Contrasting actions of diesel exhaust particles on the pulmonary and cardiovascular systems and the effects of thymoquinone

Introduction

Although air pollution consists of a heterogeneous mixture of gaseous and particulate matter, adverse cardiovascular events are most strongly associated with exposure to fine particulate matter (diameter < 2.5 µm, PM_2.5) (Brook et al., 2010). An important component of PM_2.5 is particulate matter generated during the combustion of diesel fuel (Brook et al., 2010). These particles, and particularly the nanoparticulate fraction (diameter < 100 nm), of which the combustion-derived particulates of diesel exhaust are an important component, penetrate deeply into the respiratory tract and can carry large amounts of toxic compounds, such as hydrocarbons and metals, on their surfaces (Vermylen et al., 2005; Brook et al., 2010).

While epidemiological evidence strongly supports the association between particulate air pollution and cardiopulmonary effects, the mechanism is not fully understood (Vermylen et al., 2005; Brook et al., 2010). It has been suggested that inhaled particles may lead to systemic inflammatory responses through the release of IL-6, TNFα or histamine and oxidative stress within the lungs and/or systemically (Vermylen et al., 2005; Brook et al., 2010). Additional experiments showed that exposure to air pollution is associated with rapid changes in autonomic nervous system balance, favouring sympathetic nervous system activation and parasympathetic withdrawal (Vermylen et al., 2005; Brook et al., 2010). Other lines of evidence also suggest that inhaled nanoparticulates can rapidly cross the alveolar–capillary barrier and directly affect the cardiovascular system (Nemmar et al., 2004b; Vermylen et al., 2005; Brook et al., 2010).

Epidemiological and clinical studies reported the occurrence of a cardiovascular effect of particulate air pollution within a few hours of exposure (Peters et al., 2001; 2004; Harrabi et al., 2006; Mills et al., 2007;). Indeed, acute exposure to diesel exhaust particles (DEP) caused a systemic and inflammatory response in the airways and impaired the regulation of vascular tone and endogenous fibrinolysis in healthy human volunteers (Salvi et al., 1999; Mills et al., 2005). Other human exposure studies have shown that exposure to particulate air pollution caused cardiovascular effects without significant effects on the respiratory tract (Samet et al., 2009). We have investigated the contribution of lung inflammation and/or direct particle translocation in the extrapulmonary effects of inhaled particles (Nemmar et al., 2003a,b; 2004a; 2005b; 2007b; 2009a; 2010b). To evaluate the effect of translocated particulate air pollution, several studies have assessed the direct effect of particles in vivo (Nemmar et al., 2007a; Nemmar and Inuwa, 2008), ex vivo (Nemmar et al., 2005a) and in vitro (Alfaro-Moreno et al., 2008). We have recently reported that systemic administration of DEP affected blood pressure and caused lung inflammation assessed by bronchoalveolar lavage (Nemmar et al., 2007a; 2009b; 2010a; Nemmar and Inuwa, 2008). However, with respect to pulmonary exposure, in all the animal models we studied, DEP resulted in lung inflammation in hamsters (1, 6 and 24 h) (Nemmar et al., 2003a,b; 2004a), rat (24 h) (Nemmar et al., 2010b) and mice (24 h) (Nemmar et al., 2009a). However, no measurement of pulmonary function has been reported. Thus, it is not clear whether pulmonary exposure to particulate air pollution can affect systemic and cardiovascular variables, without causing detectable pulmonary inflammation.

Thymoquinone (2-isopropyl-5-methyl-1,4-benzoquinone) is the main active principle in the oil of Nigella sativa seeds and has broad anti-inflammatory activities (Ali and Blunden, 2003), and it has been reported to prevent pulmonary inflammation in a mouse model of allergic asthma (El Gazzar et al., 2006) and to attenuate the inflammatory response in activated mast cells by blocking transcription and production of TNFα (El Gazzar et al., 2007). To our knowledge, however, no study, has addressed the effect of thymoquinone on the cardiovascular and pulmonary effects of DEP until now.

