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. Author manuscript; available in PMC: 2022 Nov 15.
Published in final edited form as: Inhal Toxicol. 2021 Nov 9;33(6-8):268–274. doi: 10.1080/08958378.2021.1989092

Diacetyl exposure disrupts iron homeostasis in animals and cells

Andrew J Ghio 1, Joleen M Soukup 1, Lisa A Dailey 1, Victor L Roggli 2, Alvin L Crumbliss 3, Scott M Palmer 4
PMCID: PMC8928485  NIHMSID: NIHMS1783998  PMID: 34752160

Abstract

Objective:

Several mechanisms have been proposed for the biological effect of diacetyl. We tested the postulate that animal and cell exposures to diacetyl are associated with a disruption in iron homeostasis.

Materials and methods:

Male, Sprague-Dawley rats were intratracheally-instilled with either distilled water or diacetyl. Seven days after treatment, animals were euthanized and the lungs removed, fixed, and embedded. Sections were cut and stained for iron, collagen, and ferritin. Human epithelial (BEAS-2B) and monocytic (THP-1) cells were exposed in vitro to ferric ammonium citrate (FAC), diacetyl, and both FAC and diacetyl. Cell non-heme iron concentrations and ferritin levels were quantified using inductively coupled plasma optical emission spectroscopy and an immunoassay respectively.

Results:

After exposure of animals to diacetyl, there were airway polypoid lesions which stained positively for both iron and the intracellular storage protein ferritin. Trichrome stain showed a deposition of collagen immediately adjacent to accumulated metal following diacetyl exposure. In in vitro cell exposures, FAC increased non-heme iron concentration but co-incubations of FAC and diacetyl elevated levels to significantly greater values. Levels of ferritin were increased with exposures of BEAS-2B and THP-1 cells to FAC but were similarly greater after co-exposure with FAC and diacetyl.

Conclusions:

Results of animal and cell studies support a disruption of iron homeostasis by diacetyl. It is proposed that, following internalization, diacetyl complexes intracellular sources of iron. The cell recognizes a loss of its requisite iron to diacetyl and imports greater concentrations of the metal.

Keywords: Diacetyl, bronchiolitis obliterans, iron, ferritin, lung diseases

Introduction

Diacetyl (2,3 butanedione) is a water-soluble, volatile, vicinal diketone (dicarbonyl) with a molecular formula of (CH3CO)2. It is a liquid found 1) naturally in alcoholic beverages as a byproduct of fermentation and 2) as a synthetic flavoring agent in foods and electronic cigarettes, added to impart a buttery flavor. In 2000, eight workers formerly employed in a microwave popcorn production facility in Missouri were observed to have lung disease following an occupational exposure to diacetyl (Kreiss et al. 2008). Implementation of surveillance programs for workers in flavoring manufacturing companies revealed additional cases of lung disease associated with diacetyl exposure (Hendrick 2008; Cavalcanti et al. 2012). Significant levels of diacetyl produced during pyrolysis and fermentation processes (e.g. during the manufacture of beer, wine, dairy products, and roasted coffee) were similarly associated with a pulmonary injury (Centers for Disease Control and Prevention 2013; Bailey et al. 2015). Further evidence of a lung disease was found among workers at a chemical plant that manufactured diacetyl (van Rooy et al. 2007). Attempts to provide substitutes for diacetyl (e.g. 2,3-pentanedione, 2,3 hexanedione, and 2,3 heptanedione) have been of questionable success as investigation demonstrated these compounds to also be hazardous (Day et al. 2011; Hubbs et al. 2012; Morgan et al. 2012). Subsequently, while lung disease following diacetyl exposure was initially called popcorn workers’ lung, flavoring-related lung disease is now favored.

There are several proposed mechanisms for the biologic effect of diacetyl and other diketones. One early proposal suggested the impact of these compounds can be attributed to a production of acetate (Frund et al. 1989). A second submitted that diacetyl acts through oxidative processes to cause biological effects (Kovacic and Cooksy 2010). A third mechanistic pathway for the biologic effect offered that electron sharing between the adjacent carbonyl groups makes diketones (dicarbonyls) particularly reactive with other compounds (Wondrak et al. 2002). Targets of this reactivity were demonstrated to include proteins and alkylamines (Espinoza-Hicks et al. 2015; Hubbs et al. 2016; Hubbs et al. 2019). A fourth hypothesized pathway was a transient inhibition of ion transport in airway epithelial cells (Zaccone et al. 2015).

