(2022) 22:233
Monsé et al. BMC Pulmonary Medicine
https://doi.org/10.1186/s12890-022-02021-y
Open Access
RESEARCH
No inflammatory effects after acute
inhalation of barium sulfate particles in human
volunteers
Christian Monsé*, Götz Westphal, Monika Raulf, Birger Jettkant, Vera van Kampen, Benjamin Kendzia,
Leonie Schürmeyer, Christoph Edzard Seifert, Eike‑Maximilian Marek, Felicitas Wiegand, Nina Rosenkranz,
Christopher Wegener, Rolf Merget, Thomas Brüning and Jürgen Bünger
Abstract
Background: Most threshold limit values are based on animal experiments. Often, the question remains whether
these data reflect the situation in humans. As part of a series of investigations in our exposure lab, this study investi‑
gates whether the results on the inflammatory effects of particles that have been demonstrated in animal models can
be confirmed in acute inhalation studies in humans. Such studies have not been conducted so far for barium sulfate
particles (BaSO4), a substance with very low solubility and without known substance-specific toxicity. Previous inhala‑
tion studies with zinc oxide (ZnO), which has a substance-specific toxicity, have shown local and systemic inflamma‑
tory respones. The design of these human ZnO inhalation studies was adopted for B
aSO4 to compare the effects of
particles with known inflammatory activity and supposedly inert particles. For further comparison, in vitro investiga‑
tions on inflammatory processes were carried out.
Methods: Sixteen healthy volunteers were exposed to filtered air and BaSO4 particles (4.0 mg/m3) for two hours
including one hour of ergometric cycling at moderate workload. Effect parameters were clinical signs, body tempera‑
ture, and inflammatory markers in blood and induced sputum. In addition, particle-induced in vitro-chemotaxis of
BaSO4 was investigated with regard to mode of action and differences between in vivo and in vitro effects.
Results: No local or systemic clinical signs were observed after acute BaSO4 inhalation and, in contrast to our previ‑
ous human exposure studies with ZnO, no elevated values of biomarkers of inflammation were measured after the
challenge. The in vitro chemotaxis induced by BaSO4 particles was minimal and 15-fold lower compared to ZnO.
Conclusion: The results of this study indicate that BaSO4 as a representative of granular biopersistent particles with‑
out specific toxicity does not induce inflammatory effects in humans after acute inhalation. Moreover, the in vitro data
fit in with these in vivo results. Despite the careful and complex investigations, limitations must be admitted because
the number of local effect parameters were limited and chronic toxicity could not be studied.
Keywords: Barium sulfate, Granular biopersitent particles, Human inhalation study, Induced sputum, Inflammatory
markers, Particle-induced chemotaxis, Zinc oxide
*Correspondence: christian.monse@dguv.de
Institute for Prevention and Occupational Medicine of the German
Social Accident Insurance, Institute of the Ruhr University Bochum (IPA),
Bürkle‑de‑la‑Camp‑Platz 1, 44789 Bochum, Germany
Background
Inhalation of granular biopersistent particles (GBP)
can lead to fatal diseases such as lung cancer in rats in
the case of chronic overload, even though GBP are only
weakly toxic, as suggested by animal studies [1, 2]. It is
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Monsé et al. BMC Pulmonary Medicine
(2022) 22:233
generally assumed that this is primarily a consequence of
chronic inflammation and that GBS in overload can also
cause serious lung diseases in humans. Therefore, the
threshold level value (TLV) for workplaces aims on the
avoidance of inflammatory particle effects [1]. Based on
this assumption, in Germany a threshold limit value of
0.3 mg/m3 was recommended for particles with a density
of 1.0 g/cm3, mainly based on toxicological data of titanium dioxide [1]. The occupational exposure limit (OEL)
for particles > 100 nm without substance-specific toxicity is based on this TLV [3] and was set at 1.25 mg/m3
for the respirable fraction and 10 mg/m3 for the inhalable
fraction for particles with a density of 2.5 g/cm3. Particles with substance-specific toxicity are regulated with an
OEL below 1.25 mg/m3.
Thus, barium sulfate (BaSO4) particles were used for
ethical reasons in this study. B
aSO4 is considered to be a
chemically inert particle without substance-specific toxicity according to animal experiments [4, 5] and in vitro
data [6, 7]. Although lysosomal solubility for BaSO4 was
reported [8, 9], it is classified as GBP in Germany based
on its water solubility. Aim of this study was to enhance
the body of evidence for the assessment of GBP using an
acute human inhalation study. Such human studies are
rare and have not been done with BaSO4.
Sikkeland and co-workers conducted an inhalation
study in humans using micro-sized aluminium oxide
(volume median diameter particle size was 3.2 μm) as a
representative of a chemical inert substance at concentrations between 3.8 and 4.0 mg/m3 for 2 h [10]. Elevated
levels of neutrophils, protein level of IL-8 and gene signature related to several pathways were found in induced
sputum collected 24 h after exposure, but no changes in
blood parameters were measured 4 h after exposure. To
our knowledge, further human inhalation- or epidemiological studies with BaSO4 are not available and it is not
known whether micro-sized
BaSO4 induces any toxic
effects in humans after acute inhalation.
A controlled inhalation study with human volunteers
at our exposure lab using nano-sized ZnO at concentrations up to 2 mg/m3 showed concentration-dependent
inflammatory effects, including an increase of body
temperature in several subjects, as well as an increase in
blood neutrophils, and acute phase protein levels [11]. In
an other ZnO study, subjects were exposed to nano- and
micro-sized ZnO for 2 h at concentration levels of 2 mg/
m3 each [12]. We could show that biological effects were
more pronounced after exposure of micro-sized ZnO
particles.