We have tested here whether and to what extent, DEP given to the lung could induce acute cardiovascular effects, in the absence of detectable effects on lung inflammation and function. We have also assessed the protective effect of thymoquinone against such effects. Thus, in the present study, we have assessed the acute (4 and 18 h) effects of DEP on a comprehensive set of indices of cardiovascular function, including blood pressure, thrombosis in pial arterioles, platelet aggregation and systemic inflammation and respiratory endpoints, including pulmonary inflammation and airway resistance measured invasively using forced oscillation. Our data show that, at 4 h, in the absence of detectable pulmonary inflammation, DEP induced systemic inflammation, decreased blood pressure and caused thrombosis in pial arterioles. However, at 18 h, DEP caused both pulmonary and cardiovascular effects. Moreover, and interestingly, pretreatment with thymoquinone significantly ameliorated DEP-induced pulmonary and cardiovascular events.

Methods

Results

Cell composition and number in BAL fluid

Depending on the i.t. treatment, the cells found in BAL were primarily macrophages and PMN (Figure 1). Lymphocytes were not found in control mice BAL. No other cells were observed microscopically.

Four hours following their i.t. administration, DEP neither induced any cellular influx nor caused an increase of either IL-6 or total protein concentrations in BAL. Thymoquinone pretreatment did not affect either the BAL cell numbers or the IL-6 and total protein concentrations. In contrast, 18 h after DEP exposure, a marked and significant increase in macrophages and PMN numbers and total protein and IL-6 concentrations were observed. The latter effects were significantly prevented by pretreatment with thymoquinone.

Histology

Sections of lung from saline-treated mice, taken at 4 h after DEP, showed a normal appearance under light microscopy (Figure 2A, B). Lung sections from DEP-treated mice showed the presence of particulates within the intralveolar and interstitial tissue with infiltration of only a few inflammatory cells (Figure 2C, D). Likewise, morphometric lung analysis showed only slight and non-significant increases of macrophage and PMNs (Figure 3).

Similarly, the lungs of the thymoquinone + saline group (Figure 2E, F) showed a normal appearance, and those of the thymoquinone + DEP group (Figure 2G, H) demonstrated the presence of particles within the intralveolar and interstitial tissue with infiltration of a few inflammatory cells (Figure 3).

At 18 h, lung sections from saline-treated mice showed a normal appearance (Figure 2I, J). However, lung sections from the DEP-treated mice showed the presence of particulates within the intralveolar and interstitial tissue with a marked and significant inflammatory cellular infiltration consisting mainly of PMN and macrophages (2, 3). While the lungs of the thymoquinone + saline group (Figure 2M, N) showed a normal appearance, those of the thymoquinone + DEP group (Figure 2O, P) demonstrated the presence of particles within the intralveolar and interstitial tissue and a significant reduction in the number of interstitial inflammatory cells that had infiltrated, following DEP exposure (2, 3).

Airway hyperreactivity to methacholine

Figure 4 shows airway resistance, measured by the FlexiVent, to increasing concentrations of methacholine, after exposure to either saline or DEP with and without thymoquinone pretreatment. The results obtained at 4 h were highly variable, and no statistical differences in airway resistance were found between the different groups (Figure 4A).

However, 18 h following the exposure of DEP, we observed a significant and dose-dependent increase in the airway resistance, compared with the saline-treated group. No statistical differences were observed between the saline and thymoquinone + saline groups. Interestingly, thymoquinone pretreatment significantly prevented DEP-induced enhancement of airway resistance after increasing concentrations of methacholine (Figure 4B).

Leukocyte numbers in whole blood, IL-6 and SOD levels in plasma

DEP induced a significant increase in the number of leukocytes in whole blood and IL-6 concentration in plasma compared with the saline-treated group (Figure 5A, B). Pretreatment of control mice with thymoquinone affected neither the number of leukocytes nor IL-6 concentrations. However, thymoquinone pretreatment significantly reduced the DEP-induced increase in leukocyte numbers and IL-6 increase in plasma (Figure 5A, B).

At 4 h after DEP, the plasma SOD activity was unchanged (Figure 5C). Similarly, thymoquinone did not affect the plasma SOD levels (Figure 5C). However, 18 h after DEP exposure, a significant decrease of SOD was observed (Figure 5C). Thymoquinone alone at this time did not significantly affect the plasma SOD levels but prevented the effect of DEP on SOD activity (Figure 5C).

