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BioMed Central
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Particle and Fibre Toxicology
Open Access
Research
The influence of hydrogen peroxide and histamine on lung
permeability and translocation of iridium nanoparticles in the
isolated perfused rat lung
James J Meiring
1
, Paul JA Borm*
1,3
, Karim Bagate
1
, Manuela Semmler
2
,
Jürgen Seitz
2
, Shinji Takenaka
2
and Wolfgang G Kreyling
2
Address:
1
Particle Research Core, Institute für Umweltmedizinische Forschung (IUF) an der Heinrich-Heine Universität gGmbH, Auf'm
Hennekamp 50 D-40225 Düsseldorf, Germany,
2
GSF Forschungszentrum für Umwelt und Gesundheit, Ingolstädter Landstr. 1, Institute for
Inhalation Biology & Focus Network Aerosols and Health, D-85746 Neuherberg / München, Germany and
3
Centre of Expertise in Life Sciences
(CEL), Zuyd University, PO Box 550, 6400 AN HEERLEN, The Netherlands
Email: James J Meiring - james.meiring@med.uni-muenchen.de; Paul JA Borm* - p.borm@hszuyd.nl; Karim Bagate - Bagate.k@wanadoo.fr;
Manuela Semmler - semmler@gsf.de; Jürgen Seitz - seitz@gsf.de; Shinji Takenaka - takenaka@gsf.de; Wolfgang G Kreyling - kreyling@gsf.de
* Corresponding author
endotheliumtranslocationultrafine particlesisolated perfused lungpermeability.
Abstract
Background: Translocation of ultrafine particles (UFP) into the blood that returns from the lungs to the heart
has been forwarded as a mechanism for particle-induced cardiovascular effects. The objective of this study was
to evaluate the role of the endothelial barrier in the translocation of inhaled UFP from the lung into circulation.
Methods: The isolated perfused rat lung (IPRL) was used under negative pressure ventilation, and radioactive
iridium particles (18 nm, CMD,
192
Ir-UFP) were inhaled during 60 minutes to achieve a lung burden of 100 – 200
µg. Particle inhalation was done under following treatments: i) control perfusion, ii) histamine (1 µM in perfusate,
iii) luminal histamine instillation (1 mM), and iv) luminal instillation of H
2
O
2
. Particle translocation to the perfusate
was assessed by the radioactivity of
192
Ir isotope. Lung permeability by the use of Tc
99m
-labeled diethylene
triamine pentaacetic acid (DTPA). In addition to light microscopic morphological evaluation of fixed lungs, alkaline
phosphatase (AKP) and angiotensin converting enzyme (ACE) in perfusate were measured to assess epithelial and
endothelial integrity.
Results: Particle distribution in the lung was homogenous and similar to in vivo conditions. No translocation of
Ir particles at negative pressure inhalation was detected in control IPL, but lungs pretreated with histamine (1 µM)
in the perfusate or with luminal H
2
O
2
(0.5 mM) showed small amounts of radioactivity (2–3 % dose) in the single
pass perfusate starting at 60 min of perfusion. Although the kinetics of particle translocation were different from
permeability for
99m
Tc-DTPA, the pretreatments (H
2
O
2
, vascular histamine) caused similar changes in the
translocation of particles and soluble mediator. Increased translocation through epithelium and endothelium with
a lag time of one hour occurred in the absence of epithelial and endothelial damage.
Conclusion: Permeability of the lung barrier to UFP or nanoparticles is controlled both at the epithelial and
endothelial level. Conditions that affect this barrier function such as inflammation may affect translocation of NP.
Published: 27 June 2005
Particle and Fibre Toxicology 2005, 2:3 doi:10.1186/1743-8977-2-3
Received: 01 December 2004
Accepted: 27 June 2005
This article is available from: http://www.particleandfibretoxicology.com/content/2/1/3
© 2005 Meiring et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0
),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Particle and Fibre Toxicology 2005, 2:3 http://www.particleandfibretoxicology.com/content/2/1/3
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Introduction
Epidemiological studies have demonstrated an increased
morbidity and mortality by particulate air pollution [1,2].
The highest relative risk for mortality and hospital admis-
sions were observed in subjects with existing pulmonary
disease including asthma and COPD [1,2]. The exact
mechanism by which PM can adversely affect humans
remains unknown, but several hypotheses have been for-
warded. These include that PM causes pulmonary inflam-
mation causing release of factors that influence blood
coagulation [3], reduced lung function [4], increased
blood plasma viscosity [5], reduced heart rate variability
[6,7] and destabilisation of atheromatous plagues [8].
Some of these effects are attributed to translocated nano-
particles based on their potential effects on vascular func-
tion [9,10], blood coagulation [11], mitochondrial
function [12] and Ca-flow [13,14].
Nanoparticles have been shown to translocate from lung
to the circulation [15-18], but most of the inhaled dose
remains in the lung interstitium [19] even up to several
years [20]. Therefore it seems that not the epithelial but
the endothelial barrier is more important in prevention of
translocation to the blood. Enhanced lung permeability
has been measured by increased Clara-cell protein in
blood [21] or enhanced DTPA clearance in the lung [22]
after ozone and hyperoxia. Recent work in rabbit isolated
perfused lungs shows that nanoparticles themselves can
influence microvascular permeability measured by weight
gain after occlusion [23]. However, since the mechanisms
of nanoparticle transport on a sub-cellular level are
unknown it remains to be determined whether the above
indices of lung permeability are related to translocation of
nanoparticles. Particles may also cause the release of
vasoactive mediators such as histamine, which was shown
to be increased in plasma of hamster after instillation of
diesel exhaust particles [24]. Histamine is well known to
induce vascular permeability through its action on
endothelial H
1
-receptor [25]. Finally, by oxidative stress
mechanisms ambient and nanoparticles can cause activa-
tion of lung alveolar macrophages and epithelial cells that
result in the production of pro-inflammatory cytokines
such as TNF and Il-1 in humans [26] and rat models [27]
that are typically associated with increased lung permea-
bility [28,29].
