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Oxidative Stress, Genotoxicity, And Vascular Cell Adhesion Molecule Expression in Cells Exposed to Particulate Matter from Combustion of Conventional Diesel and Methyl Ester Biodiesel Blends Jette Gjerke Hemmingsen,† Peter Møller,†,* Jakob Klenø Nøjgaard,‡ Martin Roursgaard,† and Steffen Loft† † ‡
Department of Public Health, Section of Environmental Health, Faculty of Health Sciences, University of Copenhagen Department of Atmospheric Environment, Faculty of Science and Technology, University of Aarhus, Frederiksborgvej 399, PO Box 358, DK-4000 Roskilde, Denmark
bS Supporting Information ABSTRACT: Our aim was to compare hazards of particles from combustion of biodiesel blends and conventional diesel (D100) in old and improved engines. We determined DNA damage in A549 cells, mRNA levels of CCL2 and IL8 in THP-1 cells, and expression of ICAM-1 and VCAM-1 in human umbilical cord endothelial cells (HUVECs). Viability and production of reactive oxygen species (ROS) were investigated in all cell types. We collected particles from combustion of D100 and 20% (w/w) blends of animal fat or rapeseed oil methyl esters in light-duty vehicle engines complying with Euro2 or Euro4 standards. Particles emitted from the Euro4 engine were smaller in size and more potent than particles emitted from the Euro2 engine with respect to ROS production and DNA damage, but similarly potent concerning cytokine mRNA expression. Particles emitted from combustion of biodiesel blends were larger in size, and less or equally potent than particles emitted from combustion of D100 concerning ROS production, DNA damage and mRNA of CCL2 and IL8. ICAM-1 and VCAM-1 expression in HUVECs was only increased by D100 particles from the Euro4 engine. This suggests that particle emissions from biodiesel in equal mass concentration are less toxic than conventional diesel.
’ INTRODUCTION Biodiesel is fuel based on alkyl esters of vegetable oils or animal fats. It has been shown that combustion of 20% (w/w) biodiesel blends is associated with lower emission of particulate matter (PM) compared to conventional diesel.1 Exposure to PM from traffic emissions is associated with higher risk of morbidity and mortality related to cancer and cardiovascular and pulmonary diseases.2,3 There is also compelling evidence from animal experimental models and humans that exposure to diesel exhaust particles (DEP) and ambient air particles is associated with accelerated progression of atherosclerotic plaques.4,5 The development of oxidative stress and inflammation are considered to be key events in health effects by exposure to PM.6,7 Indeed, earlier studies have shown that the reactive oxygen species (ROS) production of PM from combustion of biodiesel depends on the type of fuel and engine.810 Still, there is sparse knowledge about the toxicity of PM from combustion of biodiesel in terms of oxidatively damaged DNA and injury to endothelial cells. Moreover, one of the major achievements of engine improvements has been reduced emission of gases and PM, whereas the impact on the toxicity of combustion-derived PM is also an important issue in regard to hazard identification for such technology. The objective of this study was to investigate the effect of PM from combustion of biodiesel blends with 20% animal fat methyl ester (AFME20), 20% rapeseed methyl ester (RME20) r 2011 American Chemical Society
and conventional diesel (D100) in diesel engines complying with Euro2 and Euro4 emission standards. The Euro2 and Euro4 standards for passenger cars and light-duty commercial vehicles from 1998 and 2005 allow emission of 0.08 and 0.025 g/km, respectively. We compared the effect of PM from combustion of conventional and biodiesel blends with respect to ROS production, inflammation, DNA damage and expression of intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) as mechanistic intermediates of cancer and vascular disease.
