Airborne particulate matter PM_2.5 from Mexico City affects the generation of reactive oxygen species by blood neutrophils from asthmatics: an in vitro approach

Air pollutants such as particulates and exhaust gases can reach considerable levels in areas of heavy traffic or in towns near mountains that form closed valleys where air movement is restricted, significantly increasing the toxic pollutant concentration. The Mexico City Metropolitan Area (MCMA) is one of the most densely populated cities in the world with 18 million inhabitants according to the 2000 census [1]. MCMA is an elevated basin approximately 2240 meters above sea level, surrounded by mountains to the south, west and east. At this altitude, 23% less oxygen is available than at sea level, which makes combustion less efficient [2]. In view of the diurnal cycle and city size, the distribution of nitrates suggests local photochemical production. On the other hand, sulfates appear to be produced on a regional scale. There are indications of new particle formation and growth events when sulfur dioxide (SO₂) concentrations are high. The average atmospheric lifetime of sulfur emitted in Mexico City is 5.5 days, which is longer than the average lifetime of sulfur released in the rest of the world (3.9 days) [3]. Because of the altitude and the subtropical latitude of the Mexico City basin, the region receives intense solar radiation that promotes the efficient photochemical formation of pollutants. This changes their chemical composition during air transportation and results in particulate materials with different chemical properties.

For example, in the southeast zone of the city (Iztapalapa), the organic fraction of fine particles (PM_2.5) at the Centro Nacional de Investigación y Capacitación Ambiental (National Center for Environmental Research and Training, CENICA) site is estimated to represent an average of 54.6% of the total mass, with the rest consisting of inorganic compounds (mainly ammonium nitrate and sulfate/ammonium salts), black carbon (BC) and soil [4]. Since air pollution seems to be associated with respiratory and cardiac diseases, particularly in children and older people, it is likely that the particles exacerbate pre-existing diseases in susceptible populations. Acute effects occur at relatively low pollutant concentrations and are associated with particles of apparently innocuous composition (largely carbon, ammonium sulfate and nitrate) [5]. Ultra-fine particles are contained in the fine fraction and the soluble material may translocate to extrapulmonary sites [6, 7] for local cellular activation. This can increase the respiratory burst and concomitant generation of reactive oxygen species (ROS), chemical mediators and enzymes in peripheral cells, mainly neutrophils. It has been shown that activation of phagocytes both in vitro and in vivo can result in the generation of several ROS, including superoxide anion (O₂ ^.-) and hydrogen peroxide (H₂O₂), as well as the release of the heme enzyme myeloperoxidase (MPO) [8]. The increased generation of ROS due to the respiratory burst promotes an imbalance between ROS production and antioxidant defense that leads to oxidative stress leading to modification of molecules and/or disruption of cellular structures and tissue injury [9]. Due to high MPO activity, the generation of hypochlorous acid (HOCl) and reactive nitrogen species (RNS) also increases, resulting in the oxidation of tyrosine and nitrite and subsequent formation of tyrosyl and nitrogen dioxide (^.NO₂) radicals, respectively; these reactive intermediates can initiate the oxidation of lipids in the plasma membrane [10]. Another potentially important consequence of MPO activity is the consumption of nitric oxide and induction of endothelial dysfunction [8].

Although there is evidence that particulate air pollution has declined over time, epidemiological studies continue to show adverse health effects even at relatively low pollutant concentrations [11]. It is therefore likely that the increased air pollution and geographical characteristics of Mexico City have a significant impact on the health of the inhabitants [12, 13].

In view of the mechanisms that have previously been proposed for health effects of pollution, we considered a parallel mechanism involving circulating neutrophils in addition to alveolar macrophages. Because neutrophils can migrate to the lung during acute inflammation or when macrophage phagocytosis is overwhelmed by the number of particles or invading microorganisms [14], the purposes of the present work were (i) to determine plasma paraoxonase (PON) and myeloperoxidase (MPO) activities, (ii) to evaluate the susceptibility of plasma circulating phospholipids to lipoperoxidation in a group of asthmatic patients compared to healthy volunteers and (iii) to measure in vitro ROS generation by peripheral human neutrophils obtained from healthy volunteers (HV) and asthmatic patients (AP) in contact with PM_2.5 collected from MCMA.

All reagents used in this study were from Sigma Chemical Co., St. Louis, MO, unless otherwise stated.

