Journal of J Occup Health 2005; 47: 277Ð285 Occupational Health

The Prophylactic and Therapeutic Effects of Cholinolytics on Perfluoroisobutylene Inhalation Induced Acute Lung Injury

Tianhong ZHANG1, Xigang ZHANG2, Zhihua SHAO3, Rigao DING1, Shunjiang YANG3, Jinxiu RUAN1, Xiaohong SUN1, Jin XU3, Chunqian HUANG1, Zuliang HU3 and Xianchang ZHANG1

1Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, 2307 Hospital and 3Quhua Hospital, Quhua Group Corporation, P. R. China

Abstract: The Prophylactic and Therapeutic Effects cholinolytics might have prophylactic and therapeutic of Cholinolytics on Perfluoroisobutylene Inhalation roles in PFIB inhalation induced ALI. Induced Acute Lung Injury: Tianhong ZHANG, et al. (J Occup Health 2005; 47: 277–285) Institute of Pharmacology and Toxicology, Academy of Military Medical Sciences, P. R. China— Key words: Perfluoroisobutylene (PFIB), Acute lung Perfluoroisobutylene (PFIB) is a kind of fluoro-olefin injury (ALI), Cholinolytics, 3-Quinuclidinyl Benzilate that is ten times more toxic than . The (QNB), Anisodamine mechanisms of the acute lung injury (ALI) induced by PFIB inhalation remain unclear. To find possible Perfluoroisobutylene (PFIB), a colorless gas at normal pharmacological interventions, mice and rats were temperature, is a kind of fluoro-olefin that is ten times exposed to PFIB, and the prophylactic or therapeutic more toxic than phosgene1). It is usually generated as a effects of 3-quinuclidinyl benzilate (QNB) and by-product during the manufacture or the pyrolysis of anisodamine were studied and confirmed. It was (PTFE), which is widely known observed that the wet lung/body weight and the dry lung/body weight ratios at 24 h after PFIB exposure by its trade name Teflon. Accidental inhalation can cause (130 mg/m3 for 5 min) were significantly decreased severe pulmonary edema, and even death because of when a single dose of QNB (5 mg/kg) was administered pulmonary congestion. It is a notorious hazard to human intraperitoneally either 30 min before exposure or 10 h beings in chemical industry accidents, fires and other after exposure. Anisodamine was without any emergencies2). Although diverse mechanisms, such as prophylactic or therapeutic effects at single doses below hydrofluoric acid hypothesis3), nucleophilic reaction4) and 30 mg/kg. The effects of QNB against PFIB inhalation reactive intermediate species5) are supposed to be involved induced ALI were well evidenced by the significantly in PFIB inhalation induced acute lung injury (ALI), there decreased mice mortality at 72 h, the total protein is still a long way to go to fully clarify the underlying concentration in bronchoalveolar lavage fluid at 24 h toxic mechanisms of the ALI induced by PFIB inhalation. after the PFIB exposure, as well as the ultrastructural Furthermore, there is no recognized prophylactic observations. The analysis of the time courses of lung sulfhydryl concentration, myeloperoxidase (MPO) measures for human PFIB exposure although animal activity and hemorheology assay showed that the studies have indicated that increased pulmonary toxicity of PFIB may be due to consumption of lung concentrations of free radical scavengers containing thiol protein sulfhydryl, influx of polymorphonuclear groups may be of value and N-acetylcysteine has been leukocytes (PMNs) into the lung, and increased found to be effective6). Early administration of peripheral blood viscosity at a low shear rate, all of corticosteroids, as used for phosgene, seems to be the which were partially blocked by QNB intervention only clinical recommendation for pharmacological except for PMN influx. The results suggest that intervention even though proof of their beneficial effects is still lacking in general7). Received Dec 7, 2004; Accepted March 14, 2005 When comparisons are made concerning the molecular Correspondence to: R. Ding, Institute of Pharmacology and structure8, 9), the time course of ALI after the inhalation Toxicology, Academy of Military Medical Sciences, Beijing 100850, exposure10, 11) and many other toxicological P. R. China (e-mail: [email protected]) characteristics12Ð14) between PFIB and phosgene, it seems 278 J Occup Health, Vol. 47, 2005

