Distribution of molybdenum in soft tissues and blood of rats after intratracheal instillation of molybdenum(IV) sulfide nano- and microparticles

Abstract

There is still little literature data on the toxicity and safety of the commonly used molybdenum (Mo) disulfide which is present in the working as well as living environments. Thus, an experiment was carried out involving rats, with single and repeated intratracheal exposure (in the latter case, 7 administrations at 2-week intervals with the analysis performed after 90 days) to lower (1.5 mg Mo kg−1 b.w.) and higher (5 mg Mo kg−1 b.w.) doses of molybdenum(IV) sulfide nanoparticles (MoS2-NPs) and microparticles (MoS2-MPs). The analysis of Mo concentrations in the tail and heart blood as well as in soft tissues (lung, liver, spleen, brain), after mineralization and bioimaging, was meant to facilitate an assessment of its accumulation and potential effects on the body following short- and long-term exposure. The multi-compartment model with an exponential curve of Mo concentration over time with different half-lives for the distribution and elimination phases of MoS2-MPs and MoS2-NPs was observed. After 24 h of exposure, a slight increase in Mo concentration in blood was observed. Next, Mo concentration indicated a decrease in blood concentration from 24 h to day 14 (the Mo concentration before the second administration), below the pre-exposure concentration. The next phase was linear, less abrupt and practically flat, but with an increasing trend towards the end of the experiment. Significantly higher Mo concentrations in MoS2-NPs and MoS2-MPs was found in the lungs of repeatedly exposed rats compared to those exposed to a single dose. The analysis of Mo content in the liver and the spleen tissue showed a slightly higher concentration for MoS2-NPs compared to MoS2-MPs. The results for the brain were below the calculated detection limit. Results were consistent with results obtained by bioimaging technique.

Full text

Vol.:(0123456789)
1 3
Toxicol Res.
https://doi.org/10.1007/s43188-023-00213-0
ORIGINAL ARTICLE
Distribution ofmolybdenum insoft tissues andblood ofrats
afterintratracheal instillation ofmolybdenum(IV) sulfide nano‑
andmicroparticles
RenataKuraś1 · MaciejStępnik2,3· JarosławGrobelny4· EmiliaTomaszewska4· MagdalenaStanisławska1·
KatarzynaDomeradzka‑Gajda3· WojciechWąsowicz5· BeataJanasik6
Received: 7 May 2023 / Revised: 8 September 2023 / Accepted: 26 September 2023
© The Author(s) 2023
Abstract
There is still little literature data on the toxicity and safety of the commonly used molybdenum (Mo) disulfide which is present
in the working as well as living environments. Thus, an experiment was carried out involving rats, with single and repeated
intratracheal exposure (in the latter case, 7 administrations at 2-week intervals with the analysis performed after 90days) to
lower (1.5mg Mo kg−1 b.w.) and higher (5mg Mo kg−1 b.w.) doses of molybdenum(IV) sulfide nanoparticles (MoS2-NPs)
and microparticles (MoS2-MPs). The analysis of Mo concentrations in the tail and heart blood as well as in soft tissues
(lung, liver, spleen, brain), after mineralization and bioimaging, was meant to facilitate an assessment of its accumulation
and potential effects on the body following short- and long-term exposure. The multi-compartment model with an exponen-
tial curve of Mo concentration over time with different half-lives for the distribution and elimination phases of MoS2-MPs
and MoS2-NPs was observed. After 24h of exposure, a slight increase in Mo concentration in blood was observed. Next,
Mo concentration indicated a decrease in blood concentration from 24h to day 14 (the Mo concentration before the second
administration), below the pre-exposure concentration. The next phase was linear, less abrupt and practically flat, but with
an increasing trend towards the end of the experiment. Significantly higher Mo concentrations in MoS2-NPs and MoS2-MPs
was found in the lungs of repeatedly exposed rats compared to those exposed to a single dose. The analysis of Mo content in
the liver and the spleen tissue showed a slightly higher concentration for MoS2-NPs compared to MoS2-MPs. The results for
the brain were below the calculated detection limit. Results were consistent with results obtained by bioimaging technique.
Keywords Nanoparticles· Microparticles· Molybdenum(iv) disulfide· Bioimaging· Intratracheal instillation· Rat tissues
Toxicological Research
Online ISSN 2234-2753
Print ISSN 1976-8257
* Renata Kuraś
Renata.Kuras@imp.lodz.pl
Maciej Stępnik
m.stepnik@qsarlab.com
Jarosław Grobelny
jaroslaw.grobelny@chemia.uni.lodz.pl
Emilia Tomaszewska
emilia.tomaszewska@chemia.uni.lodz.pl
Magdalena Stanisławska
M.e.stanislawska@gmail.com
Katarzyna Domeradzka-Gajda
kachada@tlen.pl
Wojciech Wąsowicz
wojciech-wasowicz@wp.pl
Beata Janasik
Beata.Janasik@imp.lodz.pl
1 Central Laboratory, Nofer Institute ofOccupational
Medicine, 8 Teresy St., 91-348Łódź, Poland
2 QSAR LAB Ltd, 3 Lipy St., 80-172Gdańsk, Poland
3 Department ofToxicology andCarcinogenesis, Nofer
Institute ofOccupational Medicine, 8 Teresy St.,
91-348Łódź, Poland
4 Department ofMaterials Technology andChemistry,
Faculty ofChemistry, University ofŁódź, 163 Pomorska St.,
90-236Łódź, Poland
5 Professor Emeritus, Nofer Institute ofOccupational
Medicine, 8 Teresy St., 91-348Łódź, Poland
6 Department ofChemical Safety, Nofer Institute
ofOccupational Medicine, 8 Teresy St., 91-348Łódź, Poland

