Label-free detection and quantification of ultrafine particulate matter in lung and heart of mouse and evaluation of tissue injury

Abstract

While it is known that air borne ultrafine particulate matter (PM) may pass through the pulmonary circulation of blood at the alveolar level between lung and heart and cross the air-blood barrier, the mechanism and effects are not completely clear. In this study the imaging method fluorescence lifetime imaging microscopy is adopted for visualization with high spatial resolution and quantification of ultrafine PM particles in mouse lung and heart tissues. The results showed that the median numbers of particles in lung of mice exposed to ultrafine particulate matter of diameter less than 2.5 µm was about 2.0 times more than that in the filtered air (FA)-treated mice, and about 1.3 times more in heart of ultrafine PM-treated mice than in FA-treated mice. Interestingly, ultrafine PM particles were more abundant in heart than lung, likely due to how ultrafine PM particles are cleared by phagocytosis and transport via circulation from lungs. Moreover, heart tissues showed inflammation and amyloid deposition. The component analysis of concentrated airborne ultrafine PM particles suggested traffic exhausts and industrial emissions as predominant sources. Our results suggest association of ultrafine PM exposure to chronic lung and heart tissue injuries. The current study supports the contention that industrial air pollution is one of the causative factors for rising levels of chronic pulmonary and cardiac diseases.

Full text

Hameedetal. Particle and Fibre Toxicology (2022) 19:51
https://doi.org/10.1186/s12989-022-00493-8
RESEARCH
Label-free detection andquantication
ofultrane particulate matter inlung andheart
ofmouse andevaluation oftissue injury
Saira Hameed1,2*, Kun Pan3, Wenhua Su4, Miles Trupp5, Lan Mi4 and Jinzhuo Zhao3,6*
Abstract
While it is known that air borne ultrafine particulate matter (PM) may pass through the pulmonary circulation of blood
at the alveolar level between lung and heart and cross the air-blood barrier, the mechanism and effects are not com-
pletely clear. In this study the imaging method fluorescence lifetime imaging microscopy is adopted for visualization
with high spatial resolution and quantification of ultrafine PM particles in mouse lung and heart tissues. The results
showed that the median numbers of particles in lung of mice exposed to ultrafine particulate matter of diameter
less than 2.5 µm was about 2.0 times more than that in the filtered air (FA)-treated mice, and about 1.3 times more in
heart of ultrafine PM-treated mice than in FA-treated mice. Interestingly, ultrafine PM particles were more abundant in
heart than lung, likely due to how ultrafine PM particles are cleared by phagocytosis and transport via circulation from
lungs. Moreover, heart tissues showed inflammation and amyloid deposition. The component analysis of concen-
trated airborne ultrafine PM particles suggested traffic exhausts and industrial emissions as predominant sources. Our
results suggest association of ultrafine PM exposure to chronic lung and heart tissue injuries. The current study sup-
ports the contention that industrial air pollution is one of the causative factors for rising levels of chronic pulmonary
and cardiac diseases.
Keywords: Fluorescence lifetime imaging microscopy, Scanning electron microscopy, Ultrafine PM, Heart, Lung,
Injury
© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, 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:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco
mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.
Introduction
Air borne ultrafine particulate matter less than 2.5μm
in size (PM particles) is the leading environmental risk
factor that impairs metabolic homeostasis [1], and is
associated with the morbidity and mortality of cardiopul-
monary diseases [2].
Air borne ultrafine PM particles directly enter into the
lungs by inhalation and are transported into extrapul-
monary organs such as kidney, and liver, [3], including
brain [4]. e quantification of ultrafine PM particles can
contribute to elucidation of the effects of the PM parti-
cles on different tissues and organs. Due to nonspecific
and uncommon respiratory symptoms, clinical discover-
ies depend on evaluation of the involvement of additional
organs [5]. In recent studies magnetic iron oxide (Fe3O4)
known as magnetite has been detected in neurodegen-
erative tissues with correlation between the amount of
brain magnetite and the incidence of Alzheimer’s disease
[6, 7]. Our previous study has shown that ambient PM
particles reach mouse brain due to permeability of the
blood brain barrier, resulting in neuroinflammation, tan-
gles and plaque formation similar to Alzheimer’s disease
[4]. Carbon black particles have been detected in human
placental tissue crossing the blood placental barrier [8],
Open Access
*Correspondence: saira.hameed@umu.se; jinzhuozhao@fudan.edu.cn
1 Department of Chemistry, Umeå University, 901 87 Umeå, Sweden
3 Department of Environmental Health, School of Public Health
and the Key Laboratory of Public Health Safety, Ministry of Education,
Fudan University, 130 Dong’an Road, Box 249, Shanghai 200032, China
Full list of author information is available at the end of the article
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 2 of 12
Hameedetal. Particle and Fibre Toxicology (2022) 19:51
and magnetite has been detected in human brain [9],
indicating that ultrafine PM particles can enter into dif-
ferent organ systems [10].
However, it is difficult to detect the amount of PM
particles in different tissues because of the variable size
and complex chemical nature. Consequently, there are
few studies focused on detecting the amount of ultrafine
PM particles in different tissues to determine the rates
of deposition. It is generally accepted that there is a
large amount of black carbon in the air borne particu-
late matter. ere are several reports of carbon particles
with high two-photon absorption cross-section, ranging
between 39,000 and 48,000 GM (Goeppert-Mayer unit,
with 1 GM = 10–50 cm4s/photon) [11–13]. In contrast,
the endogenous fluorescent probe, NADH was reported
to have a much lower two-photon absorption cross-sec-
tion, around 340 GM [14]. us, black carbon can be an
efficient two-photon fluorescent indicator of ultrafine PM
particles with the femtosecond pulsed laser excitation. In
2019, Bové et al. performed fluorescence spectroscopy,
fluorescence lifetime analysis, and two-photon excitation
imaging on human placental tissues [8], which are simi-
lar to methods we used in this paper. ey confirmed the
carbonaceous nature of the identified black carbon par-
ticles and external contamination of the tissues could be
excluded. And they found a positive association between
the placental black carbon load and the mothers’ residen-
tial black carbon exposure. erefore, the two-photon
excitation fluorescence lifetime imaging method is reli-
able for measuring the carbon particles in tissues.
In this study, sixteen mice were divided into two groups
and were exposed to concentrated ultrafine PM parti-
cles (PM, dirty air), and filtered air (FA, control), using
the “Shanghai Meteorological and Environmental Ani-
mal Exposure System (Shanghai-METAS)”, located in
the School of Public Health at Fudan University at Xujia-
hui District in Shanghai. We used fieldemission scan-
ningelectronmicroscopy(FE-SEM) to visualize ultrafine
PM particles, and fluorescence lifetime imaging micros-
copy (FLIM) to quantify the PM particles in lung and
heart tissues.
Knowing the precise deposition process and the trans-
port mechanism of inhalable particles is crucial for health
risk assessment and evaluation of target organ injury
[15]. e deposition of ultrafine PM particles in different
tissues has adverse effects on the target organs includ-
ing tissue injury and inflammation [16] that are causally
linked to cardiopulmonary diseases such as atheroscle-
rosis, coronary heart disease and chronic obstructive
pulmonary disease. Under the influence of pathological
states healthy proteins lose their normal structure and
function and aggregate in tissue and organs in the form
of amyloid deposits. Amyloids can accumulate not only
in brain but also in different tissues and body organs that
result in clinical syndromes [17]. Studies have shown that
exposure to ultrafine PM particles led to influx of inflam-
matory cytokines in serum, heart, liver and lung of mice
[18–21]. Our recent study has shown that exposure to
ambient PM particles resulted in inflammation, depo-
sition of Aβ amyloids and formation of neurofibrillary
tangles and plaques in mouse brain [4]. Amyloids can
accumulate not only in brain but also in different tissues
and body organs that result in clinical syndromes [17].
