Multigenerational mitochondrial alterations in pneumocytes exposed to oil fly ash metals

https://doi.org/10.1016/j.ijheh.2010.10.003Get rights and content

Abstract

Oil fly ash (OFA), containing high amounts of transition metals, is among the most reactive airborne particulate matter emissions, which have been associated with several diseases, such as chronic obstructive pulmonary diseases (COPD), lung cancer, and cardiovascular diseases. The aim of the present study was to evaluate mitochondrial alterations in OFA-exposed cultured pneumocytes and in their progeny. Alveolar epithelial cells (A549 line) were exposed either to an OFA water solution, containing 68.8 μM vanadium (V), 110.4 μM iron (Fe), and 18.0 μM nickel (Ni), or to the individual metal solutions. Structural and functional mitochondrial parameters were determined in exposed cultures and in 3 consecutive subcultures. OFA, V and Fe solutions caused a time-dependent loss of mitochondrial enzymatic activity, glutathione depletion, generation of lipid hydroperoxides, hydrogen peroxide and other reactive oxygen species, especially in G0–G1 phase cells, accompanied by a decrease in mitochondrial mass and transmembrane potential. Mitochondrial alterations were partly transmissible to daughter cells for up to 3 generations. Fe and especially V were responsible for the observed mitochondrial alterations in pneumocytes exposed to OFA. Spread of mitochondrial dysfunctions to daughter cells is expected to amplify oxidative stress in the respiratory epithelium and to play an important role in the pathogenesis of respiratory diseases.

Introduction

Epidemiological studies have documented a relationship between exposure to airborne particulate matter (PM) and an excess short-term mortality (Chen and Lippmann, 2009), adverse reproductive effects (Lewtas, 2007), promutagenic lesions (Binkova et al., 2007), and a variety of chronic diseases (Brunekreef and Holgate, 2002, Chen et al., 2008), such as chronic obstructive pulmonary diseases (COPD) and their exacerbations (Ling and van Eeden, 2009), lung cancer (Pope et al., 2002, Lewtas, 2007), and cardiovascular diseases either due to triggering of acute cardiac events or to promotion of the chronic development of cardiovascular disorders (Franchini and Mannucci, 2009). The oil fly ash (OFA) generated from oil-fired power stations is a major component of PM in industrialized sites and is among the most reactive PM emissions (Chen and Lippmann, 2009).

The OFA inorganic residue remaining after burning contains high amounts of the metals vanadium (V), iron (Fe), and nickel (Ni) (Kim et al., 2004), in the form of water soluble salts delivering V(IV), Fe(III), and Ni(II). In particular, Ni and V, which are characteristic tracers of OFA, are particularly influential components in terms of acute cardiac function change and excess short-term mortality (Chen and Lippmann, 2009). V and Fe undergo redox cycling reactions, while the primary route for Ni toxicity is depletion of reduced glutathione (GSH) and protein-bound sulfhydryl groups of proteins (Stohs and Bagchi, 1995, Valko et al., 2006). The PM-mediated oxidative stress may arise either from direct generation of reactive oxygen species (ROS) from the surface of particles or from soluble compounds such as transition metals or altered functions of mitochondria or activation of ROS-generating inflammatory cells or depletion of endogenous antioxidants (Risom et al., 2005, Møller et al., 2010).

