Transcription profiles in BEAS-2B cells exposed to organic extracts from particulate emissions produced by a port-fuel injection vehicle, fueled with conventional fossil gasoline and gasoline-ethanol blend.

Emissions from road traffic are among the major contributors to air pollution worldwide and represent a serious environmental health risk. Although traffic-related pollution has been most commonly associated with diesel engines, increasing evidence suggests that gasoline engines also produce a considerable amount of potentially hazardous particulate matter (PM). The primary objective of this study was to compare the intrinsic toxic properties of the organic components of PM, generated by a conventional gasoline engine fueled with neat gasoline (E0), or gasoline-ethanol blend (15 % ethanol, v/v, E15). Our results showed that while E15 has produced, compared to gasoline and per kg of fuel, comparable particle mass (µg PM/kg fuel) and slightly more particles by number, the organic extract from the particulate matter produced by E15 contained a larger amount of harmful polycyclic aromatic hydrocarbons (PAHs), as determined by the chemical analysis. To examine the toxicity, we monitored genome-wide gene expression changes in human lung BEAS-2B cells, exposed for 4 h and 24 h to a subtoxic dose of each PM extract. After 4 h exposure, numerous dysregulated genes and processes such as oxidative stress, lipid and steroid metabolism, PPARα signaling and immune response, were found to be common for both extract treatments. On the other hand, 24 h exposure resulted in more distinctive gene expression patterns. Although we identified several common modulated processes indicating the metabolism of PAHs and activation of aryl hydrocarbon receptor (AhR), E15 specifically dysregulated a variety of other genes and pathways related to cancer promotion and progression. Overall, our findings suggest that the ethanol addition to gasoline changed the intrinsic properties of PM emissions and increased the PAH content in PM organic extract, thus contributing to a more extensive toxic response particularly after 24 h exposure in BEAS-2B cells.

Effects of braking conditions on nanoparticle emissions from passenger car friction brakes.

Automobile friction brakes generate, in addition to coarse particles generated by mechanical processes, highly variable amount of nanoparticles from high temperature processes. The effects of braking conditions - speed, deceleration rate, brake rotor temperatures - on nanoparticle production were investigated here, aiming to provide practical guidance for reducing emissions through driving style and traffic management. Typical brake pads and a rotor from a common passenger car were subjected, on a brake dynamometer, to three runs of the WLTP brake cycle developed for brake wear particle measurements. Additionally, four sets of common brake pads were subjected to those parts of standardized brake performance tests believed to be reasonably realistic for common driving. Particle size distributions (5.6-560 nm electric mobility diameter, without removal of volatiles) show a dominant peak at 10 nm commensurate to the severity of braking and a non-linear increase of the total particle number at higher braking powers and higher total energy dissipated. The average emissions for three runs of the WLTP brake cycle were 3.3 × 1010 particles/km, while the harshest deceleration, 175-100 km/h at 5.28 m·s-2, has produced 8.4 to 38 × 1013 particles, corresponding to 2.5-11.5 thousands of km of WLTP-like driving. While previous studies have correlated higher PN production with higher average brake rotor temperature, a more complex relationship between nanoparticle emissions and a combination of initial rotor temperature, total energy dissipated and braking power has been observed here. From a driver behavior and regulatory perspective, it appears limiting harsh braking and braking from high speeds, possibly through improved driving practices, road design and traffic management, may potentially reduce brake wear nanoparticles. From the measurement perspective, it appears that "off-cycle" braking, even if relatively infrequent, may be associated with exponentially higher emissions and non-negligible share of the total emissions, and therefore should not be neglected.

Markers of lipid oxidation and inflammation in bronchial cells exposed to complete gasoline emissions and their organic extracts.

Road traffic emissions consist of gaseous components, particles of various sizes, and chemical compounds that are bound to them. Exposure to vehicle emissions is implicated in the etiology of inflammatory respiratory disorders. We investigated the inflammation-related markers in human bronchial epithelial cells (BEAS-2B) and a 3D model of the human airways (MucilAir™), after exposure to complete emissions and extractable organic matter (EOM) from particles generated by ordinary gasoline (E5), and a gasoline-ethanol blend (E20; ethanol content 20% v/v). The production of 22 lipid oxidation products (derivatives of linoleic and arachidonic acid, AA) and 45 inflammatory molecules (cytokines, chemokines, growth factors) was assessed after days 1 and 5 of exposure, using LC-MS/MS and a multiplex immunoassay, respectively. The response observed in MucilAir™ exposed to E5 gasoline emissions, characterized by elevated levels of pro-inflammatory AA metabolites (prostaglandins) and inflammatory markers, was the most pronounced. E20 EOM exposure was associated with increased levels of AA metabolites with anti-inflammatory effects in this cell model. The exposure of BEAS-2B cells to complete emissions reduced lipid oxidation, while E20 EOM tended to increase concentrations of AA metabolite and chemokine production; the impacts on other inflammatory markers were limited. In summary, complete E5 emission exposure of MucilAir™ induces the processes associated with the pro-inflammatory response. This observation highlights the potential negative health impacts of ordinary gasoline, while the effects of alternative fuel are relatively weak.

