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Toxicology and Applied Pharmacology

journal homepage: www.elsevier.com/locate/taap

Comparative cytotoxicity of respirable surface-treated/untreated calcium

carbonate rock dust particles in vitro

Timur O. Khaliullina,b, Elena R. Kisina, Naveena Yanamalaa, Supraja Guppia, Martin Harperc,

Taekhee Leed

, Anna A. Shvedovaa,b,⁎

a Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, Health Effects Laboratory Division, Exposure Assessment Branch, 1095

Willowdale road, Morgantown, WV 26505, USA

b West Virginia University, Department of Physiology and Pharmacology, PO Box 9229, Morgantown, WV, USA

c

Zefon International, 5350 SW 1st Lane, Ocala, FL 34474, USA

d Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, Pittsburgh Mining Research Division, 626 Cochrans Mill Road,

Pittsburgh, PA 15236, USA

ARTICLE INFO

Keywords:

Rock dust

Marble

Limestone

Silica

Macrophages

Stearate

ABSTRACT

Calcium carbonate rock dust (RD) is used in mining to reduce the explosivity of aerosolized coal. During the

dusting procedures, potential for human exposure occurs, raising health concerns. To improve RD aerosolization,

several types of anti-caking surface treatments exist. The aim of the study was to evaluate cytotoxicity of four

respirable RD samples: untreated/treated limestone (UL/TL), untreated/treated marble (UM/TM), and crystalline silica (SiO2) as a positive control in A549 and THP-1 transformed human cell lines. Respirable fractions were

generated and collected using FSP10 high flow-rate cyclone samplers. THP-1 cells were differentiated with

phorbol-12-myristate-13-acetate (20 ng/ml, 48 h). Cells were exposed to seven different concentrations of RD

and SiO2 (0–0.2 mg/ml). RD caused a slight decrease in viability at 24 or 72 h post-exposure and were able to

induce inflammatory cytokine production in A549 cells, however, with considerably less potency than SiO2. In

THP-1 cells at 24 h, there was significant dose-dependent lactate dehydrogenase, inflammatory cytokine and

chemokine release. Caspase-1 activity was increased in SiO2- and, on a lesser scale, in TM- exposed cells. To test

if the increased toxicity of TM was uptake-related, THP-1 cells were pretreated with Cytochalasin D (CytD) or

Bafilomycin A (BafA), followed by exposure to RD or SiO2 for 6 h. CytD blocked the uptake and significantly

decreased cytotoxicity of all particles, while BafA prevented caspase-1 activation but not cytotoxic effects of TM.

Only TM was able to induce an inflammatory response in THP-1 cells, however it was much less pronounced

compared to silica.

1. Introduction

During the mining and transport of coal, combustible coal dust can

accumulate in the air, thus posing the threat of a large-scale explosion.

Despite implementing rigorous safety measures, coal dust explosions

are still of great concern, resulting in 65 fatalities and 18 injuries in

2002–2012 in the U.S. alone (CDC/NIOSH, 2012a). Mitigation of explosion hazard in coal mines is achieved by the spreading of fine rock

dust (RD), such as pulverized limestone or marble (both having the

chemical formulae of CaCO3) to render the coal dust inert. The Office of

Mine Safety and Health Research (OMSHR) has investigated the effect

of rock dusting on the prevention and suppression of coal dust

combustion (Harris et al., 2012; CDC/NIOSH, 2014; 2012b). OMSHR

investigators found that 80% total incombustible content of rock dust

was needed, which became a federal law in June 2011(“U.S. Code of

Federal Regulations. Title 30eMandatory safety standards. Part

75eunderground coal mines. Subpart E. Section 75.403 Maintenance of

incombustible content of rock dust.”). Rock dust must meet the following definition stated in 30 CFR 75.2: It shall be comprised of pulverized stone (limestone, dolomite etc.); when particles are wetted and

dried will not cohere to form a cake which will not be dispersed into

separate particles by a light blast of air; does not contain > 5% combustible matter or > 4% free and combined silica (SiO2) - if such silica

concentrations are not available, it must not contain > 5% free and

https://doi.org/10.1016/j.taap.2018.10.023

Received 7 September 2018; Received in revised form 20 October 2018; Accepted 23 October 2018

⁎ Corresponding author at: Centers for Disease Control and Prevention, National Institute for Occupational Safety and Health, Health Effects Laboratory Division,

Exposure Assessment Branch, 1095 Willowdale road, Morgantown, WV 26505, USA.

E-mail addresses: ved2@cdc.gov, tokhaliullin@mix.wvu.edu (T.O. Khaliullin), edk8@cdc.gov (E.R. Kisin), wqu1@cdc.gov (N. Yanamala),

mzi6@cdc.gov (S. Guppi), mHarper@zefon.com (M. Harper), fwc8@cdc.gov (T. Lee), ats1@cdc.gov (A.A. Shvedova).

