Air pollution particles and iron homeostasis

    Air pollution particles and iron homeostasis Andrew J. Ghio, Joleen M. Soukup, Lisa A. Dailey PII: DOI: Reference:

S0304-4165(16)30168-4 doi: 10.1016/j.bbagen.2016.05.026 BBAGEN 28498

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BBA - General Subjects

Received date: Revised date: Accepted date:

27 January 2016 4 May 2016 19 May 2016

Please cite this article as: Andrew J. Ghio, Joleen M. Soukup, Lisa A. Dailey, Air pollution particles and iron homeostasis, BBA - General Subjects (2016), doi: 10.1016/j.bbagen.2016.05.026

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Air pollution particles and iron homeostasis

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Andrew J. Ghio, Joleen M. Soukup, and Lisa A. Dailey

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National Health and Environmental Effects Research Laboratory, Environmental Protection Agency, Chapel Hill NC

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Correspondence should be addressed to: Andrew Ghio, Human Studies Facility, 104 Mason Farm Road, NC 27711 Telephone #: (919)-966-0670 FAX #: (919)-966-6367

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Running title: Particles and iron

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ACCEPTED MANUSCRIPT Abstract Background: The mechanism underlying biological effects, including pro-inflammatory

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outcomes, of particles deposited in the lung has not been defined.

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Major Conclusions: A disruption in iron homeostasis follows exposure of cells to all particulate matter including air pollution particles. Following endocytosis, functional groups at the surface

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of retained particle complex iron available in the cell. In response to a reduction in

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concentrations of requisite iron, a functional deficiency can result intracellularly. Superoxide production by the cell exposed to a particle increases ferrireduction which facilitates import of

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iron with the objective being the reversal of the metal deficiency. Failure to resolve the functional iron deficiency following cell exposure to particles activates kinases and transcription

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factors resulting in a release of inflammatory mediators and inflammation. Tissue injury is the end product of this disruption in iron homeostasis initiated by the particle exposure. Elevation of

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available iron to the cell precludes deficiency of the metal and either diminishes or eliminates biological effects.

General Significance: Recognition of the pathway for biological effects after particle exposure to involve a functional deficiency of iron suggests novel therapies such as metal supplementation (e.g. inhaled and oral). In addition, the demonstration of a shared mechanism of biological effects allows understanding the common clinical, physiological, and pathological presentation following exposure to disparate particles.

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ACCEPTED MANUSCRIPT Highlights: · A disruption in iron homeostasis is proposed as the pathway common to the biological effects

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of all particulate matter including air pollution particles

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· Oxidative stress, kinase and transcription factor activation, release of inflammatory mediators, and inflammation in particle-exposed cells result from a functional iron deficiency

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· Recognition of the pathway for biological effects after particle exposure to involve a functional

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deficiency of iron suggests novel therapies such as metal supplementation

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Key words: Air pollution; iron; inflammation; oxidants; oxidative stress; kinases; transcription

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factors

Abbreviations: DMT1, divalent metal transporter 1; FAC, ferric ammonium citrate; HBE, human

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bronchial epithelial; HULIS, humic-like substances; IRE, iron-responsive element; IRP, ironregulatory protein; MAP kinase, mitogen-activated protein kinase; PM, particulate matter; TfR, transferrin receptor; WSP, wood smoke particle

Acknowledgement: This report has been reviewed by the National Health and Environmental Effects Research Laboratory, United States Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendations for use.

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ACCEPTED MANUSCRIPT Introduction Inhalation of suspended particulate matter (PM) has presented a challenge to human

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health for thousands of years [1]. This PM was initially of crustal and plant origin but later

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included particles generated from the burning of biomass to meet the needs of both heating and cooking. Larger anthropogenic contributions to ambient air pollution arrived with

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industrialization and accelerated with the development of automotive transport in the 20th

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century.

Episodes of extremely high levels of ambient air pollution such as that in the Meuse

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Valley of Belgium in 1930 and Donora, Pennsylvania in 1948 were observed to be associated with biological effects in humans (e.g. acute elevations in mortality) [2, 3]. Between 1948 and

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1962, eight wintertime air pollution episodes occurred in London, England. Excess deaths during the episode in December of 1952 alone numbered in the thousands [4]. In the United

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States, increasing concerns regarding the health consequences of air pollution culminated in the Clean Air Act of 1970. Despite reductions in air pollution which followed its implementation, epidemiologic investigation during the 1980s and 1990s continued to demonstrate an association between 1) exposure to airborne PM at levels observed in cities both in the United States and worldwide and 2) indices of acute human morbidity and mortality [5]. These findings were initially met with skepticism but both re-evaluation and a plethora of new investigation provided concordant results [6, 7]. Accordingly, the National Ambient Air Quality Standards were altered in 2006 to provide new annual and daily levels of PM2.5 and PM10 (particulate matter with diameters <2.5 and <10 micron respectively). In addition to the PM in ambient air pollution, the human lung can regularly be exposed to a variety of particles including those from cigarette smoke, burning of biomass, dust storms,

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ACCEPTED MANUSCRIPT mining/processing of coal and mineral oxides, agricultural work, wild fires, environmental tobacco smoke, emissions from traffic, and gas and wood stoves (Table 1). Features of the

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clinical presentation and changes in human physiology and pathology following exposure to

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these diverse particles appear to be comparable to some extent and can include 1) respiratory symptoms of cough, wheezing, and shortness of breath, 2) an acute, reversible decrement in

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pulmonary function and elevation in bronchial hyperreactivity, 3) histopathological changes of

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acute neutrophilic inflammation, emphysema, and parenchymal fibrosis, 4) hemorheological changes with elevations in white blood cell counts and increases in C-reactive protein,

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fibrinogen, and blood viscosity, and 5) an association with cardiovascular disease [8-14]. This shared clinical, physiological, and pathological presentation associated with exposure to many

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disparate particles suggests a common mechanism for their biological effects. Therefore, a

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single pathway is proposed through which biological effects of all particles are generated.

