J Occup Health 2010; 52: 48–57

#Ge MENG and Jian ZHAO made equal contribution to the work.Received Mar 18, 2009; Accepted Nov 4, 2009Published online in J-STAGE Dec 25, 2009Correspondence to: R.-G. Ding, Institute of Pharmacology andToxicology, Academy of Military Medical Sciences, 27 TaipingRoad, Beijing 100850, P.R. China(e-mail: dingrigao@nic.bmi.ac.cn)

Journal ofOccupational Health

Cell Injuries of the Blood-Air Barrier in Acute Lung Injury Causedby Perfluoroisobutylene Exposure

Ge MENG#, Jian ZHAO#, He-Mei WANG, Ri-Gao DING, Xian-Cheng ZHANG,Chun-Qian HUANG and Jin-Xiu RUAN

Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, P.R. China

Abstract: Cell Injuries of the Blood-Air Barrier inAcute Lung Injury Caused by PerfluoroisobutyleneExposure: Ge MENG, et al. Institute of Pharmacologyand Toxicology, Academy of Military MedicalSciences, PR China—Objectives: To investigate thecomplete process of cell injuries in the blood-air barrierafter perfluoroisobutylene (PFIB) exposure. Methods:Rats were exposed to PFIB (140 mg/m3) for 5 min. Thepathological changes were evaluated by lung wet-to-dry weight ratio, total protein concentration ofbronchoalveolar lavage f lu id and HE sta in.Ultrastructural changes were observed by transmissionelectron microscope. Apoptosis was detected by insitu apoptosis detection. Changes of actin in the lungtissue were evaluated by western blot assay. Results:No significant pulmonary edema or increasedpermeability was observed within the first 4 h, post PFIBexposure. However, inflammatory cell infiltration andalveolar wall thickening were observed from 2 h.Destruction of the alveoli constitution integrity, edemaand protein leakage were observed at 8 h. The injuriesculminated at 24 h and then recovered gradually. Theultrastructural injuries of alveolar type I epithelial cells,alveolar type II epithelial cells and pulmonarymicrovascular endothelial cells were observed at 30min post PFIB exposure. Some injuries were similarto apoptosis. Compared with control, more seriousinjuries were observed in PFIB-exposed rats after 30min. At 8 h, some signs of cell necrosis were observed.The injuries culminated at 24 h and then ameliorated.The number of apoptotic cells abnormally increased at30 min post PFIB exposure, the maximum appearedat 24 h, and then ameliorated gradually. Western blotanalysis revealed that the level of actin in the lung

showed no significant changes within the first 4 h postPFIB exposure. However, it decreased at 8 h, reacheda nadir at 24 h, and then recovered gradually.Conclusions: The pathological processes were inprogress persistently post PFIB exposure. The earlyinjuries probably were the result of the direct attack ofPFIB and the advanced injuries probably arose fromthe inflammatory reaction induced by PFIB.(J Occup Health 2010; 52: 48–57)

Key words: Acute lung injury, Alveolar epithelial cell,Apoptosis, Perf luoroisobutylene, Pulmonarymicrovascular endothelial cell

Dysfunction of the blood-air barrier has recently beenidentified as a focus for the study of acute lung injury(ALI) induced by lipopolysaccharide (LPS), plateletactivating factor (PAF), thrombin, ischemia andreperfusion and mechanical ventilation etc1–5). The blood-air barrier is composed of the pulmonary alveoli epithelialbarrier, the extracellular matrix and the pulmonarymicrovascular endothelium barrier. It is responsible forgas exchange between the circulatory system and thealveolar space. Alveolar type I epithelial cells (AT-I),alveolar type II epithelial cells (AT-II) and pulmonarymicrovascular endothelila cells (PMVEC) are the maincells of the blood-air barrier6–8). It is well-known thatincreased permeability of the blood-air barrier in ALI, inwhich fluid, protein, macrophage and leukocytes moveinto the alveolar air space through the interstitial space,is the general basis for the formation of the pulmonaryedema9, 10). In some cases of severe ALI, patients die inthe early stage of pulmonary edema. If the injuries ofthe blood-air barrier are not repaired quickly, pulmonaryfibrosis eventually forms, and it is the most seriouscomplication in the advanced ALI11, 12). Along with thechanges of structure and function, if left uncontrolled,ALI will result in excessive apoptosis or necrosis13–15).