A recent study has shown multiorgan amyloidosis in a
coal miner [22]. In a Swedish study on amyloidosis, all
of the 33 cases had simultaneous pulmonary and cardiac
involvements [5]. A previous report has suggested that
amyloid protein could be produced in tissues and might
be derived from precursors in the blood circulation [23].
Results
Detection andquantication ofultrane PM particles
inlung andheart tissues byuorescence lifetime imaging
microscopy (FLIM)
Sixteen mice were divided equally into filtered air (FA)
(control) (Fig. 1A), and 2X concentrated air (Fig. 1B)
groups, and given the FA or concentrated ultrafine PM
exposure (dirty air). e mean concentrations of ultrafine
PM in dirty air and FA chambers during the exposure
were 71.20 ± 45.01 and 11.76 ± 4.40 μg/m3, respectively.
e mean outdoor ultrafine PM concentration during
the exposure was 43.00 ± 6.05 μg/m3. We hypothesize
that during respiration ultrafine PM particles from air
pollution entered into lungs and were carried to heart
via blood circulation. Figure 1C presents a graphical
illustration showing the movement of ultrafine PM par-
ticles inside the respiratory and blood circulation sys-
tems. FLIM microscopy enabled label-free detection and
quantification of ultrafine PM particles on lung and heart
tissues of mouse. Fluorescence spectra of ultrafine PM
particles in PBS and tissues showed large overlap between
450 and 550nm (Fig.1D). us, it is hard to distinguish
ultrafine PM from the tissues. However, the fluorescence
lifetime of ultrafine PM and tissues were quite different,
the lifetime of ultrafine PM was much shorter than that
of tissues (Fig.1E). Based on the different lifetime val-
ues, the green dots denote PM particles in tissues and
the red fluorescence reveals the tissue structure, and the
superimposed images showed clear distribution of PM
particles (Fig. 1F–Q). During respiration, the ultrafine
PM particles from dirty air enter directly into lungs and
alveoli lined with blood capillaries. Lung tissues from the
filtered air group showed little deposition of ultrafine PM
particles (Fig.1H). Lung tissues from the dirty air expo-
sure group showed large numbers of ultrafine PM par-
ticles (Fig.1K). e blood capillaries absorb oxygen and
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 3 of 12
Hameedetal. Particle and Fibre Toxicology (2022) 19:51
ultrafine PM particles from lung during respiration and
transport to heart (Fig.1N). Heart absorbs and collects
higher amounts of ultrafine PM particles from dirty air
(Fig.1Q). e particle densities in lung and heart of mice
were estimated (Fig.1R) accordingly. e results showed
that the median numbers of particles in lung of ultrafine
PM-treated mice (dirty air group) are 3.4 times more
than that in the FA-treated mice, and in heart of ultrafine
PM-treated mice, 1.3 times more than in FA-treated
mice. e dispersion of data in lung tissues is relatively
large across the animals.
Exposure toconcentrated ultrane PM particles caused
lung damage
Surface evaluation of tissues by field emission scanning
electron microscopy (FE-SEM) showed that lung tis-
sue sections from filtered air (control) group showed no
signs of abnormality (Fig.2A), whereas ultrafine PM par-
ticles (Fig. 2B–C), macrophages (Fig. 2D–F), and amy-
loid deposits (Fig. 2G–I), and fibrosis (Fig. 2J–L) were
detected in lung tissue sections from the dirty air expo-
sure group. e effects of ultrafine PM particles on lung
tissue sections were determined by histopathological
evaluations. Congo red (azo dye) was used as a classical
qualitative method to stain and detect amyloid buildup in
tissue sections. Congo red staining of lung tissues from
filtered air showed no signs of abnormality (Fig. 2M),
whereas lung tissue from dirty air showed salmon red
amyloid deposits under light microscopy (Fig.2N). More-
over, immunohistochemistry with Aβ antibody was used
to enhance sensitivity of amyloid detection that found no
signs of abnormality in lung tissues from filtered air (con-
trol) group (Fig.2O), whereas lung tissue from dirty air
showed immunoreactive areas with dark brown amyloid
deposits (Fig.2P). e AIF-1/IBA-1 antibody was used as
a macrophage marker in tissue injury. AIF-1 is cytosolic
actin binding protein allograft inflammatory factor also
known as IBA-1 or calcium binding adapter molecule
1s. Immunohistochemistry with IBA-1 antibody found
no signs of abnormality in lung tissues from filtered air
(Fig. 2Q), whereas macrophages were detected in lung
tissues from dirty air exposure group (Fig.2R).
Exposure toconcentrated ultrane PM particles caused
heart tissue damage
Surface evaluation of tissues by FE-SEM showed that
heart tissue sections from the filtered air (control) group
showed no signs of abnormality (Fig. 3A), whereas
Fig. 1 Mice were kept in exposure chambers for six months. Graphical illustrations A) Mouse in filtered air chamber. B) Mouse in dirty air
chamber. C) Movement of ultrafine PM particles into the respiratory track and accumulation in lungs and heart. D) Normalized fluorescence
spectra of ultrafine PM in PBS (green) and auto-fluorescence of tissues (red). E) Typical lifetime decay curves of ultrafine PM in tissues (green) and
auto-fluorescence of tissues (red). F) Auto-fluorescent image of lung tissue from filtered air. G) Ultrafine PM particles from filtered air. H) Fluorescent
deposition pattern of ultrafine PM particles in lung tissue from filtered air. I) Auto-fluorescent image of lung tissue from dirty air. J) Ultrafine PM
particles from dirty air. K) Fluorescent deposition pattern of ultrafine PM particles in lung tissue from dirty air. L) Auto-fluorescent image of heart
tissue from filtered air. M) Green dots are the ultrafine PM particles. N) Fluorescent deposition pattern of ultrafine PM particles in heart tissue from
filtered air. O) Auto-fluorescent image of heart tissue from dirty air. P) Green dots are the ultrafine PM particles. Q) Fluorescent deposition pattern
of ultrafine PM particles in heart tissue from dirty air. Scale bar: 20 µm. The resolution of FLIM images is approximately 250 nm. R) The estimated
particle density in lung and heart of mice
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 4 of 12
Hameedetal. Particle and Fibre Toxicology (2022) 19:51
ultrafine PM particles (Fig.3B, C), amyloid (Fig.3D, E),
and macrophages (Fig.3F) were detected in heart tissue
sections from dirty air exposure group. Moreover, the
effects of ultrafine PM particles on heart tissue sections
were determined by histopathological evaluations. Congo
red staining of heart tissues from filtered air (control)
group showed no signs of abnormality (Fig.3G), whereas
heart tissue sections from dirty air showed amyloid depo-
sition (Fig.3H, I). Moreover, immunohistochemistry with
amyloid marker Aβ antibody found no signs of abnor-
mality in heart tissues from filtered air (control) group
(Fig. 3J), whereas heart tissue from dirty air showed
amyloid deposition (Fig.3K, L). Immunohistochemistry
with macrophage marker IBA-1 antibody found no signs
of abnormality in heart tissues from filtered air (control)
group (Fig.3M), whereas macrophages were detected in
heart tissues from the dirty air exposure group (Fig.3N,
O).
The organic components ofultrane PM particles indirty
air andFA chambers
e organic molecular formulae in dirty air and FA were
detected by electrospray ionization mass spectrometry
(ESI–MS). Electro-spray ionization (ESI) is a powerful
technique for analysis of molecules at different polari-
ties in a complex sample mixture [24]. In positive ion
mode (ESI +) the spraying nozzle is kept at positive
potential and protonation of the analyte occurs. While
in the negative ion mode (ESI-) the spraying nozzle is
kept at negative potential and deprotonation of the ana-
lyte occurs.
e Fig.4A, B show the mass spectra of samples and
the ratios of different element compositions in FA and
dirty air groups. e numbers of organic molecular for-
mulae in dirty air and FA are shown in Tables 1 and 2.
e results indicated that dirty air group showed more
organic substances, characterized by CHON, CHNaO,
CHNNa, CHONS, CHNNaO, CHO, CHOS and CHONS
when compared with FA group.