Mechanistic pathway underlying the biological effects of particles The mechanism underlying biological effects of particles deposited in the lung has not been defined. A disruption in iron homeostasis is a pathway common to the biological effects following exposure to all PM including air pollution particles (Figure 1). Functional groups at the surface of retained particle can complex iron available in the cell. In response to a reduction in concentrations of requisite iron, the cell increases metal import attempting to reverse a functional deficiency associated with the particle complexation of metal. Superoxide production by the cell exposed to a particle increases ferrireduction which further facilitates import of iron. Incomplete resolution of the functional iron deficiency following cell exposure to particles activates kinases and transcription factors which results in a release of inflammatory mediators

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ACCEPTED MANUSCRIPT and inflammation (Figure 2). Tissue injury, including lung, cardiac, and vascular disease, is the

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end product of this disruption in iron homeostasis initiated by the particle.

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Iron homeostasis in the healthy human

Iron is an essential micronutrient required for normal cell function. A favorable

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oxidation-reduction potential and a relative abundance in nature has led to its selection for a wide

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range of functions. However, following the introduction of oxygen to the atmosphere as a product of photosynthesis, water-soluble ferrous ion (Fe2+) was effectively removed from the

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Earth’s crust. Ferric ion (Fe3+) remained but at concentrations inadequate to meet the requirements for life since it is insoluble in water; the concentrations of Fe3+ in water at

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physiologic pH values is about 10-18 M while that required for life approaches 10-6 M. To support life, greater quantities of metal had be procured. This challenge to living systems was

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achieved by 1) the chemical reduction of Fe3+ to Fe2+ (i.e. ferrireduction) with its subsequent import and utilization and 2) the complexation of Fe3+ with chelators coupled with receptors for uptake of the complex and employment of the metal. In addition to solubility limiting its availability, iron-catalyzed generation of radicals presented a potential for oxidative stress. Such reactivity mandated that iron homeostasis be tightly regulated. Living systems evolved strategies to regulate the procurement of adequate iron for cellular function and homeostasis without major damage to biological macromolecules. As a result, life exists at the interface of iron-deficiency and iron-sufficiency. Cellular iron homeostasis is maintained by a coordinated expression of proteins involved in the import, export, storage, and utilization of this metal [15]. Post-transcriptional control mediated by iron-regulatory proteins (IRPs) is essential [16-19]. IRP1, the cytosolic counterpart

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ACCEPTED MANUSCRIPT of mitochondrial aconitase, is a bifunctional protein that, through [4Fe-4S] cluster assembly/disassembly, shifts from the aconitase to the IRP form in response to decreased

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intracellular iron concentrations [20]. Accordingly, iron levels regulate RNA-binding capacity

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of IRP1. With diminished availability of the metal, IRP1 binds to cis-acting mRNA motifs termed iron-responsive elements (IREs) to impact the expression of proteins involved in uptake,

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storage, and export. This includes stabilizing the mRNA of the divalent metal transporter 1

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(DMT1) and transferrin receptor (TfR) to promote translation and increase their expression while suppressing the synthesis of the storage protein ferritin and exporter ferroportin 1 [21-23].

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The serum ferritin level is considered to be the best indicator of stored and total body iron [24]. It is assumed that there is an association between the total iron and the concentration

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available to cells, tissues, and living systems. Normally, concentrations of iron inadequate to support the function of cells, tissues, and living systems are reflected by levels of serum ferritin,

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serum iron, transferrin, transferrin receptor, and other proteins involved in iron homeostasis. Insufficient iron can result from both an absolute and a functional deficiency of the metal. In an absolute deficiency, iron concentrations are lacking as a result of decreased intake or an elevated loss; provision of the metal can improve such a condition. A functional deficiency frequently develops from the introduction of an inappropriate chelator which binds host iron decreasing concentrations available to the cell (e.g. microbial membranes, aminoglycosides, and doxorubicin). The host response to an inappropriate chelator and a functional deficiency in iron includes increased expression of importers in an attempt by the affected cells to reverse the loss of requisite metal. Concurrent with the elevated levels of metal importers following exposures to inappropriate chelators, there is an increased expression of storage protein (i.e. ferritin). Persistence of the chelator can prevent correction of the functional deficiency. Provision of

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ACCEPTED MANUSCRIPT additional iron is unlikely to reverse the associated functional deficiency of metal.

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Particles and iron homeostasis

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In an aqueous environment, inorganic particles including oxides and oxide minerals have oxygen-containing functional groups at their surface (e.g. Si-OH; silanol groups). Therefore, the

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deposition of particles with surfaces comprised of oxygen-containing functional groups

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introduces an electronegative interface into the lower respiratory tract following deprotonation at physiologic pH values. Among the cellular cations available for complexation by the particle

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surface, iron is kinetically preferred as a result of its electropositivity, high affinity for oxygencontaining functional groups, and relative abundance. Following endocytosis of an inorganic

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particle, surface functional groups react with cell iron to produce a coordination complex [25]. In response to this loss of essential metal by the cell to the particle, IRP can be activated and iron