ZnO is not an inert particle and is known to cause
metal fume fever after inhalation. In this study, the same
effect parameters as in the ZnO studies were used. We
hypothesized that the biological effect markers that were
Page 2 of 12
observed after inhalation of ZnO are not enhanced after
acute inhalation of a chemically inert substance. Based
on this assumption, we used a higher concentration for
BaSO4 (4.0 mg/m3) than for the ZnO studies. The justification of the dose selection is discussed in detail in the
discussion. Due to organizational reasons and time constraints, we were only able to perform an acute inhalation
study. In this study, the endpoints clinical signs, body
temperature, and inflammatory markers in blood and in
induced sputum were examined in the subjects.
Moreover, we investigated if in vitro data based on particle-induced chemotaxis reflect these in vivo outcomes.
This study may help to bridge the gap between animal
and human in vivo experiments.
Materials and methods
Since this study makes comparisons with previously
conducted ZnO studies, many methods were adopted,
especially from the study by Monsé et al. [12], which are
described again for clarity.
Micro‑sized BaSO4 particles
The design and technical setup of the human wholebody exposure unit at our institute was described previously in detail [13]. The unit allows the exposure of up to
4 subjets at the same time. A self-constructed nebulizer
was installed in the air conditioning duct of the exposure
unit equipped with a 7.0 L stirred tank and a self-priming two-substance nozzle (model 970, Düsen-Schlick
GmbH, Untersiemau, Germany). A suspension of 18.0 g
of purchased
BaSO4 (for medical purposes, CAS No.
7727-43-7, Merck GmbH & Co. KG, Darmstadt, Germany) in 5.0 L of water (water purification system, model
Milli-Q Advantage A 10, Merck KGaA, Darmstadt, Germany) was continuously nebulized with pressurized air
at 3.0 bar. The BaSO4 was sieved for 5 min before use,
using the < 100 μm fraction (Vibratory Sieve Shaker,
model AS 200 control, Retsch GmbH, Haan, Germany)
to exclude larger clumps in the stirred tank. The suspension was stirred during dosing (260 rounds per min). Two
flow baffles were installed in the tank to ensure a turbulent flow. The metering of the BaSO4 was controlled via
a pulse width modulation by means of a compressed
air shutdown of the two-substance nozzle. The aerosol
droplets of the sprayed suspension completely dried out
during the flight into the exposure unit and released the
desired BaSO4 particles.
Briefly, constant target concentrations of 4.0 mg/m3
micro-sized BaSO4 were planned. The homogeneity of
the particle atmosphere was given by the use of a ceiling
fan and the type of air supply and exhaust. It was confirmed in preliminary tests by determining the particle
size distributions at different locations in the exposure
Monsé et al. BMC Pulmonary Medicine
(2022) 22:233
unit (data not shown). Sham exposures (0 mg/m3 BaSO4)
were performed with filtered and conditioned air. All
exposure scenarios were carried out with an air exchange
rate at 12 per hour (360 m3/h) with a room temperature
of 23.4 °C (± 0.5 °C) and a relative humidity of 44.5%
(± 1.8%).
Characterization of BaSO4 particles
An Aerosol Particle Sizer (APS, model 3321, TSI Inc.,
Shoreview MN, USA, equipped with a 1:20 aerosol
diluter, model 3302 A, TSI Inc.) measured the microsized particle numbers and size distributions every 5 min.
(Additional file 1: Figure S1). The measurement system
was installed directly in the exposure unit near the subjects to minimize particle losses to the instrument inlet.
Mass concentration measurements of airborne B
aSO4
were recorded at 1-min intervals using a tapered elemental oscillating microbalance (TEOM, model 1400a,
Rupprecht and Patashnik, Albany NY, USA). Both the
airborne mass of B
aSO4 particles and the drying process
of the B
aSO4 suspension were confirmed by gravimetric
measurements using a total dust sampling system (model
GSP, GSA Messgerätebau GmbH, Ratingen, Germany)
with cellulose nitrate filter (Sartorius Stedim Biotech
GmbH, 8 µm pore size, 37 mm diameter). Two filters
were exposed in sequence to a volume flow of 10.0 L/min
for 96 and 69 min each (air sampler, model SG10-2, GSA
Messgerätebau GmbH, Ratingen, Germany). The deviation between the TEOM and gravimetric measurements
for the first filter was + 0.95% and for the second – 0.77%.
The specific surface area was determined using a BET
device (BET, model Gemini VII 2390a, Micromeritics
GmbH, Aachen, Germany). BaSO4 was dried at 300 °C
for 60 min as a pretreatment, and a surface area of 3.2
m2/g was determined. A second measurement showed a
slightly smaller surface area of 2.8 m2/g after further drying at 300 °C for 90 min. The measurements confirm that
the used B
aSO4 has no appreciable porosity and agree
very well with measurements known from the literature
[14].
The particles were taken directly from the chemical
packaging, put on a scanning electron microscopy pin
stub and characterized by scanning electron microscopy
(SEM, model Zeiss Supra 40VP, Carl Zeiss Microscopy
Deutschland GmbH, Oberkochen, Germany) with a
nominal resolution of 2 nm. The micro-sized BaSO4 particles consisted of individual crystals and were rounded
at their edges (Additional file 1: Figure S2).
An elemental analysis of the B
aSO4 particles (Mikroanalytisches Labor Pascher, Remagen, Germany) showed
a chemical purity of > 99% with regard to organic impurities. The measured carbon content was < 0.01% and the
hydrogen content < 0.09%. To exclude the compound
Page 3 of 12
barium carbonate, which is soluble in the body and
therefore toxic, the carbonate content was analyzed. It
was negligible at < 0.01%.