The objective of this study was to assess nanoparticle
translocation in relation to permeability changes for small
molecules and integrity of epithelial and endothelial
monolayers. In order to manipulate permeability in the
absence of neutrophil recruitment and activation we used
an isolated perfused rat lung. Several treatments to modify
lung permeability in-vitro were applied including oxida-
tive stress by instillation of hydrogen peroxide and
endothelial permeability by histamine in the perfusate.
These treatments were selected for their relevance to con-
ditions of patients with pulmonary or systemic complica-
tions. Particle translocation was assessed by the inherent
radioactivity of 18 nm size iridium nanoparticles (
192
Ir-
UFP).
Materials & methods
Animals and surgical procedure
Adult, healthy, male Wistar-Kyoto rats (WKY/Kyo@Rj
rats, Janvier, France) (200–250 g) were housed in pairs in
a humidity (55% relative humidity) and temperature
(22°C) controlled room. They were maintained on a 12-
h day/night cycle. Rats were allowed to acclimate to the
facility for a minimum of 10 days prior to use. When the
experiments were performed rats were more than 17
weeks of age. The studies were conducted under federal
guidelines for the use and care of laboratory animals and
were approved by the Oberbayern Government and by
the GSF Institutional Animal Care and Use Committee.
Surgical procedure for lung isolations was done according
the method of Uhlig and Wollin [30]. Briefly, rats were
anaesthetized intraperitoneally with 80 mg/kg ketamin.
Deep anaesthesia was characterized by a lack of response
to toe pinching. Heparin (500 IU) was injected via the tail
vein. A midline incision was made from the pelvic region
to the neck of the rat. With the ventilator operating, the
trachea was cannulated using a rigid catheter and the cath-
eter was attached to the ventilator. Therefore lung were
ventilated from the start of the whole procedure. The ani-
mals were exsanguinated opening the aorta abdominalis
after deep intraperitoneal anesthesia with ketamine (100
mg/100 g body weight) and xylazine (0.5 mg/100 g body
weight). After anesthesia a longitudinal ventral incision
was made to open the thoracic and abdominal cavity and
it was held open using clamps. The thymus was removed
and the apex of the heart was cut off to introduce a can-
nula into the pulmonary artery. A slight perfusion flow of
around 1 ml/min was maintained before inserting can-
nula. Care was taken not to introduce any air bubbles into
the pulmonary artery. The left atrium was cannulated by
advancing the venous cannula through the mitral valve. A
ligature was placed around the heart to keep both can-
nula's in place. The aortic cannula was then attached to
the lining fed through the Perspex lid of the 500 mL neg-
ative-pressure-chamber. After the Perspex lid was fully
mounted on the chamber negative pressure ventilation
started. The respiratory settings during the negative pres-
sure ventilation were 65 breaths per min. Regular sighs
were introduced (hyperventilation) to improve function
of lungs.
Isolated lung perfusion
The IPL-4401 Isolated lung ventilation perfusion system
(FMI GmbH Oberbach) was used for our study. Addi-
tional negative pressure chamber has been constructed by
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GSF -National Research Centre for Environment and
Health. The system in brief consists of a small animal ven-
tilator, jacketed upper media reservoir, negative-pressure-
chamber holding the heart-lung-bloc, perfusion lines,
peristaltic pump and pulmonary artery pressure trans-
ducer. The computer software (FMI GmbH Oberbach)
was operated by a 386 personal computer and allowed
constant monitoring of pulmonary blood pressure. The
upper media reservoir, ventilation chamber and perfusion
lines were held at 37°C by a re-circulating water-bath. The
perfusion medium was selected based on its extensive use
in isolated organ perfusion and consisted of a modified
Krebs-Ringer -bicarbonate buffer. Krebs-Ringer composi-
tion was as follows (mM): NaCl 118 ; KCl 5.9 ; CaCl
2
2.5
; MgSO
4
1.2 ; NaH
2
PO
4
1.2 ; NaHCO
3
24.9 ; glucose 11.1,
pH was adjusted at 7.4. It was then mixed at a ratio 1:1
with Haemacell solution (Hoechst Marion Roussel). The
buffer was pre-warmed and gassed with 95% O
2
and 5%
CO
2
at a rate low enough to prevent excessive frothing of
the medium. The medium flowing through the system
passed through a bubble trap prior to reaching the lungs
and the buffer pH was continuously monitored through-
out the experiment. Respiratory rate was set at 65 breaths
per min. Lungs were inflated at a maximum negative pres-
sure of -1.5 kPa in the chamber. Stroke volume was set at
10 ml to achieve a tidal volume of usually 3–4 ml
(because of the altered compliance of the lungs). The
lungs were expanded (sighed) every 4 minutes applying a
negative pressure of -2.5 kPa to the chamber. Optimal per-
fusion settings included perfusion rate of 5 ml/min and a
medium pH of 7.4. Perfusion pressure was not constant
but was kept between 10 and 14 kPa.