Toxicology and Applied Pharmacology 362 (2019) 67–76

Available online 25 October 2018 0041-008X/ Published by Elsevier Inc.

T
combined silica. Currently, in order to prevent caking in humid conditions and to increase the effectiveness of aerosolization, several types

of hydrophobic surface treatments (e.g. fatty acids or silicone) for

commercial rock dust have been developed. Experiments conducted

with treated rock dust reveled that during the application, respirable

particle concentrations in the air may exceed the permissible levels;

however, there are administrative controls in place to keep miners

minimally exposed to rock dusting during application (Harris,

Organiscak, Klima, and Perera, 2017).

A considerable body of literature consisting of studies on coal miners and the coal mining environment supports the notion that exposure to coal mine dust can lead to various respiratory diseases

causing disability and premature mortality (Wallace et al., 2011).

However, there is little data available to determine whether exposure to

calcium carbonate rock dust affects human health. In several epidemiological observations and case reports it was proposed that co-exposure with silica is most likely responsible for the adverse outcomes in

susceptible individuals, even though first-hand silica exposures were

very low (Angotzi et al., 2005; Bello et al., 2015; Crummy, Carl,

Cameron, and Heaney, 2004; Doig, 1955; Yildirim, Akgedik, Akgedik,

and Nazaroglu, 2016). We were also unable to find information as to

how surface treatments might affect the toxicological profile of the

calcium carbonate rock dusts.

In this paper we investigate the comparative cytotoxicity of respirable calcium carbonate rock dust particles with or without specific anticaking surface treatments, using a surrogate for lung epithelial cells

A549, a human lung epithelial adenocarcinoma and widely used cell

model for alveolar epithelial function (Hetland et al., 2001; Ovrevik

et al., 2006; Vuong et al., 2017). We also used the human promyelocytic

cells (THP-1) differentiated with phorbol 12-myristate 13-acetate

(PMA), to study the macrophage responses to external stimuli

(Chanput, Mes, and Wichers, 2014; Riendeau and Kornfeld, 2003;

Tedesco et al., 2018; Van der Meeren, Moureau, Laurent, Laroche, and

Angulo, 2016; Voth, Howe, and Heinzen, 2007). The dose ranges selected in this study were relevant to RD concentrations during the

dusting procedures in the mines (provided by Pittsburgh Mining Research Division). To better model the in mine scenario, we estimated

the number of alveolar epithelial cells and macrophages in the human

lungs in determining the dose ranges (See supplementary material).

2. Materials and methods

2.1. Particles

Four types of the rock dust samples were analyzed: untreated

limestone (UL, Allegheny Mining Corporation), treated limestone (TL,

private enterprise), untreated marble (UM, Micro-WhiteTM 100, Imerys

Carbonates), and treated marble (TM, a mix of KotamiteTM and MicroWhiteTM 100, Imerys Carbonates). Crystalline silica (Min-U-Sil® 5, US

Silica Corp.) served as a positive control in this project. The TL siliconebased hydrophobic surface treatment is proprietary and details are

unknown. TM samples consist of a blend of 87.5% untreated marble

with 12.5% of marble product surface-modified by stearate-based

treatment (total 0.125% of stearic acid). During the typical treatment,

stearic acid is adsorbed on the surface of CaCO3 particles by covalent

bond between the stearic acid “head” group and Ca2+, forming a

monolayer of hydrophobic molecules (Cao et al., 2016) (Fig. 1). However, depending of the method used, stearate molecules may or may not

form the monolayer or even produce multilayered surface (Shi, Rosa,

and Lazzeri, 2010; Jeon et al., 2018). Some of the stearate may also be

present in the free state.

Respirable fractions were collected from the aerosol generation

chamber with FSP10 cyclones (GSA Messgerätebau GmbH, Ratingen,

Germany) loaded with polyvinyl chloride filters (PVC, 5 μm pore size,

37 mm, SKC Inc., Eighty Four, PA), washed with a mix of 50% phosphate buffered saline and 50% isopropyl alcohol, centrifuged and dried.

For all cell-based experiments, samples were additionally washed three

times with saline, pelleted and re-suspended in United States

Pharmacopeia (USP) grade sterile Phosphate Buffer Saline (VWR

International, Radnor, PA USA) as a stock solution. Dilutions were done

in respective clear media with no phenol red, 1% fetal bovine serum

(FBS), 1% L-glutamine (HyClone), and 1% penicillin-streptomycin antibiotic mixture (HyClone). Prior to all experiments, endotoxin levels

were determined in all samples using LAL Chromogenic Endotoxin

Quantitation kit (Pierce, ThermoFisher Scientific, MA USA) according

to the manufacturer's instructions. Endotoxin was assessed in three

independent experiments in triplicates at 0.01, 0.05 and 0.2 mg/ml of

each particle sample. All RD samples contained < 1% of free silica

(Supplementary Table 1) (Soo et al., 2016).