Tables1 and 2 enumber of organic molecular for-
mulae in air samples at positive and negative modes
(ESI + and ESI-) in (A) filtered air (FA), and (B) dirty
air. An Agilent 1200 series HPLC with a C18 column
(SB-C18, 3.0 × 100 mm, 1.8 μm) was used for chro-
matographic separation. At ESI + , a: C6H11NO; b:
C6H13NO2; c: C16H22O4; d: C22H43NO; e: C22H42O4;
Fig. 2 Field emission scanning electron microscopy (FE-SEM) of lung tissues. A) Lung tissues from filtered air showed no signs of abnormality,
whereas B, C lung tissue sections from dirty air exposure group showed ultrafine PM particles, D–F macrophages. High magnification FE-SEM image
of the macrophages showed cell surface, knob like microvilli, and filopodia that extended outwards from periphery of the cells (F). Lung tissue
sections from dirty air exposure group showed amyloid deposits (G–I), and fibrosis (J–L) Congo red staining, M lung tissues from filtered air (control)
showed no signs of abnormality, whereas N lung tissue from the dirty air showed amyloid deposition. Immunohistochemistry with amyloid marker
Aβ antibody (1:500), O from lung tissue sections from filtered air (control) group showed no signs of abnormality, whereas P lung tissue from dirty
air showed immunoreactive areas with dark brown amyloid deposits. Immunohistochemistry with macrophage marker IBA-1 antibody (1:100), Q
lung tissues from filtered air showed no signs of abnormality, whereas R lung tissue from dirty air showed macrophages. Magnification (A, B, D, G,
J, and K) 20 k, scale bar: 2 µm. Magnification (E, H, I, and L) 50 k, Scale bar: 1 µm. Magnification (C and F) 100 k, Scale bar: 500 nm. Magnification (M
and N) 20X, Scale bar: 500 µm. Magnification (O, P, Q, and R) 40X, Scale bar 500 µm
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 5 of 12
Hameedetal. Particle and Fibre Toxicology (2022) 19:51
Fig. 3 Field emission scanning electron microscopy (FE-SEM) of heart tissues. A Heart tissues from filtered air (control) group showed no
signs of abnormality, whereas B, C heart tissue from dirty air exposure group showed ultrafine PM particles, D, E amyloid, and F macrophages.
Histopathological evaluation by Congo red staining, G heart tissues from filtered air (control) group showed no signs of abnormality, whereas H, I
heart tissue from the dirty air showed amyloid deposition. Immunohistochemistry with amyloid marker Aβ antibody (1:500), J from heart of filtered
air (control) group showed no signs of abnormality, whereas) heart tissue from dirty air showed immunoreactive areas with dark brown amyloid
deposition (K and L). Immunohistochemistry with macrophage marker IBA-1 antibody (1:100), M heart tissues from filtered air (control) group
showed no signs of abnormality, whereas (N and O) heart tissue from dirty air showed macrophages. Magnification (A to D) 10 k, scale bar: 5 μm.
Magnification (E) 20 k, scale bar 2 μm. Magnification (F) 100 k, scale bar: 500 nm. Magnification (G, H, I, J, L, M, N, and O) 40X, Scale bar: 500 µm.
Magnification (K) 20X, Scale bar: 500 µm
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 6 of 12
Hameedetal. Particle and Fibre Toxicology (2022) 19:51
At ESI-, A: C3H6O3; B: C16H32O2; C: C18H36O2; D:
C20H40O6; E: C22H44O6; F: C24H44N4O4; G: C30H55N5O5;
H: C35H70N2O10.
The metal components ofultrane PM particles indirty air
andFA chambers
e samples of ultrafine PM particles in dirty air and
FA chambers were collected weekly during the expo-
sure. Because of the hazards of heavy metals in air borne
particulate matter, we measured 8 heavy metals includ-
ing Zn, Bi, Cd, Ni, Fe, Mn, Cr and Cu by ICP-MS in this
study. Additional file1: Fig. S1 shows the fractograms
of the 8 metals. As shown in Additional file2: TableS1,
the concentrations of these 8 elemental components in
dirty air were about five times more than those in the FA
chamber, demonstrating that the METAS we used to give
the mice dirty air and FA exposure significantly concen-
trated the ambient ultrafine PM particles without chang-
ing its components. Of these 8 metals, Fe, Zn, Cd and Mn
were the main components.
Discussion
Numerous studies have indicated the association
between air borne particulate matter and diseases in
humans and experimental animals. is includes cardio-
pulmonary diseases, diabetes, cerebrovascular diseases
and reproductive toxicity [25, 26]. Inhalation of ultrafine
PM particles have adverse health effects. Exposure to
PM2.5 over shorter periods of time reduced lung function
in children [27].
As the main air pollutant, the association between
ultrafine particulate matter (PM particles) and health is
Fig. 4 Mass spectrometry anslyses of ultrafine PM particles
Table 1 The number of molecular formulae in FA and dirty air at
positive ion mode (ESI +)
ElementCombination FA (Mean ± SD) Dirty air (PM)
(Mean ± SD)
CHO 83.1 ± 4.6 103.8 ± 5.3
CHN 11.3 ± 1.1 16.4 ± 2.4
CHOS 1.8 ± 0.6 3.2 ± 0.8
CHNS 1.1 ± 0.4 1.2 ± 0.1
CHON 135.7 ± 11.7 236.4 ± 13.5
CHNaO 14.2 ± 2.1 20.6 ± 3.3
CHNNa 0.5 ± 0.2 0.8 ± 0.5
CHONS 3.2 ± 0.8 8.3 ± 1.2
CHNNaO 35.2 ± 3.6 33.7 ± 4.7
CHONaS 1.2 ± 0.5 0 ± 0
CHNNaS 1.3 ± 0.6 0 ± 0
CHONSNa 0.8 ± 0.2 1.3 ± 0.3
Total 288.7 ± 17.3 426.4 ± 20.7
Table 2 The number of molecular formulae in FA and dirty air at
negative ion mode (ESI-)
ElementCombination FA (Mean ± SD) Dirty air (PM)
(Mean ± SD)
CHO 89.1 ± 6.3 138.8 ± 8.4
CHS 0 ± 0 2.1 ± 0.7
CHN 0 ± 0 0 ± 0
CHOS 25.6 ± 3.3 28.9 ± 3.6
CHNS 2.2 ± 0.7 2.3 ± 0.4
CHON 71.2 ± 9.2 83.5 ± 6.9
CHONS 9.4 ± 1.5 9.3 ± 2.1
Total 196.8 ± 10.8 265.1 ± 13.5
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 7 of 12
Hameedetal. Particle and Fibre Toxicology (2022) 19:51
of great concern. is is particularly problematic in heav-
ily industrialized and populated areas, with China often
identified as a region with the highest ultrafine PM con-
centration in the world [28]. In 2020, the annual average
concentration of ultrafine PM in China was 36μg/m3,
lower than that in 2017 (43μg/m3), but still higher than
both Chinese air quality standard (35 μg/m3) and U.S
EPA standard (12μg/m3) [28].
In this study, the mice were raised in groups and were
allowed to eat food and drink water freely inside whole
body exposure chambers METAS [29], for filtered air
(control) and dirty air, between 2018 and 2019. e con-
centrations in dirty air and FA chambers were 71.20μg/
m3 and 11.76 μg/m3, respectively. e mice were sac-
rificed after six months of exposure in 2019, and their
organs were extracted for analyses. Our recent study
focused on brain of those mice [4]. In the current study,
lung and heart tissues from the same group of mice were
used for analyses.
Airborne ultrafine PM particles enter into lung tissue
by passive transport along surface liquids, and phagocy-
tosis within alveolar macrophages [30]. Although sev-
eral lines of evidence support the theory that particles
translocate from lung into the circulation, then enter into
heart, liver and kidney, quantifying the particles in these
organs took precedence in assessing particle toxicologi-
cal effects. Previous studies have used second harmonic
generation (SHG) to detect black carbon in placenta [8]
and high-resolution transmission electron microscopy
(HRTEM) to detect magnetite in brain [9], but there are
few studies to quantify the amounts of ultrafine PM par-
ticles in tissues. Itisnoteworthythat airborne ultrafine
PM particles was a mixture and not a single chemical,
which made the detection more difficult.
