First-in-human controlled inhalation of thin graphene oxide nanosheets to study acute cardiorespiratory responses.

Graphene oxide nanomaterials are being developed for wide-ranging applications but are associated with potential safety concerns for human health. We conducted a double-blind randomized controlled study to determine how the inhalation of graphene oxide nanosheets affects acute pulmonary and cardiovascular function. Small and ultrasmall graphene oxide nanosheets at a concentration of 200 µg m-3 or filtered air were inhaled for 2 h by 14 young healthy volunteers in repeated visits. Overall, graphene oxide nanosheet exposure was well tolerated with no adverse effects. Heart rate, blood pressure, lung function and inflammatory markers were unaffected irrespective of graphene oxide particle size. Highly enriched blood proteomics analysis revealed very few differential plasma proteins and thrombus formation was mildly increased in an ex vivo model of arterial injury. Overall, acute inhalation of highly purified and thin nanometre-sized graphene oxide nanosheets was not associated with overt detrimental effects in healthy humans. These findings demonstrate the feasibility of carefully controlled human exposures at a clinical setting for risk assessment of graphene oxide, and lay the foundations for investigating the effects of other two-dimensional nanomaterials in humans. Clinicaltrials.gov ref: NCT03659864.

In vitro neurotoxicity screening of engine oil- and hydraulic fluid-derived aircraft cabin bleed-air contamination.

In most airplanes, cabin air is extracted from the turbine compressors, so-called bleed air. Bleed air can become contaminated by leakage of engine oil or hydraulic fluid and possible neurotoxic constituents, like triphenyl phosphate (TPhP) and tributyl phosphate (TBP). The aim of this study was to characterize the neurotoxic hazard of TBP and TPhP, and to compare this with the possible hazard of fumes originating from engine oils and hydraulic fluids in vitro. Effects on spontaneous neuronal activity were recorded in rat primary cortical cultures grown on microelectrode arrays following exposure for 0.5 h (acute), and 24 h and 48 h (prolonged) to TBP and TPhP (0.01-100 µM) or fume extracts (1-100 µg/mL) prepared from four selected engine oils and two hydraulic fluids by a laboratory bleed air simulator. TPhP and TBP concentration-dependently reduced neuronal activity with equal potency, particularly during acute exposure (TPhP IC50: 10-12 µM; TBP IC50: 15-18 µM). Engine oil-derived fume extracts persistently reduced neuronal activity. Hydraulic fluid-derived fume extracts showed a stronger inhibition during 0.5 h exposure, but the degree of inhibition attenuates during 48 h. Overall, fume extracts from hydraulic fluids were more potent than those from engine oils, in particular during 0.5 h exposure, although the higher toxicity is unlikely to be due only to higher levels of TBP and TPhP in hydraulic fluids. Our combined data show that bleed air contaminants originating from selected engine oils or hydraulic fluids exhibit neurotoxic hazard in vitro, with fumes derived from the selected hydraulic fluids being most potent.

Neuromodulatory and neurotoxic effects of e-cigarette vapor using a realistic exposure method.

The most direct effects of inhaled harmful constituents are the effects on the airways. However, inhaled compounds can be rapidly absorbed and subsequently result in systemic effects. For example, e-cigarette vapor has been shown to evoke local effects in the lung, although little is known about subsequent effects in secondary target organs such as the brain. Traditionally, such effects are tested using in vivo models. As an alternative, we have combined two in vitro systems, which are Air-Liquid-Interface (ALI) cultured alveolar cells (A549) and rat primary cortical cultures grown on multi-well microelectrode arrays. This allows us to assess the neurological effects of inhaled compounds. We have used exposure to e-cigarette vapor, containing nicotine, menthol, or vanillin to test the model. Our results show that ALI cultured A549 cells respond to the exposure with the production of cytokines (IL8 and GROalpha). Furthermore, nicotine, menthol, and vanillin were found on the basolateral side of the cell culture, which indicates their translocation. Upon transfer of the basolateral medium to the primary cortical culture, exposure-related changes in spontaneous electrical activity were observed correlating with the presence of e-liquid components in the medium. These clear neuromodulatory effects demonstrate the feasibility of combining continuous exposure of ALI cultured cells with subsequent exposure of neuronal cells to assess neurotoxicity. Although further optimization steps are needed, such a combination of methods is important to assess the neurotoxic effects of inhaled compounds realistically. As such, an approach like this could play a role in future mechanism-based risk assessment strategies.