SBP

Figure 6 illustrates the effect of i.t. administration of DEP on SBP in mice. Compared with the blood pressure in the control group, mice exposed to DEP exhibited a significant decrease in SBP. Interestingly, while thymoquinone + saline did not affect SBP, thymoquinone pretreatment significantly prevented the decrease of SBP caused by DEP at both 4 and 18 h.

Platelet numbers in blood and photochemically induced thrombosis in pial arterioles

Platelet counts in blood were significantly decreased 4 and 18 h following DEP administration, compared with control mice (Figure 7A), indicating platelet aggregation in vivo. This effect was significantly prevented by pretreatment with thymoquinone (Figure 7A).

Consistent with the results on platelet numbers, Figure 7B illustrates that the i.t. instillation of DEP induced a shortening of the occlusion time in pial arterioles in a photochemically injured vessel. Thymoquinone pretreatment significantly reversed the DEP-induced shortening of the occlusion time in pial cerebral arterioles (Figure 7B).

In vitro platelet aggregation in whole blood

Figure 8A shows that low concentrations of DEP (0.1–1 µg·mL⁻¹ blood) caused platelet aggregation in a dose-dependent manner. The effect was significant at concentrations of 0.5 µg·mL⁻¹ (P < 0.05) and 1 µg·mL⁻¹ (P < 0.01). Thymoquinone pretreatment (0.01–0.1 mg·mL⁻¹) did not affect platelet aggregation in whole blood in vitro. For the sake of clarity, only the highest dose tested (0.1 mg·mL⁻¹) is shown in Figure 8B. Pretreatment with thymoquinone did not affect the pro-aggregatory effect of DEP, excluding any direct effect of thymoquinone on platelet aggregation (Figure 8B).

Discussion

We conclude from our results that the cardiovascular effects of DEP at 4 h were not associated with pulmonary inflammation but were more likely to be due to the entry into blood of DEP and/or their associated components. However, at 18 h, both lung inflammation and cardiovascular functions were affected by DEP. We chose to study the effects of DEP at 4 and 18 h because these times are particularly relevant, both epidemiologically and clinically, to the cardiovascular effects of particulate air pollution within a few hours of exposure (Peters et al., 2001; 2004; Harrabi et al., 2006; Mills et al., 2007).

Pulmonary function and histology were adversely affected at 18 h (but not 4 h) after DEP exposure. Using the same type of particles, dose and mouse strain, but a later time, at 24 h, pulmonary exposure to DEP (15 and 30 µg·per mouse) induced a dose-dependent macrophage and PMN influx into the BAL fluid with elevation of total proteins (Nemmar et al., 2009a). Cho et al. (2009) reported no significant increase of BAL PMN at 4 and 18 h following i.t. instillation of either fine or ultrafine airborne particulate matter in mice. Previously, PMN influx in the alveoli has been shown, which significantly increased in a time-dependent manner (peak reached at 24 h) in rats exposed to carbon black particles (6 and 24 h) (Li et al., 1997) and hamsters exposed to DEP (1, 6 and 24 h) (Nemmar et al., 2003b). These discrepancies suggest that the kinetics of the initiation of lung inflammation following particle exposure is dependent on species, strains and nature of particles.

Mechanisms of air pollution–induced cardiopulmonary morbidity and mortality are uncertain (Mills et al., 2009). Inhaled particles cause lung inflammation with release of inflammatory mediators (Nemmar et al., 2003b; 2004b) and also particle translocation (Nemmar et al., 2001; 2002a; Oberdorster et al., 2002). Consequently, it is hard to distinguish between these two processes (Nemmar et al., 2003b; 2010a). In the animal models we previously studied, pulmonary exposure to DEP caused lung inflammation in hamsters (1, 6 and 24 h), rats (24 h) and mice (24 h) (Nemmar et al., 2003b; 2009a, 2010a,b). Here, we did not observe pulmonary effects 4 h following the exposure to DEP, but this negative result encouraged us to verify whether, and to what extent, systemic inflammation and cardiovascular effects can occur in the absence of pulmonary inflammation. In humans, exposure to particulate air pollution caused cardiovascular effects without significant effects on the respiratory tract (Samet et al., 2009).