2.2. Dynamic light scattering (DLS) analysis

The average hydrodynamic diameter (Zavg) of respirable rock dust

particles was estimated using a Nanotrac 252 (Microtrac,

Montgomeryville, PA) and Microtrac particle sizing software, version

10.3.11, with a backscatter angle of 90° and a laser wavelength of

657.0 nm. Stock solutions of rock dust were suspended in ultra-pure

USP grade water to achieve 1 mg/ml samples of rock dust. Parameters

used included the Refractive indexes (RI) of 1.54 for the samples, and

1.330 for the solvent (water) at the temperature setting of 25 °C. The

mean hydrodynamic diameters reported are averages of six measurements obtained using three separate preparations of particles.

2.3. Cell cultures

A549 cells were grown in Dulbecco's modified Eagle's medium

(DMEM) supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin-streptomycin antibiotic mixture (growth medium), at 37 °C in a

5% CO2 humidified environment. THP-1 cells were obtained from the

American Type Culture Collection (ATCC®, Manassas, VA) and grown in

RPMI 1640 with 10% FBS, 2 mM L-Glutamine and 1% penicillin-streptomycin antibiotic mixture (Invitrogen, Carlsbad, CA). Following the

fourth passage, monocyte-like THP-1 were differentiated into macrophages by incubation with 20 ng/ml (32.4 nM concentration) phorbol12-myristate-13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO) for 48 h,

followed by 24 h incubation in growth medium without PMA. This

protocol induces a reproducible phenotype of a macrophage, suitable

for the study of inflammatory responses (Lund et al., 2016). Differentiated THP-1 cells were washed, detached using Accutase® enzymatic

solution (Innovative Cell Technologies Inc., San Diego, CA) and plated

into 96-well plates for further experiments.

Fig. 1. Stearate-based treatment of Calcium carbonate particulates, partially reprinted from (Cao et al., 2016), with permission from Elsevier.

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2.4. A549 and THP-1 cells' treatment and toxicological assessment

To assess time- and dose-dependent cytotoxicity, A549 cells

(15.6 × 103 per well) were seeded into 96-well plates and treated with

4 different rock dust samples and crystalline silica as a positive control

at 0.005, 0.01, 0.025, 0.05, 0.1 or 0.2 mg/ml, equivalent to 0–0.06 mg/

cm2 of the cell surface. Cell supernatants were collected 24 and 72 h

after incubation for lactate dehydrogenase (LDH) measurements and

cells were tested for viability using the Alamar Blue bioassay (Thermo

Fisher Scientific, Waltham, MA). Alamar blue reagent was added to

cells in 96-well plates (1:10 dilution in clear growth medium) followed

by 4 h of incubation. Fluorescence was measured using a Synergy H1

hybrid multimode microplate reader (BioTek Instruments, Inc.,

Winooski, VT, USA) at Ex530/Em590 nm. Cell viability, proportional to

the changes in fluorescence, was normalized to the controls and expressed as percentage viability. Cell culture supernatants were collected

for lactate dehydrogenase (LDH) measurements using Liquid LD

Reagent Set (Pointe Scientific, Lincoln Park, MI) in 2 independent experiments. Cytokines and chemokines were analyzed in the cell supernatants after 72 h of exposure using a Bio-Rad 27-plex human assay kit

(Bio-Rad, Hercules, CA). Two independent experiments were carried

out with two replicate measurements for each supernatant.

Differentiated THP-1 were collected after the 24 h resting period,

washed with clear exposure medium, seeded into 96-well plates

(15.6 × 103 per well) and treated with particles similarly to A549 cells

treatment. 24 h after incubation, supernatants were collected for LDH

and cytokines measurements, and Alamar Blue bioassay (Thermo Fisher

Scientific, MA) in the same manner as with A549 cells. Caspase-1 activity in THP-1 cells, exposed to three concentrations (0.01, 0.05,

0.1 mg/ml for RD and 0.005, 0.02, 0.05 for silica) of particles or clear

exposure media, was determined using Caspase-Glo®–1 Inflammasome

Assay (Promega Corporation, Madison, WI). We used smaller exposure

doses for silica, since it is a known activator of caspase-1 and higher

doses were too cytotoxic.

2.5. Transmission electron microscopy (TEM) of particles and cells

Transmission electron micrographs of respirable rock dust particles

were obtained on a JEOL TEM 1220 (Peabody, MA) at a working voltage of 80 kV. TEM images were obtained by placing a drop of diluted

sample on a formvar-coated copper grid to dry. Several TEM images

were analyzed to identify at least 5–10 individual particles per image to

estimate approximate dimensions of rock dust particles. For internalization studies, the cells were fixed in 0.5 ml Karnovsky's fixative

(2.5% gluteraldehyde, 2.5% paraformaldehyde in 0.1 M Sodium

Cacodylic buffer) and post-fixed in 2% osmium tetroxide for 1 h. The

cells were then dehydrated, embedded in epon, thin sectioned, and

stained with Reynold's lead citrate and uranyl acetate. The sections

were imaged on a JEOL 1220 transmission electron microscope.