Toxicol Res.
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Introduction
Currently, more and more attention is devoted to almost
all areas of life of nanoparticles (NPs) and nanostructures
sized 1‒100nm. The use of metallic NPs is wide. Molyb-
denum disulphide IV (MoS2) is exerting an increasing
impact on industrial applications. It is a transition metal
dichalcogenide, an inorganic chemical compound in the IV
oxidation state. The crystalline structure of MoS2 is a hex-
agonal layer of Mo atoms and 2 external hexagonal layers
of chalcogen sulphur (S) atoms. The unique tribological
properties of MoS2, resulting from its hexagonal crystal
structure, make it widely used as a high-temperature lubri-
cant, in dry, solid and liquid forms [1–3]. In a form of
powder, MoS2 improves the parameters and durability of
motorcycle and car engines. It is also used as a semicon-
ductor and catalyst in the fuel and petrochemical indus-
tries. Physicochemical properties of Mo make it a useful
alloy metal, both in the production of special steels as well
as non-ferrous alloys and pigments. It is also used in the
aerospace and defense industries, and in the production of
Mo wires and rods for electric bulbs and furnaces [4, 5].
In accordance with Regulation (EC) No. 1272/2008, MoS2
is classified as a hazardous substance; H332—Harmful if
inhaled [6, 7].
It is commonly known that the toxic properties of
chemical substances and their adverse health effects in
both occupationally and environmentally exposed popu-
lations may differ depending on the dose, time or route
of exposure. The state of aggregation and the chemical
form of a given metal are other factors affecting its toxic
properties. Numerous animal studies have confirmed that
NPs relatively quickly and easily overcome all the protec-
tive barriers of the body [8–11]. Works by Tjälve etal.
[12], Oberdörster etal. [13], as well as Engin and Engin
[14] have indicated that nanometer size particles reach the
brain via the olfactory nerve. Therefore, they may come
into contact with olfactory neurons in the olfactory epi-
thelium, and be transported through olfactory cell axons
to the olfactory bulb, where they directly affect the cen-
tral nervous system [13]. However, little data is available
on the distribution of MoS2-nanoparticles (MoS2-NPs)
after inhalation or intratracheal exposure [15–17]. Uncer-
tainty related to the safety and assessment of exposure to
MoS2-NPs and/or MoS2-microparticles (MoS2-MPs) dur-
ing their industrial production as well as during work in
exposure to MoS2 results from insufficient knowledge on
the mechanism of their toxicity.
The main objective of the study was the combination
of advanced and specialized analytical methodologies
[inductively coupled plasma mass spectrometry (ICP-MS),
inductively coupled plasma optical emission spectrometry
(ICP-OES), and laser ablation technique combined with
inductively coupled plasma mass spectrometry with ioni-
zation of the sample (LA-ICP-MS)] allowing for the deter-
mination of MoS2-NPs and MoS2-MPs concentration in
the blood, and mineralized tissue samples, as well as their
spatial distribution and accumulation content in selected
tissue sections (brain, lung, liver, spleen) obtained from
the rats exposed to nano- and micrometer Mo particles,
either once or 7 times at a dose of 1.5mg Mo kg−1 b.w.
and 5mg Mo kg−1 b.w. (at 2-week intervals), or to polyvi-
nylpyrrolidone (PVP) as a control substance, respectively,
for each form into the trachea.
Moreover, MoS2-NPs as well as MoS2-MPs were used
in an intratracheal instillation study in rats, in a single and
repeated exposure model at doses of 1.5 and 5mg MoS2
per kg b.w., to obtain data on the absorption and kinetics of
MoS2 particles, including their possible accumulation in the
body. It was hypothesized that biodistribution is dependent
on the primary particle size, assessed distribution, and tis-
sue accumulation at various time points, both during and
after exposure. The possible implementation of this type of
analysis in toxicological research was also assessed.
Materials andmethods
Study design
Having been approved by the Ethics Committee for Animal
Experiments (Resolution No. 6/ŁB 86/2018), the experi-
mental study was performed using albino Wistar rats. The
animals were at 6‒8 weeks of age and weighed 80‒120g.
The rats were acclimated for a week under 12-h day/12-h
night cycles with unlimited access to water, at standard air
humidity conditions as well as in temperature of 22 ± 3°C.
The general study design, including exposure time, the doses
of Mo and the number of animals, was adopted in line with
the guidelines reported by Warheit etal. [18, 19] and Ma-
Hock etal. [20]. For further assay, at least 4 animals were
selected, according to the OECD TG 417 Toxicokinetics
guidelines (Adopted: 22 July 2010). Finally, a decision was
made to follow the experimental scheme summarized in
Table1, assuming the exposure of animals to a single dose
(1.5 or 5 mg MoS2 kg−1 b.w.) with the analysis performed
after 24 h and 7 days, and a multiple dose (7 administrations
at 2-week intervals).
Reagents andstandards
Multi-element CRM Comprehensive Mix B Standard
10.00 ± 0.05 mg per l (LGC, USA), ultra-pure deionized
water from a Milli-Q water purification system (Millipore,
Milli-Q Ellix 3, resistivity of 18.2 MΩ cm−1) and 65% nitric

Toxicol Res.
1 3
acid (HNO3, ULTREX II Ultrapure Reagent, J.T.Baker™),
Triton-X (Sigma Aldrich) were used for the preparation of
calibration standard solutions. Laboratory solid standards
of agarose powder matrix (Sigma Aldrich, Darmstadt, Ger-
many) were prepared as agarose gel tablets in the range of
0.5‒50 μg g−1 for the calibration by LA-ICP-MS.
Additionally, the ICP-MS method and the analytical
procedure were verified by applying the available reference
material Seronorm™ Trace Elements Whole Blood (Sero,
Norway), as well as certified reference materials of the dog-
fish liver (DOLT-5, NRC-CNRC, Canada). Tablets of lyo-
philized reference material DOLT-5 using manual hydraulic
press (Specac Atlas™ Manual 15T) were used to check the
accuracy of LA-ICP-MS.
Preparation ofacolloid andasuspension ofMoS2
A stable aqueous dispersion, a colloid of MoS2-NPs as well
as a suspension of MoS2-MPs stabilized by PVP, with the
weight ratio of MoS2:PVP 1:1; K 90, Mw = 360 000 (Fluka)
were prepared. The size of the NPs ranged 50‒100nm, and
that of MPs 0.5‒5μm. The procedure of MoS2 particles
preparation was described by Sobańska etal. [15]. In this
paper, the authors also presented the 3-dimensional mor-
phology of particles analysis (Fig.1), including the size,
shape and possible agglomerations of MoS2 particles using
high resolution scanning electron microscopy (FEI-Nova
NanoSEM 450). In the same paper, a size distribution his-
togram was presented using the dynamic light scattering
Table 1 Study design including Mo doses, day of analysis, sampling and data collection
*The blood was collected from the same animal before and after exposure (the animal was self-controlled)
Type of exposure/dose Analysis Tissue
Blood Lung Liver Spleen Brain
1CON 7days – + + + +
2 1 administration
1.5mg MoS2-NPs kg−1 b.w
24h Tail vein* − − − −
7days + + + +
3 1 administration
1.5mg MoS2-MPs kg−1 b.w
24h Tail vein* − − − −
7days + + + +
4 1 administration
5mg MoS2-NPs kg−1 b.w
24h Tail vein* − − − −
7days + + + +
5 1 administration
5mg MoS2-MPs kg−1 b.w
24h Tail vein* − − − −
7days + + + +
6CON After 90days Venous blood from the heart during autopsy + + + +
7 7 administrations
1.5mg MoS2-NPs kg−1 b.w
After 90days Tail vein* − − − −
8 7 administrations
1.5mg MoS2-MPs kg−1 b.w
After 90days Tail vein* − − − −
9 7 administrations
5mg MoS2-NPs kg−1 b.w
After 90days Tail vein*
Venous blood from the heart during autopsy
+ + + +
10 7 administrations
5mg MoS2sMPs kg−1 b.w
After 90days Tail vein*
Venous blood from the heart during autopsy
+ + + +
Fig. 1 High-resolution scanning electron microscopy (FEI-Nova
NanoSEM 450) images of MoS2-NPs (a) and MoS2-MPs (b).The
scale bar is 200nm and 2µm, respectively

Toxicol Res.
1 3
(DLS) technique with zeta potential measurements (Fig.2).
DLS measurements revealed that the hydrodynamic diam-
eter of the MoS2 particles was: dH MoS2-NPs = 251 ± 94nm
for nanoparticles and dH MoS2-MPs = 0.7 ± 0.3 μm for
microparticles. As described earlier MoS2-NPs in solution
were dispersed and stabilized by PVP, so the high molecu-
lar weight polymer strongly increased the hydrodynamic
diameter of the MoS2-NPs nanoparticles compared to the
diameter measured by the HR-SEM technique. In the end,
the internalization of MoS2-NPs as well as MoS2-MPs was
performed by scanning transmission electron microscopy
(STEM) with energy-dispersive X-ray spectroscopy (EDS)
[15]. The volumes of the PVP solution, as well as of the
solutions of the tested nano- and micro-MoS2, administered
each time to the trachea of the rats, were calculated individu-
ally for each animal, maintaining the proportions depending
on their body weight. More specifically, 100 µL of the sub-
stance was administered per 250g of the rat’s body weight.
The physico-chemical analysis of the prepared MoS2 suspen-
sions indicates that they were nano- and micrometric forms
with satisfactory stability to perform biological tests.
Tissue sample preparation
The blood samples from the rats’ tail veins were collected
twice into EDTA tubes (SARSTEDT), before and after
intratracheal instillation. All blood samples were collected
from the right ventricle of the heart into Monovette 7.5mL
containing lithium heparin (SARSTEDT) during the dissec-
tion. All blood samples were stored at −20°C until analysis.
The soft tissue samples collected during autopsy were
weighed (approx. 100 mg each) using the analytical balance
Sartorius (BA 210S), and then they were mineralized under
the appropriate conditions using an UltraWave mineralizer
(Milestone, SpectroLab). The mineralization process was
carried out according to the program using concentrated
HNO3.
For determination and tissue distribution of Mo in the
tissue samples using LA-ICP-MS analysis, the brain, spleen,
lung and liver samples were first formalin-fixed and then
paraffin-embedded (automated Belair RVG/1 Vacuum Tissue
Processor; TES 99 Tissue Embedding System). Then, the
paraffin blocks were cut into 20 µm thick sections (HM 325
Rotary Microtome). In order to check the analyte contamina-
tion, all solutions (formalin, paraffin, agarose and the PVP
solution) were determined using the inductively coupled
plasma excitation mass spectrometry (ICP-MS) technique.
The bronchoalveolar lavage cells and lung tissues were
processed using routine histopathological protocols (light
microscopy using Giemsa and hematoxylin-eosin staining;
electron scanning microscopy with lead and osmium con-
trasting) to evaluate tissue changes and MoS2 particles dis-
tribution in the cells in different organ compartments.
Instrumentation andexperimental parameters
Molybdenum determination in blood samples was performed
by ICP-MS (ELAN DRC-e, PerkinElmer, SCIEX, USA)
using a dynamic reaction cell (DRC) with methane (CH4)
reaction gas, eliminating spectral and matrix-derived inter-
ferences. The linear calibration curve for Mo determination
in blood ranged 0.5‒50μg L−1 with the correlation coef-
ficient r = 0.9999. The analytical precision of the method
amounted to 5.4%. The repeatability of measurements using
the Seronorm™ Trace Elements Whole Blood as an internal
quality control amounted to 8.8%. The limit of detection
(LOD) based on 3*standard deviation (SD)/slope, by the
5 repetitive analysis of the response of the curve amounted
to 0.026µg L−1 and the limit of quantitation (LOQ: 6*SD/
slope) amounted to 0.052µg L−1.
Fig. 2 DLS histograms determining the hydrodynamic diameter of the MoS2 particles