Participants
Sixteen healthy nonsmoking volunteers (8 women, 8
men) with a median age of 28 (range 20–37) years participated in the study (Table 1). The subjects reported
no previous exposure to airborne BaSO4 and did not
show bronchial hyperresponsiveness to methacholine as assessed with a reservoir method [15]. The study
participants had to be able to produce sputum after
induction with 0.9% saline according to our criteria
(eosinophils < 1%, epithelial cells < 95% and neutrophils
not dominant) in order to exclude subjects with airway
inflammation and to make sure that the material originated from the lower airways. Standard baseline laboratory parameters were within normal ranges. Specific
IgE antibodies (sIgE) to ubiquitous aeroallergens (atopy
screen sx1, Phadiatop, ImmunoCAP system, ThermoFisher Scientific, Phadia AB, Uppsala, Sweden) as well
as total IgE were measured with the ImmunoCAP 250
system. A positive atopic status was assumed in case of a
sIgE concentration to sx1 > 0.35 kUA/L. Two atopic subjects (one woman, one man) without any clinical manifestation of allergies were included in the study cohort.
Study design
16 subjects were exposed according to the scheme for
2 h (Fig. 1), with a minimum interval of two weeks
between each exposure (sham and 4.0 mg/m3 BaSO4,
subjects served as their own control). The subjects were
at rest except for two periods of 30 min with moderate
physical activity set to 15 L/min/m2 (corresponding to a
median work load of 60 watts (range 43 to 90 watts)) on
a cycle ergometer. The aim of this setting was to allow
the subjects to inhale approx. 10 m3 in accordance with
Table 1 Characteristics of the study subjects
Parameters
Total
Male
Female
N = 16
N=8
N=8
Age [years]
28 (20–37)
28 (23–37)
24 (20–35)
Height [cm]
178 (160–194)
182 (175–194)
168 (160–190)
Weight [kg]
75 (50–122)
89 (64–122)
BMI [kg/m2]
24.9 (18.4–32.4) 26.3 (19.1–32.4)
22.6 (18.4–29.7)
26.1 (< 2–51.2)
19.4 (3.83–643)
66 (50–95)
Total IgE [kU/L]
23.1 (< 2–643)
sIgE to sx1 [kUA/L]
0.06 (0.03–5.71) 0.065 (0.03–0.75) 0.06 (0.04–5.71)
sIgE to sx1 > 0.35
kUA/L [n]
2
1
1
Medians and ranges are listed. BMI = body mass index. Specific IgE (sIgE) to
sx1 ≥ 0.35 kUA/L is an indicator of sensitization to environmental allergens
Monsé et al. BMC Pulmonary Medicine
(2022) 22:233
Page 4 of 12
Fig. 1 Time line of this study
the assumption of the German MAK Commission for
the respiratory volume of humans of 10 m3 per 8-h
working day [1]. The retrospective evaluation resulted
in an averaged value of 9.48 ± 1.44 m3 for the 16 subjects. Exposures were randomized and double blinded.
Medical examinations were performed before, directly
after and approximately 22 h after the start of exposure.
Baseline testing was performed before the first exposure. The following examinations were done: detailed
questionnaire-based medical history, physical examination, blood sampling, sputum induction and analysis,
lung function testing, cotinine measurement in urine,
measurements of fractional exhaled nitric oxide (FeNO),
electrocardiogram, blood pressure, spiroergometry to
assess the work load, methacholine testing and body
temperature. After the last exposure a final testing was
performed including physical examination, blood and
induced sputum sampling and analysis, electrocardiogram, blood pressure, lung function testing and body
temperature. In addition, vital functions (electrocardiogram, blood pressure) were monitored during the exposures, which were always carried out from 10 to 12 am.
Time of sample collection was recorded in order to adjust
for possible diurnal varations in the biomarker levels.
Questionnaire
All subjects answered a questionnaire addressing flulike symptoms (at least one of three symptoms: feeling
of fever, feeling sick and muscle pain) and airway irritation (throat irritation and/or cough) at different time
points (before exposure, directly after and 22 h after
exposure). To avoid any information bias we added
questions about clearing throat, shortness of breath,
fatigue, headache, feeling warm, discomfort, chills, and
feeling unwell. All symptoms were graded according
to severity (not at all (0 score point), barely (1 point),
little (2 points), moderate (3 points), strong (4 points),
very strong (5 points)). Sum scores and percentage of
sum scores were generated for each study participant,
described in detail previously [12].
Portable PSG devices (SOMNOscreen™ plus, SOMNOmedics GmbH, Randersacker, Germany) were used with
2 canal ECG at 512 samples/s, SpO2 and Cuffless Continuous Blood Pressure Measurement based on puls transit time. Additionally, the blood pressure was manually
measured before and after each exposure.
ECG and blood pressure
Body temperature
Subjects measured their own body temperatures using
a digital thermometer (model MT3001, Microlife AG,
Widnau, Switzerland) before, during and after
BaSO4
exposure and additionally every 2 h until the next day,
but not during sleep. All participants were instructed
to put the thermometer at minimum for 1 min under
the tongue (sublingual) with the mouth closed and no
drinking or eating 5 min prior to measurements. The
limit value at T = 37.5 °C was considered a fever. The
measurements were subject to a deviation of 0.1 °C. No
separate quality assessments were carried out, the plausibility of the measurement results was assessed by expert
judgment.
Blood parameters
Blood samples (12 ml each) were obtained at the baseline
examination, directly before exposures, 22 h post-exposures and at the final examination. Inflammatory markers
(differential blood cell count, C-reactive protein (CRP),
serum amyloide A (SAA)) were analyzed using standard
methods. The total and differential blood cell counts were
determined using the Coulter counter- method with UniCell DxH800 (Beckman Coulter Inc., Brea, CA, USA).
ELISA techniques were used to quantify the following
serum biomarker: SAA (Invitrogen™ Carlsbad, CA, USA;
detection of human serum amyloid A1 cluster (Hu SAA)
in the range of 9.4–600 ng/mL), and CRP (high sensitive ELISA from IBL International, Hamburg, Germany;
range 0.4–19 µg/mL). The parameters in this study were
examined using the same methods as described in [12].
