Diesel Exhaust Particles in the Lung Aggravate ...

TOXICOLOGICAL SCIENCES 113(1), 267–277 (2010) doi:10.1093/toxsci/kfp222 Advance Access publication October 1, 2009

Diesel Exhaust Particles in the Lung Aggravate Experimental Acute Renal Failure Abderrahim Nemmar,*,1 Suhail Al-Salam,† Shaheen Zia,* Javed Yasin,‡ Isehaq Al Husseni,§ and Badreldin H. Ali§ *Department of Physiology, †Department of Pathology, and ‡Department of Internal Medicine, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al Ain, United Arab Emirates 17666; and §Department of Pharmacology and Clinical Pharmacy, College of Medicine & Health Sciences, Sultan Qaboos University, Muscat, 123 Sultanate of Oman 1

Received May 11, 2009; accepted September 14, 2009

Inhaled particles are associated with pulmonary and extrapulmonary effects. Also, acute renal failure (ARF) is associated with increased mortality, related to pulmonary complications. Here, we tested the possible potentiating effect of diesel exhaust particles (DEP) in an animal model of ARF induced by a single ip injection of cisplatin (CP, 6 mg/kg) in rats. Six days later, the rats were intratracheally instilled with either DEP (0.5 or 1 mg/kg) or saline (control) and renal, systemic, and pulmonary variables were studied 24 h thereafter. CP increased the serum concentrations of urea and creatinine and reduced glutathione (GSH) concentration and superoxide dismutase activity in renal cortex. CP caused renal tubular necrosis; increased urine volume, protein concentrations, and N-acetyl-b-D-glucosaminidase (NAG) activity; and decreased urine osmolality. The combination of DEP and CP aggravated the CP-induced effects on serum urea and creatinine, urine NAG activity, and renal GSH. The arterial O2 saturation and PO2 were significantly decreased in CP 1 DEP versus CP 1 saline and CP 1 DEP versus DEP. The number of platelets was reduced in DEP compared to saline-treated rats and CP 1 DEP versus DEP alone or CP 1 saline. Increases in macrophage and neutrophils numbers in bronchoalveolar lavage were found in DEP versus saline group and CP 1 DEP versus CP. Histopathological changes in lungs of DEPtreated rats were aggravated by the combination of CP 1 DEP. These included marked interstitial cell infiltration and congestion. We conclude that the presence of DEP in the lung aggravated the renal, pulmonary, and systemic effects of CP-induced ARF. Key Words: air pollution; diesel exhaust particles; lung inflammation; acute renal failure.

Acute renal failure (ARF) is increasingly becoming more frequent and is associated with high costs and adverse clinical outcomes, including excess mortality, increased length of hospital stay, and the requirement for chronic dialysis in survivors (Hoste and Schurgers, 2008; Pannu et al., 2008). Several studies have reported consistent association between ARF and dysfunction of extrarenal organs, particularly the lungs (Hoke et al., 2007; Pierson, 2006;). Experimentally, ARF

resulting from either ischemia or bilateral nephrectomy has been reported to cause lung inflammation (Hoke et al., 2007). Furthermore, it has been recently demonstrated that the kidney plays an important role in the production and elimination of mediators of pulmonary injury and that prolonged exposure to these mediators contributes to pulmonary injury (Grigoryev et al., 2008; Hoke et al., 2007). Ischemia and toxicity are considered the main pathophysiological factors that lead to the development of ARF (Ali and Al Moundhri, 2006; Ali et al., 2007; Grigoryev et al., 2008; Hoke et al., 2007). A substance that is well known to induce toxic kidney injury is cisplatin (CP). CP is a potent anticancer drug that is commonly used against multiple solid human cancers, including testicular, cervical, ovarian, head, and neck malignancies. The drug is bioactivated to a nephrotoxicant and is also known to produce proximal tubular injury, which is thought to be due to a combination of direct cytotoxicity, intrarenal vasoconstriction, and oxidative stress (Ali and Al Moundhri, 2006; Ali et al., 2007, 2008). Inhaled particulate air pollution with particle diameter less than 2.5 lm contributes to respiratory and cardiovascular morbidity and mortality (Kunzli et al., 2005; Pekkanen et al., 2002; Peters et al., 2001; Pope et al., 2002). Diesel exhaust particles (DEP), which are the major contributors to PM2.5 and ultrafine particles (diameter 0.1 lm) in cities, have been identified in a number of epidemiological studies to cause adverse health effects, including cardiorespiratory diseases, particularly in individuals with preexisting disease (Atkinson et al., 2001; Pope et al., 1992). Experimental exposure to DEP causes systemic and inflammatory response in the airways and impairs the regulation of vascular tone and endogenous fibrinolysis in healthy human volunteers (Mills et al., 2005, 2007; Salvi et al., 1999). Moreover, we (Nemmar and Inuwa, 2008; Nemmar et al., 2003a,b, 2004a, 2007) and others (Inoue et al., 2005, 2006) have reported that exposure to DEP cause pulmonary inflammation and thrombotic complication in hamsters and mice.

Ó The Author 2009. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: [email protected]

Downloaded from toxsci.oxfordjournals.org at Sultan Qaboos University on January 18, 2011

To whom correspondence should be addressed at Department of Physiology, Faculty of Medicine and Health Sciences, United Arab Emirates University, PO Box 17666, Al Ain, United Arab Emirates. Fax: þ9713 7671966. E-mail: [email protected].