In this study, FLIM enabled label free detection and
quantification of ultrafine PM particles with high resolu-
tion on lung and heart tissues, which can provide broad
insights into the distribution of particles entering into
tissues. Although there is the limitation that FLIM can’t
completely separate the debris of cellular and tissue
injury/inflammation from ultrafine particles, compared
with transmission electron microscopy (TEM), FLIM
has several advantages: sample preparation is simple and
does not need special treatment; the imaging range of
FLIM is larger than TEM, which is convenient for large
samples statistics. In this study ultrafine PM particles
were also detected in the filtered air group because filters
do not have 100 percent efficiency, and extremely small
particles can pass through them. Our current study found
that the amounts of ultrafine PM particles was higher in
dirty air (PM) group than that in FA group. Intriguingly,
ultrafine PM particles were more in heart than lung,
probably because lung has air sacs and the PM particles
are cleared by transportation over fluid and phagocyto-
sis [30], whereas heart is the muscular organ that pumps
oxygenated blood along with dissolved PM particles from
lungs. To the best of our knowledge it is the first report
on label-free detection and quantification of ultrafine
PM particles in lung and heart tissues of mouse, and the
mechanism of particle distribution is unknown. Previ-
ous studies have shown that PM particles enter in blood
circulation just after exposure [31]. As heart is the main
pumping organ therefore it is hypothesized that the pow-
erful mechanical force of blood passing through heart
may contribute to adsorption of ultrafine PM particles
into heart muscles, which may act as a sink for inhaled
particulate matter. However, it should be taken into con-
sideration that as there is no known evidence from litera-
ture to support the hypothesis therefore future studies are
needed to give a more definitive statement how ultrafine
PM particles enter into heart tissue. As the number of
ultrafine particles were higher in heart as compared to
lung, therefore, it may have more adverse effects on heart
as compared to lung of mouse. Interestingly, a recent
report on certain cardiopulmonary ailments with patho-
logical evaluations of lung and heart tissues of 76 patients
by Mayo clinic physicians have shown that cardiac mani-
festations might occur earlier and are more frequent and
severe than pulmonary disease, and the survival rate of
patients was found to be directly related to the degree of
cardiac involvements [5].
Scanning electron microscopy provides a unique
means for examining the dynamic aspects of inflamma-
tory response. FE-SEM enabled detection of ultrafine
PM particles (Fig. 2B, C), macrophages (Fig. 2D–F),
amyloid deposition (Fig.2G–I), and fibrosis (Fig.2J–L)
on lung tissues from dirty air exposure group. Under
the influence of pathological states healthy proteins lose
their normal structure and function and aggregate in
tissue and organs in the form of amyloid deposits that
can be detected by the classical gold standard congo
red (azo dye) staining that gives salmon red color under
light microscopy [17]. Moreover, sensitivity of detec-
tion was enhanced by immunohistochemical staining
with amyloid marker Aβ antibody that detected amy-
loid deposition (Fig. 2N, P), and macrophage marker
IBA-1 antibody that detected macrophages at sites of
tissue injury (Fig.2R). Previous studies have shown that
ultrafine PM exposure was associated with the release
of inflammatory cytokines and inflammatory cell infil-
tration. A recent study has shown that activation of the
NLRP3/ caspase-1 signaling pathway by ultrafine PM
particles induced pulmonary inflammation [32]. e
mechanism of inflammation in pulmonary diseases has
been reviewed recently [33]. Inflammatory response has
been certified as a vital mechanism linking particulate
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 8 of 12
Hameedetal. Particle and Fibre Toxicology (2022) 19:51
matter and adverse effects, and our results directly veri-
fied the occurrence of inflammation. Chronic inflamma-
tion and amyloidosis directly target lungs, severely effect
alveolar structures, hamper gaseous exchange and result
in serious respiratory impairment including asthma
and other pulmonary diseases [33]. Pulmonary inflam-
mation is a risk factor for cardiovascular diseases [34].
In this study surface evaluations by FE-SEM detected
ultrafine PM particles (Fig.3B, C), amyloid (Fig.3D, E),
and macrophages (Fig.3F) in heart tissues from dirty air
exposure group. e observations were supported by his-
tochemical evaluations that also detected amyloid depos-
its (Fig.3H, L) in the lung tissues. Heart is composed of
heterogenous population of cells [35]. Macrophages are
found at the site of fibrosis, that activate reparative and
maladaptive processes that lead to organ dysfunction in
many different diseases [36, 37].
Inflammation is common in coronary heart diseases
and atherosclerosis but the mechanism is not known.
Moreover, amyloids can infiltrate heart tissues and result
in cardiac amyloidosis. Cardiac amyloidosis results in
myocardial thickening and dysfunction [38]. e results
from the current study suggests that air pollution is one
of the causative factors for rising levels of chronic pul-
monary and cardiac diseases [39, 40] and that particulate
induced amyloidosis is a potential mechanism for tar-
geted therapeutic development.
A previous human study found metal components (e.g.
Al, Fe, Ca, Ni, Cu, Pb, V and Zn) of PM2.5 significantly
decreased whole blood coagulation time in healthy sub-
jects [41]. PM2.5 rich in metal components such as nickel
(Ni) has been linked to adverse cardiopulmonary effects
[42, 43]. Moreover, other studies also found that metal
constituents such as Zn, Cd, Mn, Cu [44], Cd, Pb [45]
in PM2.5 were associated with a variety of adverse health
effects. In this study, we determined eight main (Zn, Bi,
Cd, Ni, Fe, Mn, Cr and Cu) metals in ultrafine PM par-
ticles, in which the concentration of Fe, Zn, Cd and Mn
are higher than other metals. e variations in molecu-
lar compositions of ultrafine PM particles in dirty air and
FA were also evaluated in this study. Sulphur and nitro-
gen containing organics have received the most attention
because they can be used to reveal the pollutant sources,
and aging mechanisms. CHON species that can form via
gas-phase nitrate radical initiated oxidation are also sig-
nificant components of secondary organic aerosol [46]. A
previous study indicated that CHN species were a signifi-
cant contributor to the organic matter at the Beijing site,
and high levels of CHN species and their CH2 homolo-
gous series were identified as quinoline and benzo [f]
quinoline compounds, which may have considerable
health implications [47]. erefore, chemical characteris-
tics of organic aerosols can provide a clue for exploring
the adverse effects of ambient particulate matter. More-
over, the components of air pollution are associated to
increased hospital visits for pulmonary and cardiac ail-
ments [48].
We want to emphasize that ultrafine PM particles are
complex mixture of chemicals of variable sizes that range
from course to ultrafine [49]. e nature of PM particles
may change as they enter inside the living organisms.
PM particles cause the activation of oxidative stress and
generation of reactive oxygen species [49]. Moreover, it
is very important to consider soluble components of PM
particles during interpretation of the results of this study.
e ultrafine PM particles detected in lung and heart tis-
sues could be the insoluble fraction of ultrafine particles
that once inside the body may be coated by biomolecules
such and form protein corona and soluble ions of metal
complexes. We also tried scanning electron microscopy
/ energy dispersive X-Ray spectroscopy (SEM/EDS) to
perform elemental/ component analysis of ultrafine PM
particles in lung and heart tissue sections, but it was not
successful because the PM particles were scattered over
tissue sections as tiny particles in nm size range, and no
big clusters were found.
Moreover, as mice were exposed to ultrafine PM par-
ticles with concentrations about two times higher than
ambient air in the Shanghai metropolitan areas, and the
control group was exposed to air that passed through
HEPA filters to remove most of the ultrafine particles.
While, the gaseous contents in both chambers were
same. erefore, it is suggested that the tissue damages in
the lung and heart tissues observed in dirty air exposure
group were seeded by ultrafine PM particles, which were
lower in filtered air (FA) control group.