exposure were studied. Materials and Methods 1. General 1.1. Animals Specific pathogen-free male Wistar rats (260Ð300 g b.w.) and specific pathogen-free male mice (18Ð22 g b.w.) were used in this study. All animals were obtained from the Center of Medical Experimental Animals, the Academy of Military Medical Sciences (Beijing, P. R. Fig. 1. Possible interaction between PFIB and protein. China). The animals were housed in quiet, humidified, clean rooms with a light-cycle of 12 h/12 h for 1 week before use. They were fed with laboratory pellet food clear to us that both primary (direct) injury by the and tap water ad libitum except when they were in the chemical itself and secondary (indirect) injury, possibly exposure chamber. All the animal experiments were mediated by a later signal cascade network, are the two performed in accordance with the Guidelines for Animal key aspects of PFIB inhalation induced ALI. This is Experiments at the Chinese Academy of Medical because (1) the halogen and p-π electron conjugation in Sciences, Beijing, P. R. China. the molecular structure of PFIB make it possess a much 1.2. Doses of PFIB for animal exposure stronger electrophilicity, allowing it to attack much more PFIB was obtained from the Shanghai Institute of easily the nucleophilic groups of thiol (-SH), amino (-NH2) Organic Fluorine Materials at a purity of 98%. A flow- and hydroxy (-OH), distributed widely in the alveolar past whole-body exposure apparatus was used to expose membrane (Fig. 1), which are crucial to the maintenance small animals to PFIB, as described in our earlier paper15). of the cellular structure and function; and (2) as direct injury Except for the mortality study, PFIB inhalation doses were develops, a highly complex network is initiated and 130 mg/m3 × 5 min and 140 mg/m3 × 8 min for mice and established, resulting in an overshoot in inflammation15) rats, respectively, and were chosen for the convenient followed by severe lung damage, fulminating pulmonary observation of the pathophysiological changes without edema and even death. Multiple mediators such as TNF- fatal consequences. α16), IL-1β17), IL-818), metabolites of arachidonic acid19), adhesion molecule20), reactive oxygen species21), matrix 2. Experimental Design metalloproteinases22), neutrophil elastase23) and many other Test animals were generally divided into four groups. factors24) are thought to be involved in the latter mechanism. In groups 1) normal/saline and 2) normal/test drug, animals Any medical or pharmacological measure that can experienced the same exposure procedure with only pure effectively counteract the above-mentioned factors, air circulated in the chamber, and were treated with either theoretically, would impede or reverse the toxicity due saline or the test drug. In groups 3) PFIB/saline and 4) to PFIB or phosgene exposure. In fact, the antibody of PFIB/test drug, animals were exposed to PFIB in the same 25) 26) TNF-α , the COX2 inhibitor ibuprofen , SOD induction chamber, and administered either saline or the test drug. into the lung27), and other pharmacological interventions28) In the first part of the present study, comparisons of have all shown some prophylactic or therapeutic effects the effects of anisodamine and QNB (i.p.) on pulmonary on ALI experimentally. edema induced by PFIB inhalation were made by the Anisodamine, a typical cholinolytic extract from a weighing method; that is, the wet lung/body weight and Chinese herb, has been reported to possess effects of the dry lung/body weight ratios were calculated. To obtain microcirculation improvement29), stabilization of consistent data, the effects were expressed as relative lysosomal membranes30) and inhibition of the metabolism values of both ratios, with the averages of PFIB/saline of arachidonic acid31) along with its classical anti- groups at the same exposure were regarded as 100%, i.e., cholinergic effects, and is widely used clinically in the (PFIB/test drug) /(PFIB/saline). The difference between treatment of many diseases including ALI32, 33). In an a prophylactic and a therapeutic effect rest with the endeavor to throw some light on the complex process of administration of the test drug 30 min before or 10 h ALI and seek solutions for successful medical or after the PFIB exposure. The reason for the selection of pharmacological intervention, we hypothesized, that the treatment time-point 10 h after PFIB exposure, was cholinolytics used properly might have some prophylactic in consideration of the results from the time-course studies and therapeutic value against PFIB inhalation induced on the edema development pattern in our earlier study15), ALI. Accordingly, the effects of 3-quinuclidinyl benzilate which showed edema occurs after a latent period (8Ð12 (QNB), the most potent cholinolytic available, and h), as well as the experience of medical doctors that most anisodamine administered both before and after PFIB PFIB intoxicated patients usually are unconcerned in the Tianhong ZHANG, et al.: Intervention of PFIB-Induced ALI by Cholinolytics 279 asymptomatic period, and only when obvious symptoms min. Myeloperoxidase activity per gram wet lung was occur, such as cough, asthma and suffocation, do they calculated as follows: MPO activity (u/g lung)= ∆A × come to a hospital and seek a doctor’s help. 4.05/lung weight (g). (∆A=rate of change in absorbance In the second part of the study, in which QNB was at 460 nm between 1 and 3 min) used as a typical representative of the cholinolytics, a 3.4. Lung sulfhydryl compounds concentrations assay detailed study of QNB’s beneficial effects against PFIB Six mice in each group were sacrificed at 2, 4, 8, 16 induced ALI was made by the observations of the changes and 24 h after PFIB exposure, and the isolated mice lungs in mice mortality at 72 h after an over-LCt50 dose of were frozen at Ð70°C. After thawing, the lung PFIB (190 mg/m3 × 5 min) exposure, the total protein homogenates were prepared with saline in an ice bath. concentration in bronchoalveolar lavage fluid (BALF) at Sulfhydryl compounds including total sulfhydryl (TSH), 24 h after a sublethal dose of PFIB exposure, the time nonprotein sulfhydryl (NPSH) and protein sulfhydryl course of lung sulfhydryl concentration, myeloperoxidase (PSH) were assayed according to Sedlak36) using Ellman’s (MPO) activity, hemorheological assay, as well as reagent, and a calibration curve using GSH was prepared. ultrastructural studies. 3.5. Peripheral blood hemorheological studies At 2, 8 and 16 h after PFIB exposure, six rats in each 3. Experimental methods group were anesthetized. Blood was drawn from the rat 3.1. Wet lung/body weight and dry lung/body weight ratios carotid artery into tubes containing anticlotting agent