Particle generation and exposure
Aerosols of ultrafine iridium particles (Ir-UFP) radiola-
beled with
192
Ir were produced with a spark generator as
described previously [15]. Size distribution and number
concentration were monitored continuously by a differen-
tial mobility particle sizer (DMPS 3070, TSA instruments)
and a condensation particle counter (CPC 3022A, TSI
Instruments). The size distribution of the
192
Ir-UFP was
aimed to a count median diameter of 17–20 nm (geomet-
ric standard deviation 1.6) at a particle concentration of
10
7
cm
-3
aiming for a tidal volume of 3–4 cm
3
at a fre-
quency of 65 /min. The estimated dose under these condi-
tions is 180 µg/hour. A schematical description of the
experimental system is shown in Figure 1
Experimental design
Following a 15 minute period of equilibration, during
which the lungs were already ventilated and perfused, the
experiment started by the inhalation of freshly produced
192
Ir-UFP from the aerosol line. Intratracheal instillation
of
99m
Tc-DTPA or other instillations were performed at
this starting time point. The perfusate was collected con-
tinuously and sampled at 15-min time intervals (Figure.
2). The following treatments were investigated,
group I: control group, only
192
Ir-UFP inhalation for 120
min, ;
group II: instillation of 50–100 µL
99m
Tc-DTPA, 500 µl
H
2
O
2
bolus (10 mM),
192
Ir-UFP aerosol inhalation for
120 min;
group III: instillation of 50–100 µL
99m
Tc-DTPA, hista-
mine continuously perused during the next 2 hours at
concentration 10 µM,
192
Ir-UFP aerosol inhalation for
120 min;
group IV: instillation of 50–100 µL
99m
Tc-DTPA and 500
µl histamine bolus instillation at a concentration of 10
mM,
192
Ir-UFP aerosol inhalation for 120 min;
group V: instillation of 50–100 µL
99m
Tc-DTPA.
Evaluation of
192
Ir-UFP translocation
The perfusate samples as well as the heart-lung-blocs were
analysed for
192
Ir-UFP activity in a shielded 1-L-well-type
gamma-spectrometer. Analysis of
192
Ir activity was per-
formed in those samples studied for
99m
Tc-DTPA permea-
bility when the
99m
Tc activity had decayed – see below.
Activity measurements of both isotopes were decay and
background corrected.
192
Ir activity in the perfusate sam-
ples were given as a fraction of the total activity found in
the perfusate and the heart-lung-bloc.
Evaluation of lung permeability
Technetium-99
m
labelled DTPA (
99m
Tc-DTPA; DRN 4362
TechneScan-DTPA, Malinckrodt Medical BV, The Nether-
lands) was used to evaluate lung permeability. The
lyophilised DTPA powder was dissolved in 10 ml sterile
99m
Tc activity containing saline, which was eluted from
the
99m
Tc generator. The solutions were then allowed to
equilibrate for 15 minutes at room temperature. The vol-
ume instilled in the trachea was 50–100 µL at a DTPA con-
centration of 120–250 µg. and a
99m
Tc activity of 5–10
MBq. The
99m
Tc-DTPA permeability was studied measur-
ing the activity in the heart-lung-bloc and the perfusate
samples. The
99m
Tc radioactivity was also analysed in the
shielded 1-L-well-type gamma-spectrometer at the appro-
priate photo peak of
99m
Tc. Since the
99m
Tc activity was
chosen to be at least an order of magnitude higher than
the
192
Ir deposition in the lungs, interference of Compton
rays in the
99m
Tc window originating from
192
Ir was negli-
gible. Permeated
99m
Tc activity in the perfusate samples
was given as a cumulative fraction of the total instilled
activity recovered in the perfusate and the heart-lung-bloc.
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Tissue preparation and microscopy
Immediately after the termination of the lung perfusion
the radioactive particles treated lungs were air dried with
room air at a pressure 3.5 kPa and subsequent imaging for
particle distribution. For histopathology only lungs
treated with non-radioactive iridium particles were used.
After the experiment, the trachea and pulmonary vein of
the IPL were perfused with 2.5 % glutaraldehyde in 0.1 M
phosphate buffer (pH 7.2, 340 mOsm) at 25 cm fixative
pressure. Post-fixation of the lungs was done by
Diagram of the experimental perfusion system used for this studyFigure 1
Diagram of the experimental perfusion system used for this study. The ultrafine iridium particles (Ir-UFP) radiolabelled with
192
Ir were produced in the spark generator. At the exit of spark generator the aerosol was quasi-neutralized by a radioactive
85
Kr source. The aerosol was diluted with nitrogen and with oxygen and adjusted to obtain 20% oxygen and was air condi-
tioned at 50–60 % relative humidity. The particle size distribution and number concentration were monitored by a differential
mobility particle sizer (DMPS) and a condensation particle counter (CPC).
192
Ir-UFP radioactivity of the aerosol was deter-
mined by continuous aerosol sampling of a measured volume and integral radioactivity counting. The lungs were perfused at a
perfusion rate of 5 ml/min and a stroke volume of 10 ml. Respiration rate set at 65 breaths per minute. Negative ventilation
pressure in chamber was regulated with animal ventilator. Lungs were manually expanded (sighed) every 4 minutes by applying
a negative pressure of -2.5 kPa to the chamber.
