Season linked responses to fine and quasi-ultrafine Milan PM in cultured cells

Abstract

Exposure to urbane airborne particulate matter (PM) is related to the onset and exacerbation of cardiovascular and respiratory diseases. The fine (PM1), and quasi-ultrafine (PM0.4) Milan particles collected during different seasons have been characterised and the biological effects on human epithelial lung A549, monocytes THP-1 cells and their co-culture, evaluated and compared with the results obtained on the PM10 and PM2.5 fractions. Chemical composition and transmission electron microscopy (TEM) analysis of PM0.4 showed that this fraction was very similar to PM1 for biological responses and dimension. All the winter fractions increased within 1 h the level of reactive oxygen species (ROS), while only summer PM2.5 had this effect on A549 cells. The phosphorylation of H2AX (γH2AX), a marker of double strand DNA breaks (DSBs), was increased by all the winter fractions on A549 and THP-1 cells while summer PM samples did not induced this effect. PM0.4 and PM1 biological effects are partly similar and related to the season of sampling, with effects on ROS and DNA damage induced only by winter PM fractions. The winter PM damaging effect on DNA correlates with the presence of organic compounds.

Introduction

The relationship between the exposure to airborne particulate matter (PM) and the onset of adverse effects on human health has been widely described in many epidemiological studies on the increased rates of mortality and morbidity in the population for cardiovascular and respiratory diseases (Brook et al., 2010, de Kok et al., 2006); moreover there is increasing evidence that PM2.5 exposure increases lung cancer mortality (Pope et al., 2011, Turner et al., 2011). Pollution-related cardio respiratory effects have been linked to the presence of a pulmonary and systemic inflammatory status, evidenced by the augmented levels of pro-inflammatory cytokines, in the circulating blood of PM exposed population (Pope and Dockery, 2006). In vitro studies on various lung cell lines have used different biological endpoints to demonstrate such correlation. It has been demonstrated that PM from different sources and places can promote the release of inflammatory mediators (Alfaro-Moreno et al., 2002, Hetland et al., 2004), genotoxic effects (Billet et al., 2008, Don Porto Carero et al., 2001, de Kok et al., 2005) and cell death (Alfaro-Moreno et al., 2002, Hsiao et al., 2000). Cell viability, pro-inflammatory proteins release, ROS production and DNA DSB induction have been here analysed to evaluate the effects produced by exposure to different PM fractions, variable for dimension and season of sampling.

In fact, the structure and composition of PM are complex and heterogeneous, and influence the biological properties of particles. The chemical composition varies with season and region of sampling, photochemical-meteorological conditions and sources of emissions (Brüggemann et al., 2009, Perrone et al., 2010, Pey et al., 2010). The winter Milan PM chemical composition is characterised by high levels of polycyclic aromatic hydrocarbons (PAHs), whereas summer PM is rich of elements such As, Cr, Cu, Al and Zn (Perrone et al., 2010). These data are rather representative of urban air particles: Brüggemann et al. (2009) reported a higher organic carbon (OC) concentration during winter in comparison with summer PM samples and explained the presence of such element as the contribution of domestic heating and of a lower atmospheric mixing layer.

PM size is another important parameter influencing biological effects. PM can be classified as PM10 (particles with an aerodynamic diameter less than 10 μm), PM2.5 (Ø < 2.5 μm) and PM1 (Ø < 1 μm). The fine fractions (PM2.5 and PM1) are dominated by combustion derived particles, consisting mainly of organic and inorganic elements adsorbed onto the surface of a carbonaceous core (Brüggemann et al., 2009, Zerbi et al., 2008). The coarse fraction (PM10) contains a major part of mineral compounds and some adsorbed endotoxins (Pérez et al., 2007, Schins et al., 2004).

The PM coarse fraction (2.5–10 μm) has been associated with pro-inflammatory and cytotoxic effects (Gualtieri et al., 2010, Hetland et al., 2005, Monn and Becker, 1999, Schins et al., 2002). The presence of endotoxins (Soukup and Becker, 2001, Schins et al., 2004, Becker et al., 2005, Camatini et al., 2010) in summer PM10 is partly responsible of the inflammation processes, even the release of inflammatory mediators occurs also in cells exposed to fine PM. This event has been explained with the large surface area of these small particles (Hetland et al., 2004, Donaldson et al., 2001, Donaldson et al., 2002, Oberdörster, 2001).

The PM fine fraction has been associated mainly to a higher genotoxic potential (Billet et al., 2008, de Kok et al., 2005), with the winter samples being more potent (Binková et al., 2003, Chakra et al., 2007) in relation to higher PAHs and metal contents. Moreover, positive relationships were found between the formation of reactive oxygen (ROS) and nitrogen (RNS) species and the induction of DNA damage (de Kok et al., 2006, Gualtieri et al., 2011). Depending on the amount formed, ROS may result in necrosis as well as apoptosis. The most severe DNA lesions produced by ROS are double strand breaks (DSB; Mills et al., 2003), which may be detected by the phosphorylation of histone 2AX (γH2AX; Albino et al., 2009).

Besides the increasing epidemiological data on particles with a diameter less than 1 μm (quasi-ultrafine), there are still few data on the chemical composition (Kudo et al., 2011, Chen et al., 2010) and biological effects (Steenhof et al., 2011, Val et al., 2011, Jalava et al., 2007) of the ultrafine fraction (Ø < 0.1 μm) for the difficulty in sampling such particles (Furuuchi et al., 2010).

In order to fulfil this gap of knowledge on the biological responses triggered by the finest PM fractions on human cell lines, we extended our study to the effects produced by Milan summer and winter quasi-ultrafine PM0.4 and PM1 on A549 and THP-1 cells and on the co-culture of these two lines.