’ MATERIALS AND METHODS Samples. We collected PM from missions of two light-duty diesel engines, complying with Euro2 or Euro4 emission standards. We refer to the engines as “Euro2” and “Euro4” and they are representatives of the older and newer part of the car fleet, respectively. The fuels were an ultra low sulfur reference fossil diesel (D100), RME20 and AFME20. We included standard reference material 2975 (SRM2975) in the assays as a benchmark Received: March 25, 2011 Accepted: August 15, 2011 Revised: August 12, 2011 Published: August 15, 2011 8545
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Environmental Science & Technology sample of diesel emission particles. We collected combustion particles from biodiesel for toxicological assays on 150 mm quartz filters (Frisenette, Denmark) at 250 ( 25 °C in a raw gas tunnel. PM were prepared for the experiments by scraping the PM off the quartz filters and suspend them by sonication in Milli-Q water at a concentration of 500 μg/mL. The aerosol number concentration was monitored and the concentration of polycyclic aromatic hydrocarbons (PAH) was measured in PM collected on separate filters. The collection, characterization and suspension of PM are described in the Supporting Information. Particle Size. The distribution of particle sizes in Milli-Q water and cell medium for human umbilical vein endothelial cells (HUVECs) was measured by nanoparticle tracking analysis (Nanosight LM20, Amesbury, Wiltshire, United Kingdom). It tracks and analyses particles in liquids from 10 to 1000 nm using Brownian motion and random migration models to calculate diffusion coefficients and subsequently StokesEinstein’s equation to determine particle size as hydrodynamic diameter. Cell Culture and Exposure. We assessed DNA damage in the human alveolar epithelial A549 cell line by the comet assay as DNA strand breaks (SB) and formamidopyrimidine DNA glycosylase (FPG) sensitive sites,11,12 expression of ICAM-1 and VCAM-1 in HUVECs,13 and inflammation adhesion potential in terms of mRNA expression of CCL2 and IL8 in the monocytic THP-1 cell line.14 Increased expression of ICAM-1 and VCAM-1 on HUVECs indicates activation of these cells, which can occur because of cytokine signaling and oxidative stress. The ROS production was assessed by oxidation of 20 ,70 -dichlorofluorescin diacetate as a potential mechanistic driving force for toxicity was assessed in all cell types.15 We assessed viability by the trypan blue method in all cell types. The cells were exposed to 0.78100 μg/mL because earlier studies on particles from wood smoke, diesel exhaust and ambient air have shown effect in this concentration range with effect on cytotoxicity.11,13,14 Statistics. The effects of different type of particles were analyzed by nested ANOVA analysis with the concentration nested in the type of particles. The statistical analysis on ROS production in THP-1 and A549 cells, DNA damage, expression of ICAM-1 and VCAM-1, and mRNA were carried out on logtransformed data because it yielded a residual variation with a normal distribution. We excluded results from the two highest concentrations in the analysis of ROS production in the statistical analysis because some type of the PM displayed bell-shaped concentrationresponse relationships. We analyzed the individual effects and interaction between the type of fuel (D100 versus AFME20) and engine (Euro2 versus Euro4) by a 2-factor ANOVA analysis on baseline corrected data. There were no statistically significant interactions and the P-values therefore correspond to single-factor effects of the type of engine and fuel. These effects are reported as mean ( standard error of the mean (SEM) or fold differences with 95% confidence intervals (CI). In all tests, the level of significance was 5%. The analyses were performed in Statistica version 5.5 for Windows, StatSoft, Inc. (1997), Tulsa, OK.
’ RESULTS Characterization of the Particles. The emitted particles from the Euro2 engine contained more PAH with combustion of D100 than with combustion of AFME20, whereas the concentration of PAH in PM from the Euro4 engine was lower than the detection
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Figure 1. Size distribution of particles in water (A) and HUVEC cell culture media (B) measured by Nanosight particle tracking analysis.
limit, 1.5 ng/m3 for benzo[a]pyrene (Table S-3, Supporting Information). The particle number concentration in aerosol in the range 10700 nm was slightly higher for the Euro4 engine as compared to the Euro2 engine (Table S-4, Supporting Information). The particle size profile of PM from the combustion of D100 in the Euro4 engine suspended in cell medium had a larger fraction of small particles than the particles from combustion of AFME20 and RME20 (Figure 1 and Table S-4, Supporting Information). The combustion of D100 and AFME20 in the engine complying with the Euro4 emission standards generated PM with a larger fraction of smaller particles (based on the distribution of particles in suspension in both cell medium and water) compared with PM from the engine that complied with the Euro2 emission standards. Viability. The exposure to PM from combustion of Euro2/ AFME20 was associated with 8.4% (95% CI: 3.9%13.0%), 15.3% (95% CI: 10.7%19.8%) and 32.8% (95% CI: 28.3% 37.4%) lower viability assessed by trypan blue exclusion in THP-1 cells at 25, 50, and 100 μg/mL, respectively. The other PM preparations were not associated with reduced viability in THP-1 cells. The unexposed HUVECs contained about 95% viable cells. The HUVECs exposed to 100 μg/mL of Euro2/ AFME20 and Euro4/AFME20 had about 10% more nonviable 8546
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Figure 2. ROS production in acellular conditions (A), THP-1 (B), A549 (C) and HUVECs (D) exposed for 3 h. The data represent the fold-difference as compared with the unexposed controls. The mean ( SD and statistics are outlined in Table S-6 of the Supporting Information.