Inflammatory and fibrotic lung injuries follow exposures to numerous dissimilar compounds and substances which are recognized chemically to form coordination complexes with metal cations and demonstrate a relationship with a disruption in cell, tissue, and systemic iron homeostasis (Ghio et al. 2020). Intracellular concentrations of iron immediately available for complexation after internalization of such compounds and substances are very low approximating the concentration of the labile iron pool (1–5 μM) (Cabantchik 2014). Accordingly, these compounds and substances, or their metabolites, will compete with iron utilized by the cell for functions frequently critical for survival. Following their exposure, iron previously employed in essential cell processes is absent. An absolute or functional cell iron deficiency results. If enough cell iron is lost, cell death ensues. However, prior to death, exposed cells will attempt to reverse the loss of requisite metal by increasing expression of metal importers and elevating ferrireduction necessary for uptake (e.g. superoxide generation). Following the response of increased expression of importers and ferrireduction, cell iron is altered, and a new metal homeostasis is established. This new metal homeostasis includes increased total iron concentrations in cells with metal sufficient to meet requirements for continued function.

We tested the postulate that animal and cell exposures to diacetyl are associated with a disruption in iron homeostasis and an accumulation of the metal.

Methods

Materials.

Diacetyl was from Fluka Chemical Company (Milwaukee, WI). Unless specified otherwise, all other reagents were from Sigma-Aldrich (St. Louis, MO).

Animal investigation.

The use of animals was approved by the National Institute of Environmental Health Sciences (NIEHS) Animal Care and Use Committee (NIEHS Assurance number A4149–1). NIH guidelines were taken to limit animal discomfort, distress, pain and injury. Methods of euthanasia and reason for selection are consistent with the AVMA Guidelines on Euthanasia. Veterinary care was provided by highly trained and experienced staff. A mean body weight determined on study day 1 was used in calculation of diacetyl dose. Male, Sprague-Dawley rats (295–305 g body weight; Charles River, Raleigh, NC) were anesthetized (isofluorane) and intratracheally-instilled with either control (200 μL distilled water) or diacetyl (125 mg/kg in 200 μL) (n=3/group) (Palmer et al. 2011). At 7 days after treatment, animals were euthanized (Nembutal, 60 mg/kg intraperitoneally) and the lungs removed en bloc, fixed in 10% formalin, and embedded in paraffin. The block included a lobe of the right lung. Five-micron tissue sections were cut, floated on a protein-free water bath, mounted on silane treated slides (Fisher, Raleigh, NC), and air-dried overnight. Sections were stained for iron and collagen using a Perls’ Prussian Blue and a Masson’s trichrome stain respectively. Immunohistochemistry for ferritin was performed with rabbit anti-human ferritin primary antibody (Dako, Carpinteria, CA). The images provided are representative of the lung tissue from the three animals exposed to either control or diacetyl.

Cell investigation.

BEAS-2B cells were employed in in vitro studies. These are an immortalized line of normal human bronchial epithelium derived by transfection of primary cells with SV40 early-region genes. Cells were grown to 90–100% confluence on uncoated plastic twelve-well plates in keratinocyte growth medium (KGM; Clonetics) which is essentially MCDB 153 medium supplemented with 5 ng/ml human epidermal growth factor, 5 mg/ml insulin, 0.5 mg/ml hydrocortisone, 0.15 mM calcium, bovine pituitary extract, 0.1 mM ethanolamine and 0.1 mM phosphoethanolamine. Fresh medium was provided every 48 hours. BEAS-2B cells were exposed to HBSS, 200 μM ferric ammonium citrate (FAC), and 20, 100 and 500 μM diacetyl with and without 200 μM FAC. FAC was employed as a source of metal available to the cells since 1) iron complexed to citrate is relevant to homeostasis in animals, including humans, and 2) it is buffered. After 4 and 24 hr incubation, the media and exposure were removed, and the cells washed with HBSS and scraped into 1.0 mL 3 N HCl/10% trichloroacetic acid (TCA).Following hydrolysis at 70o C for 24 hr, iron (non-heme) concentration in the supernatant was determined using inductively coupled plasma optical emission spectroscopy (ICPOES; Model Optima 4300D, Perkin Elmer, Norwalk, CT).