In either the absence (4 h) or presence (18 h) of lung inflammation, exposure to DEP caused systemic inflammation characterized by an increase of leukocyte numbers and IL-6 concentration in plasma. The SOD activities decreased only at the 18 h time point. Recently, Dubowsky et al. (2006) provided epidemiological evidence for positive associations between air pollution and indicators of systemic inflammation such as leukocyte numbers, C-reactive protein and IL-6. In animals, 24 h following particulate air pollution exposure, IL-6 levels in the BAL fluid and in blood samples were increased (Mutlu et al., 2007; Nemmar et al., 2009b). In mice, PM₁₀ exposure increased pulmonary inflammatory mediators, which entered the circulation, contributing to the systemic inflammation with downstream effects, such as vascular dysfunction (Kido et al., 2011). Our results concur with this finding at 18 h but not at 4 h. The results obtained at 4 h could be explained by translocation of DEP themselves and/or their associated components into the systemic circulation, as has been reported in humans and experimental animals (Nemmar et al., 2001; 2002a; Oberdorster et al., 2002; Elder et al., 2006).

Plasma SOD activity was significantly decreased at 18 h but not at 4 h post-DEP exposure. DEP, 24 h following their i.t. instillation, caused oxidative stress leading to systemic inflammation and pretreatment with a cysteine prodrug, l-2-oxothiazolidine-4-carboxylic acid, prevented these effects through its ability to balance oxidant–antioxidant status (Nemmar et al., 2009a). Nevertheless, the levels of IL-6 were not affected in BAL and plasma, 24 h following DEP exposure (Nemmar et al., 2009a). Here, at 4 h after exposure, IL-6 was increased markedly but by 18 h, the concentration of IL-6 in the DEP group was decreased, compared with that at 4 h, while higher than those in the control group (P < 0.005). This suggests that, after DEP, IL-6 concentrations in plasma reach a peak at 4 h, decrease by 18 h and return to control levels at 24 h. However, in BAL, the IL-6 concentration increased only at 18 h but not at either 4 or 24 h. These results highlight the importance of determining the time course of release of inflammatory mediators following exposure to particulate air pollution.

SBP significantly decreased at both 4 and 18 h following i.t. administration of DEP. While epidemiological and experimental studies found that particles may affect blood pressure in different ways (Delfino et al., 2005), our observation concurs with reports suggesting that the exposure of rats to either instilled PM_2.5 or inhaled concentrated ambient particles, decreased blood pressure at 1–2 h post exposure (Chang et al., 2004; Rodriguez Ferreira Rivero et al., 2005). Our findings concur with another study in which blood pressure fell after exposure to PM₁₀ (10 µg·m⁻³) (Harrabi et al., 2006). This association was significant only for a short time – in the order of few hours – following the increase of the atmospheric level of PM₁₀. However, one should take into account that the dose used in the present study was higher than the PM₁₀ concentrations reported by Harrabi et al. (2006). The decrease of SBP could be related to systemic inflammation and increase of IL-6 levels as there was an association between decreased blood pressure and raised IL-6 in humans with sepsis and cats exposed to endotoxin (Tateishi et al., 2007; Declue et al., 2010).

I.t. administration of particles increases the risk of thrombosis (Nemmar et al., 2002b; 2003a, 2004a; Mutlu et al., 2007), and the i.t. instillation of DEP promoted thrombosis in pial cerebral venules (Nemmar et al., 2009a). We used pial venules because a more stable thrombus is produced in the conditions of low blood flow, encountered in venules (Nemmar et al., 2009a). The present photochemical injury model of thrombosis in the pial cerebral arterioles of mice represents an improvement over our previous study. In this model, damage to endothelial cells causes platelets to adhere at the site of endothelial damage and then aggregate (Nemmar et al., 2009a). A decrease of platelet number following exposure to particulate air pollution has been reported in humans and animals, suggesting in vivo platelet aggregation (Ruckerl et al., 2007; Nemmar et al., 2009a). This effect was confirmed both by a prothrombotic effect of DEP in pial arterioles in vivo and a direct proaggregatory effect of DEP in whole blood in vitro, suggesting that translocated particles can either directly or indirectly (through systemic inflammation) activate platelets and promote thrombotic events. The possible involvement of the endothelial changes and reduction of fibrinolysis in the pial model of thrombosis remains to be determined. In fact, inhaled DEP have been reported to impair regulation of vascular tone and endogenous fibrinolysis in humans (Salvi et al., 1999; Mills et al., 2005)

Because of the occurrence of systemic inflammation following the exposure to DEP and subsequent cardiovascular effects, we aimed to assess the possible protective effect of thymoquinone, an active principle of the volatile oil of black seeds (from N. sativa) that exhibits diverse pharmacological activities (Ali and Blunden, 2003), including anti-oxidant (Erkan et al., 2008) and anti-inflammatory actions (El Gazzar et al., 2006; El Mezayen et al., 2006). However, thymoquinone has not been used in the context of prevention of particulate-induced systemic and cardiovascular complications.