Further standard clinical parameters of renal and
liver function were determined during the recruiting
process of the subjects. Determination of creatinine in
Monsé et al. BMC Pulmonary Medicine
(2022) 22:233
urine was done via the Jaffé method (measuring range
0.3—25.0 mg/dL).
Induced sputum
Sputum samples were obtained at the baseline examination, 22 h post-exposures and at the final examination, but not directly before exposures. This procedure
eliminates the possibility that repeated sputum recovery
within a short time period may induce inflammatory
effects triggered by sputum induction itself. According to the procedure used in several studies [12, 16, 17]
sputum induction was carried out by inhalation of nebulized isotonic saline solution (0.9% sodium chloride
(NaCl); Pariboy, Pari GmbH, Weilheim, Germany) for
15 min. Concentrations of interleukin-8 (IL-8), matrix
metalloproteinase-9 (MMP-9) and tissue inhibitors of
metalloproteinases-1 (TIMP-1) were determined in
the appropriate immunoassays based on monoclonal or
polyclonal antibodies (Pharmingen, Heidelberg, Germany, Assay Design and/or Bio Vendor, all: Heidelberg,
Germany) according to the recommendations of the
manufacturers. The total protein determination was carried out with bovine serum albumin as a standard with a
measuring range of 10 to 100 mg/L. The respective lower
quantification limit was 3 pg/mL for IL-8, 31.2 pg/mL for
MMP-9 and 9.76 pg/mL for TIMP-1 [18].
FeNO
Increased excretion of nitric oxide (NO) in the airways
is expected due to inflammatory processes. FeNO was
measured using a portable electrochemical analyzer
(NIOX Mino, Aerocrine, Solna, Sweden) taking into
account the guidelines of the American Thoracic Society
and European Respiratory Society [19].
Lung function testing
Lung function was recorded using both body plethysmography [20] and spirometry [21] in a linked maneuver
with a MasterLab (Vyaire Medical GmbH, Höchberg,
Germany). A battery of different parameters was evaluated (e.g. airway resistance, lung volumes, and flows).
Data analysis of effect parameters
Characteristics of subjects were expressed as medians as
well as minimum and maximum (see Table 1). Descriptive analysis was performed for each variable stratified by
exposure (sham, 4.0 mg/m3 BaSO4) and time of measurement (before, 22 h after exposure). Graphical representations were illustrated with boxplots (median and the
25–75% percentiles). Effects were compared between
before and 22 h after exposure. Exposure groups were
compared using paired Student`s t-test for normal or
Page 5 of 12
log-normal distributed variables. If normal distribution
could not be assumed Wilcoxon signed-rank test was
used.
The problem of multiple comparisons was counteracted using the Bonferroni correction [22]. Individual
descriptive analyses were performed for body temperature with a cut-off of ≥ 37.5 °C. Differences in the blood
and sputum parameters between sham and B
aSO4 exposures and time of measurements were examined using
multivariable generalized estimating equations (GEE)
logistic regression [23]. Here, we compared the differences for each parameter separately for the time of measurement and exposure using odds ratios (OR) and 95%
confidence intervals (CI). These analyses were performed
taking into account the sample time and concentration.
Statistical analysis
To represent the dose–response relation in the in vitro
experiments more precisely, model fits like the fourparameter log-logistic model or the Emax model were
used. According to O’Connell et al. [24] the four-parameter log-logistic model is for data following a sigmoidal
shaped curve and defined for concentration x as:
f (x, (b, c, d, e)) = c +
d−c
1 + exp b log (x) − log (e)
,
with b, c, d, and e as corresponding parameters. c
and accordingly d indicate the lower respectively upper
asymptote, b serves as parameter for the slope of the
curve and e is the 50% effective concentration E
C50. Furthermore on the report of Pinheiro et al. [25] the Emax
model is defined as:
f (x, (E0 , Emax , EC50 )) = E0 + Emax
x
,
EC50 + x
with E0 corresponding to the basal effect at placebo concentration x = 0, Emax representing the maximum change in effect and EC50 being the 50% effective
concentration.
Estimation of lung deposition efficiency
To estimate the B
aSO4 particle lung deposition efficiency we modified the open-source code [26] based on
the International Committee for Radiological Protection
(ICRP) Publication 66 [27]. Further information is given
in [18].
Particle induced cell migration assay
In order to assess whether ZnO and
BaSO4 differ in
their acute biological effects, we analyzed the chemotactic attraction of differentiated human leukemia
cells (dHL-60 cells) in response to cell supernatants of
Monsé et al. BMC Pulmonary Medicine
(2022) 22:233
particle- challenged NR8383 rat alveolar macrophages,
as a model for the particle-induced accumulation of neutrophils in the inflamed lung [6]. The substances used in
the in vitro experiments were the same as those used in
the human experiments (aerodynamic diameter ZnO:
1,33 µm; aerodynamic diameter BaSO4: 1,90 µm). dHL-60
cells have properties similar to that of physiological neutrophil granulocytes. Briefly, the cell supernatants were
used to investigate migration of dHL-60 cells. We challenged with compounds separately. As a positive control,
a silica (SiO2) reference sample was used (CAS No. 763186-9, Lot MKBF2889V, 99.5%, 10–20 nm, Sigma-Aldrich,
Steinheim, Germany). In order to calculate a continuous
course of each treatment, dose–response models were
fitted for each compound. Though four-parameter loglogistic models were used for ZnO and BaSO4 while SiO2
was modeled by a E
max-model. The choise of the model is
based on the known biological activity of the compound.
In preliminary tests, for example, ZnO showed a plateau
effect after a dose of 20 μg/cm2 and higher, which the fitted model maintains. A detailed description of the preliminary test can be found in the Additional file 1: Figure
S4. Modelling was performed in R [28] using the R packages Dosefinding [29] and drc [30].