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MATERIALS AND METHODS Animals. Male Wistar rats (Taconic Farms Inc., Germantown, NY), aged 10–12 weeks and initially weighing 258 ± 6 g, were given a standard laboratory chow and water ad libitum. They were randomly divided into four groups and individually housed in metabolic cages to facilitate urine collection, at a temperature of 23 ± 2°C, relative humidity of 50–60%, and a 12-h darklight cycle. An acclimatization period of 4 days was allowed for the rats before any experimentation. The rats were weighed at the beginning of the experiment and just before sacrifice. Rats were cared for under a protocol approved by the Animal Research Ethics Committee of our college and according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals, NIH publication no. 85-23, 1985. Intratracheal instillation. We used DEP (SRM 2975) from the National Institute of Standards and Technology (Gaithersburg, MD). We have recently (Nemmar et al., 2007) analyzed the size of DEP used in the present study by transmission electron microscopy and found a substantial amount of ultrafine (nano)-sized particle aggregates and larger particle aggregates (< 1 lm in largest diameter). DEP were suspended in sterile normal saline (NaCl 0.9%) containing Tween 80 (0.01%). To minimize aggregation, particle suspensions were always sonicated (Clifton Ultrasonic Bath, Clifton, NJ) for 15 min and vortexed before their dilution and prior to intratracheal (i.t.) administration. Control animals received normal saline containing Tween 80 (0.01%). Treatments. The ARF in rats was induced by a single ip injection of CP (David Bull Laboratories, PTY Ltd, Victoria, Australia) at a dose of 6 mg/kg (Ali et al., 2007, 2008). Control animals received similar volume of normal saline ip. On day 6 of treatment, the animals were anesthetized with ip injection of ketamine (75 mg/kg) and xylazine (10 mg/kg) and placed supine with extended neck on an angled board. A Becton Dickinson 18 Gauge cannula (Franklin Lakes, NJ) was inserted via the mouth into the trachea. DEP suspension (0.5 or 1 mg/kg in 150 ll) or vehicle only were instilled (150 ll) via a sterile syringe and followed by an air bolus of 100 ll. The six groups were treated as follows (n ¼ 6–8 in each group): Group 1: single normal saline (control, 500 ll/rat) given ip, and on day 6 of the treatment, a single i.t. administration of saline (150 ll per rat); Group 2: single normal saline (control, 500 ll/rat) given ip, and on day 6 of the treatment, a single i.t. administration of DEP (0.5 mg/kg); Group 3: single normal saline (control, 500 ll/rat) given ip, and on day 6 of the treatment, a single i.t. administration of DEP (1 mg/kg); Group 4: single CP (6 mg/kg) given ip, and on day 6 of the treatment, a single i.t. administration of saline (150 ll per rat); Group 5: single CP (6 mg/kg) given ip, and on day 6 of the treatment, a single i.t. administration of DEP (0.5 mg/kg); and

Group 6: single CP (6 mg/kg) given ip, and on day 6 of the treatment, a single i.t. administration of DEP (1 mg/kg). On day 6, immediately after i.t. administration of saline or DEP, rats were placed in metabolic cages and urine of each rat was collected over a 24-h period and the volume measured. Blood collection and bronchoalveolar lavage. Twenty-four hour after the i.t. administration of saline or DEP, the rats were anesthetized as described above and blood was drawn from the inferior vena cava in EDTA (4%). A sample was used for hematocrit measurement and platelets and white blood cells counts using an ABX Micros 60 counter (ABX Diagnostics, Montpellier, France). The remaining blood was left at room temperature for 2 h before it was centrifuged at 900 3 g at 4°C for 15 min to separate serum. The serum obtained was stored frozen at 80°C to await biochemical analyses. The rats were then sacrificed with an overdose of ketamine. Bronchoalveolar lavage (BAL) was then performed by cannulating the trachea, the left bronchus was clamped. The bronchi and right lung were lavaged three times with 5 ml sterile 0.9% NaCl. The BAL fluid was pooled in a plastic tube on ice. No difference in the amount of recovered fluid was observed between the different groups. BAL fluid was centrifuged (1000 3 g 3 10 min, 4°C). Cell counting was performed in a hemocytometer after resuspension of the pellets and staining with 1% gentian violet. The cell differentials were performed on cytocentrifuge preparations fixed in methanol and stained with Diff Quick (Dade Behring, Marburg, Germany). The supernatant was stored at 80°C until further analysis. Biochemical analysis and histopathology. Lungs and kidneys were excised, washed with ice-cold saline, blotted with filter paper, and weighed. Small pieces from the left lung and left kidney were fixed in 10% neutral formalin, dehydrated in increasing concentrations of ethanol, cleared with xylene, and embedded in paraffin. The cortex of the right kidney was excised from the medulla and rapidly homogenized in ice-cold saline to produce 10 (wt/vol) tissue homogenate. The concentrations of urea and creatinine in serum were spectrophotometrically measured using commercial kits (BioMerieux, Marcy-l’Etoile, France). Urine osmolality was measured by the freezing point depression method (70°C) using an osmometer (Roebling, Berlin, Germany) and N-acetyl-b-Dglucosaminidase (NAG) activity by kits from Diazyme, General Atomics, San Diego, CA. Urine protein concentration was measured spectrophotometrically using a kit from BioMerieux. In renal cortex homogenates, glutathione (GSH) concentration and superoxide dismutase (SOD) activity were measured spectrophotometrically (Randox, Antrim, UK). The concentration of CP (as platinum) in cortical tissue was measured by flameless atomic absorption spectrophotometry (Perkin-Elmer, Vernon Hills, IL; 3300 DV ICP-OES equipped with a cross-flow nebulizer, in addition to an ultrasonic nebulizer). The procedure involved mineralization of the kidney cortex tissue with a mixture of concentrated HNO3 and H2O2, followed by determination of platinum in the extract, using inductively coupled plasma optical emission spectrometry, at an emission wavelength of 265.945 nm. Five-micrometer sections were prepared from left lung and left kidney paraffin blocks and stained with hematoxylin and eosin. Staining for apoptosis in the kidney sections has been performed using signal stain cleaved caspase-3 Immunohistochemical detection Kit (Cell Signaling Technology, Boston, MA). This kit was used to detect the activation of caspase using avidin-biotin immunoperoxidase method to detect intracellular caspase-3 protein. Staining was performed on 5-lm paraffin sections from left kidney by standard technique using rabbit anti-cleaved caspase-3 (clone Asp175, 1:50) (Vielhauer et al., 2005). A known positive control sections for apoptosis were used. For negative control, primary antibody was replaced with normal rabbit serum. Blood gas measurements. Arterial blood gases were measured in separate animals following the protocol described above. Immediately after the anesthesia, arterial blood was obtained via cardiac puncture in EDTA. Analysis was performed immediately after collection with a Roche blood gas analyzer (Mannheim, Germany).


Interest in the nonpulmonary targets of particulate air pollutants has been increasing since the demonstration that inhaled ultrafine particles are able to translocate directly from the lungs to extrapulmonary tissues and cause the release of soluble inflammatory mediators into the systemic circulation, which affect other organs, such as the liver, the heart, and even the brain (Nemmar et al., 2004b; Oberdorster et al., 2005; Peters et al., 2006; Vermylen et al., 2005). However, as far as we are aware, the effect of particulate air pollution on ARF has not been yet investigated. Therefore, the aim of this study was to investigate, in vivo, the possible aggravating effect of pulmonary exposure to DEP in an animal model of ARF induced by CP, by measuring some commonly used renal, systemic, and pulmonary variables.

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Statistics. All data were analyzed with GraphPad Prism Version 4.01 for Windows software (Graphpad Software Inc., San Diego, CA). Data were analyzed for normal distribution using the D’Agostino and Pearson omnibus normality test. Data are expressed as means ± SD. Comparisons between groups were performed by one-way ANOVA, followed by Newman-Keuls test for comparing treated with control data. The p values of 0.05 are considered significant.

