key: cord-0861011-3e7eorgd
authors: Barbara A, Maher; González-Maciel, A.; Reynoso-Robles, R.; Torres-Jardón, R.; Calderón-Garcidueñas, L.
title: Iron-rich air pollution nanoparticles: an unrecognised environmental risk factor for myocardial mitochondrial dysfunction and cardiac oxidative stress
date: 2020-06-21
journal: Environ Res
DOI: 10.1016/j.envres.2020.109816
sha: aa2611c40c11d7b50b87222eaa4cfbc29a8b0301
doc_id: 861011
cord_uid: 3e7eorgd

Exposure to particulate air pollution is a major environmental risk factor for cardiovascular mortality and morbidity, on a global scale. Both acute and chronic cardiovascular impacts have so far been attributed to particulate-mediated oxidative stress in the lung and/or via ‘secondary’ pathways, including endothelial dysfunction, and inflammation. However, increasing evidence indicates the translocation of inhaled nanoparticles to major organs via the circulation. It is essential to identify the composition and intracellular targets of such particles, since these are likely to determine their toxicity and consequent health impacts. Of potential major concern is the abundant presence of iron-rich air pollution nanoparticles, emitted from a range of industry and traffic-related sources. Bioreactive iron can catalyse formation of damaging reactive oxygen species, leading to oxidative stress and cell damage or death. Here, we identify for the first time, in situ, that exogenous nanoparticles (∼15 – 40 nm diameter) within myocardial mitochondria of young, highly-exposed subjects are dominantly iron-rich, and co-associated with other reactive metals including aluminium and titanium. These rounded, electrodense nanoparticles (up to ∼ 10 x more abundant than in lower-pollution controls) are located within abnormal myocardial mitochondria (e.g. deformed cristae; ruptured membranes). Measurements of an oxidative stress marker, PrP(C) and an endoplasmic reticulum stress marker, GRP78, identify significant ventricular up-regulation in the highly-exposed vs lower-pollution controls. In shape/size/composition, the within-mitochondrial particles are indistinguishable from the iron-rich, combustion- and friction-derived nanoparticles prolific in roadside/urban environments, emitted from traffic/industrial sources. Incursion of myocardial mitochondria by inhaled iron-rich air pollution nanoparticles thus appears associated with mitochondrial dysfunction, and excess formation of reactive oxygen species through the iron-catalyzed Fenton reaction. Ventricular oxidative stress, as indicated by PrP(C) and GRP78 up-regulation, is evident even in children/young adults with minimal risk factors and no co-morbidities. These new findings indicate that myocardial iron overload resulting from inhalation of airborne, metal-rich nanoparticles is a plausible and modifiable environmental risk factor for cardiac oxidative stress and cardiovascular disease, on an international scale.

Exposure to particulate air pollution is a major environmental risk factor for cardiovascular mortality and morbidity, on a global scale. Both acute and chronic cardiovascular impacts have so far been attributed to particulate-mediated oxidative stress in the lung and/or via 'secondary' pathways, including endothelial dysfunction, and inflammation. However, increasing evidence indicates the translocation of inhaled nanoparticles to major organs via the circulation. It is essential to identify the composition and intracellular targets of such particles, since these are likely to determine their toxicity and consequent health impacts. Of potential major concern is the abundant presence of iron-rich air pollution nanoparticles, emitted from a range of industry and traffic-related sources. Bioreactive iron can catalyse formation of damaging reactive oxygen species, leading to oxidative stress and cell damage or death.

Here, we identify for the first time, in situ, that exogenous nanoparticles (~15 -40 nm diameter) within myocardial mitochondria of young, highly-exposed subjects are dominantly iron-rich, and co-associated with other reactive metals including aluminium and titanium.