Page 6 of 12
of 37.8 °C in the evening after her BaSO4 exposure, but
without reporting any symptoms, no increase of body
temperature (≥ 37.5 °C) was observed after both exposure scenarios (Additional file 1: Figure S3). The median
temperatures did not differ significantly when comparing sham and BaSO4 exposures at each time point.
The detected minimum in the second half of the night
reflected a physiological fluctuation in the temperature
course of a day. Normally, the maximum body temperature is expected in the afternoon [31]. In the subjects of
this study, it was in the morning, which may be due to the
physical exertion on the ergometer during the exposures,
which always took place in the morning.
No study participant showed clinical signs after the
BaSO4 exposures, which had also no effects on blood
pressure, FeNO and all lung function parameters (data
not shown).
Blood and sputum parameters
The evaluation of parts of the questionnaires relevant
for this study (feeling of fever, feeling sick, muscle pain,
throat irritation and/or cough) did not demonstrate an
increase of the effect rating after BaSO4 exposure compared to sham. On average, the relative symptom sum
score for all questions at each time point was 4.6%. The
highest relative sum score of 7.5% was found for “throat
irritation and/ or cough” 22 h after B
aSO4 exposure. No
significant differences were observed between BaSO4 and
sham exposures. When asked, the subjects could not distinguish the different exposure scenarios.
Table 2 shows the univariate evaluation of the time
course of blood parameters (leucocytes, neutrophils,
lymphocytes, monocytes, thrombocytes, CRP, SAA) and
sputum parameters (IL-8, MMP-9, TIMP-1, total protein, total cell number, neutrophils) of our BaSO4 study.
All blood parameters showed no significant changes
when comparing the values before and 22 h after sham
and BaSO4 exposure, respectively. Furthermore, the
obtained values from five examinations without BaSO4
exposure (one baseline examination, two examinations
before sham and BaSO4 exposure, one examination 22 h
after sham exposure and one final examination) were not
significantly different one from another. In addition, all
sputum parameters were unaffected by B
aSO4 exposure
and showed no significant differences compared to sham
exposures. Univariate calculations could be confirmed
with multivariate generalized estimating equations (GEE)
logistic regression and also showed no significant differences in blood and sputum parameters (data not shown).
As an exception, leukocytes [1/nL] and monocytes [1/
nL] showed significant differences between measurements directly before both exposure scenarios. However,
these findings were not affected by the BaSO4 exposure.
Additionally GEE logistic regression detected a significant difference between before and after exposure to
BaSO4 for monocytes [1/nL]. With regard to the abovementioned significance without exposure effect and the
univariate calculations, these effects were considered biologically/toxicologically not relevant.
Body temperature and other clinical features
Calculation of deposition rates of inhaled B
aSO4 particles
Results
Particle atmospheres
The average airborne BaSO4 concentration was 4.013 mg/
m3 (± 1.8%) (target concentration: 4.0 mg/m3). A particle
concentration of 9.5 µg/m3 (± 82.5%) was determined for
the filtered air. The particle size distribution at 4.0 mg/m3
was monomodal with a relatively small geometric standard deviation of 1.50 and yielded a median aerodynamic
diameter of 1.9 µm (± 2.1%). On average, 1130 particles
per cm3 were measured.
Questionnaire
Nearly all circadian temperature fluctuations were
inside 1.3 °C and lower than 37.5 °C. Except for one
female subject who measured an increased temperature
Figure 2 shows the estimation of the masses of deposited
BaSO4 particles of different airway regions (alveolar, tracheobronchial, and extrathoracic region) according to
Monsé et al. BMC Pulmonary Medicine
(2022) 22:233
Page 7 of 12
Table 2 Acute effects of BaSO4 (4 mg/m3) or sham (0 mg/m3) exposure on blood and induced sputum parameters at different time
points [median (minimum–maximum)]
Parameters
Baseline test
BaSO4
[mg/m3]
Directly before exposure
22 h after exposure
Final examination
6.20 (4.50–9.20)
Blood
Leukocytes [1/nL]
Neutrophils [%]
Neutrophils [1/nL]
Lymphocytes [%]
Lymphocytes [1/nL]
Monocytes [%]
Monocytes [1/nL]
Thrombocytes [1/nL]
Erythrocytes [1/nL]
CRP [mg/L]
SAA [µg/L]
5.50 (4.60–8.50)
56 (40.8–70)
3.08 (2.21–5.94)
29 (22–40)
1.76 (1.37–2.28)
9 (5–12)
0.53 (0.34–0.81)
242 (192–346)
4.75 (4.10–5.35)
0.78 (0.06–4.24)
10,873 (< 1880–51,897)
0
6.25 (4.80–13.10)
5.60 (4.30–9.50)
4
5.45 (4.00–8.40)
6.25 (4.60–13.90)
0
57.5 (38–76)
60.5 (41–69)
4
55 (41–73)
56 (48–80)
0
3.69 (2.00–9.94)
3.47 (1.76–6.50)
4
3.25 (1.75–6.07)
3.42 (2.42–11.10)
0
30 (14–45)
29 (19–44)
4
32 (18–47)
31.5 (11–38)
0
1.86 (1.59–2.49)
1.83 (1.14–2.44)
4
1.69 (1.16–2.79)
1.81 (0.92–2.82)
0
8.5 (4–12)
8.5 (5–12)
4
7.5 (5–12)
8 (5–12)
0
0.53 (0.39–0.82)
0.55 (0.3–0.72)
4
0.47 (0.30–0.65)
0.50 (0.31–1.00)
0
253 (181–339)
238 (168–339)
4
226 (172–344)
231 (178–326)
0
4.80 (4.10–5.50)
4.75 (4.10–5.40)
4
4.75 (4.10–5.60)
4.85 (3.90–5.50)
0
0.92(0.08–3.63)
1.05 (0.04–4.73)
4
0.86 (0.06–7.33)
1.14 (0.00–5.43)
0
15,366 (3262–87,932)
18,379 (3607–256,193)
4
8674 (2381–46,633)
10,318 (< 1880–107,439)
0
–
684 (164–16,177)
4
–
1128 (166–36,918)
57 (41–72)
3.63 (1.87–6.64)
32 (19–45)