Conclusion
In this study fluorescence lifetime imaging microscopy
(FLIM) enabled label-free detection and quantification of
ultrafine PM particles on lung and heart tissues of mouse.
Field emission scanning electron microscopy (FE-SEM)
presented visuals of ultrafine particles on tissues, and
histological insights on toxicological effects of ultrafine
PM exposure to chronic lung and heart injuries were pre-
sented. It suggested that rising levels of air pollution are
among the causative factors associated with increased
cardiopulmonary disorders worldwide.
Materials andmethods
Animal management
Six weeks old Mus musculus (C57BL/6 male mice) were
purchased from Shanghai Jiesijie Laboratory Animal Co.,
Ltd (Shanghai, China). ey were housed in a pathogen-
free animal facility at Fudan University, at constant tem-
perature (21°C ± 1°C) and humidity (60%) on a day and
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 9 of 12
Hameedetal. Particle and Fibre Toxicology (2022) 19:51
night cycle of 12h each, and were maintained on normal
chow diet. e procedures were approved by the Insti-
tutional Research Committees of the Fudan University,
Shanghai, China, and the methods were performed in
accordance with the set regulations and guidelines.
Exposure ofconcentrated ultrane PM particles tomice
Sixteen mice were divided into two groups and were
exposed to concentrated ultrafine PM (PM, dirty air), and
filtered air (FA, control), in whole body exposure cham-
bers, using the “Shanghai Meteorological and Environ-
mental Animal Exposure System (Shanghai-METAS)”,
located in the School of Public Health at Fudan Univer-
sity at Xujiahui District in Shanghai. Ambient air passed
through HEPA filters to get filtered air [50]. In the dirty
air exposure chambers Shanghai-METAS, only the par-
ticles with diameters less than 2.5µm entered into the
chamber. e versatile aerosol concentration enrichment
system (VACES) was used for enrichment of ultrafine PM
particles in the dirty air exposure chamber. We have used
the exposure system to perform several studies [29, 51].
In this study, the exposure lasted for 8h per day, 6days
per week, in a total of 24 weeks. e mice were freely
allowed to eat food and drink water in whole body expo-
sure chambers.
The real‑time concentration ofultrane PM particles
e real-time concentrations of ultrafine PM particles
from exposure chamber and control chamber were con-
tinuously measured by TEOM (ermo Fisher Scientific,
Waltham, MA), and ultrafine PM particles were sampled
on Teflon filters (Gelman Teflon, 37mm, 0.2 mm pore)
for subsequent measuring the accurate concentrations
and the components such as constituents of polycyclic
aromatic hydrocarbons (PAHs) and trace metals.
Metal concentration andcomponent analysis ofultrane
PM particles
e Teflon filter (Gelman Teflon, 37mm, 0.2mm pore)
with ultrafine PM particles was cut and divided into two
parts. e filters were treated with 10mL of 60% high-
purity nitric acid (HNO3) and 3 mL of 37% perchlo-
ric acid (HClO4). e solutions containing filters were
heated in microwave for 1h. ey were then stored at
4°C until analysis. e metal concentrations of ultrafine
PM particles were determined by inductively coupled
plasma mass spectrometry(ICP-MS).
Organic combination analysis ofultrane PM particles
indirty air andFA
e filters with ultrafine PM particles were cut into
pieces using scissors and extracted in 30 mL methanol
under ultrasonication for 30min. e extracted solution
was filtered (polytetrafluoroethylene membrane) through
a syringe with 0.22μm pore size. After concentration, the
final volume was 1ml prior to HPLC–DAD-Q-TOF–MS
analysis. An Agilent 1200 series HPLC with a C18 col-
umn (SB-C18, 3.0 × 100mm, 1.8μm) was used for chro-
matographic separation with an injection volume of 2μL.
e flow rate was set to 0.4 mL/min and the gradient
separation was conducted with 0.1% formic acid in water
(A) and methanol (B). e concentration of B was 5% for
the first 0.5min increased to 95% from 0.5 to 27min, and
then decreased back to 5% from 27 to 27.1min. e iden-
tification of BrC was determined with an Agilent 6520
Q-TOF–MS and an Agilent G1315D diode array detec-
tor (DAD). UV–Vis absorption was measured using the
DAD detector over the wavelength range of 190–600nm.
e TOF–MS was equipped with electrospray ionization
(ESI), operated in both positive and negative ion modes.
e drying gas flow rate was 7 L/min, and the temper-
ature and flow rate of sheath gas were 350°C and 11L/
min, respectively.
is study presents the molecular composition of
ultrafine PM particles according to the protocol pre-
sented in Daellenbach etal. [52]. For the whole compo-
nent analyses, the allowed range or the atomic number
limit of carbon, hydrogen, oxygen, nitrogen and sulphur
in the molecular formulae were 1–100, 1–200, 0–50, 0–5
and 0–2, respectively. e molecular compositions were
assigned to the signals using a tolerance level ± 2 ppm.
e generated formulas satisfied elemental rules:
O:C ≤ 1.5; 0.3 ≤ H/C ≤ 2.5; 0 ≤ N/C ≤ 0.5; 0 ≤ S/C ≤ 0.2.,
and irregular formulae were excluded.
Preparation oflung andheart tissue sections
e protocol approved by the institutional review board
was followed, the mice were sacrificed, and lung and
heart samples were collected and immediately stored at
-80°C until sectioning. e samples were attached to the
aluminum disc and 10-µm-thick tissue slices were pre-
pared using Leica CM1950 cryostat (Leica Biosystems),
attached over the surface of adhesion microscopic glass
slides, and stored in airtight falcon tubes at -80°C before
analyses.
Field emission scanning electron microscopy (FE‑SEM)
e falcon tubes containing glass slides of heart or lung
cryo-sections were dried at room temperature before
analysis without any pretreatment. e surface of the FE-
SEM aluminum sample stage was covered with carbon
conducting tape and the glass slide was attached onto
it. A Hitachi S-4800 field emission scanning electron
microscope (FE-SEM) equipped with Bruker Xflash 6160
detector was used for observation of heart or lung tissue
sections of eight mice from dirty air and eight mice from
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 10 of 12
Hameedetal. Particle and Fibre Toxicology (2022) 19:51
filtered air, at acceleration voltage of 1.0kV, and emis-
sion current of 10 µA. e vacuum level in the observa-
tion chamber was ~ 10−7Pa. e observations were made
at the working distance of 2.1mm to 2.4mm, and at the
scan speed of 20s for each figure, at 10k, 20k, 50k, and
100k magnifications.
Fluorescence lifetime imaging microscopy (FLIM)
e fluorescence spectra of ultrafine PM particles in
PBS solution and the tissues were excited by a 405nm
CW laser (BDL-405-SMC, Becker and Hickl, Berlin,
Germany) and recorded by an optical fiber spectrom-
eter (Ocean Optics, USB2000 + , Dunedin, FA, USA)
with a 420nm long-pass filter. e ultrafine PM parti-
cles in frozen tissues were imaged using a laser scanning
microscope (FV300/IX71, Olympus, Japan) equipped
with a femtosecond (fs) pulsed laser (680–1300nm tun-
able wavelength, 150fs, 80MHz, InSight X3 Dual, USA)
and a time correlated single photon counting (TCSPC)
system (SPC-150, Becker & Hickl, Germany), with a
60 × water-dipping objective (NA = 1.2). e fluores-
cence lifetime imaging microscopy (FLIM) were excited
by the 830 nm fs laser and collected with a 770 nm
(shortpass) SP filter. e ultrafine PM particles in PBS
solution were measured by FLIM as well to obtain the
fluorescence lifetime of ultrafine PM. Each figure had a
field of 123µm × 123µm with 256 × 256 pixels, collect-
ing the signal within the depth of about 2µm. At least
6 different areas were randomly imaged for each sample.
e mean lifetime of each pixel is fitted with multi-expo-
nential decay models and calculated using the commer-
cial SPC Image software package (Becker & Hickl GmbH,
Berlin, Germany). e fluorescence lifetime of ultrafine
PM solution was mostly in the range of 170–200ps with
the peak at 174ps. erefore, the FLIM images of tis-
sues with ultrafine PM were fitted by setting the short-
est fluorescence lifetime component as 174ps. en the
pixels with short lifetime were marked as green noting
ultrafine PM particles, and the pixels with long lifetime
were depicted as red denoting tissue autofluorescence.