These rounded, electrodense nanoparticles (up to ~ 10 x more abundant than in lowerpollution controls) are located within abnormal myocardial mitochondria (e.g. deformed cristae; ruptured membranes). Measurements of an oxidative stress marker, PrP C and an endoplasmic reticulum stress marker, GRP78, identify significant ventricular up-regulation in the highly-exposed vs lower-pollution controls. In shape/size/composition, the withinmitochondrial particles are indistinguishable from the iron-rich, combustion-and frictionderived nanoparticles prolific in roadside/urban environments, emitted from traffic/industrial sources. Incursion of myocardial mitochondria by inhaled iron-rich air pollution nanoparticles thus appears associated with mitochondrial dysfunction, and excess formation of reactive oxygen species through the iron-catalyzed Fenton reaction. Ventricular oxidative

Exposure to fine particulate matter (PM 2.5 , < 2.5 µm in aerodynamic diameter) in air pollution is reportedly the largest environmental risk factor contributing to cardiovascular mortality and morbidity, globally [1] [2] [3] [4] . The most recent exposure-hazard calculations indicate that the annual excess mortality rate from ambient air pollution (mainly PM 2.5 ) in Europe is 790,000 [95% confidence interval (95% CI) 645 000-934 000], of which between 40 and 80% are due to cardiovascular events, which dominate health outcomes 5 . Short-term PM 2.5 exposure raises risk of acute myocardial infarction by up to 5% within a few days 3 . Longerterm (i.e. several years) exposures incur higher risk (~20%) of cardiovascular events, ascribed partially to development of associated cardiometabolic conditions, e.g. hypertension, diabetes mellitus 4 . Exposure to higher ambient PM 2.5 concentrations has also been linked specifically with the development of high-risk coronary plaques 6 .

Traffic-derived PM, arising from both exhaust and non-exhaust emissions 7 may dominate individual exposures and cardiovascular outcomes. In a Dutch cohort, living near major roads was associated with increased cardiopulmonary mortality (relative risk, RR:

1.95, 95% CI: 1.09-3.52) 8 . German studies showed a RR of 1.85 (95% CI: 1.21-2.84) in patients living within 150 m of a major road 9 . The majority of particles emitted from trafficrelated PM sources are ultrafine (< 100 nm) in size. Most comprise (semi-) volatile carbonbearing aerosols (some with non-volatile cores). The solid, inorganic fraction is dominated by transition metals, and especially by potentially bioreactive iron oxides, produced in abundance from brake-wear and from exhaust emissions 7, 10, 11 . Despite their toxicity and potential ability to gain access to any organ of the body via ingestion, inhalation and/or the circulation, ultrafine particles are neither monitored nor regulated at the current time.

Ultrafine particle numbers show little correlation with measurements of PM 2. 5 12 .

So far, the mechanisms linking the statistical associations between exposure to PM 2.5 and cardiovascular impacts have been attributed to PM-mediated oxidative stress in the lung and/or more systemically across vascular beds, and/or to 'secondary' effector pathways, including endothelial barrier disruption, inflammation, arrhythmogenesis and pro-thrombotic processes 1, 2, 4 . However, major gaps remain in our understanding of the causality underlying the epidemiological associations between PM exposure and CVD, and, critically, of the specific causal components and CV targets of airborne PM. The translocation of air pollution nanoparticles from their portal of entry to remote tissues may constitute the key link between exposure to particulate air pollution and the observed and multiple epidemiological associations 1, 13, 14 .

Recent, landmark studies have demonstrated (post mortem), direct penetration of air pollution nanoparticles into the human brain 14 and heart 13 , and (experimentally) the systemic circulation of gold nanoparticles in mice and humans 15 . These studies indicate that exogenous nanoparticles can be translocated to major organs. Such particle translocation might constitute a key pathway accounting for the associations between exposure to airborne PM and the multiple manifestations of damage to the CV system. Critical, however, is to identify the composition and intracellular location of exogenous particles, since these will influence their reactivity and cytotoxicity in target tissues remote from their portal of entry.

Here, building upon our prior investigation 13 , we identify for the first time, in situ, the composition of exogenous nanoparticles in human myocardial mitochondria. These new findings substantiate our identification of these within-mitochondrial nanoparticles as air pollution-, combustion-and friction-derived nanoparticles, and underline their likely major impacts upon mitochondrial function and cardiac oxidative stress -even in very young cases with minimal conventional risk factors and no co-morbidities.

Here, we examined post mortem left ventricular tissue from 2 cases (3y and 26y) randomly The Instituto de Ciencias Forenses approved the collection of samples from forensic autopsies. Autopsies were performed 3.7 ± 1.9 hours after sudden death (accidents, homicides, suicides); subjects had no pathological evidence of inflammatory processes, CVD, chest trauma, head injury, or stroke. For TEM, heart sections were dissected and cut with ceramic knifes and plastic forceps, free from metal contamination, and fixed in 2% paraformaldehyde and 2% glutaraldehyde in sodium phosphate buffer.