1.95 (1.45–2.73)
8.5 (5–11)
0.47 (0.33–0.82)
231 (176–339)
4.80 (4.00–5.30)
0.89 (0.00–7.12)
11,082 (< 1880–158,188)
Induced sputum
IL-8 [ng/L]
MMP-9 [µg/L]
TIMP-1 [µg/L]
Total protein [mg/L]
Total cell number [× 105]
Neutrophils [%]
Neutrophils [× 104]
1771 (193–12,768)
161 (33–852)
9.27 (0.82–41.40)
260 (76–846)
60.33 (5.00–108.41)
8.75 (0–34)
37.85 (0–368.61)
0
–
61 (15–764)
4
–
104 (20–1329)
0
–
3.26 (1.01–30.12)
4
–
4.62 (0.91–54.50)
0
–
189 (66–698)
4
–
197 (59–931)
0
–
29.63 (8.55–212.44)
4
–
35.10 (8.86–140.10)
0
–
1.5 (0–40)
4
–
1.25 (0–32.5)
0
–
5.13 (0–484.59)
4
–
5.62 (0–455.33)
particle sizes. The mean values of the deposited masses
of all participants are shown. The whiskers represent
the minimum and maximum values. The total inhaled
mass of B
aSO4 particles is also shown (Intake). According to the ICRP-model, alveolar deposition rate (Al)
was 5.6% and 3.9% of the particles were deposited in
the tracheobronchial region (TB). Most of the particulate mass was deposited in the extrathoracic region (ET)
1303 (219–12,775)
58 (13–335)
2.65 (0.57–21.65)
146 (67–477)
28.60 (7.33–62.73)
3 (0–28)
8.2 (0–133)
and corresponded to a fraction of about 83.5%. Overall
93.0% of the total inhaled mass of B
aSO4 particles was
deposited in the airways, 7.0% of the mass was exhaled,
respectively.
Particle‑induced cell migration assay
Challenge of NR8383 cells with BaSO4 particles resulted
in cell supernatants that had a weak chemotactic
Monsé et al. BMC Pulmonary Medicine
(2022) 22:233
Page 8 of 12
models: ZnO: 8.06 [2.55, 13.57], BaSO4: 121.20 [25.00,
217.41], SiO2: 13.06 [-12.20, 38.31] μg/cm2.
The four-parameter log-logistic model for ZnO shows
a rapid steep sigmoidal curve. Additionally, the E
C50
of ZnO with 8.06 and its confidence interval shows the
highest activity of the investigated substances. Also the
confidence interval of ZnO is entirely lower than that of
BaSO4, which emphasizes the higher activity of ZnO.
Fig. 2 Calculated lung deposition efficiency of BaSO4 particles
in dependence of airway regions (Al: alveolar region, TB:
tracheobronchial region, ET: extrathoracic region) and particle size.
TD: total deposition (sum of Al + TB + ET), Intake: Total inhaled mass
of BaSO4 particles
Fig. 3 Chemotaxis (migrated cells) of the unexposed dHL-60 cells
in response to NR8383 cell supernatants obtained from incubations
with increasing compound concentrations. For comparison data of
the ZnO, induced chemotaxis are shown. Commercially available
silica (SiO2) nanoparticles were used as positive control that allows
comparison with historical data. Results represent fitted dose–
response models for the numbers of in vitro migrated cells after
treatment with ZnO and BaSO4. Results represent arithmetic means
and standard deviations of three independent experiments
activity on dHL-60 cells at very high concentrations
and were much weaker than the silica positive control.
ZnO particles showed the strongest effects, even considerably stronger than silica (Fig. 3).
The small number of data points at each dose led to
a high variation. Nevertheless, the model courses and
especially the 50% effective concentrations (EC50) values indicate a good tendency for the real courses. The
following EC50 with corresponding 95% confidence
intervals were calculated for the different compounds
based on the separately adjusted dose–response
Discussion
The deviation of TLV is often based on animal experiments. It is desirable to support these derivations by
human studies. However, the endpoints that can be
examined and the study design are limited here by ethical considerations. For this purpose, it is necessary to
identify sensitive and specific biological effect markers,
which ideally allow a differentiation between "adverse"
and "non-adverse “effects. For the acute B
aSO4 inhalation
study, we selected those parameters that had been shown
to be sensitive for the detection of airway inflammation
in our previous ZnO studies [11, 12, 18].
A controlled inhalation study with human volunteers at
our exposure lab using nano-sized ZnO at concentrations
up to 2 mg/m3 showed concentration-dependent systemic inflammatory effects, including an increase of body
temperature in several subjects, as well as an increase in
blood neutrophils, and acute phase protein levels [11].
Local effects were also detectable in induced sputum,
but without a concentration-dependent relationship [18].
In an other ZnO study, subjects were exposed to nanoand micro-sized ZnO for 2 h at concentration levels of
2 mg/m3 each [12]. We could show that biological effects
were more pronounced after exposure of micro-sized
ZnO particles. According to the final investigations 2 to
6 weeks after exposures all acute effects were completely
reversible in both studies. Furthermore, the results of
these studies support the assumption that the observed
ZnO effects were not caused by physical particle effects
in the sense of a lung overload.
Criteria for setting our
BaSO4 concentration were
based on preliminary tests without subjects. They showed
that atmospheres containing B
aSO4 with concentrations
above 6 mg/m3 become visible in the exposure unit and
blinding of both exposures (BaSO4 and sham) would not
be possible. Therefore, we set the maximum applicable
concentration to 4.0 mg/m3 with a sufficient distance to
6 mg/m3 so that both exposure conditions were indistinguishable for the subjects. For organizational reasons, the
exposure time was set at 2 h.