Ultrafine PM particle density in the lung or heart tissues
of dirty air- or FA-treated mice were calculated based on
the two-colored images.
Histological andimmunohistochemical staining
e lung and heart tissues were subjected to Congo red
staining for histological and morphological information.
Congo red dye was obtained from Ruibao and Biotech
Co., Ltd (Cat # R1029). For immunohistochemical anal-
yses the following antibodies and materials were used:
IBA-1 (Reego and Biology, 1:100), Aβ (Reego and Biology,
1:500). HRP-labelled goat anti-rabbit secondary antibody
(Reego and Biology, 1:200), DAB (DAKO, K5007), nor-
mal rabbit serum (Boster, AR1010), and BSA (Solarbio,
A8020). High resolution optical images of the stained tis-
sues were observed by Olympus CKX53 microscope and
recorded by using Olympus cellSens 2.1 [ver.2.1] imaging
software for Life Sciences (Olympus, Tokyo, Japan).
Statistical analysis
All the data were expressed as Mean ± Standard devia-
tion (SD). e difference between PM group and FA
group was analyzed using student t-test. e statistical
analysis was performed using SPSS22.0 software (IBM,
Armonk, NY). Graphpad Prism software (Version 6.0, La
Jolla, CA) and OriginPro 2021b was used for graph plot-
ting. P < 0.05 was considered significant.
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s12989- 022- 00493-8.
Additional le1: Fig. S1. Fractograms of heavy metals detected in PM
particles by ICP-MS. (A) Cr, (B) Mn, (C) Fe, (D) Ni, (E) Cu, (F) Zn, (G) Cd, and
(H) Bi.
Additional le2: TableS1. The mean concentrations of metal elements
in dirty air and FA during exposure.
Acknowledgements
We are grateful to Prof. Richard N. Zare of Department of Chemistry, Stanford
University for valuable support in study design. We are thankful to Malin
Linder Nording, Department of Chemistry, Umea University, Sweden for valu-
able assistance in drafting the manuscript.
Author contributions
Study design: SH/MT/JZ. Data collection: SH/KP/WS/LM/JZ. Data analysis and
interpretation: SH/MT/LM/JZ. Manuscript draft: SH/MT/JZ. Critical revision and
final decision to submit: all authors. All authors read and approved the final
manuscript.
Funding
SH thank the Kempe Foundation Sweden, and to Scientific Research Startup
Foundation (IDH1615113) of Fudan University to RNZ. JZ thanks the National
Natural Science Foundation of China (91543119, 81673125) for funding.
Availability of data and materials
All data generated or analyzed during this study are included in this published
article [and its additional files].
Declarations
Ethics approval and consent to participate
The animal study procedures were approved by the Institutional Research
Committees of the Fudan University, Shanghai, China, and the methods were
performed in accordance with the set regulations and guidelines.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 11 of 12
Hameedetal. Particle and Fibre Toxicology (2022) 19:51
Author details
1 Depar tment of Chemistry, Umeå University, 901 87 Umeå, Sweden. 2 Depart-
ment of Chemistry, Fudan University, Shanghai 200438, China. 3 Department
of Environmental Health, School of Public Health and the Key Laboratory
of Public Health Safety, Ministry of Education, Fudan University, 130 Dong’an
Road, Box 249, Shanghai 200032, China. 4 Department of Optical Science
and Engineering, Key Laboratory of Micro and Nano Photonic Structures (Min-
istry of Education), Shanghai Engineering Research Center of Ultra-Precision
Optical Manufacturing, Green Photoelectron Platform, Fudan University, 220
Handan Road, Shanghai 200433, China. 5 Department of Clinical Sciences,
Neurosciences, Umeå University, 90185 Umeå, Sweden. 6 IRDR ICoE on Risk
Interconnectivity and Governance on Weather/Climate Extremes Impact
and Public Health, Fudan University, Shanghai, China.
Received: 30 March 2022 Accepted: 19 July 2022
References
1. Woodward NC, Crow AL, Zhang Y, Epstein S, Hartiala J, Johnson R, et al.
Exposure to nanoscale particulate matter from gestation to adulthood
impairs metabolic homeostasis in mice. Sci Rep. 2019;9(1):1816. https://
doi. org/ 10. 1038/ s41598- 018- 37704-2.
2. Al-Kindi SG, Brook RD, Biswal S, Rajagopalan S. Environmental deter-
minants of cardiovascular disease: lessons learned from air pollu-
tion. Nat Rev Cardiol. 2020;17(10):656–72. https:// doi. org/ 10. 1038/
s41569- 020- 0371-2.
3. Li D, Li Y, Li G, Zhang Y, Li J, Chen H. Fluorescent reconstitution on deposi-
tion of PM(25) in lung and extrapulmonary organs. Proc Natl Acad Sci
USA. 2019;116(7):2488–93. https:// doi. org/ 10. 1073/ pnas. 18181 34116.
4. Hameed S, Zhao J, Zare RN. Ambient PM particles reach mouse brain,
generate ultrastructural hallmarks of neuroinflammation, and stimu-
late amyloid deposition, tangles, and plaque formation. Talanta Open.
2020;2:100013. https:// doi. org/ 10. 1016/j. talo. 2020. 100013.
5. Baqir M, Roden AC, Moua T. Amyloid in the lung. Semin Respir Crit Care
Med. 2020;41(2):299–310. https:// doi. org/ 10. 1055/s- 0040- 17080 59.
6. Pankhurst Q, Hautot D, Khan N, Dobson J. Increased levels of mag-
netic iron compounds in Alzheimer’s disease. J Alzheimer’s Dis JAD.
2008;13(1):49–52. https:// doi. org/ 10. 3233/ jad- 2008- 13105.
7. Hautot D, Pankhurst QA, Khan N, Dobson J. Preliminary evaluation of
nanoscale biogenic magnetite in Alzheimer’s disease brain tissue. Proc
Biol Sci. 2003;270(Suppl 1):S62–4. https:// doi. org/ 10. 1098/ rsbl. 2003. 0012.
8. Bové H, Bongaerts E, Slenders E, Bijnens EM, Saenen ND, Gyselaers W,
et al. Ambient black carbon particles reach the fetal side of human
placenta. Nat Commun. 2019;10(1):3866. https:// doi. org/ 10. 1038/
s41467- 019- 11654-3.
9. Maher BA, Ahmed IA, Karloukovski V, MacLaren DA, Foulds PG, Allsop D,
et al. Magnetite pollution nanoparticles in the human brain. Proc Natl
Acad Sci USA. 2016;113(39):10797–801. https:// doi. org/ 10. 1073/ pnas.
16059 41113.
10. Kreyling WG, Hirn S, Möller W, Schleh C, Wenk A, Celik G, et al. Air-blood
barrier translocation of tracheally instilled gold nanoparticles inversely
depends on particle size. ACS Nano. 2014;8(1):222–33. https:// doi. org/ 10.
1021/ nn403 256v.
11. Bové H, Steuwe C, Fron E, Slenders E, D’Haen J, Fujita Y, et al. Biocompat-
ible label-free detection of carbon black particles by femtosecond pulsed
laser microscopy. Nano Lett. 2016;16(5):3173–8. https:// doi. org/ 10. 1021/
acs. nanol ett. 6b005 02.
12. Cao L, Wang X, Meziani MJ, Lu F, Wang H, Luo PG, et al. Carbon dots for
multiphoton bioimaging. J Am Chem Soc. 2007;129(37):11318–9. https://
doi. org/ 10. 1021/ ja073 527l.