2.6. Uptake and phagolysosome acidification inhibition study

To examine uptake mechanisms involved in treated/untreated RD

particles exposures, differentiated THP-1 macrophages were pre-incubated for 30 min at 37 °C in medium containing one of the following

inhibitors: 2 μM cytochalasin D (reversible actin polymerization inhibitor) (Sigma-Aldrich, St. Louis, MO), 250 nM Bafylomycin A (inhibitor of vacuolar proton pump) (Sigma-Aldrich, St. Louis, MO) or

medium control (0.1% dimethyl sulfoxide, Sigma-Aldrich, St. Louis,

MO), used as the vehicle for the inhibitors. After treatment, cells were

exposed to 4 types of RD particles (0.1 mg/ml) or silica (0.05 mg/ml)

for 6 h, then cell viability, caspase-1 activity, and cytokine production

were measured as indicated previously.

2.7. Statistical analysis

Treatment related differences were compared by one-way ANOVA

using all pairwise multiple comparison procedures (Holm-Sidak

method). All data are presented as means plus the standard error of the

mean (SEM). P values of < 0.05 were considered statistically significant.

3. Results

3.1. Particle characterization

Average hydrodynamic diameters of 863 ± 31 nm,

1209 ± 64 nm, 707 ± 32 nm, 715 ± 21 nm, 1605 ± 107 were

found for UM, TM, UL, TL and silica, respectively, using DLS. The Zavg

values for each sample were the mean of six measurements.

Transmission Electron Microscopy analysis showed that the rock dust

particle sizes ranged from 200 nm to 3000 nm (Fig. 2). A detailed airborne rock dust characterization was previously described (Soo et al.,

2016). Assessment of rock dust particle dimensions derived from TEM

images are in good agreement with the particle sizes estimated by DLS

measurements. The level of endotoxin in the highest concentration of

washed respirable rock dust samples (1 mg/ml stock) was estimated as

0.078 ± 0.003 EU/ml (7.8 pg/ml). This is a no-observable effect level

for endotoxin on cells in culture (Ryan, 2008). The reported endotoxin

values for each sample (n = 3) represent the level of endotoxin in the

particulate dosing suspensions, but not levels of endotoxin from bulk

samples or aerosolized dusts.

3.2. Transmission electron microscopy of the cells

Rock dust and silica particles were clearly seen as electronic dense

inclusions in cytoplasm of the A549 cells 72 h after exposure

(Supplementary Fig. 1) and THP-1 cells 24 h after exposure

(Supplementary Fig. 2).

Fig. 2. Representative TEM images of respirable rock dust samples. UL - untreated limestone, TL – treated limestone, UM – untreated marble, TM – treated marble.

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3.3. Cytotoxicity of rock dust and silica in A549 cells

The cytotoxic potential of the respirable fraction of washed RD

samples was determined after exposure of A549 cells to various concentrations (0–0.2 mg/ml) of UM, TM, UL, TL and silica for 24 and 72 h.

Viability was slightly decreased in TL and TM-treated groups at 24 h

(Fig. 3A). There was a small, but statistically significant drop in viability in UL and TL-exposed samples at 72 h (0.05–0.2 mg/ml) compared to control cells (Fig. 3B). Silica exposure caused dose-dependent

reduction in viability at both time points (Fig. 3A and B). Cell damage

measured as LDH in the cell supernatants after 24 and 72 h exposure

was not significantly different between the four rock dusts and control

non-exposed cells, while silica treatment caused statistically significant

LDH increase only at the concentration of 0.2 mg/ml for both time

points (data not shown).

3.4. Cytotoxicity of rock dust and silica in THP-1 cells

24 h after exposure, we observed a significant decrease in viability

of the differentiated THP-1 cells treated with TM at 0.05, 0.1 and

0.2 mg/ml doses, while silica caused dose-dependent reduction in

viability starting at 0.025 mg/ml (Fig. 4A). Accordingly, LDH activity in

supernatants went up for Silica and TM in dose-dependent manner

(Fig. 4B).

3.5. Caspase-1 activity in THP-1 cells

Macrophages, exposed to all silica concentrations, TM (0.05 and

0.1 mg/ml) and UM (0.1 mg/ml) but not to UL or TL displayed a dosedependent statistically significant increase in caspase-1 activity (Fig. 5)

compared to control cells, which indicates the possible inflammasome

involvement.

3.6. Pro-inflammatory cytokine/chemokine secretion in A549 cells

following various rock dusts exposures

The release of cytokines in cell supernatants was assessed in the

A549 cells exposed to three different concentrations of RD and silica at

72 h post-exposure (Supplementary Table 2). Treated and untreated

marble had the most impact on the overall cytokine secretion with

statistically significant elevation in the concentrations of IL-6, Il-8, IL12(p70), TNF-α, IFN-γ and IL-17. Treated marble additionally induced

Fig. 3. Viability of the A549 cells following 24 h (A) and 72 h (B) exposure to various rock dusts and silica. Values are expressed as mean ± SEM. *p < .05 vs.

control.