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import, such as that by TfR and DMT1, is elevated to meet cell requirements. In the lower respiratory tract, retained particles have consistently demonstrated this capacity to accumulate iron from available cell sources reflecting the ability of the surface to complex host iron [26, 27]. The host responds to a loss of requisite metal with attempts to re-sequester the iron so as to make it less available to the particle surface. This is accomplished, in some measure, through storage in a less catalytically-reactive state within ferritin. Following the introduction of any particle into the lung, this disruption of iron homeostasis and the attempt to re-establish normal metal equilibrium in the host can result in formation of a ferruginous body [27, 28]. When the lungs of animals exposed to an inorganic particle are digested and the particle isolated, the concentration of surface iron is demonstrated to be increased supporting an appropriation of host iron by the PM. Furthermore, endpoints reflecting oxidative stress and biological effects correspond to

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ACCEPTED MANUSCRIPT elevated iron concentrations (both particle and tissue) following in vivo exposure [26]. As a result of its historical significance, silica was employed as a prototype of an

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inorganic particle that can be included in air pollution. After in vitro exposure of human

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bronchial epithelial (HBE) cells to silica, isolation of the particle demonstrated a concentration of iron which was measurable (0.20 ± 0.01 µg/g) and significantly elevated relative to that with no

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cell exposure (below detectable limits) [29]. While the silica had increased levels of iron

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following its endocytosis, it was not saturated with iron (6.00 ± 0.58 µg/g). Non-heme iron concentrations in the mitochondrial fraction of HBE cells decreased

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following 5 minute exposure to silica (Figure 3A). In contrast, iron in the nuclear fraction increased following the same particle exposure (Figure 3A). Mitochondrial sources of iron were

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complexed by silica which was then centrifuged into the nuclear fraction. This demonstrates the capacity of an inorganic particle to complex cell sources of iron and disrupt the homeostasis of

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the metal in the cell. There was a 3.7±1.1 fold increase in DMT1 RNA after 4 hour exposure of HBE cells to 100 µg/mL silica [29]. This change in RNA for a major iron importer supports a reduction in concentrations of available iron intracellularly after metal complexation by the endocytosed particle. Exposure of HBE cells to iron (ferric ammonium citrate; FAC) confirmed iron import (Figure 3B) [29]. However, co-incubation of HBE cells with both silica and FAC was associated with a greater accumulation of iron relative to either iron or silica exposure alone reflecting both a loss of the metal following particle exposure and increased import to correct the deficiency (Figure 3B). Following silica exposure, HBE cell ferritin concentrations increased after exposure to FAC and were further elevated with co-incubations of silica and FAC (Figure 3C) [29]. This reflects the same loss of cell metal to the silica with subsequent elevated iron import to correct the deficiency.

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ACCEPTED MANUSCRIPT Particle complexation of cell iron is especially pertinent to PM that contains organic compounds (e.g. diesel exhaust particle, wood stove particle, and PM associated with cigarette

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smoking and burning of biomass). Surface functional groups in these particles can include

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alcohol, diol, epoxide, ether, aldehyde, ketone, carboxylate, and ester groups. Following exposure to such particles, organic constituents (e.g. humic-like substances or HULIS) retained

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in the lung can complex available metal via these functional groups [30, 31] This can be

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observed in vivo with a formation of ferruginous bodies in exposed individuals (e.g. survivors of fires involving wooden structures) [32].

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Wood smoke particle (WSP) was utilized as an example of an organic particle which can be included in air pollution. However, other carbonaceous particles can impact iron homeostasis

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in a similar manner [33, 34]. Iron in a respiratory epithelial cell line (i.e. BEAS-2B cells) was complexed by WSP demonstrating a capacity to bind the metal via oxygen-containing functional

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groups. Comparable to the exposures to silica, mitochondrial concentrations of metal were shown to decrease following exposure to WSP (Figure 4A). Function in both specific individual organelles and the cell is accordingly compromised following such metal loss unless the normal intracellular iron concentration is re-established. The cell therefore responds to the WSP, its complexation of host iron, and a relative iron deficiency by up-regulating import of iron. After exposure to 100 µg/mL WSP for 4 hours, RNA for DMT1 significantly increased 1.8 ± 0.4 fold reflecting insufficient iron concentrations in the respiratory epithelial cells [35]. Following exposures to either WSP or FAC, cell non-heme iron increased relative to PBS (Figure 4B). However, cell incubations which included both WSP and FAC showed the greatest elevations in cell iron concentrations. This establishes that WSP could significantly increase metal import supporting an increased cell avidity for iron following exposure to this particle. Furthermore,

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ACCEPTED MANUSCRIPT cell concentration of the iron-storage protein ferritin was elevated following 24 hour exposure of BEAS-2B cells to either FAC or WSP but was greatest when both were included (Figure 4C).

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In addition to inorganic and organic particles, there is PM which includes significant

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levels of metal and demonstrates biological effects (e.g. oil fly ash with considerable quantities of water-soluble vanadium compounds). Comparable to inorganic and organic particles, the

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biological effects of these metals can also result from a disruption of iron homeostasis.

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Vanadium has been evaluated but other metals are comparable. In living systems, an impact of vanadium on iron homeostasis is suggested as exposures to vanadium-containing compounds

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increase iron concentrations [36, 37]. Following exposure to VOSO4, vanadium could be measured in both the nuclear and mitochondrial fractions of BEAS-2B cells and the supernatant

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at increased concentrations [38]. In contrast, non-heme iron in the nuclear and mitochondrial fractions was decreased immediately following the exposure to VOSO4 while there was an

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increased concentration of non-heme iron in the supernatant (Figure 5A) [38]. Vanadyl sulfate exposure revealed a capacity to reduce iron levels in both nuclear and mitochondrial fractions. RNA for DMT1 increased 3.4±0.7 fold following 4 hour exposure of BEAS-2B cells to 100 µM VOSO4, supporting a cell deficit in iron. Respiratory epithelial cells exposed to vanadyl sulfate showed a significantly increased import of iron (Figure 5B). Results suggest that the transport of vanadyl cation into the cell presents a potential for displacement of iron from pivotal intracellular sites. In response to such loss of iron following exposure to vanadyl sulfate, iron import was demonstrated to increase in an attempt to meet cell requirements. The insufficiency in cell iron triggered an elevation in DMT1. Co-exposure to VOSO4 and FAC also increased the expression of ferritin, the major metal storage protein in respiratory epithelial cells (Figure 5C). These observations suggest that, following an immediate deficit of iron in the cell with exposure to