Toxicol Res.
1 3
The mineralized tissue samples were diluted appropri-
ately before the analysis using deionized water. Molybdenum
determination in the mineralized tissue samples was per-
formed using the ICP-OES technique (Agilent 5100 SVDV,
MS Spectrum). The ranges of the calibration method for
Mo determination in the mineralized tissue samples ranged
2‒500μg L−1 with the correlation coefficient r = 0.9999.
The analytical precision of the method amounted to 1.6%.
The repeatability of measurements using a solution of Mo
with a concentration of 100µg L−1 amounted to 2.7%. The
LOD and LOQ estimated using 10 blank samples amounted
to 0.16µg L−1 and 0.32µg L−1, respectively.
The bioimaging of tissue slices was performed by the
LA system (J200 Tandem LA/LIBS Applied Spectra Inc.,
USA) with LA-ICP-MS used as a complementary analysis.
The ranges of the calibration method for Mo determination
in gel standards ranged 0.5‒50μg L–1. An intra- and inter-
assay coefficient of variability as well as the recovery of Mo
per DOLT-5 pellet (8 replicate ablation lines) amounted to
12.9%, 16.5% and 90%, respectively. The LOD and LOQ
estimated using 10 blank agarose samples amounted to
0.017µg g−1 and 0.034µg g−1, respectively.
Statistical analysis
Quantitative data were presented as mean ± standard devia-
tions (SDs). The mean and SD values were calculated using
the GraphPad Prism Software v.6.01 for Windows (Graph-
Pad Prism Software, Inc., USA). Due to a small number
of observations per group, the data were assumed to lack
normal distribution. Therefore, the Kruskal–Wallis test with
Dunn’s post hoc test were used for determining statistical
significance. The statistical significance was set at p < 0.05.
Results
Tissue content
Analysis ofMo inblood samples byICP‑MS
In the tail venous blood obtained from the animals before
exposure, Mo concentrations ranged 10.2‒38.4μg L−1
(mean: 22.4 ± 8.0μg L−1, median: 22.2μg L−1). Blood was
collected from the same animal before and after exposure,
i.e., changes in Mo concentrations were monitored in each
animal individually. After 24h of exposure, a slight increase
in Mo concentration was observed; however, this increase
was not dependent either on the form of MoS2 or its dose
(Fig.3a). In addition, there were no statistically significant
differences between the groups. Similarly, there were no
differences in Mo concentrations between the groups after
7days of exposure (Fig.3b).
0
5
10
15
20
25
30
35
40
45
Lgµ[liattaramorfdoolbninoitartnecnoco
M−
1]
A
0
5
10
15
20
25
30
35
40
45
Mo concentration in blood from a rat tail [µg L−1]
B
Fig. 3 Mo concentration (mean ± SD) in the venous blood collected
from the tail vein of male rats exposed to MoS2-NPs and MoS2-MPs
after a single administration, and the analysis performed after 24h (a)
and 7days (b), at a dose of 1.5 and 5mg MoS2 per kg b.w. In indi-
vidual groups, the same rat was bled before exposure, and after 24h
or 7days. The study groups were composed of 3 animals

Toxicol Res.
1 3
In the venous blood collected from the tail of animals
after exposure for 7 times at 2-week intervals and with the
analysis performed within 90days, Mo concentrations were
comparable to each other and they were not dependent either
on the form of MoS2 or its dose (Fig.4).
For MoS2-NPs (at both doses), a slight decrease in Mo
concentration was observed from the first administration,
which then slowly increased from the fourth administra-
tion. For MoS2-MPs (also at both doses), a slight increase
in Mo concentration was observed from the first adminis-
tration, which then slowly increased again from the fourth
administration.
After the determination of Mo concentration in the
venous blood taken from the right ventricle of the animals’
hearts during autopsy performed after 90days of exposure
to MoS2 at a higher dose (5mg kg−1), an approximately two-
fold increase in Mo concentration for both forms was shown
(a slightly higher concentration for MoS2-MPs: 15.3 ± 3.9
vs. 21.3 ± 7.3μg L−1, respectively, for MoS2-NPs and
MoS2-MPs. A similar relationship was observed in venous
blood, in which the Mo concentration before autopsy after
90days was slightly higher for MoS2-MPs (17.8 ± 2.6μg
L−1) than for MoS2-NPs (13.1 ± 4.3μg L−1) at a dose of
1.5mg MoS2 kg−1 b.w.
Kinetic profiles for MoS2‑MPs and MoS2‑NPs
The multiple-dose toxicokinetics in the experiment per-
formed reflects how the body responds to substances intro-
duced intratracheally. The concentration curves of various
forms of Mo in the blood over time, after repeated admin-
istration of 2 different fixed doses (1.5 and 5mg kg−1), are
presented in Fig.5.
The trend indicating the presence of 2 phases is visible.
During the first phase, the Mo concentration in the blood
decreases until day 14 (the Mo concentration before the
second administration), below the pre-exposure concentra-
tion. The second phase is linear, less abrupt and practically
flat, but with an increasing trend towards the end of the
experiment. The multi-compartment model assumes an
exponential curve of Mo concentration over time with dif-
ferent half-lives for the distribution and elimination phases
Fig. 4 Mo concentration
(mean ± SD) in the venous
blood collected from the tail
vein from male rats exposed to
MoS2-NPs (a) and MoS2-MPs
(b) after 7 administrations at
2-week intervals, and with
the analysis performed over
90days, at a dose of 1.5 and
5mg MoS2 per kg b.w. within
each group, the same rat was
bled after exposure at specific
time intervals. The study groups
were composed of 3 animals
0
5
10
15
20
25
before 1
administr.
(day 0)
before 2
administr.
(day 14)
before 3
administr.
(day 28)
before 4
administr.
(day 42)
before 5
administr.
(day 56)
before 6
administr.
(day 70)
before 7
administr.
(day 84)
before
autopsy (day
90)
L gµ[ liat tar a morf doolb ni noitartnecnoc oM−1]
MoS₂-NPs 1.5 mg kg-1 MoS₂-NPs 5 mg kg-1 A
0
5
10
15
20
25
before 1
administr.
(day 0)
before 2
administr.
(day 14)
before 3
administr.
(day 28)
before 4
administr.
(day 42)
before 5
administr.
(day 56)
before 6
administr.
(day 70)
before 7
administr.
(day 84)
before
autopsy (day
90)
L gµ[ liat tar a morf doolb ni noitartnecnoc oM−1]
MoS₂-MPs 1.5 mg kg-1 MoS₂-MPs 5 mg kg-1 B