192
Ir
192
Ir
Spark generator
CPC DMPS
Kr-85
Neutral-
lizer
N
2
O
2
Sample
Ventilator
Vacuum
pump and
Control
Ar
Lung
Effluent Affluent
Humidifier
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Experimental protocol for isolated perfused rat lungsFigure 2
Experimental protocol for isolated perfused rat lungs. All perfusions were done under negative pressure ventilation. Following
a 15 minute period of equilibration, during which the lungs were already ventilated and perfused, the experiment started by the
inhalation of freshly produced
192
Ir-UFP from the aerosol line. Intratracheal instillation of
99m
Tc-DTPA or other instillations
were performed at this starting time point. The perfusate was collected continuously and sampled at 15-min time intervals. The
following treatments were investigated, group 1: control group, only
192
Ir-UFP inhalation for 120 min; group 2: instillation of 50–
100 µL
99m
Tc-DTPA, 500 µl H
2
O
2
bolus (0.5 mM),
192
Ir-UFP aerosol inhalation for 120 min; group 3: instillation of 50–100 µL
99m
Tc-DTPA, histamine continuously pefrused during the next 2 hours at concentration 10 µM,
192
Ir-UFP aerosol inhalation for
120 min; group 4: instillation of 50–100 µL
99m
Tc-DTPA and 500 µl histamine bolus instillation at a concentration of 10 mM,
192
Ir-UFP aerosol inhalation for 120 min; group 5: instillation of 50–100 µL
99m
Tc-DTPA. For each group 3–4 animals were
used.
Group1 : Control group.
192
Ir-UFP
Group 2 : H
2
O
2
bolus +
192
Ir-UFP +
99m
Tc-DTPA group.
Group 3 : Histamine perfused +
192
Ir-UFP +
99m
Tc-DTPA group
Group 4 : Histamine bolus +
192
Ir-UFP +
99m
Tc-DTPA group
Group 5 :
99m
Tc-DTPA
Stabilization period Histamine Bolus
99m
Tc-DTPA
instillation
192
Iridium Inhalation H
2
O
2
Bolus Histamine media
0
60
0
0
0
0 60
60
60
60
135
135
135
135
135
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immersion of the whole lung in the same fixation solu-
tion for 2 hours at room temperature. t 25 cm fixative
pressure. Two slices from left- and right caudal lobes of
each animal were embedded in paraffin and 5 µm thick
sections were stained with hematoxylin and eosin. Small
portions of the left lobe of a a sub-group of 7 animals were
embedded in Epon
®
, and semithin sections (1 µm) were
stained by toluidine blue.
Biochemical analysis of the perfusate
Perfusate samples were analysed for histamine with an
ELISA kit from IBL-Hamburg (reference no. RE 59221).
The detection limit of the kit was 0,3 ng/ml when using
plasma. Angiotensin converting enzyme (ACE) was meas-
ured according the kinetic method of Maguire and Price
[31] using standards from Bühlmann Laboratories AG,
Switzerland (Reference KK-ACK), Protein determination
was done according the Bicinchonic acid (BCA) protein
assay [32]. Clara-cell protein was measured in perfusate
using a sensitive latex immunoassay [21] with a detection
limit 1 µg/l perfusate. Alkaline Phosphatase (ALP) deter-
mination was done with KIT manufactured by Diasys
Diagnostics GmbH Germany Cat no: 104019990314.
Samples measured (Beckman DU 640 spectrophotome-
ter) at 25°C at wavelength 405 nm. The increase of the
extinction was measured each minute for 3 min and
enzyme activity was measured as the difference in extinc-
tion divided by the minutes multiply a constant factor
2754 [33].
Statistical Analysis
Results are expressed as means ± SD, and/or as individual
experiments (Fig 5). Differences between treatments were
tested for statistical significance by Mann Whitney-test. A
value of P < 0.05 was considered significant. All statistics
were run with SPSS for Windows XP.
Results
Particle distribution and deposition
The first set of experiments measured the distribution of
192
Ir -UFP related radioactivity in the lungs. Deterioration
of lung performance was noted based on increasing fre-
quency of inflation to maintain tidal volume, but could
not be quantified during negative pressure perfusion in
the experimental set-up due to maintain a closed system
for radiation protection safety reasons. In a positive pres-
sure using the same equipment, tidal volume, respiration
pressure and weight did not change over a 2-hour per-
fusion period. The particle size distribution in the inhaled
aerosol was well reproducible and the count median
diameter (CMD) ranged between 16 and 18 nm of particle
diameter (Fig 3). Geometric standard deviation (GSD)
always was 1.6. Deposition of particles in isolated per-
fused lungs was compared to animals exposed parallel to
the same aerosol and showed similar homogenous distri-
bution, with somewhat lower deposition (data not
shown).
Translocation of ultrafine particles after modified
permeability
In a large set of perfusions in control lungs no transloca-
tion of
192
Ir-UFP particles was noted and the variance
between different perfusions is small (< 5 %) as shown in
Fig. 4A. Then several treatments were applied to investi-
gate the role of epithelial and endothelial permeability on
particle translocation. First, hyperinflation to double tidal
volume every minute was applied but did not lead to
increased translocation of nanoparticles (data not
shown). An initial bolus injection of H
2
O
2
into the tra-
Average particle size distribution in a typical perfusion exper-iment over entire exposure time (120 min), revealing an average size distribution of count median diameter of 16.9 nm, GSD of1.6, aerosol concentration 4.45 10
6
cm
-3
, SD 0.13 10
6
cm
-3
The example shown is from inhalation during hista-mine perfusion, as shown in Fig 3 (lower panel)Figure 3
Average particle size distribution in a typical perfusion exper-
iment over entire exposure time (120 min), revealing an
average size distribution of count median diameter of16.9
nm, GSD of1.6, aerosol concentration 4.45 10
6
cm
-3
, SD 0.13
10
6
cm
-3
The example shown is from inhalation during hista-
mine perfusion, as shown in Fig 3 (lower panel).
