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Aims Patients with acute coronary syndromes (ACSs) who are managed without coronary revascularization represent a mixed and understudied population that seems to receive suboptimal pharmacological treatment. Methods and results We assessed patterns of antithrombotic therapies employed during the hospitalization and in-hospital clinical events of medically managed patients with ACS enrolled in the prospective, multicentre, nationwide EYESHOT (EmploYEd antithrombotic therapies in patients with acute coronary Syndromes HOspitalized in iTalian cardiac care units) registry. Among the 2585 consecutive ACS patients enrolled in EYESHOT, 783 (30.3%) did not receive any revascularization during hospital admission. Of these, 478 (61.0%) underwent coronary angiography (CA), whereas 305 (39.0%) did not. The median GRACE and CRUSADE risk scores were significantly higher among patients who did not undergo CA compared with those who did (180 vs. 145, P < 0.0001 and 50 vs. 33, P < 0.0001, respectively). Antithrombotic therapies employed during hospitalization significantly differ between patients who received CA and those who did not with unfractioned heparin and novel P2Y12 inhibitors more frequently used in the first group, and low-molecular-weight heparins and clopidogrel in the latter group. During the index hospitalization, patients who did not receive CA presented a higher incidence of ischaemic cerebrovascular events and of mortality compared with those who underwent CA (1.6 vs. 0.2%, P = 0.04 and 7.9 vs. 2.7%, P = 0.0009, respectively). Conclusion Almost one-third of ACS patients are managed without revascularization during the index hospitalization. In this population, a lower use of recommended antiplatelet therapy and worse clinical outcome were observed in those who did not undergo CA when compared with those who did. Clinical Trial Registration Unique identifier: [NCT02015624][1], . [10.1093/ehjcvp/pvv017][2] [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT02015624&atom=%2Fehjcardpharm%2F1%2F3%2F168.atom [2]: /lookup/doi/10.1093/ehjcvp/pvv017

Aims In the last decades, several new therapies have emerged for the treatment of acute coronary syndromes (ACS). We sought to describe real-world patterns of use of antithrombotic treatments in the catheterization laboratory for ACS patients undergoing percutaneous coronary interventions (PCI). Methods EmploYEd antithrombotic therapies in patients with acute coronary Syndromes HOspitalized in iTalian cardiac care units was a nationwide, prospective registry aimed to evaluate antithrombotic strategies employed in ACS patients in Italy. Results Over a 3-week period, a total of 2585 consecutive ACS patients have been enrolled in 203 cardiac care units across Italy. Among these patients, 1755 underwent PCI (923 with ST-elevation myocardial infarction and 832 with non-ST-elevation ACS). In the catheterization laboratory, unfractioned heparin was the most used antithrombotic drug in both ST-elevation myocardial infarction (64.7%) and non-ST-elevation ACS (77.5%) undergoing PCI and, as aspirin, bivalirudin and glycoprotein IIb/IIIa inhibitors (GPIs) more frequently employed before or during PCI compared with the postprocedural period. Any crossover of heparin therapy occurred in 36.0% of cases, whereas switching from one P2Y12 inhibitor to another occurred in 3.7% of patients. Multivariable analysis yielded several independent predictors of GPIs and of bivalirudin use in the catheterization laboratory, mainly related to clinical presentation, PCI complexity and presence of complications during the procedure. Conclusion In our contemporary, nationwide, all-comers cohort of ACS patients undergoing PCI, antithrombotic therapies were commonly initiated before the catheterization laboratory. In the periprocedural period, the most frequently employed drugs were unfractioned heparin, leading to a high rate of crossover, followed by GPIs and bivalirudin, mainly used during complex PCI. Clinical trial registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT02015624.

Air pollution and particulate matter are recognised cause of increased disease incidence in exposed population. The toxicological processes underlying air pollution associated effects have been investigated by in vivo and/or in vitro experimentation. The latter is usually performed by exposing cells cultured under submerged condition to particulate matter concentration quite far from environmental exposure expected in humans. Here we report for the first time the feasibility of a direct exposure of air liquid interface cultured cells to environmental concentration of particulate matter. Inflammatory proteins release was analysed in cell medium while differential expression of selected genes was analysed in cells. Significant association of anti-oxidant genes was observed with secondary and aged aerosol, while cytochrome activation with primary and PAHs enriched ultrafine particles. The results obtained clearly show the opportunity to move from the lab bench to the field for properly understanding the toxicological effects also of ultrafine particles on selected in vitro models. © 2018 Elsevier Ltd

Air pollution is one of the world’s leading environmental causes of death. The epidemiological relationship between outdoor air pollution and the onset of health diseases associated with death is now well established. Relevant toxicological proofs are now dissecting the molecular processes that cause inflammation, reactive species generation, and DNA damage. In addition, new data are pointing out the role of airborne particulates in the modulation of genes and microRNAs potentially involved in the onset of human diseases. In the present review we collect the relevant findings on airborne particulates of one of the biggest hot spots of air pollution in Europe (i.e., the Po Valley), in the largest urban area of this region, Milan. The different aerodynamic fractions are discussed separately with a specific focus on fine and ultrafine particles that are now the main focus of several studies. Results are compared with more recent international findings. Possible future perspectives of research are proposed to create a new discussion among scientists working on the toxicological effects of airborne particles.

Greater Cairo (Egypt) is a megalopolis where the studies of the air pollution events are of extremely high relevance, for the geographical-climatological aspects, the anthropogenic emissions and the health impact. While preliminary studies on the particulate matter (PM) chemical composition in Greater Cairo have been performed, no data are yet available on the PM&rsquo, s toxicity. In this work, the in vitro toxicity of the fine PM (PM2.5) sampled in an urban area of Greater Cairo during 2017&ndash, 2018 was studied. The PM2.5 samples collected during spring, summer, autumn and winter were preliminary characterized to determine the concentrations of ionic species, elements and organic PM (Polycyclic Aromatic Hydrocarbons, PAHs). After particle extraction from filters, the cytotoxic and pro-inflammatory effects were evaluated in human lung A549 cells. The results showed that particles collected during the colder seasons mainly induced the xenobiotic metabolizing system and the consequent antioxidant and pro-inflammatory cytokine release responses. Biological events positively correlated to PAHs and metals representative of a combustion-derived pollution. PM2.5 from the warmer seasons displayed a direct effect on cell cycle progression, suggesting possible genotoxic effects. In conclusion, a correlation between the biological effects and PM2.5 physico-chemical properties in the area of study might be useful for planning future strategies aiming to improve air quality and lower health hazards.