Toxicol Res.
1 3
of MoS2-MPs and MoS2-NPs. Following the intratracheal
instillation, the distribution half-life was the fastest for the
lower MoS2-NPs dose (1.5mg kg−1) at T1/2 8.8days. At
the same dose, the calculated T1/2 was 2days longer for
the MoS2-MPs. MoS2-NPs and MoS2-MPs at the higher
dose (5mg kg−1) showed similar values of T1/2 11.5 vs.
11.8days, respectively. The multiple-dose elimination
trend is increasing for both formulations in the blood Mo
concentration.
Although the multiple-dose elimination trend is increas-
ing for both formulations in the blood Mo concentration,
compared to the control group at day 90, all concentrations
(except MoS2-MPs at a dose of 5mg kg−1) were lower
(Fig.6). MoS2-MPs were absorbed in higher amounts
and was more slowly removed from the bloodstream. For
MoS2-MPs it could be seen that after 90days the concen-
trations were higher than at the beginning of the experi-
ment, so it was excreted more slowly. After 90days of
the experiment, MoS2-NPs concentration returned to their
baseline thus, excretion even after a repeated doses was
fast.
Statistically, for MoS2-NPs and MoS2-MPs at the lower
dose of (1.5mg kg−1) the differences are statistically sig-
nificant p = 0.046, and despite apparent differences there
is no statistical significance for MoS2-NPs and MoS2-MPs
at a higher dose (5mg kg−1).
Analysis ofMo soft tissue samples byICP‑OES
Analysis of Mo concentration in the rat’s lung In accord-
ance with macroscopic observations indicating an uneven
distribution of Mo in the sampled tissues, the lung tissue
collected during the autopsy was completely mineralized.
Measurements of the Mo content in the lung tissue per-
formed 7days after exposure to MoS2 showed a large scat-
ter of individual values (Fig.7a). A greater accumulation
of MoS2-MPs in the lung tissue was observed. The concen-
tration of Mo in the lungs of animals repeatedly exposed
to MoS2, both MoS2-NPs and MoS2-MPs, was significantly
higher compared to a single administration of MoS2, prov-
ing the material accumulation of the tested preparations in
the lungs of the exposed rats. The determination of Mo con-
tent in the lung tissue after exposure to MoS2 in 90 days
showed and confirmed a several times higher accumulation
for MoS2-MPs compared to MoS2-NPs (Fig. 7b). Differ-
ences between the results of Mo concentrations in individ-
ual rats may be due to the actual differences in the baseline
Mo status, the animals’ natural adaptive abilities disorder
and the Mo absorbed after administration.
Analysis ofMo concentration intherat’s liver The measure-
ments of Mo content in the liver tissue performed 7days
after exposure to MoS2 showed no differences between the
Fig. 5 The mean blood con-
centration–time relationship for
MoS2-MPs and MoS2-NPs after
intratracheal instillation to rats.
The logarithm linear regression
curve equation. Each data point
represents the mean and SD of
Mo concentrations measured
(n = 6)
Linear regression curve equation
between 1‒14 days of exposure
Linear regression curve equation
between 28‒90 days of exposure
MoS2-MPs 5 mg y = ‒0.0255x + 1.5793 y = 0.0008x + 1.1783
MoS2-NPs 5 mg y = ‒0.0263x +1.5735 y = 0.0015x + 1.0173
MoS2-MPs 1.5 mg y = ‒0.0293x + 1.5193 y = 0.0014x + 1.1058
MoS2-NPs 1.5 mg y = ‒0.0342x + 1.6y = 0.0014x + 0.9441
0.9
1.0
1.1
1.2
1.3
1.4
1.5
1.6
1.7
0102030405060708
09
0
[LOG (Mo in blood)]
Days
MoS₂-MPs 5 mg kg-1 MoS₂-NPs 5 mg kg-1 MoS₂-MPs 1.5mg kg-1 MoS₂-NPs 1.5 mg kg-1

Toxicol Res.
1 3
groups of exposed animals (there was no dependence on the
administered dose) or in relation to the control group which
was administered PVP (Table2). The analysis of Mo content
performed after 90days showed a slightly higher concentra-
tion for MoS2-NPs compared to MoS2-MPs. However, these
Fig. 6 Median blood concen-
tration for MoS2-MPs and
MoS2-NPs for both doses
(5mg kg−1 and 1.5mg kg−1)
before autopsy (90days)
Mean
Mean+/-Std Er
r
Mean+/- SD
CON
MoS
2
-MPs 5mg kg-1
MoS
2
-MPs 1.5 mg kg-1
MoS
2
-NPs 5 mg kg-1
MoS
2
-NPs 1.5 mg kg-1
6
8
10
12
14
16
18
20
22
24
26
28
30
doolbnioM
1-Lgμ
*
0.4 92.1
572.2
172.2
371.7
0
500
1000
1500
2000
2500
3000
3500
4000
CON MoS₂-NPs
1.5 mg kg-1
MoS₂-NPs
5 mg kg-1
MoS₂-MPs
1.5 mg kg-1
MoS₂-MPs
5 mg kg-1
gnulehtninoitartnecnocoM(mg kg−1 bw)
7 daysA
0.4
572.2
371.7
23.4
1,216.0
3,656.0
0
500
1000
1500
2000
2500
3000
3500
4000
CON MoS₂-NPs 5
mg kg-1
MoS₂-MPs 5
mg kg-1
Mo concentraon in the lung(mg kg−1 bw)
7 days 90 days B
*
*
Fig. 7 Molybdenum concentration (mean values ± SD) in the lungs of
male rats exposed to MoS2-NPs and MoS2-MPs after: a single admin-
istration, and the analysis performed after 7days, at a dose of 1.5 and
5mg MoS2 per kg b.w., vs. CON (N = 3‒4); b 7 administrations (at
2-week intervals), and the analysis performed after 90days, at a dose
of 5mg MoS2 per kg b.w., vs. CON (N = 4); The PVP was adminis-
tered in a volume of 400 µL of PVP per kg b.w. (N = 4) (CON)

Toxicol Res.
1 3
values did not differ significantly between each other or in
relation to the control group which was administered PVP.
Analysis ofMo concentration intherat spleen The measure-
ments of Mo content in the spleen tissue performed 7days
after exposure to MoS2 showed no differences between the
groups of exposed animals (there was no dependence on
the administered dose) or in relation to the control group
which was administered PVP (Table2). Similar to the liver
tissue, the analysis of Mo content performed after 90days
showed a higher concentration for MoS2-NPs compared to
MoS2-MPs. However, these values did not differ signifi-
cantly between each other or in relation to the control group
which was administered PVP.
Analysis ofMo concentration intherat’s brain The results
for the brain were below the calculated detection limit, indi-
cating that Mo concentrations were at trace levels.
Tissue distribution
Analysis ofMo insoft tissue samples byLA‑ICP‑MS
There are few studies using laser bioimaging techniques that
can be very helpful in assessing the distribution and con-
centration of NPs in tissues as an important tool in assess-
ing toxicity. Taking into consideration the complexity and
multidirectional nature of factors determining the toxicity
of NPs, biological studies should be carried out in many
directions. Direct micro-sampling of solids allows for deter-
mining distribution, i.e., for obtaining detailed images of
specific tissue regions of the selected elements on the sur-
face of a solid sample (mapping). In this paper, the tissue
sections from soft tissues (brain, lung, liver, spleen) were
used for the confirmation of tissue distribution of both Mo
forms using the LA-ICP-MS technique as a complementary
Table 2 Molybdenum concentration (mean values ± SD) in the liver
and the spleen of male rats exposed to MoS2-NPs and MoS2-MPs
after: single administration, and the analysis performed after 7days,
at a dose of 1.5 and 5 mg MoS2 per kg b.w., vs. CON (N = 4); 7
administrations (at 2-week intervals), and the analysis performed
after 90days, at a dose of 5mg MoS2 per kg b.w., vs. CON (N = 4);
PVP was administered in a volume of 400 µL PVP per kg b.w. (N = 4)
(CON)
Organ Parameter Interval
7days 90days
Liver CON 1.19 ± 0.49 1.09 ± 0.34
MoS2-NPs 1.5mg kg−1 1.10 ± 0.49 –
MoS2-NPs 5mg kg−1 1.24 ± 0.61 1.67 ± 0.38
MoS2-MPs 1.5mg kg−1 1.28 ± 0.51 –
MoS2-MPs 5mg kg−1 1.09 ± 0.45 1.38 ± 0.23
Spleen CON 0.15 ± 0.02 0.16 ± 0.06
MoS2-NPs 1.5mg kg−1 0.27 ± 0.15 –
MoS2-NPs 5mg kg−1 0.16 ± 0.05 0.87 ± 0.09
MoS2-MPs 1.5mg kg−1 0.14 ± 0.10 –
MoS2-MPs 5mg kg−1 0.16 ± 0.05 0.32 ± 0.08
CON
97/95Mo [102 counts]
Rats exposed to MoS2-MPs
97/95Mo [106 counts]
Rats exposed to MoS2-NPs
97/95Mo [105 counts]
Fig. 8 LA-ICP-MS Mo bioimaging Mo (the 97/95Mo ratio) in the lung sections of the rats exposed to MoS2-NPs and MoS2-MPs vs. CON, nor-
malized to the size of the sample