Smoking-Associated Exposure of Human Primary Bronchial Epithelial Cells to Aldehydes: Impact on Molecular Mechanisms Controlling Mitochondrial Content and Function.

Chronic obstructive pulmonary disease (COPD) is a devastating lung disease primarily caused by exposure to cigarette smoke (CS). During the pyrolysis and combustion of tobacco, reactive aldehydes such as acetaldehyde, acrolein, and formaldehyde are formed, which are known to be involved in respiratory toxicity. Although CS-induced mitochondrial dysfunction has been implicated in the pathophysiology of COPD, the role of aldehydes therein is incompletely understood. To investigate this, we used a physiologically relevant in vitro exposure model of differentiated human primary bronchial epithelial cells (PBEC) exposed to CS (one cigarette) or a mixture of acetaldehyde, acrolein, and formaldehyde (at relevant concentrations of one cigarette) or air, in a continuous flow system using a puff-like exposure protocol. Exposure of PBEC to CS resulted in elevated IL-8 cytokine and mRNA levels, increased abundance of constituents associated with autophagy, decreased protein levels of molecules associated with the mitophagy machinery, and alterations in the abundance of regulators of mitochondrial biogenesis. Furthermore, decreased transcript levels of basal epithelial cell marker KRT5 were reported after CS exposure. Only parts of these changes were replicated in PBEC upon exposure to a combination of acetaldehyde, acrolein, and formaldehyde. More specifically, aldehydes decreased MAP1LC3A mRNA (autophagy) and BNIP3 protein (mitophagy) and increased ESRRA protein (mitochondrial biogenesis). These data suggest that other compounds in addition to aldehydes in CS contribute to CS-induced dysregulation of constituents controlling mitochondrial content and function in airway epithelial cells.

Unveiling the Toxicity of Fine and Nano-Sized Airborne Particles Generated from Industrial Thermal Spraying Processes in Human Alveolar Epithelial Cells.

High-energy industrial processes have been associated with particle release into workplace air that can adversely affect workers' health. The present study assessed the toxicity of incidental fine (PGFP) and nanoparticles (PGNP) emitted from atmospheric plasma (APS) and high-velocity oxy-fuel (HVOF) thermal spraying. Lactate dehydrogenase (LDH) release, 2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) metabolisation, intracellular reactive oxygen species (ROS) levels, cell cycle changes, histone H2AX phosphorylation (γ-H2AX) and DNA damage were evaluated in human alveolar epithelial cells at 24 h after exposure. Overall, HVOF particles were the most cytotoxic to human alveolar cells, with cell viability half-maximal inhibitory concentration (IC50) values of 20.18 µg/cm2 and 1.79 µg/cm2 for PGFP and PGNP, respectively. Only the highest tested concentration of APS-PGFP caused a slight decrease in cell viability. Particle uptake, cell cycle arrest at S + G2/M and γ-H2AX augmentation were observed after exposure to all tested particles. However, higher levels of γ-H2AX were found in cells exposed to APS-derived particles (~16%), while cells exposed to HVOF particles exhibited increased levels of oxidative damage (~17% tail intensity) and ROS (~184%). Accordingly, APS and HVOF particles seem to exert their genotoxic effects by different mechanisms, highlighting that the health risks of these process-generated particles at industrial settings should not be underestimated.

In Vitro Toxicity of Industrially Relevant Engineered Nanoparticles in Human Alveolar Epithelial Cells: Air-Liquid Interface versus Submerged Cultures.