13. Liu Q, Guo B, Rao Z, Zhang B, Gong JR. Strong two-photon-induced
fluorescence from photostable, biocompatible nitrogen-doped gra-
phene quantum dots for cellular and deep-tissue imaging. Nano Lett.
2013;13(6):2436–41. https:// doi. org/ 10. 1021/ nl400 368v.
14. Catalano IM, Cingolani A. Absolute determination of the two photon
cross section in NADH. Opt Commun. 1980;32(1):156–8. https:// doi. org/
10. 1016/ 0030- 4018(80) 90336-3.
15. Patton JS, Byron PR. Inhaling medicines: delivering drugs to the body
through the lungs. Nat Rev Drug Discov. 2007;6(1):67–74. https:// doi. org/
10. 1038/ nrd21 53.
16. Pietropaoli AP, Frampton MW, Hyde RW, Morrow PE, Oberdörster G, Cox C,
et al. Pulmonary function, diffusing capacity, and inflammation in healthy
and asthmatic subjects exposed to ultrafine particles. Inhal Toxicol.
2004;16(Suppl 1):59–72. https:// doi. org/ 10. 1080/ 08958 37049 04430 79.
17. Bustamante JGZS. Amyloidosis. Treasure Island: StatPearls Publishing;
2022.
18. Cong LH, Li T, Wang H, Wu YN, Wang SP, Zhao YY, et al. IL-17A-producing
T cells exacerbate fine particulate matter-induced lung inflammation
and fibrosis by inhibiting PI3K/Akt/mTOR-mediated autophagy. J Cell Mol
Med. 2020;24(15):8532–44. https:// doi. org/ 10. 1111/ jcmm. 15475.
19. Ge C, Tan J, Zhong S, Lai L, Chen G, Zhao J, et al. Nrf2 mitigates prolonged
PM2.5 exposure-triggered liver inflammation by positively regulating SIKE
activity: protection by Juglanin. Redox Biol. 2020;36:101645. https:// doi.
org/ 10. 1016/j. redox. 2020. 101645.
20. Yue W, Tong L, Liu X, Weng X, Chen X, Wang D, et al. Short term Pm2.5
exposure caused a robust lung inflammation, vascular remodeling, and
exacerbated transition from left ventricular failure to right ventricular
hypertrophy. Redox Biol. 2019;22:101161. https:// doi. org/ 10. 1016/j. redox.
2019. 101161.
21. Zhang S, Zhang W, Zeng X, Zhao W, Wang Z, Dong X, et al. Inhibition of
Rac1 activity alleviates PM2.5-induced pulmonary inflammation via the
AKT signaling pathway. Toxicol Lett. 2019;310:61–9. https:// doi. org/ 10.
1016/j. toxlet. 2019. 04. 017.
22. Gołębiowski T, Kuźniar J, Porażko T, Wojtala R, Konieczny A, Krajewska M,
et al. Multisystem amyloidosis in a coal miner with silicosis: is exposure to
silica dust a cause of amyloid deposition? Int J Environ Res Public Health.
2022;19(4):2297. https:// doi. org/ 10. 3390/ ijerp h1904 2297.
23. Joachim CL, Mori H, Selkoe DJ. Amyloid β-protein deposition in tissues
other than brain in Alzheimer’s disease. Nature. 1989;341(6239):226–30.
https:// doi. org/ 10. 1038/ 34122 6a0.
24. Kourtchev I, Godoi RHM, Connors S, Levine JG, Archibald AT, Godoi AFL,
et al. Molecular composition of organic aerosols in central Amazonia:
an ultra-high-resolution mass spectrometry study. Atmos Chem Phys.
2016;16(18):11899–11913. https:// doi. org/ 10. 5194/ acp- 16- 11899- 2016
25. Almetwally AA, Bin-Jumah M, Allam AA. Ambient air pollution and
its influence on human health and welfare: an overview. Environ
Sci Pollut Res Int. 2020;27(20):24815–30. https:// doi. org/ 10. 1007/
s11356- 020- 09042-2.
26. Schraufnagel DE, Balmes JR, Cowl CT, De Matteis S, Jung SH, Mortimer
K, et al. Air pollution and noncommunicable diseases: a review by the
forum of International Respiratory Societies’ Environmental Committee,
Part 1: The Damaging effects of air pollution. Chest. 2019;155(2):409–16.
https:// doi. org/ 10. 1016/j. chest. 2018. 10. 042.
27. Xu D, Chen Y, Wu L, He S, Xu P, Zhang Y, et al. Acute effects of ambient
PM(2.5) on lung function among schoolchildren. Sci Rep. 2020;10(1):4061.
https:// doi. org/ 10. 1038/ s41598- 020- 61003-4.
28. Du X, Chen R, Meng X, Liu C, Niu Y, Wang W, et al. The establishment of
National Air Quality Health Index in China. Environ Int. 2020;138:105594.
https:// doi. org/ 10. 1016/j. envint. 2020. 105594.
29. Du XH, Jiang S, Zeng XJ, Zhang J, Pan K, Zhou J, et al. Air pollution is
associated with the development of atherosclerosis via the coopera-
tion of CD36 and NLRP3 inflammasome in ApoE(-/-) mice. Toxicol Lett.
2018;290:123–32. https:// doi. org/ 10. 1016/j. toxlet. 2018. 03. 022.
30. Lippmann M, Yeates DB, Albert RE. Deposition, retention, and clearance
of inhaled particles. Br J Ind Med. 1980;37(4):337–62. https:// doi. org/ 10.
1136/ oem. 37.4. 337.
31. Nemmar A, Hoet PH, Vanquickenborne B, Dinsdale D, Thomeer M, Hoy-
laerts MF, et al. Passage of inhaled particles into the blood circulation in
humans. Circulation. 2002;105(4):411–4. https:// doi. org/ 10. 1161/ hc0402.
104118.
32. Jia H, Liu Y, Guo D, He W, Zhao L, Xia S. PM2.5-induced pulmonary inflam-
mation via activating of the NLRP3/caspase-1 signaling pathway. Environ
Toxicol. 2021;36(3):298–307. https:// doi. org/ 10. 1002/ tox. 23035.
33. Aghasafari P, George U, Pidaparti R. A review of inflammatory mechanism
in airway diseases. Inflamm Res. 2019;68(1):59–74. https:// doi. org/ 10.
1007/ s00011- 018- 1191-2.
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

Page 12 of 12
Hameedetal. Particle and Fibre Toxicology (2022) 19:51
•
fast, convenient online submission
•
thorough peer review by experienced researchers in your field
•
rapid publication on acceptance
•
support for research data, including large and complex data types
•
gold Open Access which fosters wider collaboration and increased citations
maximum visibility for your research: over 100M website views per year
•
At BMC, research is always in progress.
Learn more biomedcentral.com/submissions
Ready to submit your research
Ready to submit your research
? Choose BMC and benefit from:
? Choose BMC and benefit from:
34. Van Eeden S, Leipsic J, Paul Man SF, Sin DD. The relationship between
lung inflammation and cardiovascular disease. Am J Respir Crit Care Med.
2012;186(1):11–6. https:// doi. org/ 10. 1164/ rccm. 201203- 0455PP.
35. Lafuse WP, Wozniak DJ, Rajaram MVS. Role of cardiac macrophages on
cardiac inflammation, fibrosis and tissue repair. Cells. 2020;10(1):51.
https:// doi. org/ 10. 3390/ cells 10010 051.
36. Frangogiannis NG. Cardiac fibrosis: cell biological mechanisms, molecular
pathways and therapeutic opportunities. Mol Aspects Med. 2019;65:70–
99. https:// doi. org/ 10. 1016/j. mam. 2018. 07. 001.
37. Frangogiannis NG. Cardiac fibrosis. Cardiovasc Res. 2021;117(6):1450–88.
https:// doi. org/ 10. 1093/ cvr/ cvaa3 24.