RESULTS

Effect of CP and DEP on Renal Variables

FIG. 1. Serum urea (A) and creatinine (B) concentrations in Wistar rats treated with saline (control), DEP 0.5 mg/kg, DEP 1 mg/kg, CP þ saline, CP þ DEP 0.5 mg/kg, or CP þ DEP 1 mg/kg (n ¼ 6–8). Mean ± SD. Statistical analysis by Newman-Keuls test.

Figure 3 depicts the effect of treatment with saline and CP with or without DEP on urinalyses. Compared with saline- or DEP-treated animals, CP þ saline and CP þ DEP treatments increased the 24-h urine volume (Fig. 3A). Urinary NAG activity was significantly increased in CP þ saline compared with saline-treated controls (p < 0.001) and in rats treated with CP þ DEP compared to the DEP-treated group (p < 0.001). Interestingly, the combination of CP þ DEP 1 mg/kg significantly enhanced (p < 0.01) the NAG activity compared to CP þ saline (Fig. 3B). Urine protein concentrations were not affected by saline or DEP alone. However, CP þ saline and CP þ DEP treatments significantly increased the urine protein concentration to a similar level when compared to saline- and DEP-treated groups, respectively (Fig. 3C). CP þ saline and CP þ DEP treatments decreased urine osmolality when compared to saline- and DEP-treated groups (p < 0.001), respectively (Fig. 3D). No significant difference in urine osmolality was observed between rats treated with CP þ saline and CP þ DEP. Figure 4 shows representative micrographs of renal cortex from the six groups used. The kidney architecture was not


To induce ARF, rats were treated with a single CP injection at a dose of 6 mg/kg and this resulted in ARF, similar to previously reported studies (Ali et al., 2007; Mohan et al., 2006). In the present work, rats given saline gained about 2.6% (p < 0.05), while those receiving DEP 0.5 or 1 mg/kg gained about 6 and 0.7% (p: not significant) of their initial body weight. However, rats treated concomitantly with CP and saline lost about 6.4% (p < 0.05), whereas those administered with CP and DEP 0.5 and 1 mg/kg lost more weight, i.e., 8.6 and 9.3% (p < 0.05), respectively. The kidney weights were slightly but not significantly increased in DEP 0.5 mg/kg-exposed (7.1 ± 0.3 g/kg body weight) and DEP 1 mg/kg rats (7.2 ± 0.4 g/kg body weight) compared to saline-treated (6.6 ± 0.2 g/kg) rats. However, CP þ saline treatment significantly increased kidney weight (9.6 ± 0.9 g/kg, p < 0.01) compared to the saline group. Rats treated with CP þ DEP 0.5 mg/kg significantly increased kidney weight (8.7 ± 0.7 g/kg, p < 0.05) compared to DEP 0.5 mg/kg alone. Similarly, the combination of CP þ DEP 1 mg/kg treatment increased the weight of kidneys (8.8 ± 0.5 g/kg, p ¼ 0.05) compared to the DEP 1 mg/kg group. No statistical difference has been observed between CP þ saline and CP þ DEP. Figure 1 illustrates the effects of saline and CP with or without DEP, on the concentrations of creatinine and urea in serum. DEP did not significantly affect the concentrations of urea or creatinine compared to the saline-treated group. However, CP þ saline significantly increased the concentration of urea and creatinine compared to the saline-treated rats. Interestingly, CP þ DEP 0.5 and 1 mg/kg treatment significantly and consistently increased the concentration of urea and creatinine in serum more than in rats treated with DEP 0.5 or 1 mg/kg alone or CP þ saline. DEP 1 mg/kg alone caused a significant decrease of renal GSH compared to the saline group. On the other hand, CP þ saline decreased the level of renal GSH compared to the salinetreated rats. A more pronounced effect was seen in the CP þ DEP 1 mg/kg group in which the concentrations of renal GSH was significantly reduced compared to both DEP 1 mg/kg and saline þ CP-treated groups (Fig. 2A). However, the renal SOD concentration was only slightly and insignificantly decreased after DEP 1 mg/kg exposure. Treatment with both CP þ saline and CP þ DEP 1 mg/kg reduced the activity of renal SOD compared to values from rats treated with saline and DEP, respectively (Fig. 2B).

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platinum has been found in the kidneys of rats treated with saline, DEP 0.5 mg/kg, or DEP 1 mg/kg. Effect of CP and DEP on Systemic Variables

affected by saline (Fig. 4A), DEP 0.5 mg/kg (Fig. 4B), or DEP 1 mg/kg (Fig. 4C) treatments. However, in CP þ saline (Fig. 4D), CP þ DEP 0.5 mg/kg (Fig. 4E), and CP þ DEP 1 mg/kg (Fig. 4F), the renal cortex showed the presence of acute tubular necrosis with the presence apoptotic cells, tubular distention with eosinophilic material, interstitial edema, and congestion. No accumulation of DEP in the different kidney sections has been observed in animal exposed to DEP. The staining for the detection of apoptotic cells showed no evidence of apoptotic cells in the kidneys of saline or DEP groups (Figs. 5A–C). Apoptotic cells were seen in the kidneys of CP þ saline (Fig. 5D), CP þ DEP 0.5 mg/kg (Fig. 5E), and CP þ DEP 1 mg/kg (Fig. 5F). However, there was no significant difference in the number of apoptotic cells between these three groups. The concentration of platinum in the renal cortex of rats given CP þ saline (3.95 ± 0.17 ppm) was not significantly different from that in rats treated with CP þ DEP 0.5 mg/kg (4.07 ± 0.3 ppm) or CP þ DEP 1 mg/kg (4.08 ± 0.09 ppm). No

Effect of CP and DEP on Pulmonary Variables Depending on the treatment performed, the cells found in BAL were primarily macrophages and polymorphonuclear leukocytes (PMN) (Fig. 8) and no lymphocytes or other cells were observed microscopically. The pulmonary administration of DEP resulted in a marked influx of macrophage and PMN in the lung compared to saline-treated rats. Similarly, CP þ DEP significantly and dose-dependently increased the numbers of macrophage (0.5 mg/kg, p < 0.05 and 1 mg/kg, p < 0.005) and PMN (0.5 mg/kg, p < 0.05 and 1 mg/kg, p < 0.01) compared to CP þ saline group. Although the numbers of macrophage and PMN in CP þ DEP group were higher compared to DEPtreated rats, this difference did not reach statistical significance.