Fig. 4. Viability (A) and LDH leakage (B) following exposure to various rock dusts and silica in differentiated THP-1 cells. Values are expressed as mean ± SEM.

*p < .05 vs. control.

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significantly higher IL-10, basic Fibroblast Growth Factor (bFGF) and

platelet-derived growth factor (PDGF) secretion compared to other

samples.

3.7. Hierarchical cluster analysis of cytokine profiles in THP-1 supernatants

24 h post-exposure

The release of cytokines in cell supernatants was assessed in the

THP-1 cells exposed to four different concentrations of RD and silica at

24 h post-exposure (Supplementary Table 3). Both TM and silica induced significant dose-dependent increase in the production of proinflammatory cytokines and together formed a major cluster, separated

from other particles (Fig. 6A). Both TM and silica had caused significant

increase in the secretion of MIP-1β and eotaxin, again, forming a separate cluster (Fig. 6B), while IL-8 concentration was over the observable range in all samples. Distinct grouping of TM and silica was

observed with > 2-fold increase of IL-2, IL-4, and IL-9 concentrations in

the cell supernatants. (Fig. 6C).

3.8. Uptake-related toxicity study

Cytochalasin D (actin polymerization inhibitor) treatment blocked

the particles uptake and mitigated or significantly decreased LDH

production and caspase-1 activity in treated cells compared to respective controls. Bafilomycin A, an inhibitor of vacuolar H+-ATPase,

prevented caspase-1 activation and partially rescued the LDH leakage in

SiO2- but not TM-exposed cells (Figs. 7 and 8).

4. Discussion

Limestone and marble are rocks, mainly comprised of calcium and

magnesium carbonate mineral varieties. Since ancient times, they have

been used in building and road construction, sculpture, other arts,

agriculture, and even the food industry. In mining, the term “rock dust”

is applied to a finely crushed (pulverized) stone (e.g. limestone) used to

reduce the combustibility of coal dust and prevent an explosion from

propagating. It must contain no > 5% combustible matter and no more

than a total of 4% free and combined SiO2. The federal register requires

that the RD should be effectively applied, but if caking (due to wetting)

occurs, the dust loses its ability to disperse and therefore cannot make

the coal dust inert or prevent an explosion from propagating. Anticaking surface treatments exist that increase the dispersibility, but there

is no data on the potential health implications of such treatments.

Miners' and other workers' exposure may occur during the application

process, especially downwind from the application. In a recent study

that assessed area dust levels at particular locations throughout the

mine, treated RD has been shown to produce more respirable particulates during the dusting procedure than non-treated (Harris et al.,

2017). Authors also raised concern regarding the re-aerosolization of

rock dust covering the floor due to the routine movements of personnel

and equipment. Scoop operators using flinger rock dusting machines

are also at risk.

Scattered evidence shows little or no cytotoxicity caused by calcium

carbonate rock particles, such as limestone, marble, or dolomite (calcium magnesium carbonate). Studies in marble cutters and limestone

quarry workers (Doig, 1955; Angotzi et al., 2005; Bwalya, Bratveit, and

Moen, 2011; Neghab, Abedini, Soltanzadeh, Iloon Kashkooli, and

Ghayoomi, 2012) and several published case reports (Crummy et al.,

2004; Bello et al., 2015; Yildirim et al., 2016) discovered increases of

respiratory symptom occurrence as well as such adverse health outcomes as COPD, pneumoconiosis, and fibrosis. Co-exposure with silica

was proposed to be responsible for adverse outcomes in susceptible

Fig. 5. Intracellular caspase-1 activity following 24 h exposure to various concentrations of rock dusts and crystalline silica in THP-1 cells. Values are expressed as

mean ± SEM. *p < .05 vs. control.

Fig. 6. Hierarchical cluster analysis of inflammatory cytokines (A), chemokines

(B) and TH2-cytokines (C) in THP-1 supernatants 24 h post-exposure to various

rock dusts or crystalline silica.

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individuals, even though the exposure to silica was very low. Previously, several reported studies revealed that the respirable limestone

particles were easily taken up by macrophages causing little or no cytotoxicity as compared to silica (Sugiura et al., 1998), and instillation or

inhalation of limestone dust in rats or rabbits did not cause any pulmonary fibrosis or tumorigenic outcomes (Mashimo et al., 1998; Oyabu

et al., 1998).