cells than the nonexposed cultures; this difference was not statistically significant (P > 0.10). The exposure to 100 μg/mL of Euro2/AFME20 particles were also associated with an 8.1% (95% CI: 4.311.9%) reduced viability in A549 cells, whereas the exposure to the particle preparations were associated with less than 2% reduction in viable cells (Table S-5, Supporting Information). ROS Production. The data on ROS production are outlined in Figure 2. Particles generated by the Euro4 engine generally displayed bell-shaped concentrationresponse relationships that were strikingly different from those generated by the Euro2 engine and SRM2975. The PM derived from the Euro4 engine generated higher level of ROS than the particles generated by the Euro2 engine and SRM2975 in the A549, HUVEC and THP-1 cells as well as under acellular conditions (P < 0.05). The Euro4/ D100 PM generated higher levels of ROS than Euro4/AFME20 and Euro4/RME20 in THP-1 cells (P < 0.01), whereas there was no difference in the ROS production between Euro4-derived PM in the A549 cells, HUVECs and acellular conditions. The SRM2975 generated higher levels of ROS than Euro2/D100 and Euro2/AFME20 in acellular conditions (P < 0.001). DNA Damage in A549 cells. All types of PM generated DNA damage in terms of SB (P < 0.01) and FPG sensitive sites (P < 0.001) in A549 cells (Figure 3). The only significant difference when comparing the individual PM preparations was that Euro4/ D100 generated 1.23-fold (95% CI: 1.141.50) higher levels of
FPG sensitive sites than Euro4/RME20 (P < 0.05). In addition, there was no significant difference when comparing the fuel and engine types, except that the PM from the Euro4 engine was associated with higher levels of FPG sensitive sites than the PM from the Euro2 engine was. Expression of Cell Adhesion Proteins on HUVECs. The EU4/D100 preparation was the only type of PM that increased the expression of VCAM-1 (P < 0.01) and ICAM-1 (P < 0.05) compared with the control on HUVECs (Figure 4). Expression of Inflammatory Genes in THP-1 Cells. Figure 5 depicts the relationship between exposure to PM and gene expression of CCL2 and IL8 in THP-1 cells. There was no statistically significant effect of the individual PM samples on the CCL2 and IL8 expression levels in the nested ANOVA analysis. The expressions of CCL2 and IL8 were increased by 87-fold (95% CI: 31243 fold) and 31-fold (95% CI: 1757 fold) in the LPS-exposed THP-1 cells. We also tested whether or not the filter material gave rise to increased mRNA levels because it previously has been observed that PM obtained from quartz filters could generate an inflammatory response in cultured human macrophages.16 The THP-1 cells exposed to the filter material in the same concentration range as the PM did not have increased mRNA levels of CCL2 (0.43 ( 0.29, n = 4) and IL8 (2.6 ( 0.5, n = 4) per 106 18S (mean ( SEM). Analysis of the Interaction between Fuel and Engine. Figure 6 display the results from the assessment of effect of the 8547
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Figure 3. DNA strand breaks (A) and FPG sensitive sites (B) in A549 cells exposed for 3 h to particles. *P < 0.05 compared with the control. The letters represent statistically significant difference between particles as follows: (d) Euro4/D100, (e) Euro4/RME20.
type of engine (Euro2 or Euro4) and fuel (D100 or AFME20) on the biomarkers. Overall, the Euro4 engine generated higher level of the biomarkers of cell adhesion molecules, DNA damage and ROS than the Euro2 engine, while the expression of CCL2 and IL8 showed no difference with the PM from the Euro2 and Euro4 engine. The type of fuel had little effect on the generation of ROS, DNA damage and mRNA expression of inflammationrelated genes, whereas PM from D100 generated higher expression level of ICAM-1 and VCAM-1 on HUVECs than PM from AFME20 did.