Among the ALI models, the toxic gas inhalation modelhas been extensively used by researchers in recent years,

49Ge MENG, et al.: PFIB-Induced Impairments of Lung Blood-Air Barrier

because this model is common in chemical accidents andresults in rapid ALI. Perfluoroisobutylene (PFIB), anewly discovered chemical (chemical structure:(F

3C)

2=CF

2), is a potential pneumoedematogenic gas. It

is usually produced as the main by-product in thefluoropolymer industry and is a hazard to human beingsin chemical industry accidents, fire disasters and otheremergencies. PFIB inhalation can cause acute lung injuryor acute pulmonary edema, and even death16, 17).Unfortunately, no specific antidote or successfultherapeutic measures are currently available. Themechanisms of ALI induced by PFIB remain largelyunclear. Findings of other laboratories and ours haves h o w n t h a t a g g r e g a t i o n , s e q u e s t r a t i o n o fpolymorphonuclear leukocytes (PMN) and excessiveinflammatory reaction are the decisive factors 18–21). Theblood-air barrier is probably the main target of attack inALI induced by PFIB. Moreover, because of smallermolecular weight and stronger electrophilicity, PFIB candirectly attack the blood-air barrier in the early periodpost PFIB exposure, however literature on the changesof the blood-air barrier post PFIB exposure are rare. Inthe current paper, we will describe the complete processof cell injuries in the blood-air barrier post PFIB exposure.

Materials and Methods

General1) AnimalsEighty specific pathogen-free male Wistar rats (180–

220 g, 8 wk old) were used in this study. They were allobtained from the Center of Medical ExperimentalAnimals, the Academy of Military Medical Sciences(Beijing, P. R. China). The animals were housed in quiet,humidified, clean rooms with a light-cycle of 12 h/12 hfor 1 wk before use. They were fed with laboratory pelletfood and tap water ad libitum except when they were inthe exposure chamber. All the animal experiments wereperformed in accordance with the Guidelines for AnimalExperiments of the Chinese Academy of MedicalSciences, Beijing, P. R. China.

2) Doses of PFIB for animal exposurePFIB was obtained from the Shanghai Institute of

Organic Fluorine Materials at a purity of 98%. A head-nose exposure apparatus was used to expose rats to PFIB,as previously described17). The PFIB exposure dose was140 mg/m3 × 5 min.

3) Treatment of specimensThe rats were randomly divided into a control group

and PFIB exposure groups (30 min, 1 h, 2 h, 4 h, 8 h, 16h, 24 h, 48 h and 72 h post PFIB exposure) with 8 rats ineach group. At sacrifice, they were anesthetized by 25%ethylcarbamate (0.5 ml/100 g bw, i.p.) and exsanguinatedvia abdominal aorta transection. The right lungs were

clamped by hemostatic forceps. Each animal’s tracheawas cannulated with a blunt needle secured with silkligature. Bronchoalveolar lavage fluid (BALF) collectionwas performed with 5 ml phosphate buffered saline (PBS)that was infused and aspirated in the left lung 3 times.The lavage fluid was recovered (average fluid recoverywas 4 ml), and centrifuged (1,000 g, 4°C) for 10 min.The supernatants were removed and stored at –70°C untilthe total protein concentrations were assayed by themethod of Lowry22).