Ordinary Gasoline Emissions Induce a Toxic Response in Bronchial Cells Grown at Air-Liquid Interface.

Gasoline engine emissions have been classified as possibly carcinogenic to humans and represent a significant health risk. In this study, we used MucilAir™, a three-dimensional (3D) model of the human airway, and BEAS-2B, cells originating from the human bronchial epithelium, grown at the air-liquid interface to assess the toxicity of ordinary gasoline exhaust produced by a direct injection spark ignition engine. The transepithelial electrical resistance (TEER), production of mucin, and lactate dehydrogenase (LDH) and adenylate kinase (AK) activities were analyzed after one day and five days of exposure. The induction of double-stranded DNA breaks was measured by the detection of histone H2AX phosphorylation. Next-generation sequencing was used to analyze the modulation of expression of the relevant 370 genes. The exposure to gasoline emissions affected the integrity, as well as LDH and AK leakage in the 3D model, particularly after longer exposure periods. Mucin production was mostly decreased with the exception of longer BEAS-2B treatment, for which a significant increase was detected. DNA damage was detected after five days of exposure in the 3D model, but not in BEAS-2B cells. The expression of CYP1A1 and GSTA3 was modulated in MucilAir™ tissues after 5 days of treatment. In BEAS-2B cells, the expression of 39 mRNAs was affected after short exposure, most of them were upregulated. The five days of exposure modulated the expression of 11 genes in this cell line. In conclusion, the ordinary gasoline emissions induced a toxic response in MucilAir™. In BEAS-2B cells, the biological response was less pronounced, mostly limited to gene expression changes.

On-road detection of trucks with high NOx emissions from a patrol vehicle with on-board FTIR analyzer.

Technological advances in heavy-duty vehicle engines, allowing them to reach NOx emissions comparable to European diesel passenger cars per km driven, are being compromised by aftermarket defeat devices such as selective catalytic reduction (SCR) emulators, many of which can be quickly deactivated by the driver. In a pilot study, the prevalence of trucks with excess NOx emissions on Czech motorways was evaluated using an ordinary Customs Administration patrol vehicle temporarily fitted with a portable fast-response Fourier Transform Infra Red (FTIR) analyzer, acting as an impromptu chase vehicle. The Euro emissions category of the truck was provided from the motorway toll collection transponders. A total of 222 unique trucks were measured during a one-week pilot project. Of these, 66% were Euro VI, 25% were Euro V, and 9% were older categories. NO/CO2 ratios were calculated as a ratio of numerical integrals of the peaks of measured concentrations, as a ratio of maximum measured concentrations, and by linear regression, with the regression approach yielding most realistic results and mean calculated error of 0.2 g/kWh NO. At assumed 85% NO in NOx and 634 g/kWh mean CO2 emissions, the mean emissions of the cleanest 83% of Euro V and cleanest 63% of Euro VI trucks were within the corresponding NOx limit (2 g/kWh for Euro V, 0.46 g/kWh for Euro VI) multiplied by a factor of 1.5. Providing for some allowance for legitimate occurrences of high NOx emissions, about 10-15% of Euro V and about 10-25% of Euro VI trucks are believed to be excess emitters, with no SCR functionality on about 10-15% of Euro VI trucks. The portable FTIR, temporarily mounted on a law enforcement vehicle, can be readily used as a screening tool, identifying vehicles to be stopped for additional inspection, but also during roadside emissions inspections.

The Biological Effects of Complete Gasoline Engine Emissions Exposure in a 3D Human Airway Model (MucilAirTM) and in Human Bronchial Epithelial Cells (BEAS-2B).