THP-1 cells are a monocyte-like cell line also used in in vitro studies. These were are cultured in 75-cm2 tissue culture flasks using RPMI-1640 (Invitrogen, Carlsbad, CA) supplemented with 10% FCS (Invitrogen, Carlsbad, CA) and gentamicin solution (20 μg/ml) (Sigma, St. Louis, MO ). THP-1 cells (2.0 × 106/mL) were exposed to HBSS, 200 μM ferric ammonium citrate (FAC), and 20, 100 and 500 μM diacetyl with and without 500 μM FAC; a higher concentration of FAC was used relative to that for epithelial cell incubation since monocytic cells/macrophages may have a greater capacity for metal uptake (Ghio & Roggli 2021). After 24 hr incubation, the cell suspension was centrifuged, the media and exposure were removed, and the cells were washed with HBSS and hydrolyzed in 1.0 mL 3 N HCl/10% TCA at 70o C for 24 hr. Iron (non-heme) concentration in the supernatant was determined using ICPOES.

Cell ferritin concentrations.

BEAS-2B cells were exposed to HBSS, 200 μM FAC, 100 μM diacetyl, and 100 μM diacetyl with 200 μM FAC for 24 hr. After the media was removed, cells were washed with HBSS, scraped into 0.5 mL HBSS, and disrupted using five passes through a gauge 25 needle. The concentrations of ferritin in the cell lysates were quantified using an immunoturbidimetric assay (Kamiya Biomedical Company, Seattle, WA).

THP-1 cells were exposed to HBSS, 500 μM FAC, 100 μM diacetyl, and 100 μM diacetyl with 500 μM FAC for 24 hr. After centrifugation, cells were washed with HBSS, scraped into 0.5 mL HBSS, and disrupted using five passes through a gauge 25 needle. The concentrations of ferritin in the cell lysates were quantified using the immunoturbidimetric assay (Kamiya Biomedical Company).

Statistics.

Data are expressed as mean values ± standard error unless specified otherwise. The minimum number of replicates for all measurements was six. Differences between multiple groups were compared using one-way analysis of variance (ANOVA). The post-hoc test employed was Duncan’s Multiple Range test. Two-tailed tests of significance were employed. Significance was assumed at p<.05.

Results

In the lungs of animals exposed to control (distilled water), no cell or acellular structure stained for iron (Figure 1A). Seven days after instillation of animals with diacetyl, the most distal portions of the lung demonstrated an increased number of macrophages and these stained positively for iron, reflecting an accumulation of the metal (Figure 1B). Comparable siderophages (i.e. iron- and hemosiderin-laden macrophages) could be observed in the lung interstitium (Figure 1C). Sections of lung from animals exposed to diacetyl showed airway polypoid lesions and adjacent inflammatory cells (left side of image; Figure 1D). The lesion revealed an accumulation of iron centrally with Perls’ staining (Figure 1D). Normal lung showed limited uptake for a ferritin antibody by airway epithelial cells (Figure 2A). However, stains for ferritin in lung from animals exposed to diacetyl supported an intense expression of this iron-storage protein by cells included in the polypoid lesion and immediately adjacent to regions which stained positively for iron (Figure 2B). Trichrome stain showed little to no collagen in the lung exposed to distilled water while deposition after diacetyl instillation appeared to be enhanced and, comparable to the ferritin, immediately adjacent to an accumulation of metal (Figures 3A and 3B).

Figure 1.

Figure 1.

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Perls’ stain of lung tissue from diacetyl-exposed rats. Lung tissue from control-exposed animals demonstrated no iron which stains blue with Perls’ stain (A). After exposure to diacetyl, macrophages in the alveolar (B) and interstitial (C) regions stained positively for iron. A polypoid lesion obstructing an airway reveals cells, presumed to be macrophages, which stained positively for iron near its core (D). Images are representative of each animal exposure group (n= 3/group). Original magnification of 200x.