A part of the lobus apicalis of the right lungs wereused for ultrastructural studies, the rest were stored inliquid nitrogen and used for western blot analysis. Thelobus cardiacus was used for paraffin sections, the lobusdiaphragmaticus for assay of the lung wet-to-dry weightratio and the accessory lobus for frozen sections. Theparaffin sections were used for conventionalpathohistological studies and in situ apoptosis detectionof lung tissue.

Experimental methods1) Assay of the lung wet-to-dry weight ratio and the

total protein concentration of the BALF assayThe assay methods were described in our previous

paper19), and are described here in briefly. Assay ofthe lung wet - to -dry weight ra t io : the lobusdiaphragmaticus was weighed to obtain the wet weightthen dried in an oven at 80°C for 24 h to obtain thedry weight and the lung wet-to-dry weight ratio wascalculated as: mass

wet lung

/massdry lung

. The total proteinconcentration of BALF assay: 0.1 ml BALF was addedto 1.9 ml Na

2SO

4 (22.2%), and 0.5 ml of the mixture

was collected after being vortexed. Then, 9 ml NaOH(0.5%) was added to each sample, and 0.3 ml Folinphenol was added after standing for 30 min. Finallythe OD value was recorded at 500 nm after 10 min using0.5 ml tyrosine (0.2 mg/ml) as the standard. Totalprotein concention was calculated as: C

total protein (g/100

ml) = (ODtotal

protein sample

/ODstandard

) × 6.4. The increasedlung wet-to-dry weight ratio was used as an indicatorof pulmonary edema and the increased total proteinconcentration was used as an indicator of increasedpermeability of the blood-air barrier.

2) Hematoxylin and eosin (HE) staining of rat lungslices

The rat lungs fixed in 10% formalin solution weresectioned (4 µm) after dehydration, cleaning and paraffinembedding. The sections were flattened, pasted andheated on blank glass slides. Histological evaluationswere performed with HE staining and pathologicalexamination.

3) Ultrastructural studiesThe rat lungs were submerged in cold fixative overnight

50 J Occup Health, Vol. 52, 2010

then cut into 1- to 2-mm cubes. The lung fragments werepost-fixed in 1% buffered osmium tetraoxide (pH 7.2)and embedded in Poly-bed 812 epoxy resin. Semithinepoxy sections, 1 µm thick, differentiated with methyleneblue, azure II, and basic fuchsin, were used to select areasfor ultrathin sectioning under light microscopy. Ultrathinsections, 60–90 nm thick, were nonspecifically stainedwith uranyl acetate and lead citrate. Electronphotomicrographs were taken using a Philips CM 120transmission electron microscope at 80 kv.

4) In situ apoptosis detection of lung tissueThe Apoptag® Plus Peroxidase In Situ Apoptosis

Detection Kit was used to evaluate the apoptosis of lungtissue post PFIB exposure. The paraffin sections werewashed with dimethyl benzene, incubated with proteinaseK (20 mg/ml, 15 min) and 3% hydrogen peroxide (5 min),then treated with TdT Enzyme (1 h, 37°C), anti-digoxigenin conjugate (30 min), peroxidase substrate (3–6 min), 0.5% methyl green (10 min), followed bymounting. Same size areas were observed. The apoptosisratio was calculated as: the number of apoptotic cells/total cells × 100%.