Toxicol Res.
1 3
tool in the experiment. Figure8 shows an example of tis-
sue distribution for Mo. The higher signal intensity for the
MoS2-MPs in the lung tissue analyzed after 90days at a dose
of 5mg MoS2 per kg b.w. confirms the results obtained by
ICP-OES. Moreover, an analysis of Mo distribution in the
liver and spleen samples revealed a higher concentration of
MoS2-NPs in the same dose of 5mg MoS2 per kg b.w. after
7 administrations, which was in accordance with the results
obtained by ICP-OES. The bioimaging analysis confirmed
the different tissue distribution of Mo and its non-heteroge-
neity. The concentration of Mo in the brain tissue was the
lowest compared to other tissues and was below the calcu-
lated value of LOD (0.017µg g−1) for LA-ICP-MS, which
was also in good agreement with ICP-OES data.
Analysis of MoS2 particle distribution inBAL cells andlung
tissue
Exemplary images of the alveolar macrophages isolated
from bronchoalveolar lavage (BAL) and the lung tissues of
the rats exposed to 5mg kg−1 b.w. and analysed after 7days
are shown in Fig.9. Based on the images an efficient inter-
nalization of MoS2 particles by the alveolar macrophages
can be proven, without induction of visible morphologi-
cal changes of the macrophages as well as the cells in the
interstitial tissue. The similar changes indicating an efficient
clearance of the particles without induction of profibrotic
reaction could be observed in the animals at the end of the
experiment, i.e., after 90days of exposure.
Discussion
Toxicokinetics parameters of MoS2 inbothforms
intherats
Inhalatory route
The rate of absorption of Mo depends, inter alia, on its
solubility. In contrast to MoO3, MoS2 particles are prac-
tically insoluble in water, hence their absorption from the
lung tissue is expected to proceed at a very slow rate [21,
22]. Quantitative estimates of absorption following inhala-
tion exposure to molybdenum in humans or animals were
not identified [23]. Some evidence for absorption of molyb-
denum trioxide from the airways mucosa is available from
inhalation studies on molybdenum trioxide conducted in
rodents, i.e. in guinea pigs [24] well as rats and mice [25].
To our knowledge, our study is the only one available in the
published literature which evaluated kinetics parameters in
rats exposed via inhalatory route to MoS2 particles, hence
any reliable comparisons to the published data cannot be
made.
Oral andintravenous routes
Available data on Mo kinetic parameters after exposure via
other routes indicate its rather fast absorption, distribution
and elimination, depending to a great extent on the chemical
form of Mo. In the study conducted by Werner etal. [26]
on volunteers, the elimination of Mo from the blood after a
single intravenous injection of a trace quantity of Mo (rang-
ing 300‒450μg) occurred in the T1/2 range of 4‒70min
(half the time of the fast component of clearance) and in the
T1/2 range of 3‒30h (half the time of the slow component
of clearance) in a two-compartment model. In the presented
biokinetic model, the authors claimed that the clearance of
plasma was much faster than the literature data. In addition,
the authors showed that the volumes of distribution were
significantly higher than the plasma volumes, but smaller
than the calculated extracellular spaces. The authors further
claimed that the faster Mo clearance from plasma might be
explained by a quick uptake of Mo into tissues. This may
indicate a very fast distribution of Mo in body fluids. The
slight differences in results observed in our paper may be
explained by both physiological inter-individual differ-
ences and also by the sampling schedule. Moreover, we also
assume it may be related to the changing physiology of ani-
mals with maturation leading to lower demand for Mo in a
growing organisms and eventually lower Mo plasma concen-
tration. Unfortunately, we were not able to identify studies
which could provide any support for such hypothesis (and
as mentioned above no studies were found on relevant Mo
blood kinetic parameters after inhalation exposure to partic-
ulate forms of Mo). It can be assumed that an unknown frac-
tion of each dose administered after various time intervals
was absorbed from the lungs (not known if in the form of
particles or ions released after dissolution of the particles),
causing comparable spikes in Mo concentration, but it was
afterwards efficiently eliminated from the blood before the
next dose. It is probable that within 2weeks between admin-
istrations an equilibrium of the Mo distribution between the
tissues and the blood has been established, which did not
lead to increased Mo blood concentrations. As we calcu-
lated, the expected elimination half-life was T1/2 354 vs.
195days for the higher dose (5mg kg−1) of MoS2-MPs vs.
MoS2-NPs, respectively, compared with an elimination half-
life of T1/2 221 vs. 212days for the lower dose (1.5mg kg−1)
of MoS2-MPs vs. MoS2-NPs, respectively. Based on these
data, we hypothesize that, after repeated dosing, there is
quite a rapid absorption of a certain amount (most probably
very small) of the particles fraction administered (includ-
ing ions after dissolution), while the remaining part of the
particle fraction accumulates in the lungs. Moreover, For
MoS2-MPs it could be seen that after 90days the concentra-
tions were higher than at the beginning of the experiment,
so it was excreted more slowly. This is consistent with the

Toxicol Res.
1 3
observations of Kuraś etal. [27], as a lot of MoS2-MPs are
observed in the lungs, the same MoS2-MPs absorption into
the blood is faster, greater and excretion longer, as confirmed
in Fig.6.
Turnlund and Keyes [28] conducted a study on the clear-
ance of Mo from the blood in men after administration of
Mo, first intravenously (33μg of 97Mo) and then orally
(22μg day−1 Mo). The administration of Mo increased
MoS2-NPs MoS2-MPs
A
B
C
10 μm
25.3 μm
50 μm 50 μm
10 μm 10 μm
Fig. 9 Exemplary images of the lung tissue of the rats exposed to
5mg kg−1 bw and analysed after 7days. a Alveolar macrophages iso-
lated from BAL heavily loaded with MoS2 particles (Giemsa stain-
ing). The cells attached to the macrophages are probably monocytes.
b Light microscopy images of the lung tissue showing MoS2 particles
(dark brown or black agglomerates) present in alveolar macrophages
and interstitial tissue (routing HE staining 200×). c SEM pictures of
macrophages loaded with MoS2 particles. The particles are engulfed
in clusters in vesicular organelles (endosomes)