FIG. 2. Reduced GSH (A) and SOD (B) activity in renal cortex, in Wistar rats treated with saline (control), DEP 1 mg/kg, CP þ saline, or CP þ DEP 1 mg/kg (n ¼ 6). Mean ± SD. Statistical analysis by Newman-Keuls test.

The exposure of rats to DEP 1 mg/kg slightly but insignificantly decreased the PaO2 (Fig. 6A). Pretreatment of rats with CP þ DEP 0.5 mg/kg significantly decreased PaO2 compared to DEP 0.5 mg/kg alone (Fig. 6A). Interestingly, we found that treatment with CP þ DEP 1 mg/kg significantly decreased the PaO2 compared to treatment with CP þ saline and DEP 1 mg/kg alone (Fig. 6A). Nevertheless, no significant effect on PaCO2 has been observed between saline (39.5 ± 2.8), DEP 0.5 mg/kg (39.0 ± 4), DEP 1 mg/kg (41.2 ± 1.9), CP þ saline (41.4 ± 1.5), CP þ DEP 0.5 mg/kg (41.0 ± 2.3), or DEP þ CP (41.2 ± 0.9). Figure 6B illustrates that rats treated with DEP 1 mg/kg slightly but insignificantly decreased the arterial O2 saturation (SaO2) (Fig. 6B). However, we found that treatment with CP þ DEP 1 mg/kg significantly decreased the SaO2 compared to treatment with CP þ saline or to DEP 1 mg/kg alone (Fig. 6B). Similarly, the hematocrit in group treated with CP þ DEP 1 mg/kg slightly but significantly increased compared to treatment with CP þ saline or to DEP 1 mg/kg alone (Fig. 6C) Figure 7 depicts the effect of treatment with saline and CP with or without DEP on the numbers of leukocytes and platelets in whole blood. Although the level of significance was only reached in group treated with CP þ DEP 1 mg/kg (p < 0.05), a dose-dependent increase in leukocytes numbers was observed after exposure of rats to CP þ DEP 0.5 and 1 mg/kg compared to CP þ saline (Fig. 7A). The numbers of platelets was affected by the different treatments (Fig. 7B). Both doses of DEP caused a significant decrease of platelet number compared to the saline group. On the other hand, treatment with CP þ saline decreased the platelet number compared to saline-treated rats. An aggravating effect was seen in CP þ DEP 1 mg/kg group in which the number of platelets was significantly reduced compared to both DEP 1 mg/kg and CP þ saline-treated groups. The different treatments had no significant effect on the number of red blood cells and hemoglobin concentrations (data not shown).


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Figure 9 shows representative histological micrographs of lungs from the six groups studied. The histopathology findings described below were uniform within the same lung tissue and from rat to rat. Sections from the saline-treated rats showed normal lung architecture (Fig. 9A). In DEP-treated lungs (0.5 and 1 mg/kg), particles engulfed by macrophages in the interalveolar interstitium associated with increased interstitial cellularity and widening of the interalveolar spaces have been observed (Figs. 9B and 9C). CP þ saline caused an increase in cellularity of interalveolar interstitium and mild congestion (Fig. 9D). Importantly, these effects were aggravated by the concomitant administration of CP þ DEP, which showed the presence of aggregates of DEP in the interalveolar interstitium and marked interstitial cellular expansion (Figs. 9E and 9F). Moreover, in CP þ DEP 1 mg/kg, congestion and severe interstitial and intra-alveolar edema were observed (Fig. 9F). DISCUSSION

In this study, we provide the first experimental evidence that DEP deposited in the lungs can aggravate experimental ARF.

Our study shows that a number of renal, systemic, and pulmonary end points were potentiated by the concomitant administration of CP and DEP. ARF remains a common and critical clinical entity affecting 5– 7% of all hospitalized patients. It carries a significant morbidity and a 20–70% mortality rate (Singri et al., 2003). The number of people affected by air pollution is extremely large, and as a consequence, estimates of the public health burden of ambient air pollution are substantial. A tri-national European study attributed some 6% of all deaths and 2% of hospital admissions to ambient air pollution (Kunzli et al., 2000). Lung and kidney function are intimately related in both health and disease. Several studies have reported consistent association between ARF and pulmonary dysfunction (Hoke et al., 2007; Pierson, 2006). Indeed, it is well established that lung injury can aggravate ARF and vice versa (Pierson, 2006). When ARF and acute lung injury are combined, the mortality rate exceeds 80% (Chien et al., 2004). Experimentally, ARF has been reported to cause lung inflammation (Grigoryev et al., 2008; Hoke et al., 2007). Recently, Brook (2008) pointed out in an editorial that exposure to particulate air pollution play some role in variety of


FIG. 3. Urine volume (A), N-acetyl-b-D-glucosaminidase (NAG; B), proteins (C), and osmolality (D) in Wistar rats treated with saline (control), DEP 0.5 mg/kg, DEP 1 mg/kg, CP þ saline, CP þ DEP 0.5 mg/kg, or CP þ DEP 1 mg/kg (n ¼ 6–8). Mean ± SD. Statistical analysis by Newman-Keuls test.

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human diseases, including cardiovascular, neurological, and renal. Particulate air pollution influences susceptibility to hard events and may be particularly harmful to high-risk groups such as people with diabetes, elderly people, and people with hypertension (Brook et al., 2004). All of which are known to be associated with a high risk of developing complications, such as ARF (Leblanc et al., 2005). However, there is little information regarding renal effects of particulate air pollution, although one study (Hendryx, 2009) reported a higher kidney, heart, respiratory, disease, and mortality in coal mining areas due to environmental exposure to particulate matter and toxic agents. While the interest in extrapulmonary effects of particulate air pollution and animal model of enhanced susceptibility is increasing, the possible aggravating effect of particulate air pollution on animal model of ARF has not been reported so far. Thus, in the present study, we tested the possibility of aggravating effect of DEP on animal model of CP-induced renal failure. Despite the notion that the i.t. instillation of a bolus of particles as a model of exposure to particulate air pollution may not be an ideal model, this method of delivery has been shown to be a reliable, convenient, and valid mode of administration of foreign compounds into the airways as it permits the accurate introduction of a range of doses to the lungs within

FIG. 5. Immunohistochemical analysis of the renal tissue sections. Staining for the detection of apoptotic cells showed no evidence of apoptotic cells in the kidneys of saline (control, A), DEP 0.5 mg/kg (B), and DEP 1 mg/ kg (C) groups. Apoptotic cells with dark brown granular cytoplasmic staining for caspase-3, streptavidin-biotin complex immunohistochemistry were only seen in the kidneys of CP þ saline (D), CP þ DEP 0.5 mg/kg (E), and CP þ DEP 1 mg/kg (F) where acute tubular necrosis with apoptotic cell (arrows) and tubular distention with necrotic material (arrow heads) have been observed.

