Several epidemiologic studies have demonstrated the role of particulate matter (PM) in cardiopulmonary morbidity and mortality (Pope 1992; Dockery et al. 1993; Saldiva et al., 1995). Although there is an efficient defense mechanism against inhaled toxic substances, lung is the first target organ for adverse effects induced by air pollutants (Lohmann-Matthes et al. 1994). In addition, there are evidences suggesting that PM expo-sure can contribute to systemic responses through inflammation and/or oxidative stress process (Zanchi et al. 2008; Calderón-Garcidueñas, 2001; Rhoden et al. 2005).
A plausible mechanism by which residual oil fly-ash (ROFA) causes injury is the generation of reactive oxidative species (ROS) by its metal content (Ghio et al., 2002). These metals, especially iron, are involved in Fenton-like reactions, which result in metabolic products, such as superoxide anion, hydrogen peroxide and hydroxyl – the most deleterious radical (Halliwell & Gutteridge, 2007). Gurgueira et al. (2002) showed time-dependent increases in the steady-state concentration of oxidants in the lung and heart of rats after a short-term exposure to concentrated ambient particles (CAPs). While lung oxidants increased immediately upon exposure to CAPs, significant oxidative stress in the heart was observed only after a 1-h lag phase.
ReseaRch aRtIcle
Is cardiac tissue more susceptible than lung to oxidative effects induced by chronic nasotropic instillation of residual oil fly ash (ROFA)?
Roberto Marques Damiani1, Marcella Ody Piva1, Marcelo Rafael Petry1, Paulo Hilário Nascimento Saldiva2, Alexandre Tavares Duarte de Oliveira3, and Cláudia Ramos Rhoden1
1Laboratório de Estresse Oxidativo e Poluição Atmosférica, Universidade Federal de Ciências da Saúde de Porto Alegre, Brasil, 2Laboratório de Poluição Atmosférica Experimental, Universidade de São Paulo, Brasil, and 3Departamento de Ciências Básicas da Saúde, Universidade Federal de Ciências da Saúde de Porto Alegre, Brasil
abstractThe current study aimed to determine the role of oxidants in cardiac and pulmonary toxicities induced by chronic exposure to ROFA. Eighty Wistar rats were divided into four groups: G1 (10 µL Saline), G2 (ROFA 50 µg/10 µL), G3 (ROFA 250 µg/10 µL) and G4 (ROFA 500 µg/10 µL). Rats received ROFA by nasotropic instillation for 90 days. After that, they were euthanized and bronchoalveolar lavage (BAL) was performed for total count of leukocytes, protein and lactate dehydrogenase (LDH) determinations. Lungs and heart were removed to measure lipid peroxidation (MDA), catalase (CAT) and superoxide dismutase (SOD) activity. BAL presented an increase in leukocytes count in G4 in comparison to the Saline group (p = 0.019). In lung, MDA level was not modified by ROFA, while CAT was higher in G4 when compared to all other groups (p = 0.013). In heart, G4 presented an increase in MDA (p = 0.016) and CAT (p = 0.027) levels in comparison to G1. The present study demonstrated cardiopulmonary oxidative changes after a chronic ROFA exposure. More specifically, the heart tissue seems to be more susceptible to oxidative effects of long-term exposure to ROFA than the lung.Keywords: ROFA, chronic exposure, air pollution, cardiopulmonary oxidative stress
These authors also found a tissue-dependent increase in some antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT). Furthermore, the authors demonstrated that 24 h after CAPs exposure, lungs restored their oxidant balance (Gurgueira et al. 2002).
Based in the facts that the absolute number of deaths attributable to PM is higher for cardiac toxicity than the pulmonar adverse effects (Dockery, 2001; Frampton, 2001), we decided to investigate whether oxidants play a role in the cardiac PM toxicity induced by chronic treat-ment with different doses of ROFA, including its involve-ment in the lung inflammation.
Methods
AnimalsMale Wistar rats, aged 45 days, from the Animal Facility of Universidade Federal de Ciências da Saúde de Porto Alegre were used. The animals were kept in plastic cages (47 cm × 34 cm × 18 cm) under controlled humidity (75–85%), temperature (22 ± 2°C), with a 12 h light-dark period. They had free access to water and to a standard laboratory diet (Supra-lab, Alisul Alimentos S/A, Brazil). All animals used in the research were treated humanely, with due consideration to the alleviation of distress and discomfort. All experimental procedures were approved by the Universidade Federal de Ciências da Saúde Ethical Committee for Research (370/07).