Pretreatment with thymoquinone prevented pulmonary inflammation and the increase of airway resistance caused by DEP at 18 h. Moreover, it inhibited the increase of blood leukocyte numbers and IL-6 concentrations in plasma at 4 and 18 h. In addition, it prevented the decrease of SOD activity in plasma at 18 h. Pretreatment with thymoquinone exerted an antioxidant effect and decreased IL-6 expression in human proximal tubular epithelial cells, stimulated with advanced glycation end products (Erkan et al., 2008). Along with the prevention of the increase in WBC numbers and IL-6 concentration, and the decrease of SOD, thymoquinone significantly reversed the fall in SBP. Patients with sepsis have depressed heart rate variability in association with high IL-6 blood level and a lower blood pressure (Tateishi et al. (2007). Thus, we can hypothesize that the effect of DEP on SBP could result from the effects of raised IL-6. Further experimental studies using IL-6 knockout mice are required to answer this question. The effects of DEP at 4 h on SBP were more likely caused by the systemic inflammation caused by DEP, rather than by the DEP per se. In support of this possibility is that pretreatment with the anti-inflammatory thymoquinone prevented the systemic inflammation and the effect on SBP of DEP. At 18 h, the observed systemic inflammation and cardiovascular effects of DEP could result either from pulmonary inflammation and/or the direct translocation of DEP or their associated components into the systemic circulation. In fact, at this time point, both pulmonary and systemic inflammation, and the change in IL-6 and SOD levels, were all prevented by thymoquinone.

The decrease of platelet numbers in vivo and the prothrombotic effect caused by DEP were also prevented by thymoquinone. As thymoquinone failed to prevent platelet aggregation in vitro, a direct effect on platelets is less likely. We would therefore suggest that the prevention of thrombotic events by thymoquinone in vivo resulted from its ability to inhibit systemic inflammation and IL-6 (at 4 and 18 h) release, and SOD decrease (at 18 h). IL-6 levels and the oxidative stress have been correlated in humans with risk factors for cardiovascular disease, such as WBC count, and blood viscosity (Woodward et al., 1999) and the development of thrombosis in mice (Mutlu et al., 2007; Nemmar et al., 2009a). Thus, the observed in vivo thrombotic events may be due to the systemic inflammatory response and not an effect of the DEP per se. Indeed, platelet aggregation in vivo was reversed by the administration of thymoquinone. Whilst DEP caused aggregation in vitro, thymoquinone had no effect, suggesting that the mechanisms at play here are different. It is not known whether particles with different chemical composition and/or size would elicit a similar response, and this will be investigated in future experiments.

The i.t. instillation has been shown to be a reliable, convenient and valid, though admittedly not perfect, mode of administration of foreign compounds into the airways (Driscoll et al., 2000). Additional studies using inhalation exposures are needed to confirm our findings. The dose of particles (30 µg·per mouse) that we used here was based on our previous study (Nemmar et al., 2009a), which was comparable with those used in previous animal models of particulate air pollution (Nemmar et al., 2003a,b; Mutlu et al., 2007; Kido et al., 2011). However, when considering this level in relation to human exposure, the dose used in our experiments was relatively high (Brook et al., 2010).

In conclusion 4 h after DEP exposure, the cardiovascular effects did not appear to result from pulmonary inflammation but possibly from the blood translocation of DEP and/or their associated components. However, by 18 h after DEP, pulmonary and cardiovascular functions, as well as lung inflammation, were all affected by DEP. Pretreatment with thymoquinone ameliorated DEP-induced cardio-pulmonary effects. Our data provide biological plausibility to the clinical and epidemiological studies, reporting cardiovascular effects following exposure to particulate air pollution, in the absence or presence of pulmonary inflammation.

Conflict of interest

None of the authors have any conflict of interest.