The most important result of the present study is the
absence of any significant differences between BaSO4 and
sham exposures in all parameters investigated. There was
no evidence of local or systemic effects after inhalation of
Monsé et al. BMC Pulmonary Medicine
(2022) 22:233
micro-sized BaSO4 at 4.0 mg/m3 for 2 h. Body temperature measurements and symptom questionnaires also
showed no differences between both exposure scenarios. The study design was sufficiently sensitive to detect
ZnO effects, but failed to reveal any effects in the case
of micro-sized B
aSO4. Figure 4 shows a comparison of
neutrophils in blood from the second ZnO study and the
present BaSO4 study.
ZnO effects may have been influenced by random fluctuations and measurement inaccuracies, which however,
did not invalidate the significant changes in neutrophils
in blood. For BaSO4, on the other hand, a similar scattering was measured, but no significant changes were
detectable neither between different time points nor
between BaSO4 and sham exposure. However, the scatterings were sufficiently small to rule out coincidental
findings that would indicate a toxic property.
In contrast to this study, Sikkeland and co-workers
found elevations in several parameters in induced sputum 24 h after aluminium oxide exposure (volume
median diameter particle size 3.2 μm; 3.8–4.0 mg/m3 for
2 h) [10]. The blood parameters were measured 4 h after
exposure, but did not show any changes and may have
been overlooked: According to our experience, this time
point may have been chosen too short, effects occurred
significantly later [11, 12]. Other studies also suggest
this [32, 33]. However, since the chemical purity and the
chemical modification of the used material were unclear,
it cannot be excluded that the observable effects were
caused by non-inert containing substances that had a
weak substance-specific toxicity.
Comparison of the calculations for the estimation of the deposition efficiencies between our second
ZnO study [12] and the present study yielded a higher
Page 9 of 12
deposition ratio of BaSO4 in the airways. One explanation is that the mean particle size of BaSO4 with 1.90 µm
is larger than that of the micro-sized ZnO with 1.33 µm
and according to the ICRP model results in a higher deposition rate. This estimate is supported by the fact that
BaSO4 did not cause any detectable effects even at more
than twice the deposited mass (8.65 mg) compared to the
mass of micro-sized ZnO (3.04 mg) used in the second
ZnO study.
One possible weakness of the present study is the
restriction of the recording of systemic and local effect
parameters at specific time points. A possible absence
of effects may be caused by an earlier or later increase
of effect parameters compared to the sampling times.
Furthermore, no investigations with nano-sized BaSO4
particles were performed in this study. Using
BaSO4
nanoparticles, Molina et al. [8] and Keller et al. [9] have
shown the dissolution and release of barium ions under
physiological conditions. Therefore, it cannot be excluded
that our findings does not apply to nano-sized B
aSO4.
However, this acute study is not suitable to be extrapolated to repeated or chronic inhalation in humans, since
GBP are toxic only in case of lung overload and the doses
chosen in this study were far below this level. No conclusions can be drawn about the extent of accumulation
in case of repeated exposure, nor about the elimination
kinetics of the deposited
BaSO4 particles in the respiratory tract. An acute inhalation study in humans has
limited evidence of transferability of data from animal
studies to humans for chronic endpoints.
As demonstrated in our ZnO studies [11, 12], the
strength of this study is the lack of effects after sham
exposures (0 mg/m3 BaSO4). Many control conditions
were performed without BaSO4 exposure (five control
Fig. 4 Time courses of neutrophils in blood in the second ZnO study [12] and the B
aSO4 study
Monsé et al. BMC Pulmonary Medicine
(2022) 22:233
conditions for blood parameters: one baseline examination, two examinations before exposures, one examination 22 h after sham exposure and one final examination).
For the sputum parameters three control scenarios (one
baseline examination, one examination 22 h after sham
exposure and one final examination) were available. Thus,
accidental variabilities were minimized.
Since this study did not detect any differences of
parameters between
BaSO4 and sham exposures, the
question arises whether recruitment of 16 volunteers is
sufficient. Howevecell migration between 10 and 20r, we
conducted this study with a similar design as the ZnO
studies, where robust parameters showed clear changes
after ZnO exposures. Since the study design was sensitive
enough to show these acute ZnO effects, we can assume
that it is also true for B
aSO4 under the chosen exposure
conditions.
In vitro data can supplement in vivo data in order to
support mechanistic propositions. Therefore we confirmed the inert character of the B
aSO4 in vitro. We
used the particle-induced chemotaxis assay to do this
(PICMA) [6]. This assay mimics the particle induced
accumulation of neutrophils which is a hallmark of particle- induced inflammation. Polymorphonuclear neutrophil (PMN) count and total protein concentration are
usually the most sensitive, valid parameters that indicate particle toxicity as well in animal experiments (see
for example [34]). The assay is very sensitive down to
the subtoxic range. However, in vitro studies can in general also be carried out at very high doses. In this study,
BaSO4 causes a very slight increase in migrated cells
between 100 and 200 µg/cm2, compared to ZnO that
induces strong cell migration between 10 and 20 µg/cm2
(Additional file 1: Figure S4). Concordantly, BaSO4 of different particle sizes neither caused an inflammatory nor
a cytotoxic response investigated by the PICMA, by the
release of inflammatory mediators (CCL2, TNF-a, IL-6),
by apoptosis or necrosis up to the highest tested dose
each [7]. We used NR8383 rat alveolar macrophages for
particle challenge to produce cell- and particle supernatants that attract d-HL-60 cells—a well established model
cell line for neutrophils. Challenge of NR8383 cells with
the BaSO4 particles that were used in the inhalation study
yielded cell supernatants that acted very weakly chemotactic towards the dHL-60 cells at high doses. This effect
was about 9 times less pronounced compared to the
SiO2 positive control and about 15 times weaker than
the ZnO nanoparticles, based on the calculated E
C50
values. A comparison with toxic fibers helps to get an
idea of the strength of the effect: ZnO induced chemotaxis of d-HL60 cells is approximately 10-times weaker
than asbestos fibers and multiwalled carbon nanotubes
[35]. It is likely that the high E
C50 value of B
aSO4 even
Page 10 of 12
underestimates this difference, since the curve does not
reach a plateau, which normally leads to an underestimation of the calculated E
C50 value. This is in contrast to the
ZnO and SiO2 particles or other toxic particles or fibers
and due to the fact that BaSO4 does not show any cytotoxicity (Additional file 1: Figure S5). We could not find
any other biological or inflammatory in vitro effects in
the literature, including elevation of inflammatory signaling molecules after in vitro challenge with very high
doses of BaSO4 [7, 36], in contrast to the findings with
toxic particles and fibers [35].