Figure 2.

Figure 2.

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Stain for ferritin in lung tissue from control- and diacetyl-exposed rats. Ferritin stains brown when positive. In the animal exposed to distilled water (control), there was minor staining by the small airway epithelium (A). Macrophages in the alveolar region could also demonstrate modest uptake for the ferritin antibody. With exposure to diacetyl, polypoid lesions in the airway revealed considerable ferritin staining in the interior but not in the overlying epithelium (B). In addition, alveolar macrophages also stained positively for ferritin. Uptake of the antibody was increased in those regions and cells which stained positively for iron. Images are representative of each animal exposure group (n= 3/group). Original magnificent of 200x.

Figure 3.

Figure 3.

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Trichrome stain for collagen in lung tissue of control- and diacetyl-exposed rats. A focal deposition of collagen is evident as bluish-green color. With control-exposed animals, there was little to no positive stain in the distal lung (A). Small airways showed minimal stain for collagen in regions immediately below the epithelium. Polypoid lesions in the diacetyl-exposed rats displayed significant collagen deposition (B). Collagen deposition was increased in those regions immediately adjacent to an accumulation of metal. Images are representative of each animal exposure group (n= 3/group). Original magnification of 200x.

BEAS-2B cells were exposed to HBSS, FAC, diacetyl, and both FAC and diacetyl for 4 and 24 hr. FAC increased cell iron but co-incubations with diacetyl (100 and 500 μM) elevated cell iron levels to significantly greater values (Figures 4A and 4B). Concentrations of ferritin were elevated after incubation of BEAS-2B cells with FAC but were increased to greater levels after co-exposures to both FAC and diacetyl (Figure 4C).

Figure 4.

Figure 4.

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Cell iron import by BEAS-2B cells and ferritin levels after exposure to HBSS, FAC, diacetyl, and both FAC and diacetyl. Figures A and B include results using diacetyl concentrations of 20, 100, and 500 μM (n=6/group). Cell non-heme iron concentrations increased after 4 and 24 hr incubation of BEAS-2B cells with FAC and both FAC and diacetyl (A and B respectively). Relative to exposure to FAC alone, BEAS-2B cells had significantly increased non-heme iron after co-exposure to FAC and diacetyl at 100 and 500 μM. Levels of the intracellular storage protein ferritin were also increased with 24 hr exposures of BEAS-2B cells to FAC but were further elevated after exposure to both FAC and 100 μM diacetyl (C).

* significantly increased relative to HBSS (p<.05, one way ANOVA); ** significantly increased relative to HBSS and FAC alone (p<.05, one way ANOVA).

Comparable results were noted with THP-1 cells. Exposure to FAC increased concentrations of iron in the cells at 4 and 24 hr (Figures 5A and 5B). Co-exposures to FAC and diacetyl further elevated levels of iron in the THP-1 cells at 4 and 24 hr. With exposure to FAC, THP-1 cell ferritin concentration was elevated but levels were increased to significantly greater values with co-exposures to both FAC and diacetyl (Figure 5C).

Figure 5.

Figure 5.

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Non-heme iron and ferritin concentrations in THP-1 cells exposed to HBSS, FAC, diacetyl, and both FAC and diacetyl. Figure A and B include results using diacetyl 20, 100 and 500 μM (n=6/group). Relative to exposure to FAC alone, THP-1 cells had significantly increased non-heme iron concentrations after co-exposure to FAC and diacetyl at 20, 100 and 500 μM (A and B). With 24 hr exposures of THP-1 cells to FAC, ferritin concentrations were also increased but were further elevated after co-exposure to both FAC and 100 μM diacetyl (C).

* significantly increased relative to HBSS (p<.05, one way ANOVA); ** significantly increased relative to HBSS and FAC alone (p<.05, one way ANOVA).