5) Western blot analysisTissue specimens were pulverized in liquid nitrogen

and homogenized in ice-cold lysate buffer containing acocktail of protease inhibitors (Complete, EDTA-free:Roche, Mannheim, Germany). The homogenates werecentrifuged at 15,000 g for 1 h at 4°C and the proteinwas extracted and stored in liquid nitrogen untilelectrophoresis. Protein concentrations of the extractswere determined by the Bradford assay. Ten-microgramsamples were diluted with 2 × loading buffer (50 mmol/lTris-Cl, 50 mmol/l dithiothreitol (DTT), 2% sodiumdodecyl sulphate (SDS), 0.1% bromphenol blue and 10%glycerol) and heated in boiling water for 5 min. Theproteins were separated by 10% SDS-polyacrylamide gelelectrophoresis (PAGE), then transferred to anitrocellulose membrane. The membrane was washedfor 10 min in Tween-20 Tris buffered saline (TTBS) [20mmol/l Tris-HCl (pH 7.5), 0.15 mol/l NaCl, 0.05%Tween-20], then blocked with 10% skimmed milk for 1h at room temperature. The blocked membrane wasincubated with a primary antibody against actin (dilution1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA)for 1 h at 4°C. After incubation with the secondaryantibody (horseradish peroxidase-conjugated goat anti-rabbit IgG, dilution 1:5000; Beijing Zhong Shan-GoldenBridge Biological Technology Company, Beijing, China)for 1 h at room temperature, its was stained by ECLchemiluminescence (Amersham Life Science, ArlingtonHeights, IL). This experiment was repeated three timesusing separate samples.

6) Statistical analysisData are shown as the Mean ± SD. One-way analysis

of variance followed by Dunnett’s test was used to detectdifferences between groups unless otherwise indicated.

Results

Changes of lung wet-to-dry weight ratio and total proteinconcentration of BALF post PFIB exposure

The lung wet-to-dry weight ratio and the total proteinconcentration of BALF remained unchanged within thefirst 4 h (30 min, 1 h, 2 h and 4 h) post PFIB exposure.They increased significantly at 8 h, peaked at 24 h, anddeclined thereafter. Figure 1 shows the clinical courseof lung injury by PFIB, which included periods ofstimulation, latency, pulmonary edema and convalescence(Fig. 1).

Lung histopathological changes post FPIB exposureNo significant pathological changes were found in HE

stained lung slices within the first 1 h post PFIB exposure.However, inflammatory cell infiltration and alveolar wallthickening were observed from 2 h, and destruction ofthe alveoli constitution integrity and the protein leakagewere observed at 8 h. The most serious injuries wereobserved at 24 h, and gradual recovery ensued thereafter(Fig. 2).

Ultrastructural changes of the blood-air barrier postFPIB exposure

The normal AT-I is a kind of squamous flat cell with asmall and compact nucleus and a thin cytoplasm, in whichorganelles are rare, and mitochondrion and roughendoplasmic reticulum are occasionally observed. A fewthick short microvilli are observed on the dissociativeside of the cell, and connect tightly to AT-II. The earliestrecognized morphologic changes were compaction andsegregation of the nuclear chromatin, uniform finegranular masses that became localized on the nuclearenvelope, condensation of the cytoplasm, and swollenmitochondria at 30 min post PFIB exposure. All theseobservations indicate apoptosis. Subsequently, moreserious nuclear shrinkage, chromatin marginalization,split nucleolus, double membranes of mitochondria whichhad been destroyed, swollen, even broken cristae ofmitochondria and a great number of vacuoli in cytoplasmwere observed in temporal order of description. At 8 h,some characters of necrosis were observed: broken cellsand fragments appeared in tissues. All the above-mentioned pathological changes culminated at 24 h. After24 h, the symptoms were ameliorated (Fig. 3).

The normal AT-II is cubic, and contains abundantorganelles. The lamellar body, which can be dyed blackby osmic acid, is the most significant diagnosticcharacteristic of AT-II. The microvilli of AT-II began tofall off at 30 min post PFIB exposure. Later, microvilli

51Ge MENG, et al.: PFIB-Induced Impairments of Lung Blood-Air Barrier

fell off more in greater numbers. The most dramaticchanges were observed at 24 h, followed by a gradualamelioration. The pathological changes of nuclearchromatin and mitochondria of AT-II were similar to thoseof AT-I (Fig. 4).

The normal PMVEC is flat-like and contains variousorganelles. Swollen basal laminae were observed at 30min post PFIB exposure. Later, broken basal laminaewere observed. The maximum changes were observedat 24 h, followed by a gradual amelioration. Thepathological changes of nuclear chromatin andmitochondria pathological changes of PMVEC weresimilar to those of AT-I and AT-II (Fig. 5).