a short time (Driscoll et al., 2000). Nevertheless, additional studies using inhalation exposure are needed to verify our findings. The dose of particles we used in our study (0.5 and 1 mg/kg) are comparable to the doses of DEP we previously used in hamster (Nemmar et al., 2003a,b, 2004a) and lower than the 5 mg/kg used in rats by others (Castranova et al., 2001; Yokota et al., 2005). To induce ARF, rats were given CP, which is a potent anticancer drug that is commonly used to treat multiple solid human cancers (Zhang et al., 2006). In the present study, reduction in body weight following CP treatment may be due to injury to the renal tubules and the subsequent loss of the tubular cells to reabsorb water, resulting in dehydration and thus loss of body weight. The further loss of body weight following pulmonary exposure to DEP is a reflection of the aggravating effects of DEP on CP-induced ARF. The deterioration of kidney function when rats were exposed to both DEP and CP is supported by the results of the kidney function tests. Serum creatinine and urea concentrations were significantly higher in rats treated with CP þ DEP than those treated with CP þ saline. Compared to CP or DEP alone, the treatment of animals with CP þ DEP or CP þ saline increased the urine volume and


FIG. 4. Representative light microscopy sections of renal tissue of rats given saline (control, A), DEP 0.5 mg/kg (B), DEP 1 mg/kg (C), CP þ saline (D), CP þ DEP 0.5 mg/kg (E), and CP þ DEP 1 mg/kg (F). The micrograph showing acute tubular necrosis with apoptotic cells (arrows), tubular distention with eosinophilic material (arrow heads), and interstitial edema and congestion.


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FIG. 6. Arterial blood PO2 (A), SaO2 (B), and hematocrit (C) in Wistar rats treated with saline (control), DEP 0.5 mg/kg, DEP 1 mg/kg, CP þ saline, CP þ DEP 0.5 mg/kg, or CP þ DEP 1 mg/kg (n ¼ 6–8). Statistical analysis by Newman-Keuls test.

protein and decreased its osmolality. However, no aggravating effect was observed in rats given CP þ DEP when compared to those that have been given CP þ saline. Nevertheless, the increase in activity of NAG was significantly potentiated in rats treated with CP þ DEP 1 mg/kg when compared to those given CP þ saline. Increased enzyme activity in the urine is generally regarded as a reliable indicator of renal tubular dysfunction (Bosomworth et al., 1999). The lysosomal enzyme NAG is one of the most important marker of tubular damage most commonly used, primarily because NAG assays are sensitive enough to allow dilution of the urine, thus overcoming any enzyme inhibition (Bosomworth et al., 1999). Moreover, we found that DEP caused slight but insignificant hypoxemia. We measured arterial blood gas in anesthetized animals; therefore, a possible effect of anesthesia on arterial PO2 cannot be excluded. However, the comparison between


FIG. 7. Numbers of white blood cells (WBC; A) and platelets (B) in whole blood in Wistar rats treated with saline (control), DEP 0.5 mg/kg, DEP 1 mg/kg, CP þ saline, CP þ DEP 0.5 mg/kg, or CP þ DEP 1 mg/kg (n ¼ 6–8). Mean ± SD. Statistical analysis by Newman-Keuls test.

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FIG. 8. Macrophages (A) and PMN (B) numbers in BAL fluid in Wistar rats treated with saline (control), DEP 0.5 mg/kg, DEP 1 mg/kg, CP þ saline, CP þ DEP 0.5 mg/kg, or CP þ DEP 1 mg/kg (n ¼ 6–8). Mean ± SD. Statistical analysis by Newman-Keuls test.

the different groups, which were anesthetesized the same way, showed statistically significant differences. We found that CP þ DEP 1 mg/kg significantly decreased the PaO2 and SaO2 compared to CP þ saline or DEP 1 mg/kg alone. It has been suggested that pollution may results in hypoxemia and that these effects might be most relevant in older and sicker individuals (DeMeo et al., 2004; Pope et al., 1999). These effects are compatible with histopathological findings observed in the lungs where marked interstitial and intra-alveolar edema was observed (Fig. 9F). This effect could explain the decrease of PaO2 and SaO2 by the impairment of O2 transport through the alveolar capillary barrier. Moreover, we found slight but significant increase in hematocrit in CP þ DEP (1 mg/kg) versus DEP 1 mg/kg alone and CP þ saline. An increase of hematocrit levels following exposure to particulate matter has been reported in men (Riediker et al., 2004) and rats (2 mg/kg but not at lower dose) (Rivero et al., 2005). Additional studies are needed to establish the effect of a decrease in PaO2 in the lung on the level of gas exchange in the tissue. The PaCO2 did

not significantly change in different groups probably because to its easier diffusion as compared to O2. The kidneys are the major site for CP accumulation, and this results in necrosis of the terminal portion of the proximal renal tubules and apoptosis in the distal nephron (Ali et al., 2007). The concentrations of CP in the renal cortex in rats treated with CP þ DEP were similar to those treated with CP þ saline. This observation could explain the absence of further necrosis of the renal tissues seen in the slides of kidneys from rats treated with CP þ DEP compared to CP þ saline. Our data confirm that CP decreases renal GSH concentration and SOD activity, leaving the renal tissues vulnerable to damage by oxygen free radicals that are responsible for the induction of tubular epithelial cell death. The SOD activity was not affected by DEP administration, and no potentiating effect was observed in CP þ DEP compared to CP þ saline. Interestingly, DEP alone significantly reduced the GSH levels, and this effect was enhanced by the combination of CP þ DEP. This may suggest that SOD was a less sensitive marker for the generation of free radicals than GSH (Ajith et al., 2007). Several studies have reported that GSH has a protective effect against CP nephrotoxicity, and recently, CP has been shown to be metabolized to


FIG. 9. Representative light microscopy sections of lung tissues of rats given saline (control, A), DEP 0.5 mg/kg (B), DEP 1 mg/kg (C), CP þ saline (D), CP þ DEP 0.5 mg/kg (E), and CP þ DEP 1 mg/kg (F). The micrograph shows DEP engulfed by macrophages or free in the interalveolar interstitium (thick arrows) and the presence of marked interstitial cellular infiltration (thin arrows) and intra-alveolar edema (arrow heads).