38. McVeigh T, Tennyson C. Understanding and recognizing cardiac amyloi-
dosis. Jaapa. 2020;33(10):16–20. https:// doi. org/ 10. 1097/ 01. JAA. 00006
97236. 11386. 3a.
39. Amsalu E, Wang T, Li H, Liu Y, Wang A, Liu X, et al. Acute effects of fine
particulate matter (PM25) on hospital admissions for cardiovascular dis-
ease in Beijing, China: a time-series study. Environ Health. 2019;18(1):70.
https:// doi. org/ 10. 1186/ s12940- 019- 0506-2.
40. Hayes RB, Lim C, Zhang Y, Cromar K, Shao Y, Reynolds HR, et al. PM2.5
air pollution and cause-specific cardiovascular disease mortality. Int J
Epidemiol. 2020;49(1):25–35. https:// doi. org/ 10. 1093/ ije/ dyz114.
41. Sangani RG, Soukup JM, Ghio AJ. Metals in air pollution particles decrease
whole-blood coagulation time. Inhalation Toxicol. 2010;22(8):621–6.
https:// doi. org/ 10. 3109/ 08958 37100 35990 37.
42. Campen MJ, Nolan JP, Schladweiler MC, Kodavanti UP, Evansky PA, Costa
DL, et al. Cardiovascular and thermoregulatory effects of inhaled PM-
associated transition metals: a potential interaction between nickel and
vanadium sulfate. Toxicol Sci. 2001;64(2):243–52. https:// doi. org/ 10. 1093/
toxsci/ 64.2. 243.
43. Graff DW, Cascio WE, Brackhan JA, Devlin RB. Metal particulate matter
components affect gene expression and beat frequency of neonatal rat
ventricular myocytes. Environ Health Perspect. 2004;112(7):792–8. https://
doi. org/ 10. 1289/ ehp. 112- 12419 94.
44. Kogianni E, Kouras A, Samara C. Indoor concentrations of PM25 and
associated water-soluble and labile heavy metal fractions in workplaces:
implications for inhalation health risk assessment. Environ Sci Pollut Res
Int. 2020;1:1. https:// doi. org/ 10. 1007/ s11356- 019- 07584-8.
45. Xu P, He X, He S, Luo J, Chen Q, Wang Z, et al. Personal exposure to
PM2.5-bound heavy metals associated with cardiopulmonary function in
general population. Environ Sci Pollut Res Int. 2021;28(6):6691–9. https://
doi. org/ 10. 1007/ s11356- 020- 11034-1.
46. Faxon C, Hammes J, Le Breton M, Pathak RK, Hallquist M. Characteri-
zation of organic nitrate constituents of secondary organic aerosol
(SOA) from nitrate-radical-initiated oxidation of limonene using high-
resolution chemical ionization mass spectrometry. Atmos Chem Phys.
2018;18(8):5467–81. https:// doi. org/ 10. 5194/ acp- 18- 5467- 2018.
47. Jang KS, Choi M, Park M, Park MH, Kim YH, Seo J, et al. Assessment of
PM2.5-bound nitrogen-containing organic compounds (NOCs) during
winter at urban sites in China and Korea. Environ Pollut. 2020;265(Pt
B):114. https:// doi. org/ 10. 1016/j. envpol. 2020. 114870.
48. Sarnat SE, Winquist A, Schauer JJ, Turner JR, Sarnat JA. Fine particulate
matter components and emergency department visits for cardiovascular
and respiratory diseases in the St. Louis, Missouri-Illinois, metropolitan
area. Environ Health Perspect. 2015;123(5):437–44. https:// doi. org/ 10.
1289/ ehp. 13077 76.
49. Leikauf GD, Kim S-H, Jang A-S. Mechanisms of ultrafine particle-induced
respiratory health effects. Exp Mol Med. 2020;52(3):329–37. https:// doi.
org/ 10. 1038/ s12276- 020- 0394-0.
50. Maciejczyk P, Zhong M, Li Q, Xiong J, Nadziejko C, Chen LC. Effects of
subchronic exposures to concentrated ambient particles (CAPs) in mice.
II. The design of a CAPs exposure system for biometric telemetry monitor-
ing. Inhal Toxicol. 2005;17(4–5):189–97. https:// doi. org/ 10. 1080/ 08958
37059 09127 43.
51. Du X, Jiang S, Zeng X, Zhang J, Pan K, Song L, et al. Fine particulate
matter-induced cardiovascular injury is associated with NLRP3 inflamma-
some activation in Apo E(-/-) mice. Ecotoxicol Environ Saf. 2019;174:92–9.
https:// doi. org/ 10. 1016/j. ecoenv. 2019. 02. 064.
52. Daellenbach KR, Kourtchev I, Vogel AL, Bruns EA, Jiang J, Petäjä T,
et al. Impact of anthropogenic and biogenic sources on the seasonal
variation in the molecular composition of urban organic aerosols: a
field and laboratory study using ultra-high-resolution mass spectrom-
etry. Atmos Chem Phys. 2019;19(9):5973–91. https:// doi. org/ 10. 5194/
acp- 19- 5973- 2019.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations.
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

1.
2.
3.
4.
5.
6.
Terms and Conditions
Springer Nature journal content, brought to you courtesy of Springer Nature Customer Service Center GmbH (“Springer Nature”).
Springer Nature supports a reasonable amount of sharing of research papers by authors, subscribers and authorised users (“Users”), for small-
scale personal, non-commercial use provided that all copyright, trade and service marks and other proprietary notices are maintained. By
accessing, sharing, receiving or otherwise using the Springer Nature journal content you agree to these terms of use (“Terms”). For these
purposes, Springer Nature considers academic use (by researchers and students) to be non-commercial.
These Terms are supplementary and will apply in addition to any applicable website terms and conditions, a relevant site licence or a personal
subscription. These Terms will prevail over any conflict or ambiguity with regards to the relevant terms, a site licence or a personal subscription
(to the extent of the conflict or ambiguity only). For Creative Commons-licensed articles, the terms of the Creative Commons license used will
apply.
We collect and use personal data to provide access to the Springer Nature journal content. We may also use these personal data internally within
ResearchGate and Springer Nature and as agreed share it, in an anonymised way, for purposes of tracking, analysis and reporting. We will not
otherwise disclose your personal data outside the ResearchGate or the Springer Nature group of companies unless we have your permission as
detailed in the Privacy Policy.
While Users may use the Springer Nature journal content for small scale, personal non-commercial use, it is important to note that Users may
not:
use such content for the purpose of providing other users with access on a regular or large scale basis or as a means to circumvent access
control;
use such content where to do so would be considered a criminal or statutory offence in any jurisdiction, or gives rise to civil liability, or is
otherwise unlawful;
falsely or misleadingly imply or suggest endorsement, approval , sponsorship, or association unless explicitly agreed to by Springer Nature in
writing;
use bots or other automated methods to access the content or redirect messages
override any security feature or exclusionary protocol; or
share the content in order to create substitute for Springer Nature products or services or a systematic database of Springer Nature journal
content.
In line with the restriction against commercial use, Springer Nature does not permit the creation of a product or service that creates revenue,
royalties, rent or income from our content or its inclusion as part of a paid for service or for other commercial gain. Springer Nature journal
content cannot be used for inter-library loans and librarians may not upload Springer Nature journal content on a large scale into their, or any
other, institutional repository.
These terms of use are reviewed regularly and may be amended at any time. Springer Nature is not obligated to publish any information or
content on this website and may remove it or features or functionality at our sole discretion, at any time with or without notice. Springer Nature
may revoke this licence to you at any time and remove access to any copies of the Springer Nature journal content which have been saved.
To the fullest extent permitted by law, Springer Nature makes no warranties, representations or guarantees to Users, either express or implied
with respect to the Springer nature journal content and all parties disclaim and waive any implied warranties or warranties imposed by law,
including merchantability or fitness for any particular purpose.
Please note that these rights do not automatically extend to content, data or other material published by Springer Nature that may be licensed
from third parties.
If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner not
expressly permitted by these Terms, please contact Springer Nature at
onlineservice@springernature.com