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Composition of Diesel Particles Influences Acute Pulmonary Toxicity: An Experimental Study in MICE Daniel Laks ab; Regiani Carvalho de Oliveira ab; Paulo Afonso de André ab; Mariângela Macchione ab; Miriam Lemos ab; Debora Faffe ab; Paulo H. N. Saldiva ab; Walter A. Zin ab a Laboratory of Respiration Physiology, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil b Department of Pathology, School of Medicine, University of São Paulo, São Paulo, Brazil First Published on: 04 August 2008
To cite this Article Laks, Daniel, de Oliveira, Regiani Carvalho, de André, Paulo Afonso, Macchione, Mariângela, Lemos, Miriam, Faffe,
Debora, Saldiva, Paulo H. N. and Zin, Walter A.(2008)'Composition of Diesel Particles Influences Acute Pulmonary Toxicity: An Experimental Study in MICE',Inhalation Toxicology, To link to this Article: DOI: 10.1080/08958370802112922 URL: http://dx.doi.org/10.1080/08958370802112922
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Inhalation Toxicology, xx:1–6, 2008 c Informa Healthcare USA, Inc. Copyright ISSN: 0895-8378 print / 1091-7691 online DOI: 10.1080/08958370802112922
Composition of Diesel Particles Influences Acute Pulmonary Toxicity: An Experimental Study in MICE Daniel Laks, Regiani Carvalho de Oliveira, Paulo Afonso de Andr´e, Mariˆangela Macchione, Miriam Lemos, Debora Faffe, Paulo H. N. Saldiva, and Walter A. Zin
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Laboratory of Respiration Physiology, Carlos Chagas Filho Institute of Biophysics, Federal University of Rio de Janeiro, Rio de Janeiro and Department of Pathology, School of Medicine, University of S˜ao Paulo, S˜ao Paulo, Brazil
Ambient particles have been consistently associated with adverse health effects, yielding mainly high cardiorespiratory morbidity and mortality. Diesel engines represent a major source of particles in the urban scenario. We aimed to modify the composition of diesel particles, by means of different extraction procedures, to relate changes in chemical profile to corresponding indicators of respiratory toxicity. Male BALB/c mice were nasally instilled with saline, or with diesel particles, treated or not, and assigned to five groups: saline (SHAM), intact diesel particles (DEP), and diesel particles previously treated with methanol (METH), hexane (HEX), or nitric acid (NA). Elemental composition and organic compounds were analyzed. Twenty-four hours after nasal instillation, respiratory parameters were measured and lung tissue was collected for histological analysis. Static elastance was significantly increased in groups DEP and MET in relation to the other groups. HEX and NA were different from DEP but not significantly different from SHAM and METH groups. The difference between dynamic and static elastance was increased in DEP, METH, and NA treatments; HEX was not statistically different from SHAM. DEP and METH groups presented significantly increased upper airways resistance, while DEP, METH, and NA showed higher peripheral airways resistance values. All groups had a higher total resistance than SHAM. DEP, METH, and NA showed significant increased infiltration of polymorphonuclear cells. In conclusion, diesel particles treated with hexane (HEX) resulted in a respiratory-system profile very similar to that in SHAM group, indicating that hexane treatment attenuates pulmonary inflammation elicited by diesel particles.