At present, both tropospheric ozone (O3) and particulate matters (PM) are among the most threatening air pollutants for human health in cities. The air pollution effects over public health include increased risk of hospital admissions and mortality for respiratory and cardiovascular diseases even when air pollutant concentrations are below European and international standards. The aim of this study was to (i) estimate the burden of mortality and morbidity for cardiovascular and respiratory diseases attributed to PM2.5, PM10 and O3 in nine selected cities in France, Iran and Italy in 2015 and 2016 and to (ii) compare estimated burdens at current O3 and PM levels with pre-industrial levels. The selected Mediterranean cities are among the most affected by the air pollution in Europe, in particular by rising O3 while the selected Iranian cities rank as the most polluted by PM in the world. The software AirQ+ was used to estimate the short-term health effects, in terms of mortality and morbidity by using in situ air quality data, city-specific relative risk values and baseline incidence. Compared to pre-industrial levels, long-term exposures to ambient PM2.5, PM10 and O3 have substantially contributed to mortality and hospital admissions in selected cities: about 8200 deaths for non-accidental causes, 2400 deaths for cardiovascular diseases, 540 deaths for respiratory diseases, 220 deaths for chronic obstructive pulmonary diseases as well as 18,800 hospital admissions for cardiovascular diseases and 3400 for respiratory diseases were reported in 2015. The study supports the need of city-specific epidemiological data and urgent strategies to mitigate the health burden of air pollution.

Air pollution is the primary environmental cause of death globally. To improve air quality and reduce health impacts, the National Emission Ceilings Directive requires Member States of the European Union to provide National Air Pollution Control Programmes, including emission reduction measures aimed to achieve binding commitments for the years 2020 and 2030. Integrated assessment models are pivotal to assess the reduction of pollutants concentrations determined by measures implemented or foreseen for emission reduction. Here we discuss scenarios elaborated for year 2030 in the Italian National Air Pollution Control Programme, considering 2010 as reference year. The two scenarios, “With Measures” and “With Additional Measures”, show a significant reduction of the pollutants concentration, namely PM2.5, NO2 and O3. The scenarios are here also used to provide an integrated approach for calculating the effect of the program on health impacts (mortality) and related costs. Avoidable attributable cases and associated costs are here reported at both the national and regional level and provide a significant framework to assess air-pollution reduction measures with an integrated approach. The procedure proposed may be therefore further developed and applied to assess the overall positive benefits (environmental, health and economic) determined by air-pollution control plans or other integrated policies targeting air quality, energy and climate goals.

Across the 28 EU member states there were nearly half a million premature deaths in 2015 as a result of exposure to PM2.5, O3 and NO2. To set the target for air quality levels and avoid negative impacts for human and ecosystems health, the National Emission Ceilings Directive (NECD, 2016/2284/EU) sets objectives for emission reduction for SO2, NOx, NMVOCs, NH3 and PM2.5 for each Member State as percentages of reduction to be reached in 2020 and 2030 compared to the emission levels into 2005. One of the innovations of NECD is Article 9, that mentions the issue of “monitoring air pollution impacts” on ecosystems. We provide a clear picture of what is available in term of monitoring network for air pollution impacts on Italian ecosystems, summarizing what has been done to control air pollution and its effects on different ecosystems in Italy. We provide an overview of the impacts of air pollution on health of the Italian population and evaluate opportunities and implementation of Article 9 in the Italian context, as a case study beneficial for all Member States. The results showed that SO42− deposition strongly decreased in all monitoring sites in Italy over the period 1999–2017, while NO3− and NH4+ decreased more slightly. As a consequence, most of the acid-sensitive sites which underwent acidification in the 1980s partially recovered. The O3 concentration at forest sites showed a decreasing trend. Consequently, AOT40 (the metric identified to protect vegetation from ozone pollution) showed a decrease, even if values were still above the limit for forest protection (5000 ppb h−1), while PODy (flux-based metric under discussion as new European legislative standard for forest protection) showed an increase. National scale studies pointed out that PM10 and NO2 induced about 58,000 premature deaths (year 2005), due to cardiovascular and respiratory diseases. The network identified for Italy contains a good number of monitoring sites (6 for terrestrial ecosystem monitoring, 4 for water bodies monitoring and 11 for ozone impact monitoring) distributed over the territory and will produce a high number of monitored parameters for the implementation of the NECD. Keywords: National Emission Ceilings Directive, Air pollution impacts, Climate change, Terrestrial ecosystem, Freshwater ecosystem, Human health

The presence of deoxynivalenol (DON), a mycotoxin produced by Fusarium species, has been reported worldwide in food and feedstuffs. Even though oral intake is the main route of exposure, DON inhalation is also of concern in workers and exposed population. Particulate matter (PM) is one of the most important causes of air quality detriment and it induces several adverse health effects. Therefore it is of primary importance to understand possible combined effects of DON and PM. The alveolar type II, A549, and the bronchial epithelial, BEAS-2B, cell lines were exposed for 24 h to different concentrations of DON (10-1000 ng/ml), PM10 (5 μg/cm2, sampled in summer or winter season), and a combination of these pollutants. Cell death, interleukins release and cell cycle alteration were analysed; protein array technique was also applied to evaluate proteins activation related to MAP-kinases cascade. Our results demonstrate that low doses of PM and DON used alone have scarce toxic effects, while induce cytotoxicity and inflammation when used in combination. This observation outlines the importance of investigation on the combined effects of air pollutants for their possible outcomes on human health. © 2015 Elsevier Ltd.