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ACCEPTED MANUSCRIPT vanadium, a new iron homeostasis was achieved with augmented cell iron concentrations and ferritin concentrations (measured at 4 and 24 hours respectively). Elevated cell iron

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concentrations after exposure to VOSO4 allow for continued availability to the cell of this

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requisite metal, sustained function, and survival. The specific sources of iron displaced by the vanadyl cation and pathways of iron loss from mitochondria were not defined. Vanadium has

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demonstrated a capacity to interact with iron incorporated in several proteins including ferritin,

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transferrin, and lactoferrin [39-42]. Comparable to this pathway of biological effects proposed for vanadium, cell (e.g. mitochondrial) iron-proteins can be a target of other metals with loss of

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iron from such sites associated with toxicity [41].

Exposure to inorganic particles, organic particles, and soluble metal included in PM all

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produced a deficiency of iron in exposed cells. A new iron homeostasis was determined by the interaction between the cell (including the mitochondria and probably other organelles) and

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either the particle or metal such as vanadium. This homeostasis included: increased expression of metal importer, greater total cell iron concentrations, elevated cell ferritin levels, and metal concentrations sufficient to meet requirements for continued function. In addition, some portion of cell iron was complexed by functional groups on the surface of an intracellular particle. Accordingly, particles residing in the cell bound some portion of the total metal but available iron concentrations was adjusted and made adequate for continued cell survival. Cell pretreatment with FAC elevated available concentrations of iron and precluded the deficiency in metal following exposures to inorganic particles, organic particles, and soluble metal [29, 35, 38].

Particles, iron, and oxidant generation

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ACCEPTED MANUSCRIPT Whether the result of exposure to inorganic particle, organic particle, or soluble metal included in the particle, the disruption of host iron homeostasis contributes to the generation of

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an oxidative stress following exposure. Amplex Red fluorescence increases with oxidative stress

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and this methodology showed time-dependent oxidant generation by HBE cells following exposures to silica (Figure 6). Cells pre-treated with iron, which increased available metal

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concentrations and precluded deficiency following PM, demonstrated decreased oxidant

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production after silica exposure (Figure 6). Oxidant generation was also partially inhibited by 1.0 µM rotenone consistent with some dependence on mitochondrial function [29].

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Cell oxidant generation after exposure to organic particle was similarly measured using Amplex Red. Cellular oxidant production corresponded to the disruption in iron homeostasis

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following WSP exposure with increased metal availability decreasing the fluorescence signal (Figure 7). In cells pre-treated with 1.0 µM rotenone, cell oxidant generation was again

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determined using Amplex Red fluorescence. Rotenone-mediated interference with the electron transport chain at Complex I in the mitochondria decreased fluorescence, supporting mitochondria as a source of oxidants. Finally, VOSO4 exposure of respiratory epithelial cells is associated with increased oxidant generation in a time-dependent manner using Amplex Red fluorescence (Figure 8) [29]. When the respiratory epithelial cells were pre-treated with FAC and then exposed to VOSO4, the elevated cell iron concentrations diminished oxidant generation (Figure 8). Oxidant generation increased after particle and metal (i.e. vanadium) exposure but pretreatment with iron diminished this generation of reactive oxygen species. The mitochondrial electron transport chain has been identified as a major location of oxidant production [43]; sites include complex I and complex III [44, 45]. Cellular oxidant generation, specifically superoxide,

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ACCEPTED MANUSCRIPT follows exposure to iron chelators other than particles [46-48]. The evidence suggests that decreased cell iron concentrations following exposure to silica, WSP, and VOSO4 induced a

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response which included oxidant generation. This oxidant production can function in the

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remedial response to iron deficiency following loss of the metal induced by the particle surface and metal (i.e. vanadium). Superoxide, produced by mitochondria in response to iron deficit,

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may assist in import of the iron. This ferrireduction is an essential, and frequently limiting,

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reaction in such iron import and can be accomplished in many cell types using superoxide [4951]. Furthermore, such ferrireduction can be dependent on the electron transport chain with the

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mitochondria serving as a source of reducing equivalents [52-54].

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Particles, iron, activation of kinases and transcription factors, and mediator release Biological effects after PM exposure can be associated with an oxidative stress [55, 56].

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Oxidant generation after particle and VOSO4 exposure correspond to a disruption in iron homeostasis resulting in a functional deficiency of this metal. Accordingly, the biological effects of particles (both inorganic and organic) and metals included in PM (e.g. vanadium) can be associated with a disruption in iron homeostasis. Similar to numerous particles, exposure of respiratory epithelial cells to silica impacts an activation of MAP kinases with phosphorylation of both ERK and p38 [29, 55, 57-61]. This activation of MAP kinases was diminished by pre-treatment with FAC [35]. Silica exposure increased NF kappa B activation at least 5.0 fold (Figure 9A). While cell pre-incubation with FAC had no effect on NF kappa B after exposure to buffer only, it diminished subsequent activation induced by silica (Figure 9A). In a similar manner, particle exposure elevated nrf2 ARE activation and this was prevented by FAC pre-treatment of the respiratory epithelial cells

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ACCEPTED MANUSCRIPT (Figure 9B). The activation of NF kappa B and nrf2 ARE influence the expression of genes involved in inflammation. Accordingly, the release of inflammatory mediators after particle

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exposure was assessed. Silica exposure increased IL-6 and IL-8 protein concentrations in the

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supernatant of HBE cells while iron pre-treatment of the cells diminished release of these proinflammatory mediators (Figures 9C and 9D).