Characterization of particlesResidual oil fly ash was obtained from a steel industry placed in Sao Paulo, Brazil. The particle elements were ana-lyzed by neutron activation analysis and presented the fol-lowed composition: Br, 1.4 ± 19 μg g-1; Ce, 16.3 ± 0.3 μg g-1; Co, 9.9 ± 0.25 μg g-1; Cr, 107.7 ± 1.4 μg g-1; Fe, 1058.9 ± 2.37 μg g-1; La, 10.3 ± 0.1 μg g-1; Mn, 3.8 ± 24 μg g-1; Rb, 719.7 ± 1.0 μg g-1; Sb, 2.2 ± 0.9 μg g-1; As, 154.4 ± 0.8 μg g-1; V, 35 ± 4 μg g-1; Zn, 491.9 ± 3.1 μg g-1. The values are expressed as the means ± standard deviation. The mean aerodynamic diameter was 1.2 ± 2.24 μm (Medeiros et al., 2004).
Experimental designRats were divided into four treatment groups: ROFA 500 µg/10 µL (n = 20), ROFA 250 µg/10 µL (n = 20), ROFA 50 µg/10 µL (n = 20) and Saline 10 µL (n = 20). The animals were exposed to ROFA by intranasal instillation, once a day, during 90 days. Twenty-four hours after the last instillation, 10 rats from each group were used to obtain the bron-choalveolar lavage (BAL). The remainder were euthanized by decapitation and lung and heart were removed and immediately frozen (−80°C) to perform TBARS, superoxide dismutase and catalase determinations.
Inflammation parametersBronchoalveolar lavageThe rats were anesthetized with sodium pentobarbital (50 mg/Kg body weight) and their lungs were washed through the trachea using three aliquots of 7 mL of sterile
saline. Each aliquot represents one in-and-out recovery of fluid. The obtained fluid was centrifuged at 400 × g at 4°C. Total cell counts were determined after trypan blue stain-ing using a Neubauer chamber. Total protein levels, as a measure of vascular permeability, were measured in the first lavage from each sample using the Bradford method (Bradford, 1976). As a marker of toxicity, lactate dehy-drogenase (LDH) activity was analyzed by a colorimetric method (Labtest, Brazil). These measurements were car-ried out in a Perkin Elmer Lambda 35 spectrophotometer (Perkin Elmer Life and Analytical Sciences, Shelton, USA).
Oxidative stress parametersTissue preparationTissue samples were homogenized in 5 volumes (lung) and 7 volumes (heart) of 120 mM KCl and 30 mM sodium phosphate buffer, pH 7.4, containing 0.5 mM phenylmeth-anesulfonyl fluoride as a protease inhibitor, at 0–4°C. The suspensions were centrifuged at 600 × g for 10 min at 0–4°C to remove nuclei and cell debris. The pellets were dis-carded and the supernatant were used as homogenates.
Determination of lipid peroxidationLung and heart tissue homogenates were precipitated with 10% TCA, centrifuged and incubated with thiobar-bituric acid (0.67%) (Sigma Chem. Co., St Louis, MO) for 60 min at 100°C. Malondialdeyde (MDA) were extracted using butanol (1:1;v/v) and measured at 535 nm. The concentration of MDA was expressed in nM MDA/mg of protein. Tissue protein was quantified using the Bradford assay (Bradford, 1976).
Catalase activityThe CAT tissue activity was performed according to Aebi (1984) at 240 nm, during 120 s. Data are expressed in pmol/mg protein.
Superoxide dismutase activityThe SOD tissue activity was measured as described by Maklund (1985). This method is based on capacity of pyrogallol to autoxidize. The pyrogallol autoxidation is inhibited in presence of SOD, whose activity can be mea-sured using a double-beam spectrophotometer at 420 nm. One unit of SOD is represented as units per milligram protein.
Statistical analysisData are given as mean ± standard deviation. One-Way Analysis of Variance (ANOVA) followed by Tukey’s HSD test was used to compare data among the different groups. The level of significance was set at 5%. All statistical analyses were carried out using Sigma-Stat 2.0 Software (Jandel Corporation, 1992–1995). The sample size were based in previous studies from our laboratory which demonstrated that this number of animals is fully sufficient for a statistical analysis (Pereira et al. 2007; Zanchi, 2008).