Inflammatory responses have an important role in the onset of many lung diseases associated with urban airborne particulate matter (PM). Here we investigate effects and mechanisms linked to PM-induced expression and release of two main interleukins, IL-6 and IL-8, in human bronchial epithelial BEAS-2B cells. The cells were exposed to well characterized Milan city PM, winter PM2.5 (wPM2.5) and summer PM10 (sPM10), representing combustion and non-combustion sources, respectively. Both wPM2.5 and sPM10 increased mRNA-synthesis and intracellular protein levels of IL-6 and IL-8. Exposure to sPM10 also resulted in continuous and time-dependent increases in release of IL-6 and IL-8 for up to 48 h. By comparison, in wPM2.5-exposed cells IL-8 release was not significantly augmented, while extracellular IL-6 levels were increased but remained constant beyond 24 h exposure. Moreover, wPM2.5 also reduced the lipopolysaccharide (LPS)-increased release of IL-8. No cytotoxicity or significant adsorption of cytokines to wPM2.5 were observed. Immunofluorescence microscopy revealed an accumulation of IL-8 in intracellular vesicles and alterations in actin filament organization in wPM2.5 exposed cells, suggesting that the trafficking of vesicles carrying interleukins to the plasma membrane might be inhibited. Thus, wPM2.5 appeared to impair cytokine release in BEAS-2B cells, in particular of IL-8, possibly by damaging cytoskeletal function involved in protein secretion. © 2018 Elsevier Ltd

Background: Exposure to particulate matter (PM) is associated with various health effects. Physico-chemical properties influence the toxicological impact of PM, nonetheless the mechanisms underlying PM-induced effects are not completely understood. Objectives: Human bronchial epithelial cells were used to analyse the pathways activated after exposure to summer and winter urban PM and to identify possible markers of exposure. Methods: BEAS-2B cells were exposed for 24 h to 10 μg/cm2 of winter PM2.5 (wPM) and summer PM10 (sPM) sampled in Milan. A microarray technology was used to profile the cells gene expression. Genes and microRNAs were analyzed by bioinformatics technique to identify pathways involved in cellular responses. Selected genes and pathways were validated at protein level (western blot, membrane protein arrays and ELISA). Results: The molecular networks activated by the two PM evidenced a correlation among oxidative stress, inflammation and DNA damage responses. sPM induced the release of pro-inflammatory mediators, although miR-146a and genes related to inflammation resulted up-regulated by both PM. Moreover both PM affected a set of genes, proteins and miRNAs related to antioxidant responses, cancer development, extracellular matrix remodeling and cytoskeleton organization, while miR-29c, implicated in epigenetic modification, resulted up-regulated only by wPM. sPM effects may be related to biological and inorganic components, while wPM apparently related to the high content of organic compounds. Conclusions: These results may be helpful for the individuation of biomarkers for PM exposure, linked to the specific PM physico-chemical properties. Exposure to particulate matters determines the activation of significant biological pathways and possible markers of exposure were identified.

Particulate matter (PM) exposure is related to pulmonary and cardiovascular diseases, with increased inflammatory status. The release of the proinflammatory interleukin- (IL-) 1β, is controlled by a dual pathway, the formation of inactive pro-IL-1β, through Toll-like receptors (TLRs) activation, and its cleavage by NLRP3 inflammasome. THP-1-derived macrophages were exposed for 6 h to 2.5 μg/cm2of Milan PM10, and the potential to promote IL-1βrelease by binding TLRs and activating NLRP3 has been examined. Summer PM10, induced a marked IL-1βresponse in the absence of LPS priming (50-fold increase compared to unexposed cells), which was reduced by caspase-1 inhibition (91% of inhibition respect summer PM10-treated cells) and by TLR-2 and TLR-4 inhibitors (66% and 53% of inhibition, resp.). Furthermore, summer PM10increased the number of early endosomes, and oxidative stress inhibition nearly abolished PM10-induced IL-1βresponse (90% of inhibition). These findings suggest that summer PM10contains constituents both related to the activation of membrane TLRs and activation of the inflammasome NLPR3 and that TLRs activation is of pivotal importance for the magnitude of the response. ROS formation seems important for PM10-induced IL-1βresponse, but further investigations are needed to elucidate the molecular pathway by which this effect is mediated.

Air pollution represents a global threat leading to large impacts on health and ecosystems and many European areas still show a poor air quality. Many measures and policies have been adopted in the past decades at European, national, regional and even local level and many tools have been developed to tackle this issue. Among these tools, the European Air Quality Directive places more emphasis on the use of models for air quality assessment and management. Within this context, air quality forecasting systems play an important role in supporting decision makers when short-term actions are required to reduce human health risks by limiting population exposure. In this framework, at European level within the Copernicus Atmosphere Monitoring Service (CAMS), the regional air quality models participating to the service provide 4-day daily forecasts of the main atmospheric pollutants concentrations, in the lowest layers of the atmosphere. This work presents the development and the performances evaluation of FORAIR_IT, an high-resolution air quality forecasting system operating at both European (20 km) and Italian (4 km) scales. Its skill results are compared with CAMS_50 interim ensemble reanalysis (IRA) ones and a long lasting PM10 exceeding event, occurred in Emilia Romagna region in October 2017, is studied in more detail. Results show similar skill scores between FORAIT_IT and CAMS_50. Comparing the annual average of the monthly Root Mean Square Error Difference (RMSED) between FORAIT_IT first forecast day and CAMS_50, over the European domain the RMSED is 0.6, 1.7, 1.4 and 7.4 μg/m3 for daily mean PM10 and PM2.5 and daily maximum for NO2 and O3, respectively, while over the Italian domain it is 1.2, 0.3, 4.3 and 3.8 μg/m3. The importance of increasing model resolution in the region of interest is highlighted by the lower values of RMSED over Italy with respect to Europe. The results obtained by the detailed analysis of the PM10 exceeding event suggests the crucial role of the meteorological forcing in capturing both the timing and the intensity of the exceedances. As far as we know FORAIR_IT is the first forecasting system at high spatial resolution at Italian National level.

Background: Lockdown measures during the SARS-CoV-2 pandemic determined radical changes to behavioral and social habits, that were reflected by a reduction in the transmission of respiratory pathogens and in anthropogenic atmospheric emissions. Objective: This ecological study aims to provide a descriptive evaluation on how restrictive measures during the SARS-CoV-2 pandemic impacted Pediatric Emergency Department (PED) referrals for asthma exacerbations, and their potentially associated environmental triggers in Bologna, a densely populated urban area in Northern Italy. Methods: Files of children evaluated for acute asthma during 2015 to 2020 at the PED of Sant'Orsola University Hospital of Bologna were retrospectively reviewed. Historical daily concentration records of particulate (PM2.5, PM10) and gaseous (NO2, C6H6) air pollutants, and pollen were concurrently evaluated, including specific PM chemical tracers for traffic-related air pollution (TRAP). Results: In 2020, asthma-related PED referrals decreased compared to referral rates of the previous 5 years (p < 0.01). This effect was particularly marked during the first lockdown period (March to May), when the drastic drop in PED referrals was associated with a reduction of high-priority cases up to 85% and by 54%, on average. A concomitant reduction in the concentrations of traffic-related air pollutants was observed in the range of 40%–60% (p < 0.01). Conclusions: The lower rate of asthma exacerbations in childhood was in this study paralleled with reduced TRAP levels during the pandemic. Synergic interactions of the multiple consequences of lockdowns likely contributed to the reduced exacerbations, including decreased exposure to ambient pollutants and fewer respiratory infections, identified as the most important factor in the literature.