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Comparable to silica, WSP exposure increased phosphorylation of ERK and p38 [35].

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The activation of these MAP kinases by WSP exposure was diminished by increasing the cell concentration of available iron [35]. The activation of nrf2 ARE transcription factor by WSP

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exposure was also inhibited by elevating concentrations of cell iron (Figure 10A). Similarly, changes in protein expression for IL-6 and IL-8 after WSP exposure were diminished by pre-

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treatment with iron (Figures 10B and 10C). Finally, trans-activation of NF kappa B promoter increased approximately 3.0 fold

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following exposure of respiratory epithelial cells to VOSO4 (Figure 11A). Cell pre-treatment with FAC diminished subsequent NF kappa B activation induced by VOSO4. Release of both IL-6 and IL-8 from HBE cells after 24 h exposure to VOSO4 was shown to be increased (Figures 11B and 11C). Comparable to the other biological effects evaluated in these studies, release of IL-6 and IL-8 due to VOSO4 exposure was decreased by pre-treatment of the HBE cells with FAC (Figures 11B and 11C). These results further demonstrate that particles (both inorganic and organic) and metals included in PM (i.e. vanadium) can impact changes in activation of MAP kinases and transcription factors and release of pro-inflammatory mediators. In addition, it is evident that these biological effects can be mediated, at least in part, through a decrease in cell iron following exposure to the particle or VOSO4. It is unclear whether oxidative stress is absolutely required

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ACCEPTED MANUSCRIPT as an intermediate in producing these effects.

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Particles, iron, and inflammation

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Inflammatory injury after in vivo exposure to airborne PM is related to a disruption in iron homeostasis by the particle. Silica exposure of animal models causes a neutrophilic

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alveolitis [62]. Animals intratracheally instilled with silica in buffer showed an increased

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neutrophilic influx and total protein relative to exposure to buffer alone [29]. Pre-treatment of the animal with FAC to preclude a functional deficiency of the metal following silica exposure

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diminished the inflammatory injury. This in vivo data supports the position that silica particle disrupts iron homeostasis to produce biological effects. This is comparable to other agents

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associated with inflammatory lung injury including doxorubicin and endotoxin [63, 64]. These results do conflict with previous investigation [65, 66]. It is possible that such incongruent data

results [67].

Conclusions

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reflects disparities in methodologies. Nevertheless, the results are consistent with others’ in vitro

We conclude that the initiating event in oxidative stress and biological effects after exposure to particles, including those in air pollution, is a disruption in cell iron homeostasis. Particle-initiated iron loss from the cell increases superoxide production which facilitates ferrireduction, metal import, and reversal of functional deficiency. If adequate iron concentrations are not re-established in the cell, protracted metal deficiency and oxidant generation activate MAP kinases and transcription factors resulting in a release of inflammatory mediators. After particle exposure, inflammation is the product of a functional deficiency of

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ACCEPTED MANUSCRIPT requisite iron. This mechanism applies to nanoparticles; the distinctive feature regarding nanoparticles is their small size/increased surface area which would be associated with an

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augmented capacity to complex intracellular iron and therefore impact greater biological effect.

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Cell exposures to chelators other than particles demonstrate similar inflammation [6875]. Iron deficiency following exposures can also participate as an underlying mechanism in

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biological effect of particles in extrapulmonary tissues. Cigarette smoking is the greatest risk

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factor for heart disease in the United States and cigarette smoke particle has been shown to similarly impact iron homeostasis through a complexation and sequestration of the metal [76].

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Other compounds with the capacity to complex and decrease availability of iron similarly impact injury to the heart [77-79]. Recognition of a pathway for biological effects after particle

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exposure involving a functional deficiency of iron suggests novel therapies such as metal supplementation (e.g. inhaled and oral). This same pathway for disruption in cell iron

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homeostasis, oxidative stress and biological effects also participates in the response to xenobiotics, fibers, and numerous other metals [80-82].