0.E+00
2.E+06
4.E+06
6.E+06
8.E+06
1.E+07
10.00 100.00 1000.00
electrical mobility diameter (nm)
number density dN / d (log d)
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chea of the IPL to reach a final concentration of 0.5 mM,
caused particle translocation to start at 60 min after onset
of the inhalation of radioactive aerosol. A significant dif-
ference (P < 0.05, Mann-Whitney U-test) in particle-
related radioactivity in the perfusate was observed
between control and H
2
O
2
group at 90, 105 and 120 min-
utes after onset of inhalation (Fig. 4A). At other time-
points beyond 60 minutes the differences to untreated
lungs were of borderline significance (P < 0,1; Mann Whit-
ney-test). The variance between perfusions upon this
treatment in Fig 4A was much higher than in control per-
fusions. However, individual presentation of the experi-
ments of H
2
O
2
pretreated lungs (Figure 5A) show a similar
trend in all perfusions. Increased radioactivity in perfusate
was only detected beyond 60 minutes of perfusion. A sim-
ilar translocation versus time profile was observed in
lungs upon presence of 1 µM histamine in the vascular
perfusion fluid (Fig 4A). However, here statistical signifi-
cance in this condition versus control lungs was only
attained after 120 minutes of perfusion (Fig 4A), which is
best explained by the individual experiments shown in Fig
5B. On the other hand, in the lungs treated with a hista-
mine bolus injection no
192
Ir-UFP radioactivity was
detected in the perfusate.
Interestingly, the kinetics of translocation of DTPA (Fig.
4B) and Ir-UFP are very different. Whereas Ir-UFP only
starts to increase in perfusate after 60 min of inhalation,
DTPA is measured in perfusate within a few minutes after
intratracheal instillation. On the other hand the effects of
H
2
O
2
and vascular histamine on particle translocation are
also reflected in the DTPA -clearance (Fig. 4B). Although
not significant, both treatments caused trends of a higher
rate of translocation of DTPA one hour after administra-
tion, which is also observed for translocation of
192
Ir-UFP.
The histamine bolus injection, with a final target concen-
tration in the lumen of 0.5 mM caused a considerable
slowing-down of DTPA permeability (Fig 4B) and no
observed effects on
192
Ir-UFP translocation.
Biomarkers of epithelial and endothelial damage
Alkaline phosphatase (ALP) was measured in control and
pre-treated lungs as a marker of type II cell damage. No
significant differences in ALP activity (15–135 minutes)
were observed between perfusate of the control IPL and
H
2
O
2
pre-treated lungs after exposure to
192
Ir-UFP (Fig
6A). In the IPL perfused with vascular histamine a signifi-
cantly lower activity of ALP was seen at 15 and 30 minutes
in comparison to lung perfusions that only received
192
Ir-
UFP by inhalation. At all later time points ALP showed no
difference to control lungs (Fig 6A). The histamine bolus
group did also not differ from control group. To evaluate
endothelial damage, angiotensin converting enzyme
(ACE) was measured in the lung perfusate (Figure 6B). No
significant differences were observed in ACE activity
between the control group and isolated lungs treated with
H
2
O
2
, histamine in perfusate or histamine delivered as a
bolus in the trachea (ANOVA, post-hoc Tukey and Mann
Whitney-test). To check whether the H
2
O
2
effect was
mediated by histamine release we measured histamine in
the perfusate but did not detect significant differences to
histamine levels in control perfusions (data not shown).
Also a measure of total protein for lung permeability did
not detect differences between the different treatments
used in these experiments.
Morphology of lungs
The outcomes of the histo-pathological analyses are sum-
marised in Table 1 and illustrated in Fig. 7. Overall there
is quite extensive damage at the end of the perfusion
experiments, but there are not many differences between
the different treatments. Sub-epithelial round cell infiltra-
tion, interstitial dilation along with moderate to severe
oedema was present in all the groups. Occasionally alveo-
lar dilation, alveolar inflammation and fluid in the alveo-
lar lumen were detected. The in vitro perfusion procedure
might be responsible for the perivascular and peribron-
chial dilation in all the lungs. H
2
O
2
might be directly
Table 1: Morphological evaluation of the rat lungs after 2 hours perfusion and inhalation exposure to Ir-UFP, using lung sections and
HE- or toluidine blue staining The regions investigated included the bronchial segment, the blood vessels and the alveolar region
Treatment Bronchial region Blood vessels Alveolar region
Control Sub epithelial round cell filtration;
interstitial dilation
Interstitial dilation Small alveolar dilation in periphery
Hydrogen peroxide Desquamation of epithelial layer,
partially destruction of sub epithelial
structure
Moderate to severe dilation
Histamine Perfusate Sub epithelial round cell filtration;
interstitial dilation
Moderate to severe interstitial
dilation
Alveolar dilation in periphery
Histamine (Bolus) Sub epithelial round cell filtration;
interstitial dilation
Moderate to severe interstitial
dilation
Focal alveolar dilation, oedematous
fluid in alveolar lumen
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responsible for the epithelial damage of the proximal
bronchi in the H
2
O
2
instilled group.
Discussion
The purpose of our study was to evaluate the role of epi-
thelial and endothelial barrier in the translocation of
ultrafine particles across the lung into the systemic circu-
lation using the isolated perfused lung model. Transloca-
tion of Iridium (Ir)particles was monitored by
radioactivity of the particles themselves, and not by any
attached radioactive label. No translocation of
192
Ir-UFP
(17–20 nm) was detected in isolated perfused rat lungs.