Ambient particles have been consistently associated with adverse health effects, yielding to high cardiorespiratory morbidity and mortality (Dockery, 1993; Schwartz, 1994; Pope, 1996). Urban aerosol is a complex mixture, containing both liquid and solid components produced by several sources (Hinds, 1999). The different profiles of size and composition may influence particle toxicity and, consequently, the magnitude of adverse health effects (Takenaka et al., 2004; Diociaiuti et al., 2001; Saldiva et al., 2002). Indeed, it has been shown that lung inflammation induced by short-term exposure to concentrated ambient air particles (CAPS) is mainly related to particle composition,
supporting the concept that mass is not the only factor that regulates toxicity (Saldiva et al., 2002). Thus, the determination of the relative toxicity of particle constituents is crucial for better understanding the pathogenesis of particle-induced injury, as well as to establish emission control policies aiming to preserve public health. Diesel engines represent a major source of particles in the urban scenario and considerable efforts have been made to reformulate diesel composition and engine technology in order to reduce toxic diesel emissions (McDonald, 2004; U.S. EPA, 2000). Multiple compounds are responsible for diesel toxic potential, such as metals (Adamson et al., 2000; Gavett et al., 2003; Costa & Dreher, 1997), sulfur, and organic species, mainly polycyclic aromatic hydrocarbons (PAHs) (Li et al., 2002; Hiura et al., 1999; Penning et al., 1999). Metals and PAHs exist in diesel particles either adsorbed on the particle surface or trapped within the carbonaceous core (Li et al., 2002; Pozzi et al., 2003). Surface compounds may be peeled out by simple extraction procedures,
Received 17 March 2008; accepted 8 April 2008. Address correspondence to Prof. Walter A. Zin, MD, PhD, Universidade Federal do Rio de Janeiro, Centro de Ciˆencias da Sa´ude, Instituto de Biof´ısica Carlos Chagas Filho, Ilha do Fund˜ao, 21949-900–Rio de Janeiro–RJ, Brazil. E-mail: walter
[email protected]
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allowing the investigation of specific constituents’ harmful effect. This approach was employed in the present study to determine the magnitude of acute pulmonary changes observed in mice after nasal instillation of low doses of intact diesel particles as well as diesel particles treated by different extraction methods.
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METHODS Particle Collection A particle trap device was adapted to the exhaust pipe of a bus from the public transportation fleet of S˜ao Paulo, equipped with a Mercedes Benz MB1620, 210-hp engine, without electronic control of fuel injection, running with diesel containing 500 ppm sulfur. This particular type of bus was chosen because it is the most frequent one operating in S˜ao Paulo, based on the information given by the municipality. Briefly, a mesh made of stainless steel was inserted into the exhaust pipe line of the bus. Diesel particles were collected after 1 day of routine operation of the bus and stored for toxicological studies. Particle Treatment Total collected mass of particles was divided into four samples. One sample received no further treatment and the remaining three were submitted to different extraction protocols, as follows: (a) Acidic extraction: Particles were immersed in 160 ml HNO3 65% (pH = 3.0), sonicated during 60 min, and rested for 24 h. Thereafter, the solution was centrifuged for 20 min at 1800 rpm at 5◦ C and 10 min at 5000 rpm at 10◦ C. The supernatant was collected and particles were dried at 57◦ C in a fume hood for 48 h before use. (b) Extraction with 2 solvents with different polarities: Particles were immersed either in 160 ml of methanol or hexane (P.A. grade), sonicated during 60 min, and rested for 24 h. Solutions were centrifuged for 15 min at 3000 rpm at 10◦ C. The supernatant was discharged and the precipitate stored in a vacuum dissector for 15 h.
Particle Composition Analysis Intact and treated particles were analyzed for metals and organic content. The concentration of Ni, S, Fe V, Pb, Cd, Cr, and Cu were determined by energy-dispersive energy x-ray fluorescence spectrometer (EDX 700HS, Shimatzu Corporation Analytical Instruments Division, Kyoto, Japan). Polycyclic aromatic hydrocarbons (PAHs) were analyzed by an atomic absorption spectrophotometer (Varian, model AA-1475, Victoria, Australia). The concentrations of the following PAHs were determined: benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenz[ah]anthracene, and indeno[123-cd]pyrene.