Pulmonary inflammation parametersRats treated with 500 µg/10 µL of ROFA presented an increase in number of total cells in BAL in comparison with the Saline group (p = 0.019). However, no statis-tical significant difference was observed in total pro-tein and LDH activity among the treatment groups. (Figure 1).
Oxidative stress parametersLungThere were no differences regarding lipid peroxidation among the treatment groups. In terms of SOD, anti-oxidant enzyme, we have also not detected differences among the treatment groups. However, the pulmonary catalase activity was higher in rats treated with ROFA 500 µg/10 µL when compared to all other groups (p = 0.013). (Figure 2).
HeartInterestingly, in heart, the group which has received 500 µg/10 µL of ROFA demonstrated an increase in MDA con-centration (p = 0.016) as well as in CAT activity (p = 0.027) when compared to the Saline group (Figure 3). However, analyzing SOD, no difference was found among treat-ment groups.
Discussion
The current study aimed to determine the role of oxidants in cardiac and pulmonary toxicities induced by chronic exposure to three doses of ROFA. An increased of lipid peroxidation was detected only in the heart of rats treated with 500 µg ROFA even when CAT activity was elevated. We did not find oxidant lipid damage in the rat lungs of these treatment groups, however CAT activity was higher in the group treated with 500 µg ROFA. In addition, we
Figure 1. Bronchoalveolar lavage (BAL) analysis of rats(n = 10 per group) exposed for ninety days to three different concentrations of Residual Oil Fly Ash (ROFA) or Saline. Data are demonstrated as mean ± standard deviation of the mean A: Lactate dehydrogenase (LDH) activity in BAL. B: Protein concentration in BAL. C: Total cells count in BAL. *Statistical difference when compared with Saline group. Tukey's HSD test, p = 0.019.
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detected, in the same group, an increase in the total cell counts from bronchoalveolar lavage fluid. This is the first report that links oxidative cardiopulmonary changes and long-term exposure to ROFA.
The doses of pollutant used in our study were based in a previous work which demonstrated that a single intra-tracheal instillation of 500 µg PM was capable of inducing functional cardiopulmonary changes in rats (Rivero et al. 2005). We chose the above mentioned concentration and two lower doses (250 and 50 µg) trying to determinate a dose-response influence. ROFA has been useful as sur-rogate for ambient air PM in many biological studies because of its composition, especially rich in metals. Data suggests that ambient air and other particles emis-sion sources follow a comparable mechanism of action as ROFA including phosphorylation reactions, transcrip-tion factor activation, mediators release and inflamma-tory injury (Ghio et al., 2002).
In terms of pulmonary inflammatory parameters, we observed an increase in the count of total cells in the group of animals which received the highest dose of ROFA, when compared to Saline group. In addition, we did not detect any difference in protein and LDH concentration in BAL when compared to all treatment groups. ROFA exposure triggers an inflammatory process that includes leukocyte recruitment, activation and increased alveolar macrophages count (Becker, 2002) Alveolar macrophages are the most important cells involved in lung inflamma-tion response caused by particle inhalation. (Lohmann-Matthes et al. 1994). Oberdörster et al. have demonstrated that the alveolar macrophage recruitment and the over-flow of plasmatic proteins in alveoli, after inhalation of particles with less than 2.5 µm, are triggered by differ-ent events and could occur separately. Because higher doses resulted in an increased interstialized fraction of particles, those authors suggested that inflammatory
Figure 2. Oxidative stress in lung of rats exposed for ninety days to three different concentrations of Residual Oil Fly Ash (ROFA) or Saline (n = 10 per group). Data are demonstrated as mean ± standard deviation of the mean. A: malondialdehyde (MDA) concentration in lung. B: Superoxide Dismutase (SOD) activity in lung. C: Catalase activity in lung. *Statistical difference when compared with all others treatment groups. Tukey's HSD test, p = 0.013.
events induced by particles in the interstitial space can modify the inflammation in the alveolar space detectable by BAL (Oberdörster et al., 1992). Some experimental studies have demonstrated that exposure to PM leads to a raised number of polymorphonuclear neutrophil in BAL without detectable differences in protein and LDH levels either in toxicological (Rhoden et al. 2004; 2005) and environmental (Pereira et al., 2007) levels.
