expression of Bax. Here we show that besides preventing CS-induced oxidation and fragmentation of DNA, BT also prevents CS-induced phosphorylation of p53 (Figure 7). In fact, we observed practically no accumulation of phosphorylated p53 in the lungs of guinea pigs exposed to CS and given BT as the drink (Figure 7).

Apoptosis is regulated by expression of a number of genes, including the Bcl-2 family [39, 40]. Out of these Bax is pro-apoptotic and Bcl-2 is anti-apoptotic. So, the ratio of Bax and Bcl-2 determines whether a cell will undergo apoptotic death or not. We have shown that CS exposure to guinea pigs given water as the drink has no effect on the level of Bcl-2, whereas the Bax protein is significantly increased, resulting in an overall increase of Bax/Bcl-2 ratio (Figure 8C, column 2). When the CS-exposed guinea pigs were given BT as the drink, there was no over expression of Bax (Figure 8A, column 4). This resulted in a reversal of the Bax/Bcl-2 ratio (Figure 8C, column 4). Although densitometric measurement shows that there is an increase of Bcl-2 proteins in the presence of BT (25% in lane 3 and 13% in lane B, over that of lanes 1 and 2, Fig 8), the significance of this increase is not clear.

In conclusion, we demonstrate that there is a close link between oxidative damage, apoptosis and lung cellular damage in our guinea pig model exposed to cigarette smoke. Apparently, the initial event in the pathophysiological condition is oxidative damage of proteins. This is followed by inflammation and apoptosis leading to destruction of alveolar membranes and septal cells, resulting in increased air space in the lung. When the CS-exposed guinea pigs are given BT as the drink, oxidative damage is prevented and this is accompanied by the prevention of apoptosis and lung damage.

The present study has some limitation for consideration of the smoke-induced guinea model as a model of COPD. In human smokers with COPD, marked inflammation associated with massive neutrophil influx is often seen. However, neutrophil accumulation is not a feature of the present model. Nevertheless, besides inflammation and neutrophil influx the CS-induced lung damage produced in guinea pigs may be comparable to that of human smokers. The structure of the guinea pig lung has similarity with that of the human lung with three major lobes on the right and two major lobes on the left as well as well-defined terminal bronchiole with subtending alveolar ducts (41). Also, the guinea pig develops morphologic and physiologic alterations after exposure to CS at the same pattern as humans [41]. So the results obtained with guinea pigs in our present study would imply that regular intake of black tea may protect smokers from the risk of developing lung damage.