Inhaled particles that are deposited in the lung come into contact

first with epithelial cells and resident macrophages and induce specific

reaction patterns that depend on particle chemical composition, shape,

solubility, and surface properties. Reactions range from uneventful

clearance to cell damage and full-scale immune activation, accompanied by the release of alarmins and other bioactive compounds, including cytokines and chemokines. With that in mind, our research

question was whether the hydrophobic surface treatment of calcium

carbonate RDs may influence their bioactivity. In the present study, we

assessed cytotoxicity and specific responses to several different types of

treated and untreated respirable RDs in comparison to a known fibrogenic and proinflammatory agent, crystalline silica.

Due to the fact that little is known about respirable rock dust levels

in personal breathing zones, it is hard to estimate the real workplace

exposures. Area sampling performed in a test mine during dusting

procedures revealed variability between samples depending on the

sampling point and type of the rock dust used. Personal dust monitors

can easily become overloaded if located in the rock dust cloud and even

at 500 ft. downwind the concentrations of respirable rock dust reached

17 mg/m3

. For our calculations, we chose the overall respirable dust

standard of 1.5 mg/m3

, recently introduced by MSHA (MSHA, 2014).

Our mid-range dose (0.01 mg/ml) is equivalent to nine years of human

exposure in the case of A549 cells or 1.5 years for THP-1 macrophages.

(See supplement material).

Epithelial cell damage and reactivity plays a substantial role in the

pathology of lung diseases, including exposure to particulates (Camelo,

Dunmore, Sleeman, and Clarke, 2014; Manke, Wang, and Rojanasakul,

2013). A549 cell line models the type II alveolar epithelial cells and is

widely used for toxicity, drug metabolism, and transfection studies.

A549 cells respond to a number of airborne particulates and pollutants

by producing inflammatory mediators (Corsini et al., 2013). There is

also a solid number of publications on silica-induced toxicity in A549

cells (Hetland et al., 2001; Vuong et al., 2017; Ovrevik et al., 2006).

Previous reports indicated that A549 exhibit phagocytosis of ~1 μmsized particles, albeit at a much slower rate than macrophages (Kuhn

et al., 2014; Rothen-Rutishauser, Muhlfeld, Blank, Musso, and Gehr,

2007), indeed, at 72 h post-exposure we could clearly see the RD and

silica inside the cells by TEM. After the RD treatment, dose- and timedependent cytotoxicity as well as cell damage were only seen at 72 h

Fig. 7. LDH leakage in the supernatant (A) and intracellular caspase-1 activity (B) following 6 h of exposure to various rock dusts or silica in THP-1 cells pretreated

with Bafilomycin A, Cytochalasin D or exposure medium with 0.1% DMSO. Values are expressed as mean ± SEM. *p < .05 vs. respective control.

Fig. 8. The summary of the effects of Cytochalasin D and Bafilomycin A pretreatment on cytotoxicity (LDH leakage) and caspase-1 activation in PMA-differentiated

THP-1 macrophages upon exposure to RD or Crystalline Silica.

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post exposure with the most effect upon exposure to silica and untreated limestone. Still the viability was relatively high and the cell

damage remained low. The extent of cytokine responses, however, has

provided more sensitive data. The analysis of inflammatory cytokines/

chemokines revealed an overall stronger effect of marble, both treated

and untreated, compared to limestone samples. TM induced both

greater TH1-type cytokine response, and increased bFGF and PDGF secretion. The former may prompt the localized inflammatory reaction in

the lung with edema and recruitment of immune cells, while the latter

has been implicated in the remodeling of airways and respiratory

compartment (Kelley, 1990; Shimizu et al., 2000). Overall, our results

unveiled treatment related differences as well as material dependent

changes in biological responses of the surrogate type II alveolar epithelial cells.

Alveolar macrophages (AMs) are the housekeepers of the lung.

Aside from participating in local homeostasis, such as the recycling of

surfactant components and efferocytosis, they are equipped and armed

to deal with particles that get deep into the alveolar region from inorganic particulate matter to saprophytic and pathogenic microflora

(Laskin, Malaviya, and Laskin, 2015). Importantly, alveolar macrophages contribute to the overall tolerant immunological milieu, possessing the so-called M2-like phenotype in the resting state (Alber,

Howie, Wallace, and Hirani, 2012). Nevertheless, AMs are able to

quickly adopt a phenotype (activation) required for playing a major

role in a variety of infectious and noninfectious pathologies, including

tuberculosis, bacterial and viral pneumonias and pneumoconioses.

Taking into consideration that calcium carbonate is quickly eliminated from the lungs (Oyabu et al., 1998), we did not aim to address

long-term toxicity, but rather the acute responses to respirable RD.

Thus, we sought to determine the inflammogenic potential of the rock

dust compared to silica, using the PMA-differentiated THP-1 cells as a

model of macrophages primed toward inflammatory responses (Lund

et al., 2016; Sharma, McLeland, Potter, Stern, and Adiseshaiah, 2018).

Previously, PMA-differentiated THP-1 cells have been extensively used

as a model for alveolar macrophages (Van der Meeren et al., 2016;

Kletting et al., 2018; Riendeau and Kornfeld, 2003; Voth et al., 2007).