Regarding oxidative stress, there were no statistically significant differences in lipid peroxidation in pulmonary tissue among the groups. SOD activity was similar in the different treatment groups. Our data demonstrated that superoxide anion is not essential for the triggering of the inflammatory responses in the lung, after chronic PM exposure, while in the acute exposure it is especially involved in the oxidant inflammatory response (Rhoden et al. 2008). Based in these observations, we speculate that the elevated concentration of CAT activity in lung
of rats which received the highest dose of ROFA could act as a protector of the tissue against the oxidative lipid damage. As demonstrated by Imrich (2007), lung inflammation caused by air particle inhalation depends, at least partially, on the interaction between metal content in the particles and intracellular alveolar macrophage levels of H2O2. It has been demonstrated that catalase is significantly important in lung defense because it is the enzyme that degrades hydrogen peroxide without cellular substrate (Rahman et al. 2006). This enzyme is specially localized in the alveolar type II pneumocytes, which are the most resistant cell type in the lung and also in alveolar macrophages (Kinnula et al. 1995). Evidences demonstrated a raised CAT activity in lung after a 5-h exposure to CAPs (Gurgueira et al. 2002). However, 24 h after the exposure period, the tissue recovered its oxidant balance and interrupted the inflammatory process (Gurgueira et al. 2002). Lung has
Figure 3. Oxidative stress in heart of rats exposed for 90 days to three different concentrations of Residual Oil Fly Ash (ROFA) or Saline (n = 10 per group). Data are demonstrated as mean ± standard deviation of the mean. A: malondialdehyde (MDA) concentration in heart. *Statistical difference when compared with Saline group. Tukey's HSD test, p = 0.016. B: Catalase activity in heart. *Statistical difference when compared with Saline group. Tukey's HSD test, p = 0.027. C: Superoxide Dismutase activity in heart.
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a direct contact with the environment with respect to both injury and treatment. The alveoli are in a unique position in the body, where exogenous air encounters a thin cellular layer consisting of only about two cells beyond which immediate contact occurs with a refined organ with particular tasks, definitely requiring the structural integrity of the organ (Lohmann-Matthes, 1994).
On the other hand, the increased level of CAT observed in heart tissue of rats which received 500 µg/10 µL (ROFA) was not greater enough to protect tis-sue against ROS. This is demonstrated by the high level of MDA in heart tissue of animals that were submitted to high concentration of ROFA during ninety consecu-tive days. Similarly to muscles and brain, heart has high endogenous levels of hydrogen peroxide because of its poor concentration of CAT in physiological status (Scandalios, 2005). A large number of reports have suggested that H
2O
2 is an important mediator in the
vasculature inducing vascular constriction (Matoba et al. 2000; Jones & Morice 2000). Suvorava and Kojda (2009) described that a reduction of steady-state con-centrations of vascular hydrogen peroxide induced by an endothelial-specific overexpression of human CAT resulted in a marked reduction of systolic blood pressure in mice, demonstrating the importance of maintenance of basal levels of hydrogen peroxide in the circulatory system. Exogenous H
2O
2 also evokes airway reflexes
involving lung vagal afferents that results in changes in autonomic tonus in heart (Ruan et al. 2003) Pulmonary exposure to ROFA causes oxidative stress in heart first by autonomic stimulation (Ghelfi et al. 2008, Rhoden et al. 2005), production of ROS, release of inflammatory mediators in lung and heart (Rhoden et al. 2004; 2005) and by PM fractions that gains access to the systemic cir-culation and also by a direct interaction with the heart (Oberdörster et al. 2002). All of those observations have a common sense: lead to an increased production of H
2O
2 causing an oxidative misbalance in heart. Several
new epidemiological studies have demonstrated that living in locations with higher long-term average PM concentrations increases the risk for cardiovascular morbidity and mortality vastly exceeding the risk noted with short-term exposure (Miller et al. 2007; Puett et al. 2008). Also, PM air pollution has been linked with endo-thelial dysfunction, systemic oxidative and inflamma-tory responses and the progression of atherosclerosis (Mills et al. 2007; Sun 2005).
conclusion
The present study demonstrated cardiopulmonary oxi-dative changes after a chronic ROFA exposure. More spe-cifically, the heart tissue seems to be more susceptible to oxidative effects of long-term exposure to ROFA than the lung. These results suggest that oxidative damage medi-ated by H
2O
2, may be one of the mechanisms involved in
cardiac toxicity related to PM exposure.
Declaration of interest
This work was supported by Universidade Federal de Ciências da Saúde de Porto Alegre, Brazil; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES and Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq.
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