International audience; The consequences of indoor and outdoor air pollution on human health are of great concern nowadays. In this study, we firstly evaluated indoor and outdoor air pollution levels (CO, CO2, NO, NO2, PM10) at an urban site in Dakar city center and at a rural site. Then, the individual exposure levels to selected pollutants and the variations in the levels of biomarkers of exposure were investigated in different groups of persons (bus drivers, traders working along the main roads and housemaids). Benzene exposure levels were higher for housemaids than for bus drivers and traders. High indoor exposure to benzene is probably due to cooking habits (cooking with charcoal), local practices (burning of incense), the use of cleaning products or solvent products which are important emitters of this compound. These results are confirmed by the values of S-PMA, which were higher in housemaids group compared to the others. Urinary 1-HOP levels were significantly higher for urban site housemaids compared to semirural district ones. Moreover, urinary levels of DNA oxidative stress damage (8-OHdG) and inflammatory (interleukin-6 and -8) biomarkers were higher in urban subjects in comparison to rural ones. The air quality measurement campaign showed that the bus interior was more polluted with PM10, CO, CO2 and NO than the market and urban or rural households. However, the interior of households showed higher concentration of VOCs than outdoor sites confirming previous observations of higher indoor individual exposure level to specific classes of pollutants.

Exposure to atmospheric pollutants has been recognized as a major risk factor of respiratory and cardiovascular diseases. Fine particles (PM2.5) and a coarser fraction (PM>2.5) sampled at an urban site in Dakar (HLM), characterized by high road traffic emissions, were compared with particles sampled at a rural area, Toubab Dialaw located about 40 km from Dakar. The physicochemical characteristics of samples revealed that PMs differ for their physical (surface area) and chemical properties (in terms of CHN, metals, ions, paraffins, VOCs and PAHs) that were 65–75% higher in urban samples. Moreover the fine PMs contain higher amounts of anthropogenic related pollutants than the PM>2.5 one. These differences are sustained by the ratios reported for the analysed PAHs which suggest as predominant primary emission sources vehicle exhausts at urban site and biomass combustion at the rural site. The inflammatory response and the oxidative damages were evaluated in BEAS-2B cells by the quantification of 4 selected inflammatory cytokines (TNF-α, IL-1β, IL-6, IL-8) and of total carbonylated proteins and the oxidative DNA adduct 8-OHdG after 8 or 24 h exposure. In accordance with the different sources and different physical and chemical properties, the inflammatory response and the oxidative damages were found higher in bronchial cells exposed to urban PMs. These data confirm the importance, also for West African countries, to evaluate the correlation between PM physico-chemical properties and potential biological impacts. Dakar air pollution, influenced by anthropogenic activities, causes inflammatory responses and oxidative damages in vitro.

During the last few years, the rise in woody biomass burning (BB) for household heating has caused an increase in PM mass concentrations, particularly for the fine fraction, in Europe, as reported by the European Environmental Agency. Estimating the contribution from biomass combustion to airborne particulate matter is therefore an important issue in air quality governance, due to its potential health and environmental impacts. Wood burning’s contribution to PM10 was estimated in winter at a rural site in southern Italy by means of two independent methods: source apportionment analysis with Positive Matrix Factorization (BBPMF) and the macro tracer approach, based on levoglucosan concentrations (BBLevo). PM10 and PM2.5 samples were collected every 24 h and every 8 h, respectively, and analyzed to determine the organic and elemental carbon, levoglucosan, inorganic ions and elements. The results obtained via these methods showed good agreement (r = 0.85), with a linear correlation slope of about 1, and provide a reliable assessment of the BB contribution. Woody biomass combustion contributed significantly to the PM10 (on average, slightly less than 30% of the total mass) during winter. The combination of the independent methods proposed here may be used as a methodology for refining the BB contribution to air pollution.

Particulate matter (PM) is a mixture of many different components with local and regional variations. Traffic is a major source of PM10, PM2.5, and ultrafine particles (UFP); traffic intensity contributes significantly to PM-related health effects. Recently attention has been paid to urban PM1 concentrations (50% of PM10) and its elemental composition, and it has been demonstrated that traffic intensity influences the PM physicochemical characteristics, particularly with regard to concentrations of polycyclic aromatic hydrocarbon (PAH), metals, and radical-generating capacity. PM2.5 and the even smaller UFP penetrate deeply into the lung and reach the alveoli and are thus more likely to induce adverse health effects than larger particles, such as PM10 and total suspended particles (TSPs). Epidemiological approaches are relevant, and numerous data have been furnished by the literature from 1970: time-series analyses and cohort studies, case-crossover studies, cross-sectional studies, panel studies, and case-control studies are the records present.

The results presented summarise the ones obtained in the coordinated research project Tosca, which extensively analysed the impact of Milan urban PM on human health. The molecular markers of exposure and effects of seasonally and size-fractionated PMs (summer and winter PM10, PM2.5) were investigated in in vitro (human lung cell lines) and in vivo (mice) systems. The results obtained by the analyses of cytotoxic, pro-inflammatory and genotoxic parameters demonstrate that the biological responses are strongly dependent upon the PM samples seasonal and dimensional variability, that ultimately reflect their chemical composition and source. In fact summer PM10, enriched in crustal elements and endotoxins, was the most cytotoxic and pro-inflammatory fraction, while fine winter PMs induced genotoxic effects and xenobiotic metabolizing enzymes (like CYP1B1) production, likely as a consequence of the higher content in combustion derived particles reach in PAHs and heavy toxic metals. These outcomes outline the need of a detailed knowledge of the PMs physico-chemical composition on a local scale, coupled with the biological hazard directly associated to PM exposure. Apparently this is the only way allowing scientists and police-makers to establish the proper relationships between the respirable PM quantity/quality and the health outcomes described by clinicians and epidemiologists.