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inflammatory cells in BAL after pulmonary exposure to DEP (0.05–5 mg/kg) (Nemmar et al., 2003a,b, 2004a). Moreover, Rao et al. (2005) reported in rats a dose-dependent increase in PMN in BAL 1, 7, and 30 days following the i.t. instillation of three concentrations of DEP (5, 35, and 50 mg/kg body weight). The histologic findings revealed the presence of increased interstitial cellularity and widening of the interalveolar spaces. These findings are in agreement with previous studies, which reported the direct effect of DEP on lung inflammation (Nemmar et al., 2004a). CP pretreatment did not affect the number of BAL macrophages and PMN, but histologic examination showed an increase in cellularity of the interalveolar interstitium and mild congestion. A major finding of the present work is that the combined administration of DEP and CP aggravated pulmonary inflammation and caused interstitial congestion and severe interstitial and intra-alveolar edema (observed at 1 mg/kg). These results confirm earlier studies that showed that ARF resulting from either ischemia or bilateral nephrectomy causes lung inflammation (Grigoryev et al., 2008; Hoke et al., 2007) and thus suggest that inhaled particulate air pollution can potentiate ARF. DEP consists of an elemental carbonaceous core onto which various organic compounds are adsorbed. Consequently, additional studies are needed to establish that constituents of DEP are responsible for the observed effects and the potential effect of cytochrome P450 activity in lung, liver, and kidney on circulating levels of chemical constituents associated with particles on kidney toxicity (Pratibha et al., 2006). Our data provide novel evidence that pulmonary deposition of DEP potentiates the renal, systemic, and pulmonary effects of CP-induced ARF and highlight the importance of environmental factors such as particulate air pollution in aggravating ARF. Our findings provide a plausible explanation for both the extrarenal effect of ARF and the extrapulmonary effects of particulate air pollution.

FUNDING

Faculty of Medicine and Health Sciences grant (NP/09/04); United Arab Emirates individual grant (02-05-8-11/09).

ACKNOWLEDGMENTS

We thank Mr M. H. Mansour (College of Agriculture, Sultan Qaboos University), Ms M. Sudhadevi (Department of Pathology, Faculty of Medicine and Health Sciences, UAE University), and Mr S. Dhanasekaran (Department of Physiology, Faculty of Medicine and Health Sciences, UAE University) for their technical assistances. We are grateful to Professor David Cook, Ontario, Canada, for his critical reading of the manuscript.


a nephrotoxicant through a GSH-conjugate intermediate (Hanigan and Devarajan, 2003; Zhang et al., 2006). Oxidative stress in kidney plays an important role in CP-induced renal damage, and several antioxidants and thiol compounds have been shown to protect against CP nephrotoxicity (Ali and Al Moundhri, 2006; Ali et al., 2007). Studies in humans and in animal models have demonstrated that ARF has a significant effect on the function of extrarenal organs (Grigoryev et al., 2008; Hoke et al., 2007). Inflammation is a major component of the initiation and exacerbation of kidney injury, and local inflammation of kidney tissues could be a source of the development of inflammation and injury in extrarenal organs (Grigoryev et al., 2008). Therefore, in the present study, we sought to count the number of circulatory cells, as a marker of systemic inflammation. Although not statistically significant, we found an increase of leukocyte numbers in DEP-treated group (1 mg/ kg) compared to saline group. We made a similar observation in mice exposed to DEP (Nemmar et al., 2009). The fact that CP þ DEP dose-dependently and significantly (at 1 mg/kg) increased leukocyte numbers compared to CP þ saline suggests that ARF exacerbates systemic inflammation. This finding is in agreement with the concept that particulate air pollution effects are aggravated in people with preexisting diseases (Brook et al., 2004). We have shown that the systemic administration of DEP increases the number of leukocytes and decreases both the number of erythrocytes and the hemoglobin concentration (Nemmar and Inuwa, 2008). In contrast, DEP alone caused a significant decrease of platelet number compared to the saline-treated group. Moreover, a more pronounced reduction in platelet number was seen in the CP þ DEP group, suggesting the occurrence of platelet aggregation in vivo. These results are in agreement with animal and human studies, which have reported a decrease in platelet number following exposure to particulate air pollution (Nemmar et al., 2008; Ruckerl et al., 2007). Moreover, we have recently demonstrated a prothrombotic effect of DEP in hamsters as well as an activation of platelets (Nemmar et al., 2003a,b, 2004a). It remains to be established whether these effects are aggravated by direct effect of DEP that have presumably translocated into blood and/or through pulmonary release of proinflammatory cytokines, such as interleukin (IL)6, IL-8, or tumor necrosis factor-a. The fact that DEP could not be found in kidney sections does not totally exclude the direct effects of DEP on the kidneys. Recently, L’azou et al. (2008) reported that carbon black nanoparticles exert a cytotoxic effect on renal cells in vitro and suggested involvement of particle internalization as well as activation of intracellular mechanisms that might include generation of reactive oxygen species. Our study showed that exposure to DEP caused lung inflammation characterized by an increase in the number of macrophage and PMN in BAL. This finding corroborate with our previous studies in hamsters, which showed influx of

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REFERENCES Ajith, T. A., Nivitha, V., and Usha, S. (2007). Zingiber officinale Roscoe alone and in combination with alpha-tocopherol protect the kidney against cisplatin-induced acute renal failure. Food Chem. Toxicol. 45, 921–927. Ali, B. H., and Al Moundhri, M. S. (2006). Agents ameliorating or augmenting the nephrotoxicity of cisplatin and other platinum compounds: a review of some recent research. Food Chem. Toxicol. 44, 1173–1183. Ali, B. H., Al Moundhri, M., Eldin, M. T., Nemmar, A., Al Siyabi, S., and Annamalai, K. (2008). Amelioration of cisplatin-induced nephrotoxicity in rats by tetramethylpyrazine, a major constituent of the Chinese herb Ligusticum wallichi. Exp. Biol. Med. (Maywood) 233, 891–896.