However lungs pre-treated in-situ with histamine on the
endothelial side (1 µM) or H
2
O
2
(0.5 mM) in the alveolar
lumen showed small amounts of radioactivity in the sin-
gle pass perfusate after a lag-time of 60 min. Although
Translocation of
192
Ir-UFP particles (upper panel) and
99m
Tc-DTPA (lower panel) into perfusate of isolated perfused rat lung as a fraction of the deposited dose for inhaled particles and as fraction of the instilled dose for DTPAFigure 4
Translocation of
192
Ir-UFP particles (upper panel) and
99m
Tc-
DTPA (lower panel) into perfusate of isolated perfused rat
lung as a fraction of the deposited dose for inhaled particles
and as fraction of the instilled dose for DTPA. Both control
lungs (●) were used as well as lungs treated in-situ with
H
2
O
2
bolus 0.5 mM (O), histamine (1 µM) in perfusate (䊐)
or 0,5 mM instilled into the lungs (■). A stabilisation period
of 15 minutes is done before treatment and collection of
samples are taken place every 15 minutes for translocation
and lung markers detection. Values in the upper panel depict
the mean and SD's of 3 or 4 experiments; values in the lower
panel depict only the mean of 3 or 4 experiments indicating
the trend.
0
0,2
0,4
0,6
0,8
1
15 30 45 60 75 90 105 120
Time (min)
Cumulative DTPA-Tc-99m.
B
-0,01
-0,005
0
0,005
0,01
0,015
0,02
0,025
0,03
15 30 45 60 75 90 105 120 135
Time (min)
Relative radioactivy
A
Translocation of Iridium particles in individual perfusions after treatment with (A) H
2
O
2
(bolus) and (B) histamine (10 µM) in the perfusateFigure 5
Translocation of Iridium particles in individual perfusions
after treatment with (A) H
2
O
2
(bolus) and (B) histamine (10
µM) in the perfusate. Translocation represented as relative
radioactivity of
192
Ir in the perfusate. Data shown for individ-
ual heart-lung-blocs.
HISTAMINE
-0,01
0
0,01
0,02
0,03
0,04
0,05
0,06
15 30 45 60 75 90 105 120 135
time
Rel Radioactivity in perfusion
medium
H1 H2 H3
Peroxide Instilled
-0,01
0
0,01
0,02
0,03
0,04
0,05
15 30 45 60 75 90 105 120 135
time
Rel. Radioactivity in perfusion
medium
PI1 PI2 PI3
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kinetics of DTPA and particle translocation were different
the in-situ treatments histamine and H
2
O
2
caused unidi-
rectional in both processes, in the absence of biochemical
evidence for epithelial and endothelial damage.
In this study we applied several ex-vivo treatments of the
isolated lungs with either H
2
O
2
or histamine. Using H
2
O
2
we anticipated inducing an oxidative stress, which is also
caused by PM inhalation in the lung both by direct radical
formation by PM constituents and indirectly by recruited
inflammatory cells (review: [34]). Oxidative stress has
been forwarded as a central hypothetical mechanism in
the adverse effects of PM, including ultrafine particles
[35]. Actually, the oxidative capacity of PM was shown by
us to be a predictor of bronchial inflammatory response
to PM after installation in normal human volunteers [26].
Earlier on Rhaman et al [36] forwarded that oxidative
stress and depletion of GSH can affect lung permeability
allowing for greater particle passage via lung epithelium
into the interstitium. This concept is supported by our
data using a high concentration of H
2
O
2
(5 mM) by bolus
injection into the lung, to reach a final concentration of
0.5 mM. In fact, lung-lining fluid of COPD patients has
been shown to contain levels of H
2
O
2
up to 5 µM [37].
Although this model does certainly not meet all condi-
tions of an inflammatory response, similar models have
been applied in other ex-vivo permeability studies
[38,39]. In isolated perfused rat lungs, a low concentra-
tion of H
2
O
2
(0.25 mM) in the perfusate was shown to
increase capillary permeability in the absence of lipid per-
oxidation [38]. A short-term treatment with H
2
O
2
(100
mM) on the epithelium of human airway tubes caused a
six-fold increase in translocation of
111
In-DTPA, which
was explained by the opening of paracellular pathways
[39]. In our study, we assume that a final luminal concen-
tration of 0.5 mM H
2
O
2
is reached and we found an
increased translocation of both
192
Ir-UFP as well as a trend
of increased translocation of
99m
Tc-DTPA after a lag-time
of about 60 min. However, no temporal relationship
between both markers of translocation was seen, which
suggest that they operate through different routes.
More data on the translocation route of
192
Ir-UFP in the
lungs are given by the results obtained with histamine
administered both on the luminal side and through the
microvasculature. These data show that histamine at very
low levels in the perfusate (1 µM) caused an increased
translocation of particles as well as
99m
Tc-DTPA-permea-
tion after a lag-time of about 60 min (Fig 4). On the other
hand luminal administration of histamine (0.5 mM) did
not increase
192
Ir-UFP translocation and actually showed
a slightly reduced
99m
Tc-DTPA permeation. Histamine is a
very potent vasoactive and bronchial mediator that has
been shown to be involved in both local and systemic
effects of diesel particles [40]. First, upon mast cell degran-
ulation histamine is the major mediator of bronchial con-
striction as observed in allergic airway response [41]. This
constriction probably also explains our reduction in
99m
Tc-DTPA clearance after a histamine bolus injection
into the trachea. The approach using vascular histamine is
relevant because histamine has been shown to increase
after instillation of particles in isolated tracheally perfused
rabbit lung ex-vivo (Nemmar et al, 1999)[42], hamsters in
vivo (Nemmar et al, 2003)[11] and healthy human volun-
teers (Salvi et al, 1999)[43]. We assume that histamine at
low concentrations (10
-6
M) in our system increases
endothelial permeability [44] and allows an increase in
intercellular transport of
192
Ir-UFP located in the intersti-
tium through the endothelium into the perfusate. A simi-
lar effect of 10
-4
M vascular histamine was found recently
in isolated perfused rabbit lungs (Nemmar et al,
2005)[45]. In the latter study latex particles (24–190 nm)
translocated from the vascular compartment into the
lumen, as observed by subsequent bronchoalveolar lav-
age. The amount of reverse translocation was about 2.5 %
of administered dose within 2 hrs of perfusion. No trans-
location of latex particles with different size (24–190 nm)
and surface chemistry (carboxylate versus amine) and
charge were seen under normal physiological conditions
in the rabbit lungs. In our studies 18 nm iridium particles
also did not translocate through rat lung barriers,
although we used negative pressure ventilation which
caused considerable damage to the lung tissue.