’ DISCUSSION In this study PM emitted from the Euro4 engine were smaller in size and more potent than particles emitted from the Euro2 engine with respect to ROS production and DNA damage, but similarly potent concerning mRNA expression of inflammation related genes. Particles emitted from combustion of biodiesel blends were larger in size, and less or equally potent than particles emitted from combustion of D100 concerning ROS production, DNA damage and mRNA expression. ICAM-1 and VCAM-1 expression in HUVECs was only increased by D100 particles from the Euro4 engine. PM from the Euro4 engine showed an initially steep concentration-dependent increase in ROS generation with decreasing response at higher concentrations, whereas the PM derived from the Euro2 engine and SRM2975 had concentrationresponse relationship with a smaller but steadily increasing slope. The differences in intracellular ROS production between the cell types probably related to differences in cellular redox status as well as esterase activity, which is required to activate the DCFHDA probe. The fold increase in ROS signal was higher in acellular
Figure 4. Expression of VCAM-1 and ICAM-1 in HUVECs exposed for 24 h to particles. In the “control” panel, the white, gray and black columns are HUVECs that have been exposed to 0.1, 1, or 100 ng/mL of TNF. The letters indicate statistically significant differences compared with other type of particles; *P < 0.05 compared with the control. The letters represent statistically significant difference between particles as follows: (a) SRM2975, (b) Euro2/D100, (c) Euro2/AFME20, (d) Euro4/D100, (e) Euro4/RME20, and (f) Euro4/AFME20.
conditions than for the intracellular assays, which could be because the former is a nonreducing environment with more probe available for oxidation after it has been chemically deacetylated and some light emitted intracellularly might be absorbed. The results suggest that PM from the Euro4 engine can generate more ROS production because of the larger surface area indicated by the smaller size found in suspension as compared with the PM from the Euro2 engine. This is in keeping with the notion that exposure to small sized particles is associated with oxidative stress as was observed by a strong correlation between the particle size and depletion of intracellular glutathione levels.17 We have previously observed bell-shaped concentrationresponse relationships for nanosized particles such as carbon black and singlewalled carbon nanotubes.15,18 It is possible that the apparently reduced ROS production at high concentration of some type of PM is related to scavenging of ROS by the particles or agglomeration. In addition, the different ROS production potential of the particles may also depend on the chemical composition of substances absorbed on the surface of soot particles such as quinone compounds capable of redox cycling.6 The magnitude of ROS production was mainly dependent on the type of engine, whereas there was little difference between the ROS production of PM derived from combustion of D100 and AFME20 8548
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Figure 6. Effects of the type of engine (Euro4 versus Euro2) and fuel (D100 versus AFME20) on the generation of ROS, DNA damage and expression of ICAM-1, VCAM-1 and cytokines. The data are fold increase and 95% CI.
Figure 5. Gene expression of CCL2 (A) and IL8 (B) in THP-1 cells exposed for 24 h to particles or LPS (200 μg/mL for 18 h).
or RME20. A recent study on conventional diesel and 100% soybean biodiesel also showed that the type of engine was more important for acellular ROS production than the fuel.8 However, the same study showed that the ROS production in rat alveolar macrophages was lower in cells exposed to liquid suspensions of exhaust from combustion of soybean-based biodiesel in a Euro2 compliance engine compared to a conventional Euro 1 diesel engine.8 It should be noted that these and our results cannot be directly compared because of differences in fuel, engine configurations and sampling of PM emissions. Firm conclusions about the differences in ROS production between soybean and other types of biodiesels and blends require that the PM be tested in the same experiment as we have employed in our study. Still, the subtle differences in the ROS production between AFME20 and RME20 in our experiment may indicate that the type of biodiesel is a minor contributing factor for production of ROS. We observed a substantial contrast in the ROS production potential between the PM, whereas there was less contrast between the PM in regard to the expression of the cell adhesion molecules, DNA damage and inflammation potential. The DCFH-DA is a broad spectrum redox dye that detects hydroxyl radicals and other small oxidizing compounds.19 It is generally acknowledged that oxidative stress is implicated in the development of endothelial dysfunction and inflammation. The types of DNA damage that is detected by the comet assay may have been generated because of increased ROS production (e.g., FPG sensitive sites), whereas the level of DNA SB represents a mixture of oxidatively and nonoxidatively derived DNA lesions. Our results indicate that the particle-elicited ROS