We used TEM (FEI Titan3 Themis 300, X-FEG S/TEM with S-TWIN objective lens, monochromator (energy spread approx. 0.25 eV) and multiple HAADF/ADF/BF STEM detectors, operated at 300 kV) and energy dispersive X ray analysis (EDX, FEI Super-X 4detector system) to examine the intra-cellular location of electrodense nanoparticles, and their shape, size, and, perhaps most critically in terms of toxicity and biological impact, their elemental composition. To limit beam-induced damage, a probe current of 60 pA was used to acquire the elemental maps. To ensure that that the nanoparticles identified and analysed can be considered representative of the samples, samples were analysed blind to case, and grids/tissue sections and grid areas were randomly selected and methodically scanned.

Whereas left ventricular tissue and mitochondria from a control subject were normal ( Fig. 1A) , TEM imaging in the Mexico City 3y old (Fig. 1B) The TEM/EDX data identify that the composition of these electrodense, rounded, within-mitochondrial nanoparticles ( Fig. 2A , B, D and E) is metal-rich, and dominated by iron (oxides) (Fig. 2C and F) . Additional, trace exogenous species co-associated with the iron oxide nanoparticles include aluminium and titanium (Fig. 2C) . The mitochondria containing these iron-rich nanoparticles appear damaged, with deformed, fragmented or missing cristae and altered, sometimes ruptured membrane structures (Fig. 1B) . Even in the very young Mexico City cases examined here, cardiac oxidative stress is evident, with significant up-regulation of the normal cellular isoform of the prion protein, PrP C , which acts as a key oxidative stress response. PrP C values are 3.16 (3 y old) and 9.25 (26 y old), against an average of 1.06 (variance 0.004) for 9 controls (the mean of the 63 exposed cases = 8.7 x mean 9 controls, p = 0.0003) 13 . Significant up-regulation of GRP78, a marker of endoplasmic reticulum stress, is also evident in the Mexico City cases: 1.485 (3 y old) and 4.227 (26 y old) versus an average of 1.05 (variance 0.00489) for 9 controls, (mean, 63 exposed cases = 3.2 x mean 9 controls, p = 0.0001) 13 . These data are shown in Supplementary Table 1 .

That the composition of these within-mitochondrial nanoparticles is dominated by the presence of iron is likely to have major implications for mitochondrial function and cardiac oxidative stress. Mitochondria are particularly abundant in ventricular tissue, driving energy supply, and modifying cell signalling by continuous production of reactive oxygen species (ROS), such as the superoxide anion (O 2 (−) ) and hydrogen peroxide (H 2 O 2 ). But with iron overload, the iron-catalyzed Fenton reaction can transform these less oxidising products into the highly reactive hydroxyl radical (HO·) which can attack proteins, lipids and DNA, leading to cell and tissue damage. Indeed, chronic and acute increases in myocardial mitochondrial ROS production can lead to a catastrophic cycle of mitochondrial DNA damage, functional decline, and further oxygen radical generation [16] [17] [18] . Resultant cellular injury can include myocyte hypertrophy, endothelial dysfunction, apoptosis, and thrombosis 18 , and progressive modification of myocardial gene responses to oxidative and endoplasmic reticulum stress 16, 19 . Thus, iron-driven myocardial mitochondrial dysfunction and uncontrolled ROS production can play a major role in initiation and progression of cardiovascular disease. That cardiac myopathy can result from mitochondrial iron overload is evident, for example, in Friedreich's ataxia, an inherited neurological disease associated with iron dysregulation 20 . For exposed urbanites, excess mitochondrial iron may be an environmental, rather than a genetic, risk factor; iron-rich air pollution particles readily available in acute and/or chronic doses via inhalation.