At 24 h after PFIB exposure, mice were anesthetized by K2EDTA. Blood cells counts were performed by routine an intraperitoneal injection of pentobarbital (50 µg/g) and laboratory procedures. Blood viscosity was measured at exsanguinated via abdominal aorta transection. The 37°C with a coaxial cylinder microviscometer (BV-100, tracheae and lungs were then excised en bloc and cleared China) at a shear rate of 1 sÐ1, followed by plasma of all extrapulmonary tissue. Total lung wet weight was viscosity determination after centrifugation. determined and the wet lung to body weight ratio was 3.6. Ultrastructural studies calculated. After drying at 80°C for over 24 h and At 12 h after PFIB exposure, a mouse was sacrificed weighing, the dry lung to body weight ratio was calculated. and the thorax was opened. The lung was rapidly 3.2. Bronchoalveolar lavage fluid (BALF) collection and removed and immersed in 3% glutaraldehyde in 0.1 M its total protein concentration assay phosphate buffer, pH 7.4, which had been cooled to 4°C At 24 h after PFIB exposure, mice were anesthetized to fix overnight. Paramedian transverse slice, 3-mm and exsanguinated, and tracheae were exposed. Each thick, from the lower left lobe were removed and further animal’s trachea was cannulated with a blunt needle diced into 1-mm cubes. Subsequent postfixation, secured with a silk ligature. BALF collection was dehydration, infiltration, polymerization and the like performed with 0.6 ml PBS that was infused and aspirated procedures were as described in our earlier paper15). into each lung 3 times. The lavage fluid was recovered (average fluid recovery was 0.45 ml), and centrifuged at 4. Statistical analysis 1,000 g, 4°C, for 10 min. The supernatants were removed Data are shown as the mean ± SD unless particularly and stored at Ð70°C until the total protein concentrations indicated. Student’s t-test or one-way analysis of variance were determined by the method of Lowry et al.34) followed by Dunnett’s test were used to detect differences 3.3. Lung myeloperoxidase activity assay35) between groups. Mortality data were compared by the At 2, 4, 8, 16 and 24 h after PFIB exposure, six mice Chi-square test. in each group were sacrificed, and lungs isolated from the mice were rinsed with saline and blotted dry. After Results weighing, lung tissue was homogenized in a 1.2 ml 20 1. Comparison of anisodamine and QNB on PFIB- mmol/l potassium phosphate buffer, pH 7.4, and induced changes of wet lung/body weight and dry/body centrifuged for 30 min at 30,000 × g, at 4°C. The pellet weight ratios in mice was resuspended in 1.2 ml 50 mmol/l potassium It was observed that the wet lung/body weight and the phosphate buffer, pH 6.0, containing 0.5% dry lung/body weight ratios at 24 h after the PFIB hexadecyltrimethylammonium bromide (HTAB). The exposure were significantly decreased when a single dose resuspended pellets were frozen at Ð70°C until the MPO of QNB (1 mg/kg) was administered intraperitoneally 30 activity assay was performed. Frozen samples were min before the PFIB (130 mg/m3 for 5 min) exposure, thawed, sonicated, incubated in a 60°C water bath for 2 but no significant changes were observed when the same h, and centrifuged at the same condition. Supernatant, dosage of QNB was given 10 h after the PFIB exposure. 0.1 ml, was added to 2.9 ml of 50 mmol/l potassium When the QNB dosage was increased to 5 mg/kg, phosphate buffer, pH 6.0, containing 0.167 mg/ml o- however, it was found that both ratios significantly dianisidine and 0.0005% hydrogen peroxide, and the decreased by administration either before or after the absorbance of 460 nm visible light was measured for 3 PFIB inhalation intoxication, and the most exceptional 280 J Occup Health, Vol. 47, 2005

