The acute toxic effects of particulate matter in mouse lung are related to size and season of collection

Abstract

The toxicity of size-fractionated particulate matter (PM10 and PM2.5) collected in Milano during two different seasons (summer and winter) has been evaluated in vivo. The focus is on time related (3 h, 24 h and 1 week) lung response following a single intratracheal aerosolization in BALB/c mice. The bronchoalveolar lavage fluid (BALf) and the lung parenchyma were screened for different markers of inflammation and cytotoxicity. Histology and immunohistochemistry were performed on excised fixed lungs to assess the effects produced by the different PM fractions. All the analyzed inflammatory markers (PMNs percentage, TNF-α, Hsp70 in the BALf, HO-1 in lung parenchyma), increased after summer PM10 administration; on the contrary winter PM10 and PM2.5 specifically increased the amount of the Cyp1B1, a protein putatively involved in the induction of pro-carcinogenic effect. Moreover, we detected an intensification of LDH activity in the BALf after the administration of winter PM10 and PM2.5, potentially related to an in progress necrotic process while after summer PM10 and PM2.5 administration, the initiation of the caspase cascade suggested a cytotoxic effect sustained by apoptosis. Our results evidenced the toxicity mechanisms elicited by size fractionated PM samples, collected in winter and summer seasons, which differs for dimensions, chemical and microbiological composition. PM10 has been indicated to elicit above all a pro-inflammatory response, linked to its specific biological components, while PM2.5 is supposed to be more harmful due to its smaller dimension and the ability to distribute into the lung alveolar districts. We hypothesized that adverse health effects observed after a single dose of winter PM2.5 is at least partly caused by specific winter PM components, i.e. PAH and transitional metals.

Introduction

Particulate matter (PM) is a mixture of airborne particles derived from the combustion of fossil fuels or from the breakdown of crustal components. Currently, PM standards are based on total mass and size, which can range from few nanometers to tens of micrometers.

Particles below 10 μm in diameter have the potential to reach and be deposited in the alveoli, while those greater than 10 μm are likely to land in proximal airways and be removed by the ciliary activity (Kim et al., 1994). Particles smaller than 2 μm in diameter show a clear tendency to achieve greater peripheral deposition than those greater than 2 μm (Conway et al., 1998). Numerous epidemiological studies (Calderón-Garcidueñas et al., 2007, Pope et al., 2004) presented convincing evidence of the association between increase in respiratory disease morbidity and increased levels of coarse PM, especially among susceptible populations (Pope et al., 1995). Some researches suggest that this fraction may contribute predominantly to possible toxic and pro-inflammatory effects by its constituents, which include biological agents (Camatini et al., 2010), metals (Gerlofs-Nijland et al., 2009) and organic compounds (Li et al., 2002).

Recently, attention has been focused on the fine fraction, constituted of particles with an aerodynamic diameter below 2.5 μm (PM2.5). These particles, which are usually present in high number in PM samples, may be more harmful than larger ones, as they are more efficiently retained in the alveolar lung portion. As a general rule, the primary biological targets of inhaled particles are cells of the pulmonary epithelium as well as resident macrophages (Cohen and Pope, 1995). Among these cells, alveolar epithelial cells are affected in a number of respiratory PM induced diseases (Floreani et al., 2003, Pourazar et al., 2004). The fine fraction is frequently suggested to be responsible of cardiac disorders and acute cardiovascular events, such as myocardial infarction (Dockery and Stone, 2007, Pope et al., 2006, Zanobetti and Schwartz, 2005), as well as inflammatory pathologies (Scapellato and Lotti, 2007). Increased plasma viscosity and other changes in blood-related parameters have been detected after inhalation of fine PM (Peters et al., 1997). However, the biological mechanisms of such events are still unclear; even the pulmonary endothelium appears to be the initial target site for ultrafine particles, which can translocate into the blood vessels, and reach other organs (Peters et al., 2006).

LPS, a constituent of the outer membrane of Gram-negative bacteria associated with PM, and polycyclic aromatic hydrocarbons (PAHs), an organic class of PM components, differ in PM concentration between summer and winter seasons (Camatini et al., 2010, Lonati et al., 2005, Marcazzan et al., 2001). PM composition seasonal variation has been related to the different biological responses, and cytokine release is one of the parameters affected (Hetland et al., 2005).

In vivo studies have been performed by means of various pollutant exposure methods (e.g. inhalation, intratracheal or intranasal instillation). Inhalation mimics the most natural route of exposure, even though it presents some experimental limitations (Costa et al., 2006). One of these is the estimation of the real dose delivered into the lungs, which is one of the most powerful advantages of intratracheal aerosolization. This method is very useful for the analysis of dose–response effects in comparative studies of exposure to various PM collected in different areas (Gerlofs-Nijland et al., 2009, Wegesser and Last, 2008). Recently, we used intratracheal aerosolization to assess the lung toxicity of a single dose of tire debris and PM (10 and 2.5) and we found that the effects produced were related both to the particle dimension and composition (Mantecca et al., 2009, Mantecca et al., 2010).

To analyze if the dimension and the seasonality of PM may elicit different lung responses, we examined, in intratracheally aerosolized mice, the acute effect of PM10 and PM2.5 collected in Milano during summer 2008 and winter 2009.

Bronchoalveolar lavage fluid (BALf) was screened to evaluate total and differential cell counts, inflammatory markers (tumor necrosis factor α, TNF-α; heat shock protein 70, Hsp70; secretory phospholipase A₂, sPLA₂) and markers of cell damage (total protein content; lactate dehydrogenase, LDH; alkaline phosphatase, ALP).

Lung pathologic effects were assessed by immunoblotting analyses and histological observations. In lung parenchyma we evaluated the levels of Caspase8-p18, involved in the programmed cell death, of heme oxygenase-1 (HO-1), an enzyme induced by stimuli such as oxidative stress, cytokines and hypoxia, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB-p50), involved in inflammatory pathways, cytochrome 1B1 (Cyp1B1), a member of cytochrome P450 superfamily involved in pro-oxidant action and heat shock protein (Hsp70), an heat shock protein involved in cytokines release.