Diverse industries have already incorporated within their production processes engineered nanoparticles (ENP), increasing the potential risk of worker inhalation exposure. In vitro models have been widely used to investigate ENP toxicity. Air-liquid interface (ALI) cell cultures have been emerging as a valuable alternative to submerged cultures as they are more representative of the inhalation exposure to airborne nano-sized particles. We compared the in vitro toxicity of four ENP used as raw materials in the advanced ceramics sector in human alveolar epithelial-like cells cultured under submerged or ALI conditions. Submerged cultures were exposed to ENP liquid suspensions or to aerosolised ENP at ALI. Toxicity was assessed by determining LDH release, WST-1 metabolisation and DNA damage. Overall, cells were more sensitive to ENP cytotoxic effects when cultured and exposed under ALI. No significant cytotoxicity was observed after 24 h exposure to ENP liquid suspensions, although aerosolised ENP clearly affected cell viability and LDH release. In general, all ENP increased primary DNA damage regardless of the exposure mode, where an increase in DNA strand-breaks was only detected under submerged conditions. Our data show that at relevant occupational concentrations, the selected ENP exert mild toxicity to alveolar epithelial cells and exposure at ALI might be the most suitable choice when assessing ENP toxicity in respiratory models under realistic exposure conditions.

Optimization of an air-liquid interface in vitro cell co-culture model to estimate the hazard of aerosol exposures.

Inhalation exposure to environmental and occupational aerosol contaminants is associated with many respiratory health problems. To realistically mimic long-term inhalation exposure for toxicity testing, lung epithelial cells need to maintained and exposed under air-liquid interface (ALI) conditions for a prolonged period of time. In addition, to study cellular responses to aerosol particles, lung epithelial cells have to be co-cultured with macrophages. To that aim, we evaluated human bronchial epithelial Calu-3, 16HBE14o- (16HBE), H292, and BEAS-2B cell lines with respect to epithelial morphology, barrier function and cell viability under prolonged ALI culture conditions. Only the Calu-3 cells can retain the monolayer structure and maintain a strong tight junction under long-term ALI culture at least up to 2 weeks. As such, Calu-3 cells were applied as the structural barrier to create co-culture models with human monocyte-derived macrophages (MDMs) and THP-1 derived macrophages (TDMs). Adhesion of macrophages onto the epithelial monolayer was allowed for 4 h with a density of 5 × 104 macrophages/cm2. In comparison to the Calu-3 mono-culture model, Calu-3 + TDM and Calu-3 + MDM co-culture models showed an increased sensitivity in inflammatory responses to lipopolysaccharide (LPS) aerosol at Day 1 of co-culture, with the Calu-3 + MDM model giving a stronger response than Calu-3 + TDM. Therefore, the epithelial monolayer integrity and increased sensitivity make the Calu-3 + MDM co-culture model a preferred option for ALI exposure to inhaled aerosols for toxicity testing.

Toxicity assessment of industrial engineered and airborne process-generated nanoparticles in a 3D human airway epithelial in vitro model.

The advanced ceramic technology has been pointed out as a potentially relevant case of occupational exposure to nanoparticles (NP). Not only when nanoscale powders are being used for production, but also in the high-temperature processing of ceramic materials there is also a high potential for NP release into the workplace environment. In vitro toxicity of engineered NP (ENP) [antimony tin oxide (Sb2O3•SnO2; ATO); zirconium oxide (ZrO2)], as well as process-generated NP (PGNP), and fine particles (PGFP), was assessed in MucilAir™ cultures at air-liquid interface (ALI). Cultures were exposed during three consecutive days to varying doses of the aerosolized NP. General cytotoxicity [lactate dehydrogenase (LDH) release, WST-1 metabolization], (oxidative) DNA damage, and the levels of pro-inflammatory mediators (IL-8 and MCP-1) were assessed. Data revealed that ENP (5.56 µg ATO/cm2 and 10.98 µg ZrO2/cm2) only caused mild cytotoxicity at early timepoints (24 h), whereas cells seemed to recover quickly since no significant changes in cytotoxicity were observed at late timepoints (72 h). No meaningful effects of the ENP were observed regarding DNA damage and cytokine levels. PGFP affected cell viability at dose levels as low as ∼9 µg/cm2, which was not seen for PGNP. However, exposure to PGNP (∼4.5 µg/cm2) caused an increase in oxidative DNA damage. These results indicated that PGFP and PGNP exhibit higher toxicity potential than ENP in mass per area unit. However, the presence of a mucociliary apparatus, as it occurs in vivo as a defense mechanism, seems to considerably attenuate the observed toxic effects. Our findings highlight the potential hazard associated with exposure to incidental NP in industrial settings.