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[20] M.C. Kennedy, L. Mende-Mueller, G.A. Blondin, H. Beinert, Purification and characterization of cytosolic aconitase from beef liver and its relationship to the iron-responsive element binding protein, Proc Natl Acad Sci U S A, 89 (1992) 11730-11734. [21] M.P. Mims, J.T. Prchal, Divalent metal transporter 1, Hematology, 10 (2005) 339-345. [22] J.B. Fishman, J.B. Rubin, J.V. Handrahan, J.R. Connor, R.E. Fine, Receptor-mediated transcytosis of transferrin across the blood-brain barrier, J Neurosci Res, 18 (1987) 299-304. [23] N. Aziz, H.N. Munro, Iron regulates ferritin mRNA translation through a segment of its 5' untranslated region, Proc Natl Acad Sci U S A, 84 (1987) 8478-8482. [24] P.C. Kruger, M.F. Leahy, J.K. Olynyk, Assessing iron overload: are we there yet?, Clin Cancer Res, 18 (2012) 6395-6397. [25] S.J. Dugger DL, Irby BN, McConnell BL, Cummings WW, Mattman RW, The exchange of twenty metal ions with the weakly acidic silanol group of silica gel., Journal of Physical Chemistry, 68 (1964) 757-760. [26] A.J. Ghio, R.H. Jaskot, G.E. Hatch, Lung injury after silica instillation is associated with an accumulation of iron in rats, Am J Physiol, 267 (1994) L686-692. [27] H.K. Koerten, P. Brederoo, L.A. Ginsel, W.T. Daems, The endocytosis of asbestos by mouse peritoneal macrophages and its long-term effect on iron accumulation and labyrinth formation, Eur J Cell Biol, 40 (1986) 25-36. [28] A.J. Ghio, A. Churg, V.L. Roggli, Ferruginous bodies: implications in the mechanism of fiber and particle toxicity, Toxicol Pathol, 32 (2004) 643-649. [29] A.J. Ghio, H. Tong, J.M. Soukup, L.A. Dailey, W.Y. Cheng, J.M. Samet, M.J. Kesic, P.A. Bromberg, J.L. Turi, D. Upadhyay, G.R. Scott Budinger, G.M. Mutlu, Sequestration of mitochondrial iron by silica particle initiates a biological effect, Am J Physiol Lung Cell Mol Physiol, 305 (2013) L712-724. [30] A.J. Ghio, Stonehuerner, J., Pritchard, R.J., Piantadosi, C.A., Quigley, D.R., Dreher, K.L., Costa, D.L., Humic-like substances in air pollution particulates correlate with concentrations of transition metals and oxidant generation., Inhalation Toxicology, 8 (1996) 479-494. [31] A.J. Ghio, J. Stonehuerner, D.R. Quigley, Humic-like substances in cigarette smoke condensate and lung tissue of smokers, Am J Physiol, 266 (1994) L382-388. [32] T. Sporn, V.L. Roggli, Pneumoconioses, Mineral and Vegetable, in: J.F.Tomashefski, Cagle, P.T., Farver, C.F., and Fraire, A.E. (Ed.) Dail and Hammar's Pulmonary Pathology. Volume I. Nonneoplastic Lung Disease, Springer, Place Published, 2008, pp. 933. [33] A.J. Ghio, D.R. Quigley, Complexation of iron by humic-like substances in lung tissue: role in coal workers' pneumoconiosis, Am J Physiol, 267 (1994) L173-179. [34] A.J. Ghio, J.H. Richards, J.D. Carter, M.C. Madden, Accumulation of iron in the rat lung after tracheal instillation of diesel particles, Toxicol Pathol, 28 (2000) 619-627. [35] A.J. Ghio, J.M. Soukup, L.A. Dailey, H. Tong, M.J. Kesic, G.R. Budinger, G.M. Mutlu, Wood Smoke Particle Sequesters Cell Iron to Impact a Biological Effect, Chem Res Toxicol, 28 (2015) 2104-2111. [36] M.D. Cohen, M. Sisco, C. Prophete, K. Yoshida, L.C. Chen, J.T. Zelikoff, J. Smee, A.A. Holder, J. Stonehuerner, D.C. Crans, A.J. Ghio, Effects of metal compounds with distinct physicochemical properties on iron homeostasis and antibacterial activity in the lungs: chromium and vanadium, Inhal Toxicol, 22 (2010) 169-178. [37] A. Scibior, H. Zaporowska, Effects of vanadium(V) and/or chromium(III) on L-ascorbic acid and glutathione as well as iron, zinc, and copper levels in rat liver and kidney, J Toxicol Environ Health A, 70 (2007) 696-704. 19

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involvement of p38 and extracellular signal-regulated kinase signaling pathways, J Immunol, 172 (2004) 7069-7077. [69] S.K. Lee, H.J. Jang, H.J. Lee, J. Lee, B.H. Jeon, C.D. Jun, S.K. Lee, E.C. Kim, p38 and ERK MAP kinase mediates iron chelator-induced apoptosis and -suppressed differentiation of immortalized and malignant human oral keratinocytes, Life Sci, 79 (2006) 1419-1427. [70] E.Y. Choi, Z.Y. Park, E.J. Choi, H.M. Oh, S. Lee, S.C. Choi, K.M. Lee, S.H. Im, J.S. Chun, C.D. Jun, Transcriptional regulation of IL-8 by iron chelator in human epithelial cells is independent from NF-kappaB but involves ERK1/2- and p38 kinase-dependent activation of AP1, Journal of cellular biochemistry, 102 (2007) 1442-1457. [71] J. Antosiewicz, W. Ziolkowski, J.J. Kaczor, A. Herman-Antosiewicz, Tumor necrosis factor-alpha-induced reactive oxygen species formation is mediated by JNK1-dependent ferritin degradation and elevation of labile iron pool, Free Radic Biol Med, 43 (2007) 265-270. [72] H.J. Lee, J. Lee, S.K. Lee, S.K. Lee, E.C. Kim, Differential regulation of iron chelatorinduced IL-8 synthesis via MAP kinase and NF-kappaB in immortalized and malignant oral keratinocytes, BMC cancer, 7 (2007) 176. [73] X. Huang, J. Dai, C. Huang, Q. Zhang, O. Bhanot, E. Pelle, Deferoxamine synergistically enhances iron-mediated AP-1 activation: a showcase of the interplay between extracellularsignal-regulated kinase and tyrosine phosphatase, Free Radic Res, 41 (2007) 1135-1142. [74] B.M. Kim, H.W. Chung, Desferrioxamine (DFX) induces apoptosis through the p38caspase8-Bid-Bax pathway in PHA-stimulated human lymphocytes, Toxicol Appl Pharmacol, 228 (2008) 24-31. [75] A. Klettner, S. Koinzer, V. Waetzig, T. Herdegen, J. Roider, Deferoxamine mesylate is toxic for retinal pigment epithelium cells in vitro, and its toxicity is mediated by p38, Cutaneous and ocular toxicology, 29 (2010) 122-129. [76] A.J. Ghio, E.D. Hilborn, J.G. Stonehuerner, L.A. Dailey, J.D. Carter, J.H. Richards, K.M. Crissman, R.F. Foronjy, D.L. Uyeminami, K.E. Pinkerton, Particulate matter in cigarette smoke alters iron homeostasis to produce a biological effect, Am J Respir Crit Care Med, 178 (2008) 1130-1138. [77] G. Link, R. Tirosh, A. Pinson, C. Hershko, Role of iron in the potentiation of anthracycline cardiotoxicity: identification of heart cell mitochondria as a major site of iron-anthracycline interaction, J Lab Clin Med, 127 (1996) 272-278. [78] Y. Ichikawa, M. Ghanefar, M. Bayeva, R. Wu, A. Khechaduri, S.V. Naga Prasad, R.K. Mutharasan, T.J. Naik, H. Ardehali, Cardiotoxicity of doxorubicin is mediated through mitochondrial iron accumulation, J Clin Invest, 124 (2014) 617-630. [79] G. Corna, P. Santambrogio, G. Minotti, G. Cairo, Doxorubicin paradoxically protects cardiomyocytes against iron-mediated toxicity: role of reactive oxygen species and ferritin, J Biol Chem, 279 (2004) 13738-13745. [80] Z. Elias, O. Poirot, M.C. Daniere, F. Terzetti, S. Binet, M. Tomatis, B. Fubini, Surface reactivity, cytotoxicity, and transforming potency of iron-covered compared to untreated refractory ceramic fibers, J Toxicol Environ Health A, 65 (2002) 2007-2027. [81] K. Salnikow, X. Li, M. Lippmann, Effect of nickel and iron co-exposure on human lung cells, Toxicol Appl Pharmacol, 196 (2004) 258-265. [82] N. Ueda, B. Guidet, S.V. Shah, Gentamicin-induced mobilization of iron from renal cortical mitochondria, Am J Physiol, 265 (1993) F435-439. [83] J.W. Greenawalt, The isolation of outer and inner mitochondrial membranes, Methods Enzymol, 31 (1974) 310-323. 22