Apoptosis of lung tissue post PFIB exposureThe number of apoptotic cells at 30 min post PFIB

exposure exceeded normal. The number significantlyincreased at 8 h vs that within the first 4 h post PFIBexposure. The greatest number of apoptotic cells wasseen after which they gradually declined in number (Fig.6).

Changes of actin in the blood-air barrier post FPIBexposure

Western blot analysis revealed that the level of actinin the blood-air barrier had no significant changes withinthe first 4 h post PFIB exposure. However, it decreasedat 8 h, and the minimum level was seen at 24 h, andthereafter gradually recovered (Fig. 7).

Discussion

In this paper, we focused on the complete process of

Fig. 1. The lung wet-to-dry weight ratio and the total protein concentration of rats at different times pre- and post-PFIB (140 mg/m3 × 5 min) exposure. A: lung wet-to-dry weight ratio; B: the total protein concentration.Mean ± SD, N=8, **: p

52 J Occup Health, Vol. 52, 2010

cell injuries in the blood-air barrier post PFIB exposure.No significant pulmonary edema or increasedpermeability were found within the first 4 h post PFIBexposure. However slight inflammatory cell infiltrationand alveolar wall thickening were observed at 2 h in HEstained lung slices and ultrastructural injuries of theblood-air barrier were observed at 30 min. Pulmonary

edema and increased permeability were observed at 8 h,peaked at 24 h, and then recovered. These results indicatethat the pathological process develops persistently postPFIB exposure, although there was no significantpulmonary edema during the early period of PFIBexposure. The early period is known as the latency periodin the clinical stage and is easily overlooked.

Fig. 3. Representative electronmicroscope photomicrographs of AT-I from rats sacrificed at different times pre- and post- PFIB(140 mg/m3 × 5 min) exposure. The results from left to right are control, 30 min, 1 h, 2 h, 4 h, 8 h, 16 h, 24 h, 48 h, 72 hpost PFIB exposure. Nuclear chromatin compaction, segregation and localization on the nuclear envelope ( ), swollenmitochondrion ( ), destroyed double membranes ( ) and swollen or broken cristae ( ) were observed in temporal orderof description post PFIB exposure. The most serious injuries were observed at 24 h, with gradual recovery thereafter.

53Ge MENG, et al.: PFIB-Induced Impairments of Lung Blood-Air Barrier

Fig 4. Representative electronmicroscope photomicrographs of AT-II from rats sacrificed at different times pre- and post- PFIB(140 mg/m3 × 5 min) exposure. The results from left to right are control, 30 min, 1 h, 2 h, 4 h, 8 h, 16 h, 24 h, 48 h and 72h post PFIB exposure. Nuclear chromatin compaction, segregation and localization on the nuclear envelope ( ), swollen

mitochondrion ( ), destroyed double membranes ( ), swollen or broken cristae ( ) and fallen off microvilli ( ) wereobserved in temporal order of description post PFIB exposure. The most serious injuries were observed at 24 h, and werefollowed by gradual recovery.

54 J Occup Health, Vol. 52, 2010

Consequently, the best time for treatment is often missed,the reason being that injuries of AT-I, AT-II and PMVECin this period are, in fact, in progress according to theultrastructure results. Other research teams also observedthe infiltration of inflammatory cells under an electronmicroscope in this period23, 24). Therefore we suggest thatthe term “the latency period” should be substituted by“the early damage period” in order to describe early ALImore objectively and remind doctors to adopt propertherapeutic measures during this period.

The changes of ultrastructural injuries of the blood-airbarrier, which were observed under a transmissionelectron microscope as early as 30 min post PFIBexposure in the present study, were consistent with thoseof the lung wet-to-dry weight ratio, the total proteinconcentration of BALF and the HE stained lung slicesfrom 8 h post PFIB exposure. Accumulated dataconcerning this early damage of the blood-air barrierprobably indicate the direct effect of PFIB: theconcentration of fluorion in blood significantly increased

Fig. 5. (continue.)