Toxicol Res.
1 3
both the natural intrinsic Mo in plasma and the total Mo
in plasma during the first minutes (6.9 vs. 6.9nmol L−1,
respectively) up to 1h (13.0nmol L−1 vs. 17.1nmol L−1,
respectively), then it again decreased to near baseline after
24h of uptake (5.7nmol L−1 vs. 6.0nmol L−1, respectively).
Eventually, 48h after infusion Mo concentration remained
at a similar but only slightly lower level (5.1mmol L−1 vs.
5.3mmol L−1, respectively). After 72days, Mo concentra-
tion remained unchanged (5.8 ± 2.5nmol L−1). These find-
ings on urinary excretion are in agreement with the data
obtained by Werner etal. (2000). The authors of the study
suggest that the introduced Mo disturbed the overall Mo
metabolism at the beginning of the experiment. It resulted
in an increased level of Mo after exposure, combined with
the physiological level of natural Mo. More specifically,
Mo could have been absorbed by the tissues that released
the pool of bioavailable intrinsic Mo in the body increas-
ing its concentration in the blood. Further exposure to low
dietary Mo may have resulted in physiological adaptation
[28]. Another study concerning compartmental modeling
to explain the alteration in Mo distribution and excretion
with the urine showed a positive correlation in the studied
men, where increased Mo intake was associated with both
increased Mo absorption and urinary excretion. The frac-
tion deposited in tissues was inversely correlated [29]. It
is known that Mo is mainly excreted in the urine and it is
a key pathway for modulating exposure to Mo in the body.
Molybdenum from feces is eliminated in lower amounts. In
humans, it is up to 17‒80% of the total absorbed Mo dose
[30, 31], but Giussani etal. [32] and Novotny and Turnlund
[29] reported that this excretion was on the level of 75–90%
of the absorbed Mo dose. Urinary Mo excretion, according
to the results obtained by Bell etal. [33] after oral adminis-
tration to rats, showed that 90% of the dose was eliminated
by the kidneys. The lack of multiple urine collection from
the freely moving rats may be considered a limitation of this
article. This was not included in the study implementation
schedule because attention was focused on the intratracheal
instillation exposure and on following the Mo metabolism
and key pathways of its regulation connected with blood
kinetics and tissue distribution.
Induction ofpro‑inflammatory reactions inthelung
The latest research has revealed that MoS2 has the ability
to cause inflammatory reactions [31, 34]. It was shown
that MoS2-MPs as well as MoS2-NPs deposited in the lung
tissue of the rats after intratracheal instillation may cause
inflammatory reactions, although a stronger response was
observed for MoS2-MPs. The authors observed inflam-
mation in the respiratory system in the rats after a sin-
gle administration. The difference in the inflammatory
response was statistically significant for both doses (1.5
and 5mg MoS2 kg−1 b.w.) 7days after the autopsy for
MoS2-MPs compared to control (PVP) rats [15]. More-
over, the authors showed interstitial inflammation at a
higher dose, both 24h after the autopsy (for both forms)
and 7days after the autopsy for MoS2-MPs. This data is
confirmed by the results presented in our paper. STEM
with EDS unambiguously revealed multiple alveolar mac-
rophages loaded with plate-shaped Mo-MPs as well as
agglomerates of Mo-NPs. The characteristically expanded
lysosomes in these macrophages containing similar clus-
ters of particles were observed in the cytoplasm of mac-
rophages. The authors also showed the presence of NPs
in epithelial cells, which may suggest that the process of
internalization indicates the possibility of NPs penetration
through the epithelium and systemic circulation extended
clearance [15]. Chng etal. [35] noticed that disk-shaped
particles were conducive to proinflammatory reactions
in the respiratory system. Moreover, the histopathologi-
cal assessment after chronic inhalation of 6.6mg MoO3
mg/m3 in mice revealed significantly greater instances
of adenoma or carcinoma of alveolar/bronchiolar in the
exposed groups in comparison to control ones [36]. Fur-
thermore, in the same study the authors pointed to mar-
ginally greater incidents of lung tumor in male rats. The
initial histopathological lung damages were observed
already at 10mg/m3. In another study, Huber and Cerreta
[37] reported an increase in the neutrophils and multinu-
cleated macrophages in BAL fluid in hamsters after one
day of inhalation of 5mg Mo/m3, and lymphocytes after
7days of exposure. The increase in neutrophils in BAL
fluid was also observed in mice after inhalation of 90mg
Mo/m3 [16]. What is more, the tidal volume was already
decreased after the lowest exposure level (8mg MoS2/
m3). Another study conducted by Peña etal. [17] also con-
firmed a lung inflammation caused by MoS2 nanosheets
after a single inhalation in mice. Inflammatory cytokines
and extracellular vesicles as well as immune cells detected
in BAL fluid effected on inflammatory status.
Another study assessed the toxicity of Mo-NPs on rat
BRL3A, i.e., rat liver cells, after 24-h exposure [38]. The
authors of this study observed a significant increase in the
lactate dehydrogenase enzyme release at the Mo-NPs con-
centration of 250μgm L−1—much higher than in the study
conducted by Braydich-Stolle etal. [39]. Also, an increase in
mitochondrial activity reduction occurred at a much higher
concentration—250μgm L−1 [38]. Mo supplementation sig-
nificantly increased the activity of xanthine dehydrogenase/
xanthine oxidase, sulfite oxidase and superoxide dismutase
in the liver [40]. This is also confirmed by a study conducted
by Yang and Yang [41]. The authors investigated an effect
of Mo supplementation (0.1mg Mo L−1) of rats on the
concentration of hepatic Mo, which was increased signifi-
cantly compared to controls. Thus, it is very likely that the

Toxicol Res.
1 3
increased concentration of Mo in this study, observed after
7 administrations, caused disturbances in the metabolism of
liver enzymes due to tissue retention.
Moreover, Mo is an essential trace element, which, as an
enzyme component, supports iron metabolism and thus con-
tributes to hematopoesis. Accumulation of Mo in tissues can
cause the risk of anemia [42, 43]. In our study, we observed
deviations in basic hematological parameters in exposed
animals. Similar to Sobańska etal. [15] study. Therefore,
lower Mo concentrations in blood after exposure are associ-
ated with hematological changes and damage to the vascular
system during material collection, that directly affects hema-
tological parameters (decrease in red blood cells, lower Mo
concentrations). Kusum etal. [44] obtained similar results.
According to authors, oral exposure to Mo in goats may
altered haematological profile, because it causes a state of
secondary copper deficiency. As a consequence, the study
revealed significant reduce in mean hemoglobin, packed cell
volume, total leukocyte as well as erythrocyte count. The
mean of corpuscular hemoglobin concentration was also
significantly decreased. Lyubimov etal. [45] confirmed a
decrease in erythrocyte count as well as hematocrit in rats
after gavage administered by 4.4mg Mo/kg/day. Moreover,
in the study Asadi etal. [46] the number of white blood
cells increased with increasing levels in Mo NP dosage, after
intraperitoneal injections in rats. NPs cause inflammation
due to disorders in the lymphatic system.
In the described study, rats were exposed by intratracheal
administration to nano- and micro-metric forms of Mo. It
can be concluded that, after such exposure, MoS2-NPs as
well as MoS2-MPs were mostly retained in the lung tissues.
Distribution of the administered molybdenum disulfide par-
ticles was also observed in extrapulmonary tissues. Repeated
exposure resulted in a significant accumulation of particles
in both lungs and other tissues, with the following order of
concentration: liver > spleen > brain. The distribution expo-
nent was the fastest for the lower nanoparticle dose at T1/2
8.8days. The calculated elimination half-life was also faster
for the nano-forms of Mo in comparison to the micro-forms,
regardless of the dose.
The present results provide a solid basis for further
research on the fate of nanoparticles in the body. Addi-
tional studies, such as information on the extent of oral
exposure after inhalation exposure, are necessary to clarify
the routes of exposure. In addition, this is the first study in
which 3 techniques were used to complement each other
for the evaluation of the effects of intratracheal instillation
of MoS2-MPs and MoS2-NPs on tissue distribution in rats.
The LA-ICP-MS technique was proposed as a complemen-
tary tool for ICP-OES and ICP-MS, for the identification
as well as bioimaging of different sizes of Mo particles
in rat tissue. The impact of the particle size and form was
investigated, which may be an important tool in further
internal biokinetics studies. Intratracheal exposure to Mo
particles showed their retention and deposition, mainly in
the lung tissue, in the form of MoS2-MPs, and to a lower
extent in the liver and spleen, but mainly in the form of
MoS2-NPs. Taking into consideration the complex nature
of factors determining the toxicity of NPs and MPs, bio-
logical as well as toxicological studies should be carried out
multidirectionally.
Acknowledgements The authors would like to thank K. Ranoszek-
Soliwoda, G. Celichowski from the University of Łódź, (Department
of Materials Technology and Chemistry, Faculty of Chemistry, Łódź,
Poland) for the chemical synthesis of MoS2, as well as K. Sitarek, R.
Świercz, Z. Pisarek, B. Pawlak and K. Mader for their assistance during
the animal experiments, and T. Podsiadły and W. Kuszka for the deter-
mination of Mo concentration in blood. Moreover, the authors would
like to thank C. Derrick Quarles Jr. (Elemental Scientific, Inc.) and
Charles Sisson (Applied Spectra, Inc.) for their valuable suggestions
and excellent technical assistance during the training and optimization
of the J200 Tandem LA/LIBS.
Funding This study was funded by the National Science Centre (Grant
no. 2019/33/N/NZ7/02215) and was supported by the Central Institute
for Labour Protection—National Research Institute (the fourth stage of
the national program entitled “Improvement of safety and working con-
ditions,” supported in 2017–2019, coordinated by the Central Institute
for Labour Protection—National Research Institute).
Data availability Data generated and/or analyzed during the current
study are available from the corresponding authors upon reasonable
request.
Declarations
Conflict of interest The authors declared no conflict of interest.
Ethical approval and consent to participate Research involving rats.
Ethics Committee for Animal Experiments (Resolution No. 6/ŁB
86/2018).
Consent for publication All authors agree to be published.
Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
References
1. Meng FM, Cui ZT, Cheng ZT, Han HL (2018) Experimental study
on tribological properties of graphite-MoS2 coating on GCr15. J
Tribol 140:051303. https:// doi. org/ 10. 1115/1. 40397 96