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Particle-related exposures in humans

Particle burden

Composition

Cigarette smoking

Tens of thousands µg/cigarette

Organic, combustion product

Burning of biomass

Tens of thousands µg /m3

Organic, combustion product

Dust storms

Hundreds to thousands of µg/m3

Mining

Legally allowable levels of

Usually inorganic but coal is

100 and 2000 µg/m3 for

organic

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Exposure

Inorganic

silica and coal respectively Hundreds to thousands of µg/m3

Inorganic and organic

Wildfires, wood burning

Hundreds of µg/m3

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Agricultural work

Organic, combustion product

Hundreds of µg/m3

Organic, combustion product

Traffic-related emissions

Tens to hundreds of µg/m3

Organic, combustion product

Gas stoves

Tens of µg/m3

Organic, combustion product

Tens of µg/m3

Inorganic and organic

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stoves Environmental tobacco smoke

Ambient air pollution

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ACCEPTED MANUSCRIPT Figure legends Figure 1. Schematic for changes in iron homeostasis following particle exposure. An iron

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homeostasis exists in respiratory epithelial cells with metal (red spheres denote iron)

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concentrations appropriate for organelle function (Figure 1A). Functional groups at the surface of the particle complex cell iron impacting a functional deficiency (Figure 1B). In response to

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the reduction in intracellular iron following particle exposure, the cell upregulates importers (e.g.

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DMT1) in an attempt to reacquire requisite metal (Figure 1C). The outcome of particle exposure is an altered iron homeostasis with an accumulation of the metal with sufficient

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concentrations to reverse the deficiency; some of this metal is stored in ferritin (Figure D). Figure 2. Schematic for oxidative stress and biological effects following particle exposure.

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Following particle exposure, a functional iron deficiency can result if adequate metal concentrations are not imported. In response to a reduction in intracellular iron, the cell

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generates superoxide as a ferrireductant to facilitate metal import. Continued iron deficiency and protracted oxidant generation activate MAP kinases and transcription factors resulting in a synthesis and release of inflammatory mediators and inflammation. Figure 3. Non-heme iron concentrations in nuclear and mitochondrial fractions and cells after exposure to silica [29]. HBE cells in flasks were exposed to 100 µg/mL silica (Minusil, American Silica, Berkeley Springs, WV) for 5 minutes (Figure 3A). Nuclear and mitochondrial fractions were isolated [83], hydrolyzed in 1.0 mL 3 N HCl/10% trichloroacetic acid (with the latter used to precipitate heme), and non-heme iron concentrations measured using inductively coupled plasma optical emission spectroscopy (ICPOES; Model Optima 4300D, Perkin Elmer, Norwalk, CT) operated at a wavelength of 238.204 nm. After silica exposure, the nuclear fraction showed elevated non-heme iron concentrations (*denotes a significant increase relative

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200 µM FAC and 100 µg/mL silica for 4 hours (Figure 3B). With FAC exposure, HBE cells

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imported iron. Inclusion of silica also elevated cell iron concentrations and FAC and silica together increased metal levels further. Exposures in 12-well plates were repeated for 24 hours

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and ferritin measured in cell lysate using an immunoturbidimetric assay (Kamiya Biomedical

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Company, Seattle, WA) (Figure 3C). Exposures to FAC, silica, and both increased cell ferritin concentration. (In Figures 3B and 3C, * denotes a significant increase relative to PBS exposure

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with no FAC, ** denotes a significant increase relative to PBS exposure with no FAC, and *** denotes a significant increase relative to all other exposures)

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Figure 4. Non-heme iron concentrations in nuclear and mitochondrial fractions and cells after exposure to WSP [35]. BEAS-2B cells in flasks were exposed to 100 µg/mL WSP for 15