Exposure Protocol Forty-six male BALB/c adult healthy mice (body weight between 20 and 25 g) were assigned to five groups: Animals received a single nasal instillation of 10 µl saline solution (0.9% NaCl) or 15 µg diesel particles in saline (10 µl), either intact (DEP) or previously treated with methanol (MET), hexane (HEX), or nitric acid (NA). All animals received humane care in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the “Guiding Principles in the Care and Use of Animals” approved by the Council of the American Physiological Society, and our Institutional Animal Care and Use Committee approved all protocols in this study. Lung Mechanics Twenty-four hours after the instillation, the animals were sedated with 5 mg diazepam, ip, anesthetized with 20 mg/kg pentobarbital sodium, ip, and a snugly fitting cannula (0.8 mm ID) was introduced into the trachea. A pneumotachograph was connected to the tracheal cannula for the measurements of airflow (V ’) and changes in lung volume (VT ). The pressure gradient across the pneumotachograph was determined by means of a Validyne MP45-2 differential pressure transducer (Engineering Corp., Northridge, CA). Tracheal pressure (Ptr) was measured with a Validyne MP-45 differential pressure transducer (Engineering Corp., Northridge, CA). All signals were conditioned and amplified with a Beckman type R Dynograph (Schiller Park, IL). Flow and pressure signals were also passed through 8pole Bessel filters (902LPF, Frequency Devices, Haverhill, MA, USA) with the corner frequency set at 100 Hz, sampled at 200 Hz with a 12-bit analog-to-digital converter (DT2801A, Data Translation, Marlboro, MA), and stored on a microcomputer. All data were collected using LABDAT software (RHT-InfoData, Inc., Montreal, Quebec, Canada). Muscle relaxation was achieved with pancuronium bromide (0.04 mg/kg body weight, iv), and a constant-flow ventilator provided artificial ventilation (Samay VR15, Universidad de la Republica, Montevideo, Uruguay). Special care was taken to keep tidal volume (VT = 0.2 ml) and flow (V ’ = 1 ml/s) constant in all instances in order to avoid the effects of different flows, volumes, and inspiratory durations on the measured variables. Pulmonary mechanics were measured by the end-inflation occlusion method. In an open chest preparation, Ptr corresponds to transpulmonary pressure. Pulmonary resistive (P1), viscoelastic/inhomogeneous (P2) pressures, Ptot (= P1 + P2), static elastance (Est), dynamic elastance (Edyn), and the difference between dynamic and static elastances (E) were determined. Histopathology Heparin (1000 IU) was intravenously injected immediately after the determination of respiratory mechanics. The trachea was clamped at the end of expiration, and the abdominal aorta and vena cava were sectioned, yielding a massive hemorrhage
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that quickly sacrificed the animals. Then the lungs were removed en bloc at the end of expiration and fixed for 48 h in buffered 10% formalin solution at a constant pressure of 20 cm H2 O. Fixed lung slices were embedded in paraffin and processed according to routine histological procedures. Four-micrometerthick slices were prepared and stained with hematoxylin and eosin. Two investigators, who were unaware of the origin of the material, examined the samples microscopically. Morphometric measurements were performed using a standard point-counting procedure, at 1000×, with the aid of a coherent test system of 100 points and 50 lines contained within a square of 10,000 µm2 at this magnification. The relations of polymorphonuclear and mononuclear numbers of cells and pulmonary tissue area were assessed counting grid points falling on polymorphonuclear and mononuclear cells and dividing the results by the total number of points falling on tissue area in 10 noncoincident random microscopic fields. Statistical Analysis Comparison among groups was performed by one-way analysis of variance (ANOVA). When necessary, transformations of the dependent variables, such as logarithmic and square root, were employed, in order to normalize distribution and homogenize variances. In fact, significance of the observed differences (set at the level of 5%) was robust to both types of transformations. Post hoc analyses were conducted using Tukey’s test. RESULTS Particle Composition The treatment of diesel particles with the substances used in our protocols changed appreciably their composition, as shown in Tables 1 and 2. In terms of elemental analysis, Ni, S, V, and Pb (Table 1) were the elements more affected by the different extraction procedures, whereas Fe, Cd, and Cu did not change appreciably. Interestingly, after hexane treatment, the concentration of some elements (S, Ni, Cd, and Cu) tended
TABLE 2 Contents (ng/g) of polycyclic aromatic hydrocarbons of intact diesel particles and after extraction procedures PAHs Naphthalene Acenaphthylene Fluorene Anthracene Pyrene Benz[a]anthracene Benzo[b]fluoranthene Benzo[k]fluoranthene Benzo[a]pyrene
DEP
METH
HEX
NA
49.23 179.48 683.94 94.73 12838.27 1162.73 789.93 562.28 1642.28
ND 5.08 45.75 ND 631.31 107.23 201.21 10.58 26.42
ND ND ND ND 5.90 0.57 ND 0.03 0.15
ND 35.79 80.37 1.43 4457.30 346.27 749.51 55.80 68.11
Note. Concentrations of polycyclic aromatic hydrocarbons (PAHs) determined by atomic absorption spectrophotometry. DEP, METH, HEX, and NA represent the untreated group, and those treated with methanol, hexane, and nitric acid, respectively. Methanol treatment was able to extract acenaphthylene, fluorine, and pyrene. Hexane was efficient in removing pyrene, benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, and benzo[a]pyrene. Acidic treatment (HNO3 ) extracted all PAHs except for naphthalene. The most efficient acidic PAHs extraction was anthracene.