Taken together these findings suggest that the transloca-
tion of ultrafine particles in the lung occur through differ-
ent routes. Among different uptake routes we can
discriminate transcytosis and para(inter)-cellular trans-
port. Hermans et al [46] stated that radiolabelled tracers
such as
99m
Tc-labelled DTPA permeate the epithelial bar-
rier by passing through intercellular junctions. Since
kinetics of DTPA and
192
Ir-UFP translocation in untreated
lungs is so different, we suggest that
192
Ir-UFP are translo-
cated along different pathways. In fact, ultrafine particles
may use different transcytotic pathways such as clathrin-
coated pits, pinocytosis and non-coated pits, called cave-
olae [47]. Caveolae are the most likely route of uptake for
the
192
Ir-UFP used in our study (18 nm) as derived from
studies by Gumbleton [47]. The alveolar epithelial cells,
comprising 95 % of the lung surface have a cell thickness
of 400 nm and about 600.000 to 900.000 caveolae that
are 50–60 nm wide. Such a high number of possible trans-
port units lead to assumption that NP smaller than 60 nm
can be rapidly taken up and transported through the epi-
thelial cell layer. Recently Kato et al (2003)[48] showed
that lecithin-coated polystyrene latex beads (240 nm) got
incorporated into the Type I and II alveolar epithelial cells
as well as in the capillary lumen. It was suggested that
these latex beads move from the alveolar epithelial cells to
the capillary lumen via transcytosis. Kapp et al (2004)[49]
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found Ti0
2
(29 nm) particles as intracellular clusters form-
ing needle shape particles or rounded shape particles. This
may also explain the relatively low and variable transloca-
tion as observed in other, previous in vivo studies [15-18].
We therefore used histamine to modify or facilitate trans-
endothelial passage and indeed found that this enhanced
translocation of
192
Ir-UFP to the perfusate. However, we
cannot discriminate between transcytosis and para-cellu-
lar transport in endothelium. We assume that the long
lag-time (60 min) detected which is needed before pas-
sage is caused by the fact that an interstitial load has to be
built by epithelial passage of
192
Ir-UFP in the lung. The
methods used in our study and the study by Nemmar et al
(2005)[45] do not allow to evaluate whether transloca-
tion has occurred through primary particles or by aggre-
gates. The inhalation in our study assured single UFP
deposition in the alveolar region and virtually no agglom-
eration on the epithelium because of the alveolar surface
and the number of deposited particles. However, upon
vascular injection aggregates are formed, unless surface
modifications are used that impede this process. It may
therefore be that translocation observed in Nemmar's
study occurs as aggregates by above mechanisms, or
facilitation by phagocytic cells but not in our study. With
this respect the studies by Heckel et al (2004)[50] have
demonstrated by TEM that 4 nm gold-particles really pass
membranes and reach the lumen as single particles. The
difference between 4 and 20 nm particles may however be
huge since 4 nm AU-particles are not recognized by the
reticulo-endothelial system.
Our findings may be criticized due to a number of factors
that are associated to our experimental design and per-
formance. First, it must be taken into account that isolated
and perfused lungs are not under physiological condi-
tions since, for example, lymph flow is altered, bronchial
perfusion is suppressed, autonomic innervation is discon-
nected and no blood cells (including inflammatory cells)
are present in the perfusate. However the artificial nega-
tive pressure perfusion of isolated lung resembles respira-
tory conditions and the time span of experiments was
limited up to 2 hours maximum to avoid excessive
decrease of function. Knowing this, the lack of concomi-
tant physiological measurement in the negative pressure
ventilation is a major shortcoming in our data. The obvi-
ous reason for this is that given the use of radioactive
192
Ir-
UFP inclusion of measurement devices was allowed for
radiation protection safety reasons, since they could lead
to an open system and radioactive particle emissions. We
have tried to compensate for this lack of know-how by
measurement of biochemical indices of damage in per-
fusate and performing histology in lung after the experi-
ment. No evidence for extensive lung damage in control
conditions or after in-situ treatment to the essential barri-
ers of the lung was noted. The release of ALP and ACE as
biomarkers of epithelial and endothelial integrity are not
elevated during the perfusion and do not correspond to
the small translocation of
192
Ir after a lag-time of about 60
min -UFP. Although microscopical analysis in lung sec-
tions showed desquamation of the epithelial layer in the
lungs treated in situ with hydrogen peroxide, ALP levels in
perfusate were not different from that in the control
group. The experimental conditions also did not affect the
Release of Alkaline phosphatase (A) and Angiotensin con-verting enzyme (B) measured in lung perfusate during and after particle inhalation and different pre-treatmentsFigure 6
Release of Alkaline phosphatase (A) and Angiotensin con-
verting enzyme (B) measured in lung perfusate during and
after particle inhalation and different pre-treatments. Both
control lungs (●) were used as well as lungs treated with
H
2
O
2
(O), histamine (10 µM) in perfusate (䊐) or injected
into the lungs (■). Values depict the mean of 3 or 4
experiments.