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minutes (Figure 4A) [35]. The nuclear fraction showed elevated non-heme iron concentrations (* denotes a significant increase relative to PBS exposures) while metal was diminished in the mitochondrial fraction (** denotes a significant decrease relative to all PBS exposures). BEAS2B cells in 12-well plates were exposed to 200 µM FAC and 100 µg/mL WSP for 4 hours (Figure 4B). With FAC exposure, iron was imported. Inclusion of WSP also elevated cell iron concentrations and FAC and WSP co-exposures increased metal levels further. Exposures in 12well plates were repeated for 24 hours and ferritin measured in cell lysate (Figure 4C). Exposures to FAC, silica, and both increased cell ferritin concentration. (In Figures B and C, * denotes a significant increase relative to PBS exposure with no FAC and ** denotes a significant increase relative to all other exposures) Figure 5. Non-heme iron concentrations in nuclear and mitochondrial fractions and cells after

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demonstrated decreased non-heme iron concentrations (* denotes a significant decrease relative

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to PBS exposures) (Figure 5B). With exposure of HBE cells in 12-well plates to 200 µM FAC, iron was imported. Inclusion of VOSO4 increased cell iron concentrations and both FAC and

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VOSO4 elevated metal levels further. Exposures were repeated in 12-well plates for 24 hours

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and ferritin measured in cell lysate (Figure 5C). FAC, silica, and both increased ferritin concentration in HBE cells at 24 hours. (In Figures B and C, * denotes a significant increase

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relative to PBS exposure with no FAC and ** denotes a significant increase relative to all other exposures)

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Figure 6. Cell oxidant generation after exposure to silica [29]. Oxidant generation by HBE cells was determined using Amplex Red (Molecular Probes, Eugene, OR) fluorescence. Cells grown

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on 96 well Co-Star (Corning Corp., Corning, NY) white-walled tissue culture plates to confluence were pre-loaded with the dye prior to exposure for 20 min at 37o C/5% CO2. HBE cells were pre-treated with either PBS or 200 µM FAC and then exposed to PBS and 100 µg/mL silica in PBS. The reported value is fold-change over control cells which were exposed to buffer at time zero. Oxidant generation by HBE cells was significantly increased by 100 µg/mL silica. Pre-treatment of cells with 200 µM FAC diminished the oxidant generation following exposure to both buffer and silica. Figure 7. Cell oxidant generation after exposure to WSP [35]. The reported value is foldchange over control cells which were exposed to buffer at time zero. Oxidant generation by BEAS-2B cells was significantly increased by 100 µg/mL WSP. Pre-treatment of cells with 200 µM FAC diminished the oxidant generation following exposure to both buffer and WSP.

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ACCEPTED MANUSCRIPT Figure 8. Cell oxidant generation after exposure to VOSO4 [38]. The reported value is foldchange over control cells which were exposed to buffer at time zero. Exposure to 100 µM

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VOSO4 significantly increased Amplex Red fluorescence. Pre-treatment of cells with 200 µM

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FAC diminished the oxidant generation following exposure to both buffer and VOSO4. Figure 9. Activation of transcription factors and release of pro-inflammatory mediators after

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exposures to silica [29]. To measure trans-activation of NF- kappa B and Nrf2 ARE promoters,

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BEAS-2B cells were co-transfected with either 0.1 µg of kappa B-luciferase reporter or Nrf2 ARE-luciferase reporter plasmid according to the manufacturer’s recommendations. After 24

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hours, cells were incubated with and without 200 µM FAC for 4 hours and then exposed to PBS or 100 µg silica/mL PBS for 1 hr. Cells were then pelleted and lysed in passive lysis buffer

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(Promega, Madison, WI). The trans-activation activity was measured as luciferase light units as described previously [84]. Silica increased activation of both NF kappa B (Figure 9A) and nrf2

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ARE (Figure 9B). However, incubation with FAC decreased activation of both. IL-6 and IL-8 concentrations in cell media were measured using ELISA (R&D Systems, Minneapolis, MN). Silica increased the release of IL-6 (Figure 9C) and IL-8 (Figure 9D) by HBE cells while FAC pre-treatment diminished the response. (* denotes a significant increase relative to all other exposures while ** denotes a significant increase relative to PBS exposures) Figure 10. Activation of transcription factor nrf2 ARE and release of pro-inflammatory mediators after exposures to WSP [35]. While exposure of BEAS-2B cells to 100 µg/mL WSP for 4 hours activated nrf2 ARE, treatment with 200 µM FAC diminished the effect (Figure 10A). Exposure to 100 µg/mL WSP for 24 hours increased release of IL-6 and IL-8 by BEAS-2B cells (Figures 10B and 10C respectively). FAC treatment diminished the release of interleukins exposed to WSP. (* denotes a significant increase relative to all other exposures while **

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mediators after exposure to VOSO4 [38]. Activation of NF kappa B in BEAS-2B cells increased

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after the 4 hour treatment with 100 µM VOSO4 (Figure 11A). Pre-treatment of BEAS-2B cell with 200 µM FAC for 4 hours decreased activation of NF kappa B associated with VOSO4.

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Release of IL-6 (Figure 11B) and IL-8 (Figure 11C) by HBE cells was increased following 24

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hour exposure to 100 µM VOSO4. Pre-treatment of cells with 200 µM FAC for 4 hours diminished the cell response after VOSO4. (* denotes a significant increase relative to all other

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· A disruption in iron homeostasis is proposed as the pathway common to the biological effects of all particulate matter including air pollution particles · Oxidative stress, kinase and transcription factor activation, release of inflammatory mediators, and inflammation in particle-exposed cells result from a functional iron deficiency · Recognition of the pathway for biological effects after particle exposure to involve a functional deficiency of iron suggests novel therapies such as metal supplementation

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