The ability of silica to induce cell death in macrophages, which

contributes to the pathogenesis of silicosis, is well known (Gilberti,

Joshi, and Knecht, 2008; Costantini, Gilberti, and Knecht, 2011; Joshi

and Knecht, 2013; Hamilton Jr, Thakur, and Holian, 2008). Unsurprisingly, we saw a marked dose-dependent cytotoxicity in the positive control group. Of note, silica-induced inflammation and disease

progression is heavily dependent on innate immune components, rather

than lymphocyte-mediated responses (Beamer et al., 2010). Studies of

silicosis in humans and animal experiments describe the latency period

before the onset of the active lung inflammatory reaction (Kawasaki,

2015), which is also separate from the long-term outcomes, such as

pulmonary fibrosis. (Rabolli et al., 2011; Re et al., 2014). A recent review hypothesize that this delay and the following exacerbation depends on the concerted action of different immune cell subsets, including inflammatory and so-called alternatively activated, tolerogenic

macrophages (Kawasaki, 2015). Among the rock dust samples, only the

TM caused a statistically significant decrease in cell viability and increase in extracellular LDH activity in THP-1 cells. Cytokine profiling

revealed significant dose-dependent increase in the production of

proinflammatory cytokines for both silica and TM, which formed a

cluster, separated from control cells, UM and other rock dust particles.

With the exception of TM, there was no apparent distinction in cytokine

responses between coated and uncoated RD particles, which were

clustered together on the concentration basis, lower doses being very

close to control samples. These results prompted the question of the

possible mechanism responsible for a partial semblance between TM

and silica responses. Since we could clearly observe the phagocytosis of

all particles and visualize them inside the phagolysosomes, we first

wanted to explore the NLRP3 inflammasome pathway, intrinsic in

macrophages and implicated in a variety of cell-particle interactions,

including asbestos, nanoparticles and crystalline silica exposures

(Hornung et al., 2008; Sharma et al., 2018; Cui et al., 2014; Palomaki

et al., 2011). Indeed, the activity of caspase-1, an enzyme responsible

for the cleavage of IL-1β precursor into the active form and processing

of several other cytokines, was also significantly dose-dependently

elevated in both TM and silica-exposed cells, as well as in cells exposed

to high UM concentration which indicates possible inflammasome involvement (Petrilli, Dostert, Muruve, and Tschopp, 2007).

To test if the particles' toxicities were uptake-related, we first pretreated cells with Cytochalasin D, which inhibits the actin polymerization and disrupts phagocytosis in macrophages. As expected, this

completely prevented the cytotoxic effect observed in TM-exposed

group six hours post exposure. Silica-treated cells still showed slight but

statistically significant elevated caspase-1 activity even after

Cytochalasin D administration. This could be the result of silica crystals

binding to the cell surface, which has been previously shown to be

enough stimulus for inflammasome activation in larger particles (Hari

et al., 2014). Several studies have reported that crystalline silica exerts

its toxicity through the destabilization of lysosomal membranes

(Hornung et al., 2008; Jessop, Hamilton Jr., Rhoderick, Fletcher, and

Holian, 2017). The specific mechanisms are still debated, although the

recent articles pointed out the importance of phagolysosome acidification in lysosomal membrane permeabilization and increased

NLRP3 inflammasome activity (Jessop et al., 2017; Hornung et al.,

2008; Kusaka et al., 2014). To investigate this particular mechanism,

we utilized the inhibitor of vacuolar proton pump (vATPase) Bafilomycin A. The pump produces a proton gradient across the lysosomal

membrane through the ATP hydrolysis, thus creating the low-pH environment in lysosomes and phagolysosomes, required for the macrophage pathogen and particle clearance function. Bafilomycin A treatment prevented caspase-1 activation, indicating the importance of the

phagolysosomes acidification for the activation of inflammasome

(Fig. 9). At the same time, Bafilomycin A substantially decreased the

LDH leakage in silica-exposed cells, but had no effect on marked LDH

release in the supernatant of TM-exposed macrophages, which requires

further investigation of TM behavior while inside the phagolysosome.

Taken together, the data suggests that stearate surface treatment of

the innocuous calcite particles increases cytotoxicity (although on a

much lesser scale compared to silica) upon the phagocytosis of those

particles by macrophages (Fig. 4A). The exact reason for that is currently unclear. American Conference of Governmental Industrial Hygienists (ACGIH®) considers stearates to be of low toxicity, however,

ACGIH® opinion was formed based mostly on studies that did not use

the inhalation route. One may consider that this low toxicity rating was

a factor in the selection of the coating material by the company-producer. It has to be pointed out that the coated marble rock dust product

is originally intended to be used as a polymer additive to facilitate the

dispersion in organic liquids/solvents, but not for the procedures involving aerosolization.