PM10 was collected in a Milan urban site, representative of the city air quality, during winter and summer 2006. Mean daily PM10 concentration was 48 mug m(-3) during summer and 148 mug m(-3) during winter. Particles collected on Teflon filters were chemically characterized and the endotoxin content determined by the LAL test. PM10-induced cell toxicity, assessed with MTT and LDH methods, and proinflammatory potential, monitored by IL-6 and IL-8 cytokines release, were investigated on the human alveolar epithelial cell line A549 exposed to increasing doses of PM. Besides untreated cells, exposure to inert carbon particles (2-12 mum) was also used as additional control. Both cell toxicity and proinflammatory potency resulted to be higher for summer PM10 with respect of winter PM10, with IL-6 showing the highest dose-dependent release. The relevance of biogenic components adsorbed onto PM10 in eliciting the proinflammatory mediators release was investigated by inhibition experiments. Polymixin B (Poly) was used to inhibit particle-bind LPS while Toll-like receptor-2 antibody (a-TLR2) to specifically block the activation of this receptor. While cell viability was not modulated in cells coexposed to PM10 and Poly or a-TLR2 or both, inflammatory response did it, with IL-6 release being the most inhibited. In conclusion, Milan PM10-induced seasonal-dependent biological effects, with summer particles showing higher cytotoxic and proinflammatory potential. Cytotoxicity seemed to be unaffected by the PM biogenic components, while inflammation was significantly reduced after the inhibition of some biogenic activated pathways. Besides, the PM-associated biogenic activity does not entirely justify the PM-induced inflammatory effects. (c) 2010 Wiley Periodicals, Inc. Environ Toxicol 2010.

Tire debris (TD) and its organic components were identified as a main source of PM10 atmospheric and water pollution. Because few data are available on the embryotoxic effects of TD organic components, the lethal and teratogenic potential of tire debris organic extract (TDOE) was evaluated using the frog embryo teratogenesis assay-Xenopus (FETAX), coupled with a histopathological screening of the survived larvae. From stage 8 to stage 47, Xenopus laevis embryos were exposed to TDOE at concentrations of 50, 80, 100, 120 and 140 mg/L. The results showed 50 mg/L TDOE to be the non-observable effect concentration (NOEC). TDOE mortality at 80 mg/L was significantly higher than the control, but did not increase further with higher concentrations. A good concentration-response was observed for percentages of malformed larva and from 80 mg/L on these percentages were significantly higher than the control. Therefore, probit analysis gave a 144.6 mg/L TC50. At 120 and 140 mg/L, many larvae were plurimalformed. The most frequent alterations observed were abnormal gut coiling, microphthalmia, monolateral anophthalmia, and narrowing eyes. The histological screening mainly revealed ocular malformations such as double retina, retina nervous cell layer coiling, and altered lens. Moreover severe vacuolisation and necrosis were scored in liver and axial musculature. These results strongly support the assumption that TDOE is a powerful teratogen for X. laevis. Keywords: Tire debris organic extract, FETAX, Histology, Teratogenicity

Background: This study explores and characterizes cell cycle alterations induced by urban PM2.5 in the human epithelial cell line BEAS-2B, and elucidates possible mechanisms involved. Methods: The cells were exposed to a low dose (7.5 μg/cm2) of Milan winter PM2.5 for different time points, and the cell cycle progression was analyzed by fluorescent microscopy and flow cytometry. Activation of proteins involved in cell cycle control was investigated by Western blotting and DNA damage by 32P-postlabelling, immunostaining and comet assay. The formation of reactive oxygen species (ROS) was quantified by flow cytometry. The role of PM organic fraction versus washed PM on the cell cycle alterations was also examined. Finally, the molecular pathways activated were further examined using specific inhibitors. Results: Winter PM2.5 induced marked cell cycle alteration already after 3 h of exposure, represented by an increased number of cells (transient arrest) in G2. This effect was associated with an increased phosphorylation of Chk2, while no changes in p53 phosphorylation were observed at this time point. The increase in G2 was followed by a transient arrest in the metaphase/anaphase transition point (10 h), which was associated with the presence of severe mitotic spindle aberrations. The metaphase/anaphase delay was apparently followed by mitotic slippage at 24 h, resulting in an increased number of tetraploid G1 cells and cells with micronuclei (MN), and by apoptosis at 40 h. Winter PM2.5 increased the level of ROS at 2 h and DNA damage (8-oxodG, single- and double stand breaks) was detected after 3 h of exposure. The PM organic fraction caused a similar G2/M arrest and augmented ROS formation, while washed PM had no such effects. DNA adducts were detected after 24 h. Both PM-induced DNA damage and G2 arrest were inhibited by the addition of antioxidants and α-naphthoflavone, suggesting the involvement of ROS and reactive electrophilic metabolites formed via a P450-dependent reaction. Conclusions: Milan winter PM2.5 rapidly induces severe cell cycle alterations, resulting in increased frequency of cells with double nuclei and MN. This effect is related to the metabolic activation of PM2.5 organic chemicals, which cause damages to DNA and spindle apparatus. © 2013 Longhin et al.; licensee BioMed Central Ltd

Metal oxide NPs are abundantly produced in nanotech industries and are emitted in several combustion processes, suggesting the need to characterize their toxic impact on the human respiratory system. The acute toxicity and the morphological changes induced by copper oxide and titanium dioxide NPs (nCuO and nTiO2) on the human alveolar cell line A549 are here investigated. Cell viability and oxidative stress have been studied in parallel with NP internalization and cell ultrastructural modifications. TiO2 NPs were abundantly internalized by cells through the endocytic pathway, even they did not induce cell death and ultrastructural lesions. Only after 24h cells were affected by an abundant NP internalization presenting a consequent altered morphology. High cytotoxicity, oxidative stress and severe ultrastructural damages were produced by nCuO, since cell membrane and mitochondria resulted to be heavily affected, even at early exposure time. nCuO-induced toxicity has been interpreted as a consequence of both NPs reactivity and copper ions dissolution in lysosomal compartments, even the free NPs, scattered throughout all the cell compartments, might contribute to the toxicity. The antioxidant N-acetylcysteine was effective in recovering nCuO exposed cells viability and Bafilomycin A1 inhibited copper ions release in phagolysosomes and significantly rescued cells, suggesting a relevant cytotoxic mechanism relative to oxidative damages and authophagic cell death, together with NP internalization and dissolution. Our results support the previous data reporting CuO NPs are highly cytotoxic and genotoxic, and associate their toxic effects with their cell penetration and interaction with various compartments. In conclusion, the so-called "Trojan horse" mechanism and autophagy, are involved in nCuO-induced cell death, even a further research is needed to explain the events occurring at early exposure time. © 2013 Elsevier Ireland Ltd.