Toxicol Res.
1 3
2. Hou K, Han M, Liu X, Wang J, He Y, Yang S (2018) Insitu
formation of spherical MoS2 nanoparticles for ultra-low friction.
Nanoscale 10:19979–19986. https:// doi. org/ 10. 1039/ C8NR0
6503A
3. Zhang Y, Li P, Li Ji L, Liu X, Wan H, Chen L, Li H, Jin Z
(2021) Tribological properties of MoS2 coating for ultra-long
wear-life and low coefficient of friction combined with addi-
tive g-C3N4 in air. Friction 9:789–801. https:// doi. org/ 10. 1007/
s40544- 020- 0374-3
4. Zan W, Geng W, Liu H, Yao X (2016) Electronic properties of
MoS2 on monolayer, bilayer and bulk SiC: a density functional
theory study. J Alloys Compd 666:204–208. https:// doi. org/ 10.
1016/j. jallc om. 2016. 01. 108
5. Shanmugavel R, Chinthakndi N, Selvam M, Madasamy N, Shan-
mugakani SK, Nair A, Prakash C, Buddhi D, Dixit S (2022) Al-
Mg-MoS2 reinforced metal matrix composites: machinability
characteristics. Materials 15:4548. https:// doi. org/ 10. 3390/ ma151
34548
6. European Commission, Directorate-General for Environment
(1999) Guidelines for setting specific concentration limits for
carcinogens in Annex I of Directive 67/548/EEC: inclusion of
potency considerations. Publications Office. https:// op. europa.
eu/ en/ publi cation- detai l/-/ publi cation/ 33b0a e1e- bba6- 4fcc- 8005-
d137f dd761 13. Accessed 24 Apr 2023
7. Regulation (EC) No 1272/2008 of the European Parliament and of
the Council of 16 December 2008 on classification, labelling and
packaging of substances and mixtures, amending and repealing
Directives 67/548/EEC and 1999/45/EC, and amending Regula-
tion (EC) No 1907/2006 (Text with EEA relevance). https:// eur-
lex. europa. eu/ legal- conte nt/ EN/ TXT/? uri= celex% 3A320 08R12
72. Accessed 24 Apr 2023
8. Park MV, Neigh AM, Vermeulen JP, de la Fonteyne LJ, Verharen
HW, Briedé JJ, van Loveren H, de Jong WH (2011) The effect
of particle size on the cytotoxicity, inflammation, developmental
toxicity and genotoxicity of silver nanoparticles. Biomaterials
32:9810–9817. https:// doi. org/ 10. 1016/j. bioma teria ls. 2011. 08. 085
9. Saraiva C, Praça C, Ferreira R, Santos T, Ferreira L, Bernardino
L (2016) Nanoparticle-mediated brain drug delivery: overcoming
blood-brain barrier to treat neurodegenerative diseases. J Control
Release 235:34–47. https:// doi. org/ 10. 1016/j. jconr el. 2016. 05. 044
10. Omarch G, Kippie Y, Mentor S, Ebrahim N, Fisher D, Murilla
G, Swai H, Dube A (2019) Comparative invitro transportation
of pentamidine across the blood-brain barrier using polycapro-
lactone nanoparticles and phosphatidylcholine liposomes. Artif
Cells Nanomed Biotechnol 47:1428–1436. https:// doi. org/ 10.
1080/ 21691 401. 2019. 15969 23
11. Costa A, de Souza C-W, Seabra V, Sarmento B, Lehr CM (2019)
Triple co-culture of human alveolar epithelium, endothelium and
macrophages for studying the interaction of nanocarriers with the
air-blood barrier. Acta Biomater91:235–247.https:// doi. org/ 10.
1016/j. actbio. 2019. 04. 037
12. Tjälve H, Henriksson J, Tallkvist J, Larsson BS, Lindquist NG
(1996) Uptake of manganese and cadmium from the nasal mucosa
into the central nervous system via olfactory pathways in rats.
Pharmacol Toxicol 79:347–356. https:// doi. org/ 10. 1111/j. 1600-
0773. 1996. tb000 21.x
13. Oberdörster G, Sharp Z, Atudorei V, Elder A, Gelein R, Kreyling
W, Cox C (2004) Translocation of inhaled ultrafine particles to the
brain. Inhal Toxicol 16:437–445. https:// doi. org/ 10. 1080/ 08958
37049 04395 97
14. Engin AB, Engin A (2019) Nanoparticles and neurotoxicity: dual
response of glutamatergic receptors. Prog Brain Res 245:281–303.
https:// doi. org/ 10. 1016/ bs. pbr. 2019. 03. 005
15. Sobańska Z, Sitarek K, Gromadzińska J, Świercz Ś, Szparaga
M, Domeradzka-Gajda K, Kowalczyk K, Zapór L, Wąsowicz W,
Grobelny J, Ranoszek-Soliwoda K, Tomaszewsk E, Celichowski
G, Roszak J, Stępnik M (2020) Assessment of acute toxicologi-
cal effects of molybdenum(IV) disulfide nano- and microparticles
after single intratracheal administration in rats. Sci Total Environ
742:140545. https:// doi. org/ 10. 1016/j. scito tenv. 2020. 140545
16. Sørli JB, Jensen ACØ, Mortensen A, Szarek J, Gutierrez CAT,
Givelet L, Loeschner K, Loizides C, Hafez I, Biskos G, Vogel U,
Hadrup N (2023) Pulmonary toxicity of molybdenum disulphide
after inhalation in mice. Toxicol 485:153428. https:// doi. org/ 10.
1016/j. tox. 2023. 153428
17. Peña NO, Cherukula K, Even B, Ji DK, Razafindrakoto S, Peng
S, Silva AKA, Moyon CM, Hillaireau H, Bianco A, Fattal E,
Alloyeau D, Gazeau F (2023) Resolution of MoS2 nanosheets-
induced pulmonary inflammation driven by nanoscale intra-
cellular transformation and extracellular-vesicle shuttles. Adv
Mater35:2209615.https:// doi. org/ 10. 1002/ adma. 20220 9615
18. Warheit DB, Webb TR, Sayes CM, Colvin VL, Reed KL (2006)
Pulmonary instillation studies with nanoscale TiO2 rods and dots
in rats: toxicity is not dependent upon particle size and surface
area. Toxicol Sci 91:227–236. https:// doi. org/ 10. 1093/ toxsci/
k140
19. Warheit DB, Webb TR, Colvin VL, Reed KL, Sayes CM (2007)
Pulmonary bioassay studies with nanoscale and fine-quartz par-
ticles in rats: toxicity is not dependent upon particle size but on
surface characteristics. Toxicol Sci 95:270–280. https:// doi. org/
10. 1093/ toxsci/ kfl128
20. Ma-Hock L, Burkhardt S, Strauss V, Gamer AO, Wiench K, van
Ravenzwaay B, Landsiedel R (2009) Development of a short-term
inhalation test in the rat using nano-titanium dioxide as a model
substance. Inhal Toxicol 21:102–118. https:// doi. org/ 10. 1080/
08958 37080 23610 57
21. Bailey MR, Ansoborlo E, Guilmette RA, Paquet F (2007) Updat-
ing the ICRP human respiratory tract model. Radiat Prot Dosim-
etry 127:31–34. https:// doi. org/ 10. 1093/ rpd/ ncm249
22. ICRP (2012) Annals of the ICRP: Compendium of dose coef-
ficients based on ICRP publication 60. International Commis-
sion on Radiological Protection. ICRP Publication 119. http://
www. icrp. org/ publi cation. asp? id= ICRP% 20Pub licat ion% 20119.
Accessed 24 Apr 2023
23. Agency for Toxic Substances and Disease Registry (ATSDR)
(2020) Toxicological profile for Molybdenum. U.S. Department
of Health and Human Services, Public Health Service, Atlanta,
GA
24. Fairhall LT, Dunn RC, Sharpless NE, Pritchard EA (1945) The
toxicity of molybdenum. Public Health Bull 293(36):52
25. National Toxicology Program (NTP) (1997) Toxicology and car-
cinogenesis studies of molybdenum trioxide (CAS No. 1313–27-
5) in F344 Rats and B6C3F1 Mice (Inhalation Studies). Natl
Toxicol Program Tech Rep Ser 462:1–269
26. Werner E, Roth P, Heinrichs U, Giussani A, Cantone MC, Zilker
TH, Felgenhauer N, Greim H (2000) Internal biokinetic behaviour
of molybdenum in humans studied with stable isotopes as trac-
ers. Isotopes Environ Health Stud 36:123–132. https:// doi. org/ 10.
1080/ 10256 01000 80329 38
27. Kuraś R, Stępnik M, Domeradzka-Gajda K, Janasik B (2024) The
use of LA-ICP-MS as an auxiliary tool to assess the pulmonary
toxicity of molybdenum(IV) sulfide (MoS2) nano- and micropar-
ticles. IJOMEH 37. https:// doi. org/ 10. 13075/ ijomeh. 1896. 02305
28. Turnlund JR, Keyes WR (2004) Plasma molybdenum reflects
dietary molybdenum intake. J Nutr Biochem 15:90–95. https://
doi. org/ 10. 1016/j. jnutb io. 2003. 10. 003
29. Novotny JA, Turnlund JR (2007) Molybdenum intake influences
molybdenum kinetics in men. J Nutr 137:37–42. https:// doi. org/
10. 1093/ jn/ 137.1. 37
30. Engel RW, Price NO, Miller RF (1967) Copper, manganese,
cobalt, and molybdenum balance in preadolescent girls. J Nutr
92:197–204. https:// doi. org/ 10. 1093/ jn/ 92.2. 197