In a recent study (Di Pietro et al., 2009), we demonstrated that the transition metals adsorbed to OFA cause DNA damage as well as a marked lipid peroxidation in the cytoplasm of cultured human pneumocytes. In biological tissues lipid peroxidation mainly affects the polyunsaturated side chains of membrane phospholipids, particularly susceptible to free radical-initiated oxidation (Valko et al., 2006). Fatty acid peroxidation results in lipid peroxides and lipid hydroperoxides that are transient intermediate species in the living cell. This species can either interact with enzymatic or nonenzymatic antioxidants or form much more toxic breakdown by-products reacting with metal ions or iron-containing proteins (Szweda et al., 1993). The presence in the respiratory epithelium of alkoxy radicals (RO·), peroxy radicals (ROO·) and reactive aldehydes, such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), are mainly due to the OFA V content. These reactive compounds affect biological systems by causing protein and DNA damage and by inactivating antioxidant enzymes (Møller et al., 2010). In addition, MDA forms adducts to DNA bases, while 4-HNE forms exocyclic etheno-DNA-base adducts and protein adducts having powerful effects on signal transduction pathways (Nair et al., 2007). Lipid peroxidation is involved in aging and in the pathogenesis of age-related diseases (Spiteller, 2007), and lipid peroxidation products were detected in COPD patients (Nowak et al., 1999, Rahman, 2005). The hydrocarbon ethane, a by-product of 9,12,15-linolenic acid peroxidation was negatively correlated with lung functions (Paredi et al., 2000).

Mitochondrial membrane phospholipids are important targets for the lipid peroxides that involve highly polyunsaturated side chains. The organelle respiratory chain is the major cellular site of ROS production and cellular oxygen sensing (Hamanaka and Chandel, 2009). However, under physiological conditions mitochondria do not produce large amounts of ROS, which have been estimated to be equal to the 0.2% of the consumed oxygen (St-Pierre et al., 2002). Since the harmful effect of lipid peroxidation could cause an impairment of mitochondrial metabolism, coupled with endogenous ROS generation, the aim of the current study was to assess mitochondrial function in pneumocytes exposed to bioavailable OFA transition metals, and in their progeny. In fact, the accumulation of defective mitochondria transmitted to daughter cells could perpetuate and amplify the metal-induced damage to the respiratory epithelium by causing an overproduction of radical species even in the absence of direct exposure.

The results obtained provide evidence that exposure of A549 alveolar cells either to an OFA solution or to individual metal solutions, especially V(IV) and Fe(III), results in depletion of cellular GSH and impairment of mitochondrial enzymatic activity. Moreover, in synchronized cell cultures an enhanced generation of ROS, which was significant in G0–G1 cells, was accompanied by a decrease in mitochondrial mass and a loss of membrane potential. These alterations were found to be partly transmissible to the subsequent generations of cells.

Section snippets

Oil fly ash sample

An OFA water solution, obtained as previously reported (Di Pietro et al., 2009), was used at a preliminarily assessed subtoxic dose. This solution contained 68.8 μM V(IV), 110.4 μM Fe(III), and 18.0 μM Ni(II). Several experiments were also performed by using individual metal solutions at the same molarity as they were present in the examined OFA sample.

Biochemicals and reagents

All chemicals and reagents were obtained from Sigma–Aldrich (Sigma–Aldrich-Italia, Milan, Italy) unless otherwise specified.

Cells and culture conditions

The alveolar basal

Mitochondrial enzymatic activity

Table 1 shows the time course dependence of MTT reduction in the interval 0.5–24 h. A fast metabolic inhibition was observed in cells exposed either to the OFA sample or to V(IV). Compared with controls, after 1 h of exposure the activity of mitochondrial dehydrogenases was below 60% (P < 0.05) in spite of the fact that, as assessed by trypan blue exclusion, lower amounts of dead cells were present (80.5% and 87.6%, respectively; not significant). Since 0.5 h, a significant metabolic collapse was

Discussion

OFA and OFA metals caused a moderate loss of cell viability, a transient decrease of mitochondrial dehydrogenases, and a significant depletion of GSH. In addition, different experimental approaches showed that OFA as well as V(IV) and Fe(III) were able to induce a marked increase of intracellular ROS levels and a decrease both of the mitochondrial mass and of its transmembrane potential. Generation of ROS was particularly intense in cells in G0–G1 phase, while it was less evident in G2–S phase,

Acknowledgment

This work was supported by grants from the University of Messina (Fondi di Ateneo 2005).

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