to increase, probably as a result of the relative enrichment of the inorganic fraction of particles, due to the removal of part of the organic fraction. The amount of organic compounds investigated in our study decreased with the different extraction procedures (Table 2), the greater efficiency being achieved with hexane. Lung Mechanics Static elastance was significantly increased after intranasal instillation of diesel particles (DEP, p = .0001) and methanol treated particles (MET, p = .007) when compared to SHAM mice. HEX and NA were not statistically different when compared with the SHAM group and MET. HEX and NA were
TABLE 1 Metal content (ppm) of intact diesel particles and after different extraction procedures Metals Nickel (Ni) Sulfur (S) Iron (Fe) Vanadium (V) Lead (Pb) Cadmium (Cd) Chromium (Cr) Copper (Cu)
DEP
METH
HEX
NA
0.181 ± 0.037 0.626 ± 0.416 74.556 ± 2.266 0.037 ± 0.013 0.050 ± 0.047 0.029 ± 0.008 0.161 ± 0.116 0.017 ± 0.001
0.140 ± 0.009 0.000 ± 0.000 82.604 ± 0.785 0.026 ± 0.001 0.035 ± 0.016 0.128 ± 0.020 0.156 ± 0.005 0.075 ± 0.030
0.319 ± 0.014 0.985 ± 0.145 63.996 ± 0.544 0.016 ± 0.021 0.037 ± 0.021 0.125 ± 0.044 0.166 ± 0.023 0.147 ± 0.008
0.106 ± 0.026 0.000 ± 0.000 89.311 ± 1.863 0.026 ± 0.034 0.010 ± 0.013 0.052 ± 0.003 0.159 ± 0.008 0.068 ± 0.013
Note. Concentrations of metals were determined by energy-dispersive x-ray fluorescence spectrometry. Values are mean ± SEM. DEP, particles as found; METH, HEX, and NA, particles treated with methanol, hexane, and nitric acid, respectively.
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FIG. 1. Static elastance (Est) and difference between dynamic and static elastance (E) in animals intranasally instilled with saline (SHAM), intact diesel particles (DEP), or particles previously treated with methanol, hexane, or nitric acid (METH, HEX, or NA, respectively). Static elastance was significantly increased after intranasal instillation of diesel particles (DEP) when compared to the sham group ( p = .0001). Hexane treatment was not significantly different from SHAM group. E values were increased after DEP ( p = .002), MET ( p = .001), and NA ( p = .032) treatments when compared to SHAM. Values are mean + SD of 9–10 animals/group. Different letters indicate significantly different values. different from DEP ( p = .002 and p = .005, respectively), as depicted in Figure 1. E, which represents the difference between dynamic and static elastance, was increased in DEP ( p = .002), MET ( p = .001), and NA ( p = .032) treatments when compared with the SHAM group (Figure 1). HEX was not statistically different from SHAM mice. DEP and MET treatment presented significantly higher pressures used to overcome airways resistance ( p = .001 and p = .003, respectively), as represented by P1, when compared to the SHAM group, as shown in Figure 2. Figure 2 shows that DEP, MET, and NA treatments presented significantly pressures spent against pulmonary inhomogeneities/viscoelasticity ( p = .001, p = .001, and p = .021, respectively), as represented by P2, when compared to the SHAM group. Total pulmonary resistance (Ptot) in all treatments presented higher values when compared to SHAM ( p < .05). As a general rule, hexane treatment was the most efficient in reducing adverse mechanical effects promoted by inhalation of diesel particles (Figures 1 and 2). Polymorphonuclear and Mononuclear Cells Counting Intact diesel (DEP) and particles treated with methanol (MET) and nitric acid (NA) significantly increased the infil-