These in vitro data thus appear to mirror the data from
the controlled human BaSO4 and ZnO inhalation studies,
which showed systemic and local inflammatory effects,
including elevation of blood neutrophils following inhalation of ZnO but not B
aSO4. This comparison suggests
that weak chemotaxis in vitro at such high particle concentrations is consistent with the assessment as a chemically inert particle.
Even if limitations of our study design have to be considered, in particular that only an acute study was performed, the results allow the conclusion that acute
exposures at the said level rule out irritative effects with
some degree of certainty. As similar inhalation studies with GBP were not available previously, such studies
could not be used for the setting of a TLV. It is certainly
difficult to transfer results from acute to chronic inhalation exposures to GBP. Longer or repeated inhalation
studies in humans are too laborious and thus not considered a realistic option. This study should stipulate
discussions in committees which recommend TLVs like
the American Conference of Governmental Industrial
Hygienists (ACGIH), the German MAK-commission, or
others.
Conclusions
In summary, there was no evidence of local or systemic
effects after acute inhalation of micro-sized BaSO4 in
humans at a concentration of 4.0 mg/m3 for 2 h. However, this study does not allow extrapolation to chronic
exposures. The inhalation data in humans together with
the particle-induced chemotaxis data in vitro study add
support to the hypothesis that GBP are toxic under lung
overload conditions which cannot be reached in acute
human exposures. This study supports the results of animal experiments that micro-sized BaSO4 is an ‘inert’ particle, in contrast to ZnO.
Abbreviations
EC50: 50% Effective concentration; APS: Aerosol particle sizer; ACGIH: American
Conference of Governmental Industrial Hygienists; BaSO4: Barium sulfate; BMI:
Body mass index; BET: Brunauer Emmet Teller; CCL2: CC-chemokine ligand
2; CRP: C-reactive protein; CI: Confidence intervals; ECG: Electrocardiogram;
FeNO: Fractional exhaled nitric oxide; GEE: Generalized estimating equations;
Monsé et al. BMC Pulmonary Medicine
(2022) 22:233
GBP: Granular biopersistent particles; IL-8: Interleukin-8; IL-6: Interleukin-6;
ICRP: International Committee for Radiological Protection; MMP-9: Matrix met‑
alloproteinase-9; NO: Nitric oxide; OEL: Occupational exposure limit; OR: Odds
ratios; SpO2: Oxygen saturation; PMN: Polymorphonuclear neutrophil; PSG:
Polysomnography; SEM: Scanning electron microscopy; SAA: Serum amyloide
A; SiO2: Silica; TEOM: Tapered elemental oscillating microbalance; TLV: Thresh‑
old limit value; TIMP-1: Tissue inhibitors of metalloproteinases-1; TNF-a: Tumor
necrosis factor alpha; ZnO: Zinc oxide.
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12890-022-02021-y.
Additional file 1. Figure S1. Averaged particle size distribution of
airborne BaSO4 particles and filtered air. Figure S2. SEM image of BaSO4
particles. Figure S3. 24 h temperature profile of all subjects. Figure S4.
Chemotaxis (migrated cells) of the unexposed dHL-60 cells in response to
NR8383 cell supernatants. Figure S5. Cytotoxicity of ZnO towards NR8383
cells according to the alamarBlueTM test.
Acknowledgements
We acknowledge support by the Open Access Publication Funds of the RuhrUniversität Bochum. We are grateful to our volunteers for their participation.
The authors thank Sabine Bernard, Gerda Borowitzki, Anja Deckert, Jennifer
Gili, Evelyn Heinze, Claudia Litzenberger, Ursula Meurer, Melanie Ulbrich, and
Susann Widmer for their excellent assistance. We acknowledge the techni‑
cal support of Michael Kirchner (Institute for the Research on Hazardous
Substances (IGF), Bochum, Germany) for the determination of BET and Bianca
Gasse (Institute for Occupational Safety and Health of the German Social Acci‑
dent Insurance (IFA) St. Augustin, Germany) for recording the SEM images.
Author contributions
Conception and design: CM, GW, NR, MR, JB, TB. Analysis and interpretation:
BJ, VK, BK, LS, EM, CS, FW, CW, NR, CM, MR, RM, GW, JB. Drafting the manuscript
for important intellectual content: CM, MR, RM, VK, GW, JB. All authors read
and approved the final manuscript. All authors consent to publication of this
manuscript. All authors read and approved the final manuscript.
Funding
The authors declare that no funding has received from outside sources.
Availability of data and materials
The datasets generated and/or analysed during the current study are not
publicly available due to the very extensive data collection, but are available
from the corresponding author on reasonable request.
Declarations
Ethics approval and consent to participate
The study was performed in accordance with the Declaration of Helsinki
for Human Research. It was approved by the local Ethics Committee of the
Ruhr University Bochum, Germany (Reg.-No. 18-6393). All study participants
gave written informed consent and received a financial compensation for
participation.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Received: 1 April 2022 Accepted: 25 May 2022
Page 11 of 12
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