Toxicol Res.
1 3
31. Schroeder HA, Balassa JJ, Tipton IH (1970) Essential trace metals
in man: molybdenum. J Chronic Dis 23:481–499. https:// doi. org/
10. 1016/ 0021- 9681(70) 90056-1
32. Giussani A (2008) A recycling systemic model for the biokinet-
ics of molybdenum radionuclides. Sci Total Environ 404:44–55.
https:// doi. org/ 10. 1016/j. scito tenv. 2008. 06. 019
33. Bell MC, Diggs BG, Lowrey RS, Wright PL (1964) Comparison
of Mo99 metabolism in swine and cattle as affected by stable
molybdenum. J Nutr 84:367–372. https:// doi. org/ 10. 1093/ jn/ 84.4.
367
34. Appel JH, Li DO, Podlevsky JD, Debate A, Green AA, Wang
QH, Chae J (2016) Low cytotoxicity and genotoxicity of two-
dimensional MoS2 and WS2. ACS Biomater Sci Eng 2:361–367.
https:// doi. org/ 10. 1021/ acsbi omate rials. 5b004 67
35. Chng EL, Sofer Z, Pumera M (2014) MoS2 exhibits stronger toxic-
ity with increased exfoliation. Nanoscale 6:14412–14418. https://
doi. org/ 10. 1039/ C4NR0 4907A
36. Chan PC, Herbert RA, Roycroft JH, Haseman JK, Grumbein SL,
Miller RA, Chou BJ (1998) Lung tumor induction by inhalation
exposure to molybdenum trioxide in rats and mice. Toxicol Sci
45:58–65. https:// doi. org/ 10. 1006/ toxs. 1998. 2497
37. Huber EA, Cerreta JM (2022) Mechanisms of cell injury induced
by inhaled molybdenum trioxide nanoparticles in Golden Syrian
Hamsters. Exp Biol Med 247:2067–2080. https:// doi. org/ 10. 1177/
15353 70222 11040 33
38. Hussain SM, Hess KL, Gearhart JM, Geiss KT, Schlager JJ (2005)
Invitro toxicity of nanoparticles in BRL 3A rat liver cells. Toxicol
InVitro 19:975–983. https:// doi. org/ 10. 1016/j. tiv. 2005. 06. 034
39. Braydich-Stolle L, Hussain S, Schlager JJ, Hofmann M-C (2005)
Invitro cytotoxicity of nanoparticles in mammalian germline stem
cells. Toxicol Sci 88:412–419. https:// doi. org/ 10. 1093/ toxsci/
kfi256
40. Wang X, Oberleas D, Yang MT, Yang SP (1992) Molybdenum
requirement of female rats. J Nutr 122:1036–1041. https:// doi. org/
10. 1093/ jn/ 122.4. 1036
41. Yang MT, Yang SP (1989) Effect of molybdenum supplementa-
tion on hepatic trace elements and enzymes of female rats. J Nutr
119:221–227. https:// doi. org/ 10. 1093/ jn/ 119.2. 221
42. Arrington LR, Davis GK (1953) Molybdenum toxicity in the rab-
bit. J Nutr 51:295–304. https:// doi. org/ 10. 1093/ jn/ 51.2. 295
43. Pandey R, Kumar R, Singh SP, Srivastava SP (2002) Molybdenum
in rat tissue. Hum Exp Toxicol 21:33–35. https:// doi. org/ 10. 1191/
09603 27102 ht203 oa
44. Kusum RR, Verma PK, Pankaj NK, Kant V, Kumar J, Srivastava
AK (2010) Haematolohical profile of subacute oral toxicity of
molybdenum and ameliorative efficacy of copper salt in goats.
Toxicol Int 17:82–85. https:// doi. org/ 10. 4103/ 0971- 6580. 72676
45. Lyubimov AV, Smith JA, Rousselle SD, Mercieca MD, Tomasze-
wski JE, Smith AC, Levine BS (2004) The effects of tetrathio-
molybdate (TTM, NSC-714598) and copper supplementation on
fertility and early embryonic development in rats. Reprod Toxicol
19:223–233. https:// doi. org/ 10. 1016/j. repro tox. 2004. 07. 006
46. Asadi F, Mohseni M, Dadashi Noshahr K, Soleymani FH, Jalil-
vand A, Heidari A (2017) Effect of molybdenum nanoparticles
on blood cells, liver enzymes, and sexual hormones in male
rats. Biol Trace Elem Res 175:50–56. https:// doi. org/ 10. 1007/
s12011- 016- 0765-5