Exposure to engineered nanoparticles (NPs) and to ambient particles (PM) has increased significantly. During the last decades the application of nano-objects to daily-life goods and the emissions produced in highly urbanized cities have considerably augmented. As a consequence, the understanding of the possible effects of NPs and PM on human respiratory system and particularly on the air-blood barrier (ABB) has become of primary interest. The crosstalk between lung epithelial cells and underlying endothelial cells is indeed essential in determining the effects of inhaled particles. Here we report the effects of metal oxides NPs (CuO and TiO2) and of PM on anin vitromodel of the ABB constituted by the type II epithelial cell line (NCI-H441) and the endothelial one (HPMEC-ST1.6R). The results demonstrate that apical exposure of alveolar cells induces significant modulation of proinflammatory proteins also in endothelial cells.

Background Particulate matter (PM) has been associated to adverse health effects in exposed population and DNA damage has been extensively reported in in vitro systems exposed to fine PM (PM2.5). The ability to induce gene expression profile modulation, production of reactive oxygen species (ROS) and strand breaks to DNA molecules has been investigated in A549 cells exposed to winter and summer Milan PM2.5. Results A549 cells, exposed to 10 μg/cm 2 of both winter and summer PM2.5, showed increased cytotoxicity at 24 h and a significant increase of ROS at 3 h of treatment. Despite these similar effects winter PM induced a higher number of gene modulation in comparison with summer PM. Both PMs modulated genes related to the response to xenobiotic stimuli (CYP1A1, CYP1B1, TIPARP, ALDH1A3, AHRR) and to the cell – cell signalling (GREM1) pathways with winter PM2.5 inducing higher fold increases. Moreover the winter fraction modulated also JUN ( cell – cell signalling ), GDF15, SIPA1L2 ( signal transduction ), and HMOX1 ( oxidative stress ). Two genes, epiregulin (EREG) and FOS-like antigen1 (FOSL1), were significantly up-regulated by summer PM2.5. The results obtained with the microarray approach have been confirmed by qPCR and by the analysis of CYP1B1 expression. Comet assay evidenced that winter PM2.5 induced more DNA strand breaks than the summer one. Conclusion Winter PM2.5 is able to induce gene expression alteration, ROS production and DNA damage. These effects are likely to be related to the CYP enzyme activation in response to the polycyclic aromatic hydrocarbons (PAHs) adsorbed on particle surface.

A comparison of the effects produced by size-fractionated tire particles (TP10 and TP2.5) and similar-sized urban particulate matter (PM10 and PM2.5), collected in Milan in 2007, on the lungs of mice has been performed. The focus is on early acute lung responses following intratracheal instillation of aerosolized particles at a 3-h recovery period. Together with bronchoalveolar lavage (BAL) conventional endpoints like total and differential cell counts, total protein, alkaline phosphatase, lactate dehydrogenase and pro-inflammatory cytokines (TNF-alpha, MIP-2), the expression of different stress protein markers (caspase8, Hsp70, H0-1, NF-kB) was evaluated 3h after particle instillation into Balb/c mice. The TP2.5 fraction reached the alveolar spaces and produced an acute inflammatory response as evidenced by increased LDH and AP activities, total protein and Hsp70 content. TNF-alpha and MIP-2 production was significantly increased and polymorphonuclear neutrophils (PMN) recruitment was apparent. The TP10 fraction distributed mainly in the bronchial district and the only modified BAL parameter was the expression of MIP-2. PM2.5 induced an inflammatory response lesser in magnitude than that produced by PM10 fraction. The TNF-alpha increase was not significant, and HO-1, though significantly increased with respect to the control, was unable to reduce NF-kB activation, suggesting a role of the endotoxin component of PM in stimulating a pro-inflammatory limited response. This response was maximized by the PM10 that induced a significant increase in MIP-2, TNF-alpha, and HO-1. Lung immunohistochemistry showed fine particles, TPs in particular, being able to deeply penetrate and rapidly induce inflammatory events in the parenchyma, even involving endothelial cells, while PM10 produced a strong pro-inflammatory response mediated by the bronchiolar cells and residential macrophages of the proximal alveolar sacs, likely as a consequence of its larger dimension and endotoxin content. These results provide evidence of variable inflammatory mechanisms in mouse lungs in response to both urban PM and tire particles.

Air pollution in Milan causes health concern due to the high concentrations of particulate matter (PM10 and PM2.5). The aim of this study was to investigate possible seasonal differences in PM10 and PM2.5 chemical composition and their biological effects on pro-inflammatory cytokine release and cytotoxicity. The PM was sampled during winter and summer seasons. The winter PMs had higher levels of PAHs than the summer samples which contained a greater amount of mineral dust elements. The PM toxicity was tested in the human pulmonary epithelial cell lines BEAS-2B and A549. The winter PMs were more cytotoxic than summer samples, whereas the summer PM10 exhibited a higher pro-inflammatory potential, as measured by ELISA. This inflammatory potential seemed partly due to biological components such as bacterial lipopolysaccharides (LPS), as evaluated by the use of Polymixin B. Interestingly, in the BEAS-2B cells the winter PM2.5 reduced proliferation due to a mitotic delay/arrest, while no such effects were observed in the A549 cells. These results underline that the in vitro responsiveness to PM may be cell line dependent and suggest that the PM different properties may trigger different endpoints such as inflammation, perturbation of cell cycle and cell death.

Samples of PM(2.5) were gravimetrically collected during the winter 2005/2006 in the urban area of Milan (North Italy). Samples were chemically characterized and the particles were detached from filters to determine their cytotoxic effects on the A549 cell line. Based on the potential toxicological relevance of its components, Milan winter PM(2.5) contained high concentrations of pro-oxidant transition metals and PAHs, while re-suspended particles showed a relatively high frequency of dimensional classes ranging from 40 nm to 300 nm. A549 cells exposed to particle suspensions showed a concentration-dependent decrease in viability, starting from 10 microg/cm(2). Phagocytosis of particles by A549 cells and particle aggregates were morphologically characterized and seemed to depend on both particle concentration and exposure time, with the majority of particles being engulfed in membrane-bound vacuoles after 24h of exposure. The ability of ultrafine particles to penetrate and spread throughout the cells was also verified. Cell membrane lysis and mitochondrial ultrastructural disruption appeared to be the main modifications induced by PM(2.5) on A549 cells. Concomitantly to the adverse effects observed in terms of cell mortality and ultrastructural lesions, a significant intracellular production of reactive oxygen species (ROS) was observed, suggesting that the cytotoxicity, exerted by the winter PM(2.5) in Milan, derived also from its oxidative potential, probably associated with particle-adsorbed metals and PAHs.