The existing toxicity data on metallic stearates is scarce. One subchronic study in rats given calcium stearate via intratracheal instillation

(for 4 to 8 months) revealed pulmonary damage and alveolar emphysema (Tarasenko, Shabalina, and Spiridonova, 1976). Accidental zinc

stearate powder aspiration has been implicated in a number of respiratory distress cases in infants (Elder, 1982). TiO2 nanoparticles

when coated with stearate exhibited higher cytotoxicity in mouse benign fibrosarcoma cells compared to untreated ones, even facilitating

their transformation into aggressive tumor cells (Onuma et al., 2009).

Use of nickel ferrite nanoparticles covered with oleic acid also produced

increased cytotoxicity observed in neuroblastoma cells (Yin, Too, and

Chow, 2005). The surface hydrophobicity itself could impart hazardous

properties (Gareth, 2017), however, it is probably not an issue in this

case, since TL silicone-based treatment had no observable cytotoxic

effects in our study; additionally, other studies observed the beneficial

effect of hydrophobic coating on lysosomal damage and overall particulate toxicity (Wang et al., 2012; Hohr et al., 2002). The specific TM

T.O. Khaliullin et al. Toxicology and Applied Pharmacology 362 (2019) 67–76

73
biological effects may occur through the formation of the interface

between the coating and biomacromolecules and subsequent interaction with lysosomal membrane components (Nel et al., 2009), but

without the exact knowledge of particle preparation (due to the proprietary character of the treatment) our speculations are limited. For

instance, depending of the method used, stearate molecules may or may

not form the monolayer or even produce multilayered surface (Shi

et al., 2010; Jeon et al., 2018), with a various degree of (non)polarity,

which plays a major role in the cytotoxicities of particulates (Fröhlich,

2012).

Overall, our results support the previously published data showing

that respirable calcium carbonate particles themselves probably have

little to no effect on epithelial cells or macrophages. Surface treatment

of these particles may have no effect either or alter their cytotoxicity

profile in an unexpected way. Limitations of our study should be taken

into consideration, including the use of transformed cell lines and little

information on the specifics of the RD coating. In the context of real-life

application, exposure to aerosols with high free silica content still poses

more threat than rock dusting. Nevertheless, a precautionary approach

is warranted to avoid miners' exposure downwind of rock dust applications (Harris et al., 2017). Prospective studies can be expanded to

include more relevant cell types and in vivo experimental models that

would take into account the realistic exposure scenarios. For example,

future research should benefit from understanding and evaluating the

combined effects of rock dust, silica and/or coal dust.

5. Conclusions

None of the rock dust samples induced significant cytotoxicity in

A549 cells following 24 or 72 h of exposure. Rock dust particles were

also able to induce some inflammatory cytokine production in A549

cells, however, the response was less pronounced compared to silica.

Untreated limestone (UL), untreated marble (UM) and silicone-treated

limestone (TL) particles were readily internalized by THP-1 macrophages but caused very little or no toxicity even at high doses. Exposure

of THP-1 cells to the blend of untreated (87.5%) and stearate-treated

(12.5%) marble (TM) led to significant dose-dependent LDH increase,

inflammatory and TH2-type cytokines/chemokines release along with

the elevated caspase-1 activity, however the observed effects were

substantially less prominent compared to silica at the same mass concentrations. Inhibition of particles uptake by cells using Cytochalasin D

prevented the adverse effects of both TM and silica. The use of

Bafilomycin A that prevents the acidification of phagolysosomes, reduced the toxic effects of silica treatment, but not TM, perhaps due to

different mechanisms of action. Altogether, the obtained results showed

that out of all tested rock dust samples only stearate-treated marble

caused moderate toxicity in vitro, however still significantly below the

effect of silica.

6. Disclaimer

The content and conclusions of this publication are those of the

authors and do not necessarily reflect the views or policies of the

National Institute for Occupational Safety and Health, Centers for

Disease Control and Prevention, nor does mention of trade names,

commercial products, or organizations imply endorsement by the U.S.

government. The authors declare no competing financial interest.

Acknowledgements

We would like to thank Sherri Friend for TEM pictures and consultation, Dr. Donald Beezhold and Mackenzie Newman for the helpful

review of the manuscript, Dr. Steve Mischler, Dr. Eranda Perera, and

Marcia Harris from NIOSH/PMRD Explosions Prevention Group for

supplying the rock dust samples and valuable inputs.

Funding

This work was supported by the National Institute for Occupational

Safety and Health (NIOSH) 939054L and National Occupational

Research Agenda (NORA) 939051G grants.

Fig. 9. The schematic illustrating the effect of Cytochalasin D and Bafilomycin A on caspase-1 activation in PMA-differentiated THP-1 macrophages upon exposure to

treated marble (TM) OR crystalline silica.

T.O. Khaliullin et al. Toxicology and Applied Pharmacology 362 (2019) 67–76

74
Appendix A. Supplementary data

Supplementary data to this article can be found online at https://

doi.org/10.1016/j.taap.2018.10.023.

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