The biological effects induced by complete engine emissions in a 3D model of the human airway (MucilAirTM) and in human bronchial epithelial cells (BEAS-2B) grown at the air-liquid interface were compared. The cells were exposed for one or five days to emissions generated by a Euro 5 direct injection spark ignition engine. The general condition of the cells was assessed by the measurement of transepithelial electrical resistance and mucin production. The cytotoxic effects were evaluated by adenylate kinase (AK) and lactate dehydrogenase (LDH) activity. Phosphorylation of histone H2AX was used to detect double-stranded DNA breaks. The expression of the selected 370 relevant genes was analyzed using next-generation sequencing. The exposure had minimal effects on integrity and AK leakage in both cell models. LDH activity and mucin production in BEAS-2B cells significantly increased after longer exposures; DNA breaks were also detected. The exposure affected CYP1A1 and HSPA5 expression in MucilAirTM. There were no effects of this kind observed in BEAS-2B cells; in this system gene expression was rather affected by the time of treatment. The type of cell model was the most important factor modulating gene expression. In summary, the biological effects of complete emissions exposure were weak. In the specific conditions used in this study, the effects observed in BEAS-2B cells were induced by the exposure protocol rather than by emissions and thus this cell line seems to be less suitable for analyses of longer treatment than the 3D model.

The genotoxicity of organic extracts from particulate truck emissions produced at various engine operating modes using diesel or biodiesel (B100) fuel: A pilot study.

An analysis of the toxic effects of emissions should reflect real traffic conditions. The exhaust emissions of particulate matter from diesel engines strongly depend on their operating conditions, with low-speed, low-load "urban creep" conditions, common for truck traffic in heavily congested urban areas, being one of the worst. We aimed to detect the genotoxicity of organic extracts from particulate matter in the exhaust of the diesel engine Zetor 1505 running on diesel and biodiesel (B100) fuels at characteristic modes of extended "urban creep", typical for transit truck traffic in Prague, comparing the first 5 min of idling with extended (20-80 min) idling, full load after idle, "stabilized" full load, and 30% load. The diluted exhaust was sampled with high volume samplers on glass fiber fluorocarbon coated filters. The filters were extracted with dichloromethane and DNA damage was analyzed in A549 cells using comet assay, with the inclusion of formamidopyrimidine DNA glycosylase (FPG) and endonuclease III (ENDOIII) to recognize oxidized DNA bases. The cells were exposed to extractable organic matter (EOM) for 4 and 24 h at non-cytotoxic dose corresponding to 0.001 m3 of undiluted exhaust gas per ml cell media. At the 4 h exposure interval, all samples from B100 and diesel emissions induced DNA damage. EOM from the extended idle engine mode exerted the strongest genotoxic effect for both fuels. Twenty hours later, the cells exposed to diesel EOM exhibited a further increase of DNA strand breaks compared to the preceding interval. In contrast, DNA damage seemed to be fully repaired in cells treated with EOM derived from biodiesel B100. The preliminary results suggest that (i) diesel emissions are more genotoxic than the emissions from B100, (ii) biodiesel induced DNA lesions are repaired within 24 h.

Comparison of hydrogenated vegetable oil and biodiesel effects on combustion, unregulated and regulated gaseous pollutants and DPF regeneration procedure in a Euro6 car.

The effects of traditional biodiesel (fatty acid methyl-esters, FAME) and a hydrotreated vegetable oil (HVO) were comprehensively investigated on a production Euro 6 diesel car, including fuel injection rate and timing, combustion analysis, emissions of regulated and unregulated pollutants, and regeneration of the diesel particle filter. The use of both biofuels is a part of the efforts to reduce emissions of greenhouse gases and health-relevant pollutants and to improve energy security and sustainability. HVO, albeit more expensive, offers benefits relative to FAME in terms of oxidation stability, injector fouling, energy content and cetane number. The car was fitted with an on-board instrumentation and subjected to a range of driving cycles on a chassis dynamometer. The fuel consumption calculated from instantaneous emissions data based on exhaust gas composition measured by an on-board FTIR and calculated exhaust flow matched directly measured fuel consumption within several percent on all fuels; differences in the consumption among the fuels correspond to different heating values. The combustion onset and maximum heat release rate were comparable for diesel and FAME but were advanced on HVO due to its higher cetane number, causing, at times, multiple distinct heat release peaks, suggesting that optimization of fuel injection timing for HVO might be beneficial. Emissions of methane and ammonia were negligible, of N2O were measurable and slightly lower for HVO than for other fuels, of formaldehyde were limited to cold engine accelerations and highest for FAME and negligible for HVO, of NO and NO2 were high on all fuels during all operating conditions except for the type approval test. The results confirm several relative advantages of HVO over RME, with penetration into engine lubricating oil during particle filter regeneration to be further investigated. The effects of HVO lubricity and other long-term effects were not evaluated here.