Oxidant generation is part of normal metabolism in many cell types and is critical for homeostasis. To protect against noxious oxidants, the lung has a well-developed antioxidant system [23] that includes a systemic response against air pollution. We previously demonstrated increased superoxide dismutase (SOD) activity and TBARS production during the first week of exposure to air pollutants in Mexico City among 21 volunteers who had never lived there [24]. Four months of exposure to air pollutants resulted in increased plasma antioxidant capacity that decreased lipoperoxidation, as measured by TBARS concentration [25]. An important factor for the mechanisms involved in cells death an injury, is the production of free radicals. Experimental and clinical data suggest that oxidants play a role in the pathogenesis of several respiratory disorders, including bronchial asthma [26]. In particular, increasing evidence shows that chronic airway inflammation typical of asthma results in increased oxidative stress in the airways. Moreover, many asthma triggers including viral infections and air pollutants may activate the production of ROS, thus resulting in inflammation in addition to the asthmatic symptoms [26].

The maintenance of basal ROS generation in response to the pollutant particles used to challenge neutrophils from healthy volunteers might be due to the efficient uptake of the particles by these cells, which rapidly engulf insoluble particles [27]. Although the response was not statistically significant, neutrophils from asthmatic patients showed an almost three-fold increase in in vitro ROS generation when exposed to PM_2.5. This might be related to the activation of pro-inflammatory cytokines such as TNFα and IL-6 [28, 29], which decreases the phagocytic and/or scavenger capacity [30, 31] of neutrophils from these patients [27]. The exact mechanism by which particulate matter alters the phagocytic capacity is not fully understood and is a matter of great controversy. Some researchers have argued that this damage could be related to the cationic charge on the PM_2.5 particles arising from the content of transition metals such as Fe and Cu [32–34]; other groups emphasize that organic and black carbon components found mainly in ultra-fine particles confer greater in vivo and in vitro toxicity than fine particles, and this effect is said to be independent of the soluble metal content [35]. The importance of charge in toxic xenobiotic molecules is related to the affinity of scavenger receptors for foreign material [36]; internalization seems to be increased in cells previously exposed to particulate matter. Furthermore, significantly increased MPO activity in plasma from asthmatics was observed when compared to the control group (Table 2). This may suggest an increased risk for development of asthmatic crises in these patients because of decreased bioavailability of nitric oxide. Otherwise, H₂O₂ is utilized by MPO [37] to generate reactive intermediates capable of initiating lipoperoxidation and protein damage through hypochlorite oxidation that generates reactive toxic aldehydes, increasing the likelihood of cellular injury [38]. In addition, asthmatic patients showed a significant decrease in paraoxonase activity; the presence of these markers is considered a risk factors for acute coronary syndromes [39–42]. Epidemiological, clinical and experimental evidence relates current levels of ambient air pollution to both respiratory and cardiovascular conditions. Oxidative stress, inflammation, induction of a pro-coagulatory state and dysfunction of the autonomic nervous system appear to play major roles [40]. Acute toxic effects resulting from ambient air pollution include changes in lung function, heart rate, blood pressure and an inflammatory state. The clinical consequences of such effects include respiratory symptoms, thrombosis, myocardial infarction, arrhythmia and stroke, all of which are related to acute oxidative stress caused by increased ROS and RNS, as well as inflammatory enzymes and other factors [43]. This suggests that some components of PM_2.5 interact with membrane receptors, leading to activation of NADPH oxidase and increasing ROS generation in the NAP group. Unlike the NHV group, the NAP group was likely unable to counteract ROS generation due to asthma-mediated inflammation and concomitant oxidative stress, demonstrated by increased MPO activity and susceptibility to lipid oxidation, in addition to reduced PON activity. Collectively, the increased generation of ROS in these patients might be related to a concomitant decrease in nitric oxide bioavailability, thus increasing their susceptibility to asthmatic crises induced by air pollution.

In summary, we observed a dual response in the generation of ROS and RNS by neutrophils from both asthmatic patients and healthy volunteers exposed to PM_2.5. These findings suggest that PM_2.5 pollutant materials affect blood neutrophils directly, inducing increased ROS and RNS generation in asthmatic patients. These individuals are unable to modulate this response due to their precarious oxidative stress condition, shown by increased MPO activity, reduced PON activity, and higher susceptibility to lipid oxidation, which can favor bacterial infection and increase the risk of asthmatic crises. Indeed, greater and more prolonged exposure to pollution is likely to induce more molecular damage in the exposed population; such damage includes the well-documented effects of oxidative stress, modification of circulating hormones and effects on their biological functions [44, 45], abolished recognition of low density lipoprotein (LDL) receptors [46], cell damage and tissue injury. Further studies concerning the interactions of signaling pathways that specifically induce the release of different granule populations or bacterial internalization mechanisms of fine and ultra-fine particles may provide a better understanding about their toxicity.