Tire particles (TP) represent a significant component of urban air pollution (PM), constituting more than 10% of PM10 mass at urban locations with heavy traffic. The purpose of this study was to evaluate the effects of size-fractionated TP in an animal exposure model frequently used to assess the health effects of air pollutants. Potential pro-inflammatory and toxic effects of TP2.5 (2.5 microm) and TP10 (10 microm) were investigated through instillation of suspensions of these materials in BALB/c mice. Bronchoalveolar lavage fluid (BALF) was screened for total protein, lactate dehydrogenase (LDH), alkaline phosphatase (AP), and beta-glucuronidase (B-Gluc) as markers of cytotoxicity; glutathione (GSH) and superoxide dismutase (SOD) as markers of oxidative potential; and tumor necrosis factor-alpha (TNF-alpha), macrophage inflammatory protein-2 (MIP-2), and inflammatory cells as markers of inflammation. Concomitantly, histological analysis of TP-exposed lungs was performed. A single intratracheal instillation of 10 microg/100 microl, 100 microg/100 microl or 200 microg/100 microl was performed, and after 24h mice were euthanized and BALF examined. Inflammatory cellular profiles showed dose-dependent responses after TP10 exposure, while strong cytotoxic effects, including increases in total protein, LDH and AP, were observed to be associated to TP2.5 exposure. Histologically, TP10-treated lungs mainly showed inflammatory tissue infiltration, in contrast to TP2.5-treated lungs, where lysis of the alveolar barrier appeared to be the most characteristic lesion. Our biochemical, cytological, and histological results indicated differential lung toxicity mechanisms elicited by size-fractionated TP, in agreement with other studies performed in in vivo systems that have shown that lung responses to inhaled or instilled particles are affected by particle size. We conclude that lung toxicity induced by TP10 was primarily due to macrophage-mediated inflammatory events, while toxicity induced by TP2.5 appeared to be related more closely to cytotoxicity.

Debris produced from the attrition of tires of motor vehicles constitutes 5–7% of the atmospheric particulate matter (PM10). Debris particles are indeed small enough to enter human lung and thus morphological and chemical characterization has been performed.We demonstrated that the organic fraction of tire debris induces a dose-dependent increase in cell mortality, DNA damage, as well as a significant modification of cell morphology at the dose of 60 μg/ml, which may correspond to the quantity present in the air humans inhale daily. The present research aims at investigating if reactive oxygen species (ROS) production and Hsp70 expression are involved in the cascade of toxic effects produced on the A549 cell line, as it has been suggested for the ultrafine atmospheric particles and diesel exhaust. To this end, cells were exposed at the doses of 10, 50, 60, 75 μg/ml of TD organic extract (TDOE) and analyzed at different exposure time. ROS were detected by the oxidation of 2′7′-dichlorodihydrofluorescein diacetate to dichlorofluorescein, and fluorescence was measured by flow cytometry. Hsp70 protein expression was determined by immunochemical analysis, and protein expression quantification performed by optical densitometry. ROS production was analysed after 2 h of treatment. A statistically significant increase in fluorescence was observed and the intensity of the stress response was parallel to the increasing concentrations used. An evident increase of Hsp70 expression at lower doses (10, 50 μg/ml) and at longer exposure times (72 h) was observed, during the time that our previous studies showed that cell viability, plasma membrane integrity, and DNA molecules were not affected. Thus it can be deduced that the increase in Hsp70 expression protected the cells from those damages, which became evident at the higher doses, and that this parameter might be used as a sensitive indicator of exposure. These data suggest that ROS production may be the first event caused by A549 exposure to TDOE and this result is in line with other evidences provided for the role of ROS generation in ultrafine PM toxicity. It can be suggested that this event induces an overexpression of Hsp70 only at the lower doses and longer exposure time, when cells still appear unaffected. Subsequently when ROS generation reaches high levels, a general inhibition of protein synthesis probably occurs, culminating in cell toxicity. Keywords: A549 cell line, Fine particles, PM10, PM2, Hsp70, ROS, Tire debris

The potential toxicity of tire debris organic extracts on human alveolar epithelial cells (A549) was investigated. We analysed time- and dose dependent modifications produced on plasma membrane molecular composition and on lipid microdomains expression (caveolae and lipid rafts) that represent specific signalling platforms. Cells were exposed to increasing organic extract concentrations (10, 60 and 75 μg/ml) for 24, 48 and 72 h. An up to three fold dose and time dependent increase in specific protein markers of lipid microdomains was found, suggesting a corresponding increase in signalling platforms. Since the total pool of these plasma membrane markers was unchanged, we supposed that these proteins were translocated within the plasma membrane as to assemble the newly formed lipid microdomains. Despite no major modifications in lipid bilayer composition, a time- and dose dependent toxic effect was documented at 48 h of exposure by an increase of cells positive to Trypan Blue assay. After 48 h a dose dependent increase in the cell medium of the cytosolic enzyme lactate dehydrogenase was also observed, indicating greater damage of the plasma membrane as prenecrotic sign. The overall ultrastructural morphology of the plasma membrane of treated cells was not greatly modified, suggesting that organic extracts from tire debris cause focalized discontinuities on cell surfaces.

PURPOSE To determine whether asymptomatic 11778 LHON carriers demonstrated impairments in (1) chromatic red/green (R/G) and blue/yellow (B/Y) contrast sensitivity functions (CSF) and in (2) luminance contrast sensitivity functions in the spatial CSF (SCSF) and temporal CSF (TCSF) domains. METHODS Twenty-five carriers (8 male, 17 female; 34.1 +/- 15.1 years of age) of homoplasmic 11778 LHON from the same well-described family and 30 age-matched controls (17 male, 13 female; 29.2 +/- 7.1 years of age) were tested in one eye, randomly selected. Of the 25 eyes tested, 18 had normal fundus, 5 had swelling and microangiopathy, and 2 had temporal pallor. The R/G and B/Y CSFs were obtained after equiluminance correction with bichromatic horizontal sinusoidal gratings at 0.3, 0.7, and 2 cycles per degree (cpd); the SCSFs were obtained with achromatic gratings at 0.3, 2, 6, and 12 cpd; and the TCSFs were obtained at 2, 10, 20, and 33 Hz with sinusoidal modulation of a 2.7 degrees field with a superimposed spatial Gabor function. RESULTS Differences between carriers and controls were statistically significant for all spatial frequencies of chromatic and luminance SCSFs, but not for the TCSFs. R/G equiluminance settings of carriers differed from those of controls (P < 0.001), requiring higher luminance in the green; B/Y equiluminance settings were not statistically different in carriers and controls. Fundus findings did not correlate with CS results. CONCLUSIONS Luminance and chromatic spatial CS losses that affected all tested spatial frequencies, are reported in LHON asymptomatic carriers with the mtDNA 11778 mutation. No losses were found in the temporal CSF. An intriguing finding is that the blue system is substantially spared in this LHON family. These represent subclinical visual impairments in otherwise asymptomatic LHON carriers.