Atkinson, R. W., Anderson, H. R., Sunyer, J., Ayres, J., Baccini, M., Vonk, J. M., Boumghar, A., Forastiere, F., Forsberg, B., Touloumi, G., et al. (2001). Acute effects of particulate air pollution on respiratory admissions: results from APHEA 2 project. Air pollution and health: a European approach. Am. J. Respir. Crit. Care Med. 164, 1860–1866. Bosomworth, M. P., Aparicio, S. R., and Hay, A. W. (1999). Urine N-acetylbeta-D-glucosaminidase—a marker of tubular damage? Nephrol. Dial. Transplant. 14, 620–626. Brook, R. D. (2008). Potential health risks of air pollution beyond triggering acute cardiopulmonary events. JAMA 299, 2194–2196. Brook, R. D., Franklin, B., Cascio, W., Hong, Y. L., Howard, G., Lipsett, M., Luepker, R., Mittleman, M., Samet, J., Smith, S. C., et al. (2004). Air pollution and cardiovascular disease—a statement for healthcare professionals from the expert panel on population and prevention science of the American Heart Association. Circulation 109, 2655–2671. Castranova, V., Ma, J. Y., Yang, H. M., Antonini, J. M., Butterworth, L., Barger, M. W., Roberts, J., and Ma, J. K. (2001). Effect of exposure to diesel exhaust particles on the susceptibility of the lung to infection. Environ. Health Perspect. 109(Suppl. 4), 609–612. Chien, C. C., King, L. S., and Rabb, H. (2004). Mechanisms underlying combined acute renal failure and acute lung injury in the intensive care unit. Contrib. Nephrol. 144, 53–62. DeMeo, D. L., Zanobetti, A., Litonjua, A. A., Coull, B. A., Schwartz, J., and Gold, D. R. (2004). Ambient air pollution and oxygen saturation. Am. J. Respir. Crit. Care Med. 170, 383–387. Driscoll, K. E., Costa, D. L., Hatch, G., Henderson, R., Oberdorster, G., Salem, H., and Schlesinger, R. B. (2000). Intratracheal instillation as an exposure technique for the evaluation of respiratory tract toxicity: uses and limitations. Toxicol. Sci. 55, 24–35. Grigoryev, D. N., Liu, M., Hassoun, H. T., Cheadle, C., Barnes, K. C., and Rabb, H. (2008). The local and systemic inflammatory transcriptome after acute kidney injury. J. Am. Soc. Nephrol. 19, 547–558. Hanigan, M. H., and Devarajan, P. (2003). Cisplatin nephrotoxicity: molecular mechanisms. Cancer Ther. 1, 47–61. Hendryx, M. (2009). Mortality from heart, respiratory, and kidney disease in coal mining areas of Appalachia. Int. Arch. Occup. Environ. Health 82, 243–249. Hoke, T. S., Douglas, I. S., Klein, C. L., He, Z., Fang, W., Thurman, J. M., Tao, Y., Dursun, B., Voelkel, N. F., Edelstein, C. L., et al. (2007). Acute renal failure after bilateral nephrectomy is associated with cytokine-mediated pulmonary injury. J. Am. Soc. Nephrol. 18, 155–164. Hoste, E. A., and Schurgers, M. (2008). Epidemiology of acute kidney injury: how big is the problem? Crit. Care Med. 36, S146–S151. Inoue, K., Takano, H., Sakurai, M., Oda, T., Tamura, H., Yanagisawa, R., Shimada, A., and Yoshikawa, T. (2006). Pulmonary exposure to diesel

Inoue, K., Takano, H., Yanagisawa, R., Ichinose, T., Shimada, A., and Yoshikawa, T. (2005). Pulmonary exposure to diesel exhaust particles induces airway inflammation and cytokine expression in NC/Nga mice. Arch. Toxicol. 79, 595–599. Kunzli, N., Jerrett, M., Mack, W. J., Beckerman, B., LaBree, L., Gilliland, F., Thomas, D., Peters, J., and Hodis, H. N. (2005). Ambient air pollution and atherosclerosis in Los Angeles. Environ. Health Perspect. 113, 201–206. Kunzli, N., Kaiser, R., Medina, S., Studnicka, M., Chanel, O., Filliger, P., Herry, M., Horak, F., Jr.., Puybonnieux-Texier, V., Quenel, P., et al. (2000). Public-health impact of outdoor and traffic-related air pollution: a European assessment. Lancet 356, 795–801. L’azou, B., Jorly, J., On, D., Sellier, E., Moisan, F., Fleury-Feith, J., Cambar, J., Brochard, P., and Ohayon-Courte`s, C. (2008). In vitro effects of nanoparticles on renal cells. Part. Fibre. Toxicol. 5, 22. Leblanc, M., Kellum, J. A., Gibney, R. T., Lieberthal, W., Tumlin, J., and Mehta, R. (2005). Risk factors for acute renal failure: inherent and modifiable risks. Curr. Opin. Crit. Care 11, 533–536. Mills, N. L., Tornqvist, H., Gonzalez, M. C., Vink, E., Robinson, S. D., Soderberg, S., Boon, N. A., Donaldson, K., Sandstrom, T., Blomberg, A., et al. (2007). Ischemic and thrombotic effects of dilute diesel-exhaust inhalation in men with coronary heart disease. N. Engl. J. Med. 357, 1075–1082. Mills, N. L., Tornqvist, H., Robinson, S. D., Gonzalez, M., Darnley, K., MacNee, W., Boon, N. A., Donaldson, K., Blomberg, A., Sandstrom, T., et al. (2005). Diesel exhaust inhalation causes vascular dysfunction and impaired endogenous fibrinolysis. Circulation 112, 3930–3936. Mohan, I. K., Khan, M., Shobha, J. C., Naidu, M. U., Prayag, A., Kuppusamy, P., and Kutala, V. K. (2006). Protection against cisplatininduced nephrotoxicity by Spirulina in rats. Cancer Chemother. Pharmacol. 58, 802–808. Nemmar, A., Al Maskari, S., Ali, B. H., and Al Amri, I. S. (2007). Cardiovascular and lung inflammatory effects induced by systemically administered diesel exhaust particles in rats. Am. J. Physiol. Lung Cell. Mol. Physiol. 292, L664–L670. Nemmar, A., Al Salam, S., Dhanasekaran, S., Sudhadevi, M., and Ali, B. H. (2009). Pulmonary exposure to diesel exhaust particles promotes cerebral microvessel thrombosis: protective effect of a cysteine prodrug l-2oxothiazolidine-4-carboxylic acid. Toxicology 263, 84–92. Nemmar, A., Hoet, P. H., Dinsdale, D., Vermylen, J., Hoylaerts, M. F., and Nemery, B. (2003a). Diesel exhaust particles in lung acutely enhance experimental peripheral thrombosis. Circulation 107, 1202–1208. Nemmar, A., Hoet, P. H. M., Vermylen, J., Nemery, B., and Hoylaerts, M. F. (2004a). Pharmacological stabilization of mast cells abrogates late thrombotic events induced by diesel exhaust particles in hamsters. Circulation 110, 1670–1677. Nemmar, A., Hoylaerts, M. F., Hoet, P. H., and Nemery, B. (2004b). Possible mechanisms of the cardiovascular effects of inhaled particles: systemic translocation and prothrombotic effects. Toxicol. Lett. 149, 243–253. Nemmar, A., and Inuwa, I. M. (2008). Diesel exhaust particles in blood trigger systemic and pulmonary morphological alterations. Toxicol. Lett. 176, 20–30. Nemmar, A., Melghit, K., and Ali, B. H. (2008). The acute proinflammatory and prothrombotic effects of pulmonary exposure to rutile TiO2 nanorods in rats. Exp. Biol. Med. (Maywood) 233, 610–619. Nemmar, A., Nemery, B., Hoet, P. H. M., Vermylen, J., and Hoylaerts, M. F. (2003b). Pulmonary inflammation and thrombogenicity caused by diesel particles in hamsters—role of histamine. Am. J. Respir. Crit. Care Med. 168, 1366–1372.