Comparative toxicity of ultrafine particles around a major airport in human bronchial epithelial (Calu-3) cell model at the air-liquid interface.

Relatively high concentrations of ultrafine particles (UFPs) have been observed around airports, in which aviation and road traffic emissions are the major sources. This raises concerns about the potential health impacts of airport UFPs, particularly in comparison to those emitted by road traffic. UFPs mainly derived from aviation or road traffic emissions were collected from a location near a major international airport, Amsterdam-Schiphol airport (AMS), depending on the wind direction, along with UFPs from an aircraft turbine engine at low and full thrust. Human bronchial epithelial cells (Calu-3) model in combination with an air-liquid interface (ALI) cloud system was used for the in vitro exposure to UFPs at low doses ranging from 0.09 to 2.07 µg/cm2. Particle size distribution was measured. Cell viability, cytotoxicity and inflammatory potential (interleukin (IL) 6 and 8 secretion) on Calu-3 cells were assessed after exposure for 24 h. The biological measurements on Calu-3 cells confirm that pro-inflammatory responses still can be activated at the high cell viability (> 80%) and low cytotoxicity. By the Benchmark Dose (BMD) analysis, Airport and Non-Airport (road traffic) UFPs as well as UFPs samples from a turbine engine have similar toxic properties. Our results suggest that UFPs from aviation and road traffic in airport surroundings may have similar adverse effects on public health.

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing.

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Differences in cytotoxicity of lung epithelial cells exposed to titanium dioxide nanofibers and nanoparticles: Comparison of air-liquid interface and submerged cell cultures.

Air Liquid Interface (ALI) system has emerged as a useful tool for toxicity evaluation of nanomaterials related to inhalation since the system mimics the aerosol exposure. We compared the biological responses of lung epithelial cells exposed to titanium dioxide (TiO2) nanofibers and nanoparticles in ALI and submerged cell cultures systems. Cells were exposed to 2 and 10 µg/cm2 for 24 h, 48 h and 72 h and LDH release, TiO2 internalization, DNA-double strand breaks (DSBs) and ROS production were assessed. LDH release was similar in both systems and particles had higher cytoplasmic uptake in submerged systems. Both TiO2 types were located in the cytoplasm but nanofibers had nuclear uptake regardless to the system tested. Cells exposed to TiO2 nanofibers had higher DSBs in the ALI system than in submerged cell cultures but cells exposed to TiO2 nanoparticles had similar DSBs in both systems. ROS production was higher in cells exposed to TiO2 nanofibers compared to cells exposed to TiO2 nanoparticles. In conclusion, cytotoxicity of lung epithelial cells was similar in ALI or submerged cell cultures, however cells exposed to TiO2 nanofibers displayed higher toxicity than cells exposed to TiO2 nanoparticles.

Silver nanoparticles inhaled during pregnancy reach and affect the placenta and the foetus.