Particulate matter (PM) exposure is related to diverse health effects, ranging from respiratory and cardiovascular diseases, to cancer induction; the determination of the elicited effects can be consequence of PM characteristics. We have previously reported (Longhin et al., 2013) that Milan PM composition is related to the sampling season and the particles size, and these properties are responsible for different in vitro effects. Briefly, PM2.5 is dominated by combustion derived particles, consisting of a carbonaceous core with organic and inorganic compounds adsorbed on its surface. In this fraction, organic components are mostly present in winter season, and as a consequence, winter PM2.5 induces a high level of ROS formation and a DNA damaging potential. PM10 derives from abrasion and resuspension processes, and is enriched in crustal material (metals) and biological components (endotoxins), especially during summer season; thus human cells exposed to summer PM10 have a strong pro-inflammatory response. We have now investigated the global gene and microRNA expression changes by GeneChip technology in human bronchial epithelial cells (BEAS-2B) exposed to single dose of Milan winter PM2.5 or summer PM10 for 24 h PM2.5 and PM10 respectively modulated 162 and 144 genes, with 2 fold values respect to the control. Gene prioritization analysis by Toppgene revealed that PM exposure modulates molecular pathways such as TNF and NFKB signalling. Moreover season of sampling and PM size fraction have a specific impact in the regulation of miRNAs expression associated to inflammation and cancer.

Helicobacter-like organisms are frequently observed in the stomach of dogs but the relationship between these microorganisms and gastric pathology has not been clearly established. Different species of helicobacters are known to be present in the canine stomach but their specific prevalence in naturally infected dogs is unknown. The aims of this study were to isolate and characterize helicobacters in canine gastric biopsies, to compare the commonly used tests for the identification of Helicobacter spp. and to determine the occurrence of these species in dogs. Twenty-three out of 25 dogs (92%) were positive for Helicobacter-like organisms in cytological screening. Culture was successful from biopsies of 5/25 dogs. The isolates were analyzed by electron microscopy, biochemical and physiological tests, whole protein analysis and 16S rDNA sequencing. Helicobacter felis was identified in four samples and Helicobacter bizzozeronii in one sample. Only the whole protein analysis in combination with electron microscopy was able to clearly discriminate the two species. Compared to the high prevalence of Helicobacter-like organisms, the occurrence of H. felis and H. bizzozeronii, was low (17 and 4%, respectively). No Flexispira rappini-like organisms or H. salomonis were detected. Electron microscopy revealed that H. bizzozeronii- like microorganisms were present in three additional biopsies where we were unable to culture any Helicobacter-like organisms. These observations indicate that in the stomach of dogs not all helicobacters are culturable. The unculturable bacteria appeared to be the prevalent ones and may represent different spiral organisms. The presence of distinct helicobacters with different characteristics can reflect different roles in the pathogenesis of canine gastric disease.

We examined achromatic contrast discrimination in asymptomatic carriers of 11778 Leber‘s hereditary optic neuropathy (LHON 18 controls) and 18 age-match were also tested. To evaluate magnocellular (MC) and Parvocellular (PC) contrast discrimination, we used a version of Pokorny and Smith's (1997) pulsed/steady-pedestal paradigms (PPP/SPP) thought to be detected via PC and MC pathways, respectively. A luminance pedestal (four 1° × 1° squares) was presented on a 12 cd/m2 surround. The luminance of one of the squares (trial square, TS) was randomly incremented for either 17 or 133 ms. Observers had to detect the TS, in a forced-choice task, at each duration, for three pedestal levels: 7, 12, 19 cd/m2. In the SPP, the pedestal was fixed, and the TS was modulated. For the PPP, all four pedestal squares pulsed for 17 or 133 ms, and the TS was simultaneously incremented or decremented. We found that contrast discrimination thresholds of LHON carriers were significantly higher than controls' in the condition with the highest luminance of both paradigms, implying impaired contrast processing with no evidence of differential sensitivity losses between the two systems. Carriers’ thresholds manifested significantly longer temporal integration than controls in the SPP, consistent with slowed MC responses. The SPP and PPP paradigms can identify contrast and temporal processing deficits in asymptomatic LHON carriers, and thus provide an additional tool for early detection and characterization of the disease.

PURPOSE. Leber’s hereditary optic neuropathy (LHON) is a maternally inherited disease resulting in loss of central vision and dyschromatopsia, caused by mitochondrial DNA point mutations. However, only a subset of the mutation carriers becomes affected, with a higher penetrance in males. This study was conducted to investigate chromatic losses in asymptomatic carriers of the LHON mutation. METHODS. Monocular chromatic discrimination was studied with the Cambridge Colour Test (CCT; Cambridge Research Systems, Ltd., Rochester, UK) along the protan, deutan, and tritan cone isolation axes in 46 LHON carriers (15 men) belonging to the same LHON maternal lineage and 74 agematched control subjects (39 men). Inclusion criteria were absence of ophthalmic complaints and clear ocular media. A detailed neuro-ophthalmic examination was performed in all the LHON carriers. RESULTS. The differences in threshold between carriers and control subjects were significant for the three cone isolation axes at P 0.0001. Sixty-five percent of the carriers had abnormal protan and/or deutan thresholds; some of those with higher thresholds also had elevated tritan thresholds (13%). The male thresholds were higher and more frequent than those of the women for the protan and deutan axes (ANOVA; P 0.05), but not for tritan thresholds. In the most severe losses, the women had instances of diffuse defect whereas all the men displayed a red– green defect. CONCLUSIONS. Male LHON asymptomatic carriers had color vision losses with the red– green pattern of dyschromatopsia typical of patients affected with LHON, which includes elevation of tritan thresholds as well. This predominantly parvocellular (red– green) impairment is compatible with the histopathology of LHON, which affects mostly the papillomacular bundle. In contrast with male losses, female losses were less frequent and severe. These gender differences are relevant to understanding LHON pathophysiology, suggesting that hormonal factors may be of great importance. (Invest Ophthalmol Vis Sci. 2007;48:2362–2370) DOI:10.1167/iovs.06-0331