Ali, B. H., Al Moundhri, M. S., Tag, E. M., Nemmar, A., and Tanira, M. O. (2007). The ameliorative effect of cysteine prodrug L-2-oxothiazolidine-4carboxylic acid on cisplatin-induced nephrotoxicity in rats. Fundam. Clin. Pharmacol. 21, 547–553.

exhaust particles enhances coagulatory disturbance with endothelial damage and systemic inflammation related to lung inflammation. Exp. Biol. Med. (Maywood) 231, 1626–1632.

DIESEL PARTICLES AND RENAL FAILURE Oberdorster, G., Oberdorster, E., and Oberdorster, J. (2005). Nanotoxicology: an emerging discipline evolving from studies of ultrafine particles. Environ. Health Perspect. 113, 823–839. Pannu, N., Klarenbach, S., Wiebe, N., Manns, B., and Tonelli, M. (2008). Renal replacement therapy in patients with acute renal failure: a systematic review. J. A. M. A. 299, 793–805. Pekkanen, J., Peters, A., Hoek, G., Tiittanen, P., Brunekreef, B., de Hartog, J., Heinrich, J., Ibald-Mulli, A., Kreyling, W. G., Lanki, T., et al. (2002). Particulate air pollution and risk of ST-segment depression during repeated submaximal exercise tests among subjects with coronary heart disease: the Exposure and Risk Assessment for Fine and Ultrafine Particles in Ambient Air (ULTRA) study. Circulation 106, 933–938. Peters, A., Dockery, D. W., Muller, J. E., and Mittleman, M. A. (2001). Increased particulate air pollution and the triggering of myocardial infarction. Circulation 103, 2810–2815.

Pierson, D. J. (2006). Respiratory considerations in the patient with renal failure. Respir. Care 51, 413–422. Pope, C. A., III., Burnett, R. T., Thun, M. J., Calle, E. E., Krewski, D., Ito, K., and Thurston, G. D. (2002). Lung cancer, cardiopulmonary mortality, and long-term exposure to fine particulate air pollution. JAMA 287, 1132–1141.

mediators in rat lung after diesel exhaust particle exposure. Environ. Health Perspect. 113, 612–617. Riediker, M., Cascio, W. E., Griggs, T. R., Herbst, M. C., Bromberg, P. A., Neas, L., Williams, R. W., and Devlin, R. B. (2004). Particulate matter exposure in cars is associated with cardiovascular effects in healthy young men. Am. J. Respir. Crit. Care Med. 169, 934–940. Rivero, D. H., Soares, S. R., Lorenzi-Filho, G., Saiki, M., Godleski, J. J., Antonangelo, L., Dolhnikoff, M., and Saldiva, P. H. (2005). Acute cardiopulmonary alterations induced by fine particulate matter of Sao Paulo. Brazil. Toxicol. Sci. 85, 898–905. Ruckerl, R., Phipps, R. P., Schneider, A., Frampton, M., Cyrys, J., Oberdorster, G., Wichmann, H. E., and Peters, A. (2007). Ultrafine particles and platelet activation in patients with coronary heart disease—results from a prospective panel study. Part. Fibre. Toxicol. 4, 1. Salvi, S., Blomberg, A., Rudell, B., Kelly, F., Sandstrom, T., Holgate, S. T., and Frew, A. (1999). Acute inflammatory responses in the airways and peripheral blood after short-term exposure to diesel exhaust in healthy human volunteers. Am. J. Respir. Crit. Care Med. 159, 702–709. Singri, N., Ahya, S. N., and Levin, M. L. (2003). Acute renal failure. JAMA 289, 747–751. Vermylen, J., Nemmar, A., Nemery, B., and Hoylaerts, M. F. (2005). Ambient air pollution and acute myocardial infarction. J. Thromb. Haemost. 3, 1955–1961.

Pope, C. A., III., Schwartz, J., and Ransom, M. R. (1992). Daily mortality and PM10 pollution in Utah Valley. Arch. Environ. Health 47, 211–217.

Vielhauer, V., Stavrakis, G., and Mayadas, T. N. (2005). Renal cell-expressed TNF receptor 2, not receptor 1, is essential for the development of glomerulonephritis. J. Clin. Invest. 115, 1199–1209.

Pope, C. A., III., Verrier, R. L., Lovett, E. G., Larson, A. C., Raizenne, M. E., Kanner, R. E., Schwartz, J., Villegas, G. M., Gold, D. R., and Dockery, D. W. (1999). Heart rate variability associated with particulate air pollution. Am. Heart J. 138, 890–899.

Yokota, S., Seki, T., Furuya, M., and Ohara, N. (2005). Acute functional enhancement of circulatory neutrophils after intratracheal instillation with diesel exhaust particles in rats. Inhal. Toxicol. 17, 671–679.

Pratibha, R., Sameer, R., Rataboli, P. V., Bhiwgade, D. A., and Dhume, C. Y. (2006). Enzymatic studies of cisplatin induced oxidative stress in hepatic tissue of rats. Eur. J. Pharmacol. 532, 290–293. Rao, K. M., Ma, J. Y., Meighan, T., Barger, M. W., Pack, D., and Vallyathan, V. (2005). Time course of gene expression of inflammatory

Zhang, L., Cooper, A. J., Krasnikov, B. F., Xu, H., Bubber, P., Pinto, J. T., Gibson, G. E., and Hanigan, M. H. (2006). Cisplatininduced toxicity is associated with platinum deposition in mouse kidney mitochondria in vivo and with selective inactivation of the alphaketoglutarate dehydrogenase complex in LLC-PK1 cells. Biochemistry 45, 8959–8971.


Peters, A., Veronesi, B., Calderon-Garciduenas, L., Gehr, P., Chen, L. C., Geiser, M., Reed, W., Rothen-Rutishauser, B., Schurch, S., and Schulz, H. (2006). Translocation and potential neurological effects of fine and ultrafine particles a critical update. Part. Fibre. Toxicol. 3, 13.

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