In size, shape and composition, the within-mitochondrial, iron-dominated nanoparticles identified here in the Mexico City cases (Fig. 2) are indistinguishable from the iron-rich nanoparticles so abundant and pervasive in urban and roadside air pollution (Fig. 3B − F). Of key potential concern for cardiovascular outcomes is that, almost invariably, iron is the most abundant metal in the solid (non-volatile) fraction of the ultrafine particles present in urban air pollution (Fig. 3A) . Figure Strongly magnetic nanoparticles thus comprise ~1% of the total roadside particle numbers (much of the remainder being composed of elemental carbon and volatile organic carbon compounds) and ~10% of the primary (non-volatile) nanoparticles 21 .

It is likely that many components of airborne PM can elicit oxidative stress, whether singly and/or synergistically with other components. In the case of iron, it is also likely that the particle size, specific iron phase, and co-associated metal species, together with any surface corona formed upon interaction with the biological substrates encountered, will play important roles in governing subsequent impacts in the intra-cellular environment. Because iron can have many different particulate sources (including natural, windblown dust), it may be that some previous studies have not discriminated between them, producing an 'umbrella' analysis of total iron, rather than of its more bioreactive, ultrafine components. (In fact, there are numerous studies of PM composition which do not provide Fe content, presumably because of the catholic nature of its sources). In the increasing number of studies examining the impacts of iron oxide nanoparticles on cells, it is clear that those impacts vary widely depending on both the properties of the iron oxide particles used, and the types of cell lines and antioxidants used. Overall, more, and more specific, information and data are necessary in order to resolve the question of the relative potential impacts of different PM species.

Evident from our current findings, however, is that iron dominates the composition of the within-mitochondria nanoparticles; perhaps unsurprising since iron is frequently the dominant metal species in the solid fraction of the ultrafine particles produced and emitted in urban airborne pollution.

The abundance of iron-rich mitochondrial air pollution nanoparticles in myocardial cells, even in a 3 y old, indicates their direct translocation to the heart following repeated inhalation exposure in urban environments. The myocardial iron-rich nanoparticles are coassociated with other potentially bioreactive, exogenous metals (e.g. Al 28 , Ti 29 , Fig. 2 Finally, given that many of the iron-rich nanoparticles in the left ventricular tissues of the exposed cases are strongly magnetic (magnetite and/or maghemite) 13 , then such particles may respond to external magnetic fields (e.g. from household appliances, like hair dryers 31 and mobile phones, and from occupational exposures, such as those reported for welders, and power line engineers 32 ). Depending on their location and ferrimagnetic response (the latter determined by magnetite particle size, concentration and inter-particle interactions), magnetic pollution particles translocated to the heart might feasibly induce heart electrical dysfunction and cell damage, whether by magnetic rotation or hyperthermia.

In conclusion, these findings indicate that acute and chronic exposures specifically to iron-rich airborne nanoparticles, which are abundant in the urban environment, constitute a plausible, pervasive risk factor for cardiac mitochondrial dysfunction and subsequent CVD development, from earliest childhood. Critically, exposure to such air pollution represents a modifiable risk factor for CVD, on a global scale, reinforcing the urgent need for individual and government actions not just to reduce PM 2.5 but to monitor, regulate and reduce emissions of these specific, ultrafine components of the urban air pollution 'cocktail'. Given that exposure to air pollution is estimated to cause > 3 million premature, CVD-related deaths annually (more than arise from conventional cardiac risk factors such as smoking, obesity or diabetes) 33 , it seems essential that health care professionals assess cardiovascular risk from iron-rich air pollution exposure at every life stage, including (and perhaps especially) the prenatal and childhood phases.

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The authors thank Zabeada Aslam and Mark S'Ari of Leeds University's EPSRC Nanoscience and Nanotechnology Facility (LENNF) and Partha S. Mukherjee from the Indian Statistical Institute, Kolkata, India for their support and assistance in this work. This work was partially supported by E022 Instituto Nacional de Pediatría.

JaguarLandRover, UK. The remaining authors have nothing to disclose.

Air pollution nanoparticles inside human myocardial mitochondria are iron-richThe within-mitochondrial particles match iron-rich particles from traffic/industrial sources Myocardial iron-rich air pollution nanoparticles abundant even in young children Ventricular upregulation of oxidative and ER stress markers in exposed subjects Inhalation of iron-rich air pollution a risk factor for cardiovascular disease

☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☒The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:There is no competing financial interest but Barbara Maher receives funding for a PhD Studentship currently from JaguarLandRover, UK< for research on brakewear emissions.