FIG. 2. Pulmonary resistive (P1), viscoelastic/inhomogenei ties (P2), and total pressure (Ptot) in animals intranasally instilled with saline (SHAM), intact diesel particles (DEP), or particles previously treated with methanol, hexane, or nitric acid (METH, HEX, or NA, respectively). Values are mean + SD of 9–10 animals/group. Different letters indicate significantly different values ( p < .05). tration of polymorphonuclear cells ( p = .001, p = .005, and p = .001, respectively) when compared to the SHAM group. On the other hand, particles treated with hexane (HEX) presented a pattern very similar to SHAM, indicating that hexane treatment attenuates pulmonary inflammation elicited by diesel particles. DISCUSSION This study aimed to modify the composition of diesel particles, by means of different extraction procedures, trying to associate changes in their chemical profile to corresponding indicators of respiratory toxicity. As depicted in Tables 1 and 2, treating particles with different substances was effective in modifying particle composition. We administered low doses of diesel particles (15 µg by intranasal instillation) because we reasoned that higher doses could promote pulmonary alterations due to particle overload, which might hinder the characterization of the effects elicited by different particle compositions. More specifically, high doses of particles in the alveolar space are expected to cause inflammation, regardless of their chemical nature. We sampled our particles directly from the tailpipe of a regular operating bus, whose driving cycle and operation depend on traffic conditions and drivers’ profiles. In addition, engine technology and fuel characteristics of our source of particles probably reflect the average situation of fleets that operate in developing countries. Thus, the adverse findings detected after instillation of intact diesel particles do not represent the toxic potential of diesel using the present technology, but probably indicate the consequences of using bad fuel with low-technology engines (Leung & Harrison, 1999).
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FIG. 3. Photomicrographs of pulmonary parenchyma stained with hematoxylin-eosin. Typical lungs in animals that received saline solution (A), diesel particles as found (B), and diesel particles pretreated with methanol, hexane, and nitric acid (C, D and E, respectively). Original magnification 200×. Particle suspensions were administered to the mice by nasal instillation. This procedure affects not only size distribution but also deposition pattern of particles. However, this strategy allowed us to perform the chemical extraction procedures without the necessity of collecting a large amount of particles, which implies long sampling time and the possibility of chemical modifications during particle collection. Thus, our results may not reflect accurately the interaction of particles with the respiratory system when an aerosol delivers the particles, but it is convenient, nevertheless, to conduct comparative toxicity studies. TABLE 3 Percentage of cells contained in lung tissue in different diesel treatments GROUP
MN (%)
SHAM DEP HEX METH NA
24.7 ± 2.2 10.9 ± 0.5a 23.2 ± 6.3 B 18.4 ± 2.2 17.3 ± 1.8
PMN (%) 13.7 ± 2.2 35.1 ± 3.5a 19.1 ± 4.3b c a b c 24.1 ± 2.4 32.6 ± 2.9a
TOT CEL (%) 38.3 ± 3.2 46.0 ± 3.2 44.6 ± 5.8 42.5 ± 2.2 47.6 ± 3.4
Note. MN, mononuclear cells; PMN, polymorphonuclear cells; TOT CEL, total number of cells in lung tissue of animals intranasal instilled with saline (sham) intact diesel particles (DEP) or those previously treated with methanol, hexane, or nitric acid (METH, HEX, or NA, respectively). Values are mean ± SD of 9–10 animals/group. Different superscript letters indicate significantly different values ( p < .05).