production is a contributing factor to the toxicity, but DNA damage, expression of cell adhesion molecules and inflammation may occur independent of the oxidative stress reactions detected by DCFH-DA. All the particles generated DNA damage in A549 cells, whereas there was only a difference between the fuels for the Euro4 engine where the combustion of RME20 generated particles that were associated with less generation of FPG sensitive sites than the D100 particles. The type of engine had a stronger effect than the fuel on the generation of DNA damage, possibly partly due to the different ROS production. The oxidative stress cannot be explained by the level of PAH which was much lower in PM from the Euro4 than from the Euro2 engine. Similarly, carbon black of small size but with very low levels of PAH is highly capable of inducing oxidative stress and related DNA damage.20 Numerous studies have reported increased levels of oxidatively damaged DNA in cultured cells and lung tissue of animals exposed by inhalation or intratracheal instillation. In addition, several studies in humans living in areas with emissions from traffic or exposed in controlled studies have generally found increased levels of oxidatively damaged DNA bases in leukocytes or urine.21 Interestingly, we have previously observed that authentic particles collected from the air in a traffic street had the same ability to generate SB and FPG sensitive sites in cultured A549 cells as the benchmark SRM2975 particles.22 This implies that increased levels of oxidatively damaged DNA by the biodiesel-generated PM are relevant as hazard identification. The expression of ICAM-1 and VCAM-1 was significantly increased compared to the control only in HUVECs exposed to D100 generated by the Euro4 engine, although the differences between the samples were related to the type of fuel rather than the engine. Interestingly, the results from the expression of ICAM-1 and VCAM-1 suggest that PM from combustion of 20% biodiesel is less potent than conventional diesel. It has recently been reported that inhalation of emission from combustion of 50 or 100% soybean-based biodiesel or reference diesel (a blend containing 3% biodiesel) in a diesel electrical generator showed that the biodiesel promoted stronger cardiovascular effects as well as pulmonary and systemic inflammation in mice.9 Our finding that biodiesel appears less potent than conventional diesel is not necessarily at odds with this result because the fuel was notably different in terms of origin and content of biodiesel, and the types of engines are substantially different. However, 8549
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Environmental Science & Technology it should also be acknowledged that the concentrations were larger than the exposure in the animal studies. For instance we have previously shown that intratracheal instillation of 0.5 mg/kg of nanosized carbon black did not increase the expression of ICAM-1 or VCAM-1 in the brachiocephalic artery of mice.23 This corresponds to a maximal plasma concentration of 10 μg/mL, assuming a highly unrealistic complete translocation. The same type of nanosized carbon black increased the expression of ICAM-1 or VCAM-1 in HUVECs at 50 μg/mL, whereas there was unaltered expression at 1 and 10 μg/mL.13 We found no inflammatory responses in terms of mRNA expression of cytokines, which is in contrast to their ability to induce oxidative stress. At high levels oxidative stress can induce inflammatory responses.24 The inflammation response related to the PM from the Euro2 and 4 engines was also different from that of LPS which increased the expression of IL8 and CCL2 in our system. We have previously found increased mRNA expression of IL8 and CCL2 in THP-1 cells exposed to different wood smoke particles under similar conditions as in the present study.14 We have also found increased mRNA expression of IL8 in cells exposed to diesel emission particles for 224 h at concentrations up to 500 μg/mL.25 A parallel determination of cytokine protein levels could add further information, although we did not measure this because the levels can be affected by the presence of PM.26 The overall assessment of the results in our study indicates that the PM collected from the combustion of D100 by the Euro4 light-duty diesel engine was more hazardous in terms of expression of cell adhesion molecules, than PM collected from combustion of AFME20 or RME20. The Euro4 diesel engine is representative of the present day type of engine and the results thus suggest that benefits could be achieved by promoting the use of biodiesel instead of conventional diesel. It should be acknowledged that our experimental evidence stems from in vitro studies of PM that have been collected on a filter, scraped off and suspended in cell medium, which might not be representative of in vivo exposure. We believe that the differences in toxicological profiles of the conventional diesel and biofuel blends can be assessed in cultured cells exposed to suspension of particles, whereas the extrapolation of the data to real life inhalation exposures should be done with caution. In addition, cell cultures of nonpulmonary origin were used because of observations that exposure to traffic-generated PM is associated with systemic effects such as increased morbidity and mortality of cardiovascular diseases. The pathway from pulmonary exposure to PM to cardiovascular effects might be related to systemic oxidative stress and inflammation by either direct translocation of a small fraction of the inhaled particles or yet unresolved secondary mechanisms evoked by pulmonary effects. In addition, it should be emphasized that our assessment was based on equal mass concentration and the health benefit could be substantially larger if the total emission of PM is reduced by combustion of biodiesel. Similarly, breathing air from traffic emissions of Euro4 type of engine is likely to be less hazardous than Euro2-derived PM because the maximum emission is 0.025 and 0.08 g per km, respectively, even though the former generated particles of smaller size and more potency in terms of ROS production and genotoxicity than the PM collected from the latter Euro engine. Advanced after-treatment technologies especially filter in diesel engines is even more efficient than cleaner fuels in reducing PM emissions.27 Indeed, we attempted to collect PM from a similar Euro4 engine equipped with a particle filter, but this was
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so efficient that it would have required an extended period of time to collect a sufficient amount of material for the analysis in cell cultures.