Recently, interest for the potential impact of consumer-relevant engineered nanoparticles on pregnancy has dramatically increased. This study investigates whether inhaled silver nanoparticles (AgNPs) reach and cross mouse placental barrier and induce adverse effects. Apart from their relevance for the growing use in consumer products and biomedical applications, AgNPs are selected since they can be unequivocally identified in tissues. Pregnant mouse females are exposed during the first 15 days of gestation by nose-only inhalation to a freshly produced aerosol of 18-20 nm AgNPs for either 1 or 4 h, at a particle number concentration of 3.80 × 107 part./cm-3 and at a mass concentration of 640 µg/m³. AgNPs are identified and quantitated in maternal tissues, placentas and foetuses by transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy and single-particle inductively coupled plasma mass spectrometry. Inhalation of AgNPs results in increased number of resorbed foetuses associated with reduced oestrogen plasma levels, in the 4 h/day exposed mothers. Increased expression of pregnancy-relevant inflammatory cytokines is also detected in the placentas of both groups. These results prove that NPs are able to reach and cross the mouse placenta and suggest that precaution should be taken with respect to acute exposure to nanoparticles during pregnancy.

Inhaled Nanoparticles Accumulate at Sites of Vascular Disease.

The development of engineered nanomaterials is growing exponentially, despite concerns over their potential similarities to environmental nanoparticles that are associated with significant cardiorespiratory morbidity and mortality. The mechanisms through which inhalation of nanoparticles could trigger acute cardiovascular events are emerging, but a fundamental unanswered question remains: Do inhaled nanoparticles translocate from the lung in man and directly contribute to the pathogenesis of cardiovascular disease? In complementary clinical and experimental studies, we used gold nanoparticles to evaluate particle translocation, permitting detection by high-resolution inductively coupled mass spectrometry and Raman microscopy. Healthy volunteers were exposed to nanoparticles by acute inhalation, followed by repeated sampling of blood and urine. Gold was detected in the blood and urine within 15 min to 24 h after exposure, and was still present 3 months after exposure. Levels were greater following inhalation of 5 nm (primary diameter) particles compared to 30 nm particles. Studies in mice demonstrated the accumulation in the blood and liver following pulmonary exposure to a broader size range of gold nanoparticles (2-200 nm primary diameter), with translocation markedly greater for particles <10 nm diameter. Gold nanoparticles preferentially accumulated in inflammation-rich vascular lesions of fat-fed apolipoproteinE-deficient mice. Furthermore, following inhalation, gold particles could be detected in surgical specimens of carotid artery disease from patients at risk of stroke. Translocation of inhaled nanoparticles into the systemic circulation and accumulation at sites of vascular inflammation provides a direct mechanism that can explain the link between environmental nanoparticles and cardiovascular disease and has major implications for risk management in the use of engineered nanomaterials.

Maternal exposure to diluted diesel engine exhaust alters placental function and induces intergenerational effects in rabbits.

BACKGROUND: Airborne pollution is a rising concern in urban areas. Epidemiological studies in humans and animal experiments using rodent models indicate that gestational exposure to airborne pollution, in particular diesel engine exhaust (DE), reduces birth weight, but effects depend on exposure duration, gestational window and nanoparticle (NP) concentration. Our aim was to evaluate the effects of gestational exposure to diluted DE on feto-placental development in a rabbit model. Pregnant females were exposed to diluted (1 mg/m(3)), filtered DE (NP diameter ≈ 69 nm) or clean air (controls) for 2 h/day, 5 days/week by nose-only exposure (total exposure: 20 days in a 31-day gestation). RESULTS: DE exposure induced early signs of growth retardation at mid gestation with decreased head length (p = 0.04) and umbilical pulse (p = 0.018). Near term, fetal head length (p = 0.029) and plasma insulin and IGF1 concentrations (p = 0.05 and p = 0.019) were reduced. Placental function was also affected, with reduced placental efficiency (fetal/placental weight) (p = 0.049), decreased placental blood flow (p = 0.009) and fetal vessel volume (p = 0.002). Non-aggregated and "fingerprint" NP were observed at various locations, in maternal blood space, in trophoblastic cells and in the fetal blood, demonstrating transplacental transfer. Adult female offspring were bred with control males. Although fetoplacental biometry was not affected near term, second generation fetal metabolism was modified by grand-dam exposure with decreased plasma cholesterol (p = 0.008) and increased triglyceride concentrations (p = 0.015). CONCLUSIONS: Repeated daily gestational exposure to DE at levels close to urban pollution can affect feto-placental development in the first and second generation.