On-road and laboratory emissions of NO, NO2, NH3, N2O and CH4 from late-model EU light utility vehicles: Comparison of diesel and CNG.

Exhaust emissions of eight Euro 6 light duty vehicles - two station wagons and six vans - half powered by diesel fuel and half by compressed natural gas (CNG) were examined using both chassis dynamometer and on-road testing. A portable on-board FTIR analyzer was used to measure concentrations of reactive nitrogen compounds - NO, NO2 and ammonia, of CO, formaldehyde, acetaldehyde and greenhouse gases CO2, methane and N2O. Exhaust flow was inferred from engine control unit data. Total emissions per cycle were compared and found to be in good agreement with laboratory measurements of NOX, CO and CO2 during dynamometer tests. On diesel engines, mean NOX emissions were 136-1070mg/km in the laboratory and 537-615mg/km on the road, in many cases nearly an order of magnitude higher compared to the numerical value of the Euro 6 limit. Mean N2O emissions were 3-19mg/km and were equivalent to several g/km CO2. The measurements suggest that NOX and N2O emissions from late-model European light utility vehicles with diesel engines are non-negligible and should be continuously assessed and scrutinized. High variances in NOX emissions among the tested diesel vehicles suggest that large number of vehicles should be tested to offer at least some insights about distribution of fleet emissions among vehicles. CNG engines exhibited relatively low emissions of NOX (12-186mg/km) and NH3 (10-24mg/km), while mean emissions of methane were 18-45mg/km, under 1g/km CO2 equivalent, and N2O, CO, formaldehyde and acetaldehyde were negligible. The combination of a relatively clean-burning fuel, modern engine technology and a three-way catalyst has resulted in relatively low emissions under the wide variety of operating conditions encountered during the tests. The on-board FTIR has proven to be a useful instrument capable of covering, with the exception of total hydrocarbons, essentially all gaseous pollutants of interest.

Genotoxic potential of organic extracts from particle emissions of diesel and rapeseed oil powered engines.

The present study was performed to identify possible genotoxicity induced by organic extracts from particulate matter in the exhaust of two typical diesel engines run on diesel fuel and neat heated fuel-grade rapeseed oil: a Cummins ISBe4 engine tested using the World Harmonized Steady State Test Cycle (WHSC) and modified Engine Steady Cycle (ESC) and a Zetor 1505 engine tested using the Non-Road Steady State Cycle (NRSC). In addition, biodiesel B-100 (neat methylester of rapeseed oil) was tested in the Cummins engine run on the modified ESC. Diluted exhaust was sampled with high-volume samplers on Teflon coated filters. Filters were extracted with dichlormethane (DCM) and DNA adduct levels induced by extractable organic matter (EOM) in an acellular assay of calf thymus DNA coupled with (32)P-postlabeling in the presence and absence of rat liver microsomal S9 fraction were employed. Simultaneously, the chemical analysis of 12 priority PAHs in EOM, including 7 carcinogenic PAHs (c-PAHs) was performed. The results suggest that diesel emissions contain substantially more total PAHs than rapeseed oil emissions (for the ESC) or that these concentrations were comparable (for the WHSC and NRSC), while c-PAHs levels were comparable (for the ESC) or significantly higher (for the WHSC and NRSC) for rapeseed oil emissions. DNA adduct levels induced by diesel and rapeseed oil derived EOM were comparable, but consistently slightly higher for diesel than for rapeseed oil. Highly significant correlations were found between 12 priority PAHs concentrations and DNA adduct levels (0.980; p<0.001) and these correlations were even stronger for c-PAHs (0.990; p<0.001). Metabolic activation by the microsomal S9 fraction resulted in several fold higher genotoxicity, suggesting a major contribution of PAHs to genotoxicity. Directly acting compounds, other than c-PAHs, and not requiring S9 to exhibit DNA reactivity were also significant. Generally, DNA adduct levels were more dependent on the type of engine and the test cycle than on the fuel. Our findings suggest that the genotoxicity of particulate emissions from the combustion of rapeseed oil is significant and is comparable to that from the combustion of diesel fuel. A more detailed study is ongoing to verify and extent these preliminary findings.