’ ASSOCIATED CONTENT
bS
Supporting Information. Information about the characteristics of the fuel and engines, sampling of PM, particle size, cell culture conditions, ROS production, DNA damage, expression of ICAM-1 and VCAM-1 and mRNA expression. Ancillary information on the mean ( SEM of results on cell viability and ROS production are also reported. This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected].
’ ACKNOWLEDGMENT This work was supported by the Strategic Research Council. ’ REFERENCES (1) McCormick, R. L. The impact of biodiesel on pollutant emissions and public health. Inhalation Toxicol. 2007, 19, 1033–1039. (2) Brook, R. D.; Rajagopalan, S.; Pope, C. A., III; Brook, J. R.; Bhatnagar, A.; Diez-Roux, A. V.; Holguin, F.; Hong, Y.; Luepker, R. V.; Mittleman, M. A.; Peters, A.; Siscovick, D.; Smith, S. C., Jr.; Whitsel, L.; Kaufman, J. D. Particulate matter air pollution and cardiovascular disease: An update to the scientific statement from the American Heart Association. Circulation 2010, 121, 2331–2378. (3) Eisner, M. D.; Anthonisen, N.; Coultas, D.; Kuenzli, N.; PerezPadilla, R.; Postma, D.; Romieu, I.; Silverman, E. K.; Balmes, J. R. Committee on Nonsmoking COPD, Environmental and Occupational Health Assembly An official American Thoracic Society public policy statement: Novel risk factors and the global burden of chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 2010, 182, 693–718. (4) Araujo, J. A.; Nel, A. E. Particulate matter and atherosclerosis: role of particle size, composition and oxidative stress. Part. Fibre Toxicol. 2009, 6, 24. (5) Møller, P.; Mikkelsen, L.; Vesterdal, L. K.; Folkmann, J. K.; Forchhammer, L.; Roursgaard, M.; Danielsen, P. H.; Loft, S. Hazard identification of particulate matter on vasomotor dysfunction and progression of atherosclerosis. Crit Rev. Toxicol. 2011, 41, 339–368. (6) Risom, L.; Møller, P.; Loft, S. Oxidative stress-induced DNA damage by particulate air pollution. Mutat. Res. 2005, 592, 119–137. (7) Loft, S.; Møller, P. Oxidative DNA damage and human cancer: need for cohort studies. Antioxid. Redox Signaling 2006, 8, 1021–1031. (8) Cheung, K. L.; Polidori, A.; Ntziachristos, L.; Tzamkiozis, T.; Samaras, Z.; Cassee, F. R.; Gerlofs, M.; Sioutas, C. Chemical characteristics and oxidative potential of particulate matter emissions from gasoline, diesel, and biodiesel cars. Environ. Sci. Technol. 2009, 43, 6334–6340. (9) Brito, J. M.; Belotti, L.; Toledo, A. C.; Antonangelo, L.; Silva, F. S.; Alvim, D. S.; Andre, P. A.; Saldiva, P. H.; Rivero, D. H. Acute cardiovascular and inflammatory toxicity induced by inhalation of diesel and biodiesel exhaust particles. Toxicol. Sci. 2010, 116, 67–78. (10) Tzamkiozis, T.; Stoeger, T.; Cheung, K.; Ntziachristos, L.; Sioutas, C.; Samaras, Z. Monitoring the inflammatory potential of exhaust particles from passenger cars in mice. Inhal. Toxicol. 2010, 22 (Suppl 2), 59–69. (11) Danielsen, P. H.; Loft, S.; Kocbach, A.; Schwarze, P. E.; Møller, P. Oxidative damage to DNA and repair induced by Norwegian wood 8550
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