55Ge MENG, et al.: PFIB-Induced Impairments of Lung Blood-Air Barrier

Fig. 5. Representative electronmicroscope photomicrographs of PMVEC from rats sacrificed at different time pre- and post- PFIB(140 mg/m3 × 5 min) exposure. The results from left to right are control, 30 min, 1 h, 2 h, 4 h, 8 h, 16 h, 24 h, 48 h and 72h post PFIB exposure. Nuclear chromatin compaction, segregation and localization on the nuclear envelope ( ), swollenmitochondrion ( ), destroyed double membranes ( ), swollen or broken cristae ( ) and swollen or broken basal lamina ( )were observed in temporal order of description post PFIB exposure. The most serious injuries were observed at 24 h, withgradual recovery thereafter.

Fig. 6-1. Light photomicrographs of paraffin-embedded andin situ apoptosis detection sections of representativelungs from rats sacrificed at different times pre- andpost- PFIB (140 mg/m3 × 5 min) exposure. Thewhite arrows point to apoptotic cells (the nuclei arestained brown). The results from left to right arecontrol, 30 min, 1 h, 2 h, 4 h, 8 h, 16 h, 24 h, 48 h, 72h post PFIB exposure.

Fig. 6-2. Apoptosis of rat lung tissue at different times pre-and post- PFIB (140 mg/m3 × 5 min) exposure. Theresults from left to right are control, 30 min, 1 h, 2 h,4 h, 8 h, 16 h, 24 h, 48 h, 72 h post PFIB exposure.Mean ± SD, N=8, **: p

56 J Occup Health, Vol. 52, 2010

at 0 h post PFIB exposure, and was still higher thannormal at 1 h post PFIB exposure25); the aggregation andsequestration of a great number of PMNs were observedat 1 h post PFIB exposure17); and the level of interleukin-1 (IL-1β) in sera of PFIB-exposed mice dramaticallyincreased at 30 min and reached its maximum at 1 h postPFIB exposure. The level of IL-8 in sera of PFIB-exposedmice climbed to a peak at 1 h, followed by a plateau till 4h post PFIB exposure18). The direct injuries probablyarise from PFIB itself. The halogen and p-π electronconjugation in the molecular structure of PFIB makes itvery electrophilic, allowing it to easily attack the thiol (-SH), amino (-NH

2) and hydroxy (-OH), nucleophilic

groups, which are widely distributed in the alveolarmembrane and are crucial to the maintenance of thecellular structure and function30). Moreover, as amicromolecular gas, PFIB can pass through the blood-air barrier easily to attack the epithelial barrier andendothelial barrier together. However, later injuries inALI due to PFIB exposure are probably attributable tothe inflammatory cascade or network of the inflammatorycells and their secretion substances26): the concentrationof fluorion in blood returned to normal levels at 1.5 hpost PFIB exposure25); and multiple mediators, especiallyPMN, probably became a key factor in the period. Elastaseand collagenase, which are important damage factors, weredelivered to attack the blood-air barrier27, 28); andmacrophages also probably participated in the process ofinjury in the later period17), but the details are still

Fig. 7. The changes of actin in the blood-air barrier atdifferent times pre- and post- PFIB (140 mg/m3 × 5min) exposure. The results from left to right arecontrol, 30 min, 1 h, 2 h, 4 h, 8 h, 16 h, 24 h, 48 h, 72 hpost PFIB exposure. Mean ± SD, N=3, *: p

57Ge MENG, et al.: PFIB-Induced Impairments of Lung Blood-Air Barrier

induced ALI calls for much more detailed clarification.

Acknowledgments: This work was supported by theNational Natural Science Foundation of China(30800904) and The National Key Technologies R&DProgram for New Drugs (2008ZX09305-003).

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