0,0
1,0
2,0
3,0
4,0
5,0
6,0
7,0
8,0
9,0
10,0
15 30 45 60 75 90 105 120 135
Time (min)
ALP (U/ml)
0,0
1,0
2,0
3,0
4,0
5,0
6,0
7,0
8,0
9,0
10,0
15 30 45 60 75 90 105 120 135
Time (min)
ACE (U/ml)
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integrity of the endothelial layer since perfusate levels of
ACE did not change during perfusion. However, in most
lungs oedema was noted in microscopy as interstitial dila-
tion (Table 1) at the end of the experiment. The formation
of oedema is due to the imbalance between fluid transvas-
cular filtration and clearance. Also the excess fluid causes
an overhydration of the interstitium associated with accu-
mulation of oedema fluid in the loose connective tissue
[51]. It could be argued that oedema might affect the
translocation of
192
Ir-UFP from the lumen via the intersti-
tium across the endothelium. In fact, a recent study using
iv injection of colloidal gold particles (4 nm) in rabbits,
showed that a small but significant percentage (7 %) of
the UFP was taken up in endothelial and epithelial cells of
the lung [50]. After LPS infusion, causing mild pulmonary
oedema, transendothelial transport was boosted five-fold,
while a significant amount of gold particles accumulated
in the interstitium (14 %) and even reached the alveoli
(11 %). Although this suggests a potential interference for
oedema in our study, one should realize that in the above
study [50] oedema was present at the very beginning and
translocation is followed in a different direction. Never-
theless an effect of oedema on particle translocation is
considered unlikely in our experimental setup since no
192
Ir-UFP translocation was observed in the control
groups and after a histamine bolus for up to 2 hours after
onset of inhalation.
The minute translocation of
192
Ir-UFP in the isolated lung
perfusion system conforms to our previous in vivo find-
ings [15] using the same particles by inhalation at similar
dose in rats. Other studies however reported very different
amounts and kinetics of translocation. Nemmar et al [16]
studied particle translocation after intratracheal instilla-
tion of uF particles in hamsters in vivo and observed a
rapid (3 % within 5 min) translocation of 80 nm albumin
particles coated with
99m
Tc but no translocation was
observed after the 15 minutes. On the other hand, the
same group could not find latex (24–190 nm) particle
translocation isolated perfused rabbit lungs at positive
pressure [45]. Surface chemistry did not affect this proc-
ess. In a similar approach using latex fluorescent beads
and positive pressure ventilation, we also did not find par-
ticle translocation (data not shown). In contrast Brooking
et al [48] showed a continuous increase in translocation
with time up to 180 minutes during nasal inhalation of
latex particles between 50 and 250 nm by rats. The latter
study highlighted the importance of particle size as the
smallest ultrafine particles showed higher uptake rates
than the larger particles. In addition they demonstrated
that particle surface chemistry was an important character-
istic [52]. Oberdorster et al [18] reported quite extensive
(> 20 %) translocation of uF carbon particles (18 nm)
after short-term inhalation. Whatever the mechanism or
particle properties involved in passing the lung barriers,
the question remains what particle translocation means in
terms of systemic effects. The mainstream hypothesis is
that lung inflammation causes and facilitates the release
of mediators that adversely affect cardiovascular parame-
ters [3]. Alternatively, translocation of particles to the
brain (Oberdorster et al, 2004)[53] or the systemic circu-
lation may also explain effects of PM exposure on heart
and vascular tissue. The blood that leaves the lung first
enters the heart before it is pumped to the other organs. In
our previous work we showed that suspensions or filtrates
of PM
10
could have direct effects on vessels [9]. However,
the effects were rather due to the soluble components (i.e.
transition metals) than to particles themselves [9] and are
in contrast to in vivo findings on endothelial function with
PM [54,10] 2004).
Although these data do not allow quantitative conclu-
sions on the exact mechanism and the importance of sys-
Examples of histopathological lesions encountered in lungs after 2 hour perfusion and inhalation of non-radioactive
192
Ir-UFP in control lungs (A), lungs pretreated with a bolus of H2O2 in the lumen (B, D), showing septum and bronchus reduction, and (C) lung perfused with histamine, showing oedema and infiltrationFigure 7
Examples of histopathological lesions encountered in lungs
after 2 hour perfusion and inhalation of non-radioactive
192
Ir-
UFP in control lungs (A), lungs pretreated with a bolus of
H2O2 in the lumen (B, D), showing septum and bronchus
reduction, and (C) lung perfused with histamine, showing
oedema and infiltration.
A
LP- Ir- No pretreatment
LP- Ir- Histamine (perfusate)
oedema + infiltration
Ir- H2O2-pretreatment
septum reduction
LP- Ir- No pretreatment
Ir- H2O2-pretreatment
bronchus reduction
B
D
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Page 12 of 13
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temic translocation of UFP as a mechanism in adverse
effects of PM, we do confirm that ultrafine particles can
translocate from the lung into the circulation using the
isolated perfused rat lung upon pharmacological media-
tion. Permeability of the lung barrier to ultrafine particles
seems to be controlled both at the epithelial and endothe-
lial level and conditions that affect this barrier function
such as inflammation may affect translocation of UFP.
The conditions under which this does occur mimic condi-
tions that are met in diseased, susceptible subjects includ-
ing asthmatics and COPD-patients.
Acknowledgements
This study is supported by the German Ministry of Environment (BMU) and
the Baden-Württemberg Environmental research program BW Plus,
project number 20018. We thank dr Doris Hoehr and Erich Jermann for
technical assistance during the start-up of the project, and dr Roel Schins
for his valuable suggestions during the completion of the study.
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