Low doses of intact diesel particles promoted significant alterations in lung parenchyma, as characterized by two independent estimators: respiratory mechanics and lung histology. The observed changes were indicative of injury to both airways and distal airspaces, as shown in Figures 1 and 2 and Table 3, confirming that diesel particles present in our scenario exhibit an important toxic potential. Among the different extraction procedures employed, hexane-treated diesel particles presented the less harmful outcome. As expected, they contain the lowest concentrations of the measured PAHs, as shown in Table 2. Methanol, a solvent that extracts organic compounds with intermediate polarity, did not substantially influence particle toxicity. Our findings indicate that low-polarity organic compounds play a pivotal role in determining particle toxicity. Indeed, Figure 3 shows that the administration of hexane-treated particles resulted in a lung parenchyma similar to that in the SHAM group, methanol-treated particles did not substantially produce alterations, and the harmful effect of inhaled particles could be easily perceived in the DEP and NA groups. In agreement with this finding, Li et al. (2002) showed that the organic fraction of DEP induces particle-associated oxidative stress in alveolar macrophages, especially for particles containing the high amounts of PAHs. Aromatic hydrocarbons of urban PM have also been associated with mutagenesis and chromosomal aberrations (Somers et al., 1992; Bocskay et al., 2005). In our study, metals and sulfur affected particle toxicity to a smaller extent than organic compounds (Figures 1 and 2 and Table 3). Indeed, the relative concentration of metals and sulfur
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had the same or higher order of magnitude after hexane extraction (Table 1). This finding is not in line with the known role of metals in determining particle toxicity. Gurgueira et al. (2002) showed that rats exposed to residual oil fly ash presented elevated oxidative stress in the heart and lungs, and it was strongly associated with the metal fraction of those particles. Gavett et al. (2003) showed that metal content of PM led to an increase in airway responsiveness in mice with induced allergic airway disease, especially zinc, magnesium, lead, copper, cadmium, and arsenic. The smaller effect of metals and sulfur in modulating particle toxicity observed in our case may be due to the intrinsic characteristics of the particles employed in the present study. Low-quality diesel and incomplete burning due to the absence of electronic control of fuel injection result in higher content of organic compounds. (Khan et al., 2006) Thus, it is possible that the nature and amount of organic molecules in our sample dominated particle toxicity, allowing less space for metals to play their toxic role. CONCLUSION We showed that low doses of diesel particles can acutely elicit pulmonary toxicity in mice. Moreover, the extraction procedures applied herein were able to modify particle chemical characteristics and modulate the functional changes and inflammation after diesel particle intranasal instillation. We attribute the in vivo response to treated diesel particles to the high concentration of organic compounds, thus competing with metal-dependent toxic aggression. These findings can contribute to the establishment of new quality control policies for diesel compounds, as well as to stimulating public transportation authorities to renew their bus fleets in order to avoid health costs in developing countries. REFERENCES Adamson, I. Y., Prieditis, H., Hedgecock, C., and Vincent, R. 2000. Zinc is the toxic factor in the lung response to an atmospheric particulate sample. Toxicol. Appl. Pharmacol. 166:111–119. Bocskay, K. A., Tang, D., Orjuela, M. A., Liu, X., Warburton, D. P., and Perera, F. P. 2005. Chromosomal aberrations in cord blood are associated with prenatal exposure to carcinogenic polycyclic aromatic hydrocarbons. Cancer Epidemiol. Biomarkers Prev. 14:506–511. Costa, D. L., and Dreher, K. L. 1997. Bioavailable transition metals in particulate matter mediate cardiopulmonary injury in healthy and compromised animal models. Environ. Health Perspect. 105:1053– 1060. Diociaiuti, M., Balduzzi, M., De Berardis, B., Cattani, G., Stacchini, G., Ziemacki, G., Marconi, A., and Paoletti, L. 2001. The two PM(2.5) (fine) and PM(2.5-10) (coarse) fractions: Evidence of different biological activity. Environ. Res. 86:254–262. Dockery, D. W., Pope, C. A., Xu, X., Spengler, J. D., Ware, J. H., Fay, M. E., Ferris, B. G., and Speizer, F. E. 1993. An association between air pollution and mortality in six U.S. cities. N. Engl. J. Med. 329:1753–1759. Gavett, S. H., Haykal-Coates, N., Copeland, L. B., Heinrich, J., and Gilmour, M. I. 2003. Metal composition of ambient PM2.5 influences
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