Identification of the appropriate dose metric for pulmonary inflammation of silver nanoparticles in an inhalation toxicity study.

A number of studies have shown that induction of pulmonary toxicity by nanoparticles of the same chemical composition depends on particle size, which is likely in part due to differences in lung deposition. Particle size mostly determines whether nanoparticles reach the alveoli, and where they might induce toxicity. For the risk assessment of nanomaterials, there is need for a suitable dose metric that accounts for differences in effects between different sized nanoparticles of the same chemical composition. The aim of the present study is to determine the most suitable dose metric to describe the effects of silver nanoparticles after short-term inhalation. Rats were exposed to different concentrations (ranging from 41 to 1105 µg silver/m(3) air) of 18, 34, 60 and 160 nm silver particles for four consecutive days and sacrificed at 24 h and 7 days after exposure. We observed a concentration-dependent increase in pulmonary toxicity parameters like cell counts and pro-inflammatory cytokines in the bronchoalveolar lavage fluid. All results were analysed using the measured exposure concentrations in air, the measured internal dose in the lung and the estimated alveolar dose. In addition, we analysed the results based on mass, particle number and particle surface area. Our study indicates that using the particle surface area as a dose metric in the alveoli, the dose-response effects of the different silver particle sizes overlap for most pulmonary toxicity parameters. We conclude that the alveolar dose expressed as particle surface area is the most suitable dose metric to describe the toxicity of silver nanoparticles after inhalation.

Particle size dependent deposition and pulmonary inflammation after short-term inhalation of silver nanoparticles.

BACKGROUND: Although silver nanoparticles are currently used in more than 400 consumer products, it is not clear to what extent they induce adverse effects after inhalation during production and use. In this study, we determined the lung burden, tissue distribution, and the induction and recovery of adverse effects after short-term inhalation exposure to 15 nm and 410 nm silver nanoparticles. METHODS: Rats were nose-only exposed to clean air, 15 nm silver nanoparticles (179 µg/m³) or 410 nm silver particles (167 µg/m³) 6 hours per day, for four consecutive days. Tissue distribution and the induction of pulmonary toxicity were determined at 24 hours and 7 days after exposure and compared with the internal alveolar dose. Presence of silver nanoparticles in lung cells was visualized by transmission electron microscopy (TEM). RESULTS: Exposure to 15 nm silver nanoparticles induced moderate pulmonary toxicity compared to the controls, indicated by a 175-fold increased influx of neutrophils in the lungs, a doubling of cellular damage markers in the lungs, a 5-fold increase in pro-inflammatory cytokines, and a 1.5-fold increase in total glutathione at 24 hours after exposure. All the observed effects disappeared at 7 days after exposure. No effects were observed after exposure to 410 nm silver particles. The internal alveolar mass dose of the 15 nm nanoparticles was 3.5 times higher compared to the 410 nm particles, which equals to a 66,000 times higher particle number. TEM analysis revealed 15 nm nanoparticles in vesicles and nuclei of lung cells, which were decreased in size to <5 nm at 24 hours after exposure. This demonstrates substantial dissolution of the silver nanoparticles. CONCLUSION: The results show a clear size-dependent effect after inhalation of similar mass concentrations of 15 nm and 410 nm silver (nano)particles. This can be partially explained by the difference in the internal alveolar dose between the 15 nm and 410 nm silver (nano)particles as well as by a difference in the release rate of silver ions.

Combustion-derived nanoparticulate induces the adverse vascular effects of diesel exhaust inhalation.