Discussion

In the initial cohort of individuals with lung disease following exposure to diacetyl, three of eight employees at a microwave popcorn production facility in Missouri had a lung biopsy and at least one of these three biopsies demonstrated findings consistent with bronchiolitis obliterans (Akpinar-Elci et al. 2004; Kreiss et al. 2008) Animal investigation has confirmed that exposure to diacetyl, including inhalation, can result in airway lesions that are histopathologically similar to bronchiolitis obliterans in humans (Harber et al. 2006; Hubbs et al. 2008; Morgan et al. 2008; Morgan et al. 2012; Morgan et al. 2016; Flake and Morgan 2017). The histopathology of bronchiolitis obliterans is characterized by polypoid obliteration of the bronchiole lumen without involvement of the distal lung parenchyma by inflammation or organizing pneumonia (King 2003). Injury of the bronchiolar epithelium is considered to be the initial event in the pathogenesis of bronchiolitis obliterans O’Koren et al. 2013). Exposure to diacetyl leads to an initial necrotic epithelial injury which is followed by aberrant repair and fibroproliferation (Flake and Morgan 2017). Epithelial cell regeneration to repair the damage and cover the denuded surface can result in an intraluminal mound or polyp associated with fibrous tissue intraluminally and/or circumferentially. This repair process compromises the airway which can be partially or totally obliterated by fibrosis. In this study, pathologic inspection of lung tissue from the rats exposed to diacetyl demonstrated histopathological changes of intraluminal polyp formation with fibrous tissue intraluminally. Comparable changes were previously observed and considered consistent with bronchiolitis obliterans (Harber et al. 2006; Flake and Morgan 2017). Staining for iron and ferritin supported a disruption of iron homeostasis with accumulation of both the metal and its storage protein in the alveolar region, interstitium, and in the polypoid lesions of the airway. In the animals instilled with diacetyl, the accumulation of iron and ferritin appear to be predominantly in macrophages comparable to numerous exposures (e.g. particles and fibers) (Ghio et al. 2019). Trichrome staining confirmed the injury with collagen deposition in the polypoid lesion. This fibrosis demonstrated a relationship to the accumulation of both metal and ferritin being contiguous to and indistinguishable from cells/tissue which stained for both iron and ferritin. These observations suggest a relationship between a disruption in iron homeostasis and the fibrotic lung injury after diacetyl.

Cell investigation into a disruption of iron homeostasis associated with diacetyl revealed that exposure of respiratory epithelial and THP-1 cells to both FAC and diacetyl increased metal import and ferritin levels. It is proposed that, following internalization, diacetyl complexes intracellular sources of iron which are initially bound by host molecules. The cell recognizes a loss of its metal to diacetyl and, if iron is available, greater concentrations are imported to meet requirements for continued function and survival. Both respiratory epithelial and THP-1 cells exposed to FAC imported iron. With exposure to diacetyl, there was significantly greater metal uptake when FAC was included in the incubation. Some of the imported metal impacts the expression of iron responsive proteins to increase levels of cell ferritin which were increased with co-exposures to both iron and diacetyl. In the media without added FAC, the concentration of iron approximates 10 μM and subsequently there is little to no metal to import following these incubations. In support of such a pathway, diacetyl is a diketone and similar compounds with carbonyl functional groups (e.g. aldehydes, ketones, and carboxylates) participate in the formation of coordination complexes with numerous metals including iron. In addition, metabolic pathways for biotransformation of diketones include reduction to hydroxyketones (Anders 2017). These carbonyl-containing compounds are also predicted to participate in metal complexation. Furthermore, reaction of diacetyl with water followed by deprotonation will produce an alkoxide with a capacity to participate in iron complexation (using some combination of hydroxyl(s) and ketone groups; Figure 6) and disrupt the homeostasis of this critical metal. Finally, additional metabolic pathways of diacetyl produce carboxylates, hydroxycarboxylates, and diols (Anders 2017). These compounds similarly can complex iron and disrupt the homeostasis of this metal in the host.

Figure 6.

Figure 6.

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Reaction of diacetyl with water.

This investigation supports a disruption of iron homeostasis as a potential mechanistic pathway for the biological effect of diacetyl. Diverse pathological patterns of involvement in lung inflammatory and fibrotic injuries (e.g. bronchiolitis obliterans and usual interstitial pneumonitis) possibly reflect dissimilarities in exposure (e.g. total dose, solubility, deposition, clearance, and retention) rather than divergent mechanistic pathways for biological effect.

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