AIM: Exposure to road traffic and air pollution may be a trigger of acute myocardial infarction, but the individual pollutants responsible for this effect have not been established. We assess the role of combustion-derived-nanoparticles in mediating the adverse cardiovascular effects of air pollution. METHODS AND RESULTS: To determine the in vivo effects of inhalation of diesel exhaust components, 16 healthy volunteers were exposed to (i) dilute diesel exhaust, (ii) pure carbon nanoparticulate, (iii) filtered diesel exhaust, or (iv) filtered air, in a randomized double blind cross-over study. Following each exposure, forearm blood flow was measured during intra-brachial bradykinin, acetylcholine, sodium nitroprusside, and verapamil infusions. Compared with filtered air, inhalation of diesel exhaust increased systolic blood pressure (145 ± 4 vs. 133 ± 3 mmHg, P< 0.05) and attenuated vasodilatation to bradykinin (P= 0.005), acetylcholine (P= 0.008), and sodium nitroprusside (P< 0.001). Exposure to pure carbon nanoparticulate or filtered exhaust had no effect on endothelium-dependent or -independent vasodilatation. To determine the direct vascular effects of nanoparticulate, isolated rat aortic rings (n= 6-9 per group) were assessed in vitro by wire myography and exposed to diesel exhaust particulate, pure carbon nanoparticulate and vehicle. Compared with vehicle, diesel exhaust particulate (but not pure carbon nanoparticulate) attenuated both acetylcholine (P< 0.001) and sodium-nitroprusside (P= 0.019)-induced vasorelaxation. These effects were partially attributable to both soluble and insoluble components of the particulate. CONCLUSION: Combustion-derived nanoparticulate appears to predominately mediate the adverse vascular effects of diesel exhaust inhalation. This provides a rationale for testing environmental health interventions targeted at reducing traffic-derived particulate emissions.

Pulmonary and cardiovascular effects of traffic-related particulate matter: 4-week exposure of rats to roadside and diesel engine exhaust particles.

Traffic-related particulate matter (PM) may play an important role in the development of adverse health effects, as documented extensively in acute toxicity studies. However, rather little is known about the impacts of prolonged exposure to PM. We hypothesized that long-term exposure to PM from traffic adversely affects the pulmonary and cardiovascular system through exacerbation of an inflammatory response. To examine this hypothesis, Fisher F344 rats, with a mild pulmonary inflammation at the onset of exposure, were exposed for 4 weeks, 5 days/week for 6 h a day to: (a) diluted diesel engine exhaust (PM(DEE)), or: (b) near roadside PM (PM(2.5)). Ultrafine particulates, which are largely present in diesel soot, may enter the systemic circulation and directly or indirectly trigger cardiovascular effects. Hence, we assessed the effects of traffic-related PM on pulmonary inflammation and activity of procoagulants, vascular function in arteries, and cytokine levels in the heart 24 h after termination of the exposures. No major adverse health effects of prolonged exposure to traffic-related PM were detected. However, some systemic effects due to PM(DEE) exposure occurred including decreased numbers of white blood cells and reduced von Willebrand factor protein in the circulation. In addition, lung tissue factor activity is reduced in conjunction with reduced lung tissue thrombin generation. To what extent these alterations contribute to thrombotic effects and vascular diseases remains to be established. In conclusion, prolonged exposure to traffic-related PM in healthy animals may not be detrimental due to various biological adaptive response mechanisms.

Diesel engine exhaust initiates a sequence of pulmonary and cardiovascular effects in rats.

This study was designed to determine the sequence of events leading to cardiopulmonary effects following acute inhalation of diesel engine exhaust in rats. Rats were exposed for 2 h to diesel engine exhaust (1.9 mg/m(3)), and biological parameters related to antioxidant defense, inflammation, and procoagulation were examined after 4, 18, 24, 48, and 72 h. This in vivo inhalation study showed a pulmonary anti-oxidant response (an increased activity of the anti-oxidant enzymes glutathione peroxidase and superoxide dismutase and an increase in heme oxygenase-1 protein, heme oxygenase activity, and uric acid) which precedes the inflammatory response (an increase in IL-6 and TNF-α). In addition, increased plasma thrombogenicity and immediate anti-oxidant defense gene expression in aorta tissue shortly after the exposure might suggest direct translocation of diesel engine exhaust components to the vasculature but mediation by other pathways cannot be ruled out. This study therefore shows that different stages in oxidative stress are not only affected by dose increments but are also time dependent.