Fig. 2. Relative values of wet lung/body weight ratio and dry lung/body weight ratio with different doses of anisodamine (A)(B) and QNB (C)(D) at 24 h after exposure to PFIB (130 mg/m3) for 5 min; the averages of PFIB/saline groups at the same exposure were regarded as 100%, QNB or anisodamine was administered i.p. at 30 min before exposure, pretreatment group, or at 10 h post exposure, treatment group. *p<0.05 versus PFIB group at the same exposure, n=12.

Table 1. Effects of QNB on PFIB-induced mortality in mice

Group n survival death mortality

Normal 16 16 0 0/16 Normal/QNB 16 16 0 0/16 PFIB 16 6 10 10/16* pretreatment 16 11 4 4/16# treatment 16 13 3 3/16#

Mice were exposed to PFIB at 190 mg/m3 for 5 min. QNB (5 mg/kg) i. p. was administered at 30 min before exposure, pretreatment group, or at 10 h post exposure, treatment group. Mortality was evaluated at 72 h after exposure. *p<0.01 versus normal group, #p<0.05 versus PFIB group by Chi-square test. result was that the above ratios decreased to the levels of mice mortality after 5 min exposure to PFIB (190 mg/ the normal control group when QNB was administered m3). After 3 d of observation, no mice died or showed twice, both before and after the PFIB inhalation (Fig. any signs of illness in the normal control or normal/QNB 2C, 2D). Anisodamine, the less potent cholinolytic, was groups. In contrast, mortality was dramatically elevated without any prophylactic or therapeutic effects at single in the PFIB group, while QNB, whether administered 30 doses below 30 mg/kg (Fig. 2A, 2B). min before or 10 h after the PFIB exposure, significantly decreased the mortality (p<0.05). 2. Effects of QNB administration on PFIB-induced In mice at 24 h after the inhalation of PFIB (130 mg/ changes in mortality and BALF total protein m3) for 5 min, a significantly high protein concentration in concentration in mice BALF was observed, which was effectively reversed by Table 1 shows the effect of QNB (5 mg/kg i.p.) on QNB (5 mg/kg i.p.) either given 30 min before or 10 h Tianhong ZHANG, et al.: Intervention of PFIB-Induced ALI by Cholinolytics 281

after the PFIB exposure (Fig. 3).

3. Effects of QNB administration on PFIB-induced changes in lung MPO activity, sulfhydryl compounds concentrations and peripheral blood hemorheology Inhalation of PFIB (130 mg/m3) for 5 min led to a slow increase in the lung MPO activity which peaked at 16 h after the exposure. Although there was some decrease in lung MPO activity after QNB pretreatment (5 mg/kg i.p.), it was not statistically significant. Interestingly, however, there were significant increases in the normal/QNB group at 4 h, and the PFIB/QNB group Fig. 3. Effects of QNB (5 mg/kg) i.p. on PFIB-induced at 2 h after PFIB exposure (Table 2). increases in mice total protein concentration in BALF. In general QNB (5 mg/kg i.p.) itself had no positive or The parameters were determined at 24 h post exposure negative effects on the lung sulfhydryl concentration. A to PFIB (130 mg/m3) for 5 min. QNB (5 mg/kg) i. p. marked depletion in PSH and TSH of lung tissue (p<0.05) was administered at 30 min before exposure, was found at 8 and 16 h in the PFIB group, which was pretreatment group or at 10 h after exposure, treatment group. *p<0.05 versus normal group, #p<0.05 versus partly blocked by QNB (5 mg/kg i.p.) pretreatment. In PFIB group, n=9. contrast, no change in the NPSH concentration was found

Table 2. Effects of QNB (5 mg/kg i.p. at 30 min before exposure) on PFIB (130 mg/m3 for 5 min) inhalation-induced changes in mice lung MPO activity (µ/g)

Time (h) 24 81624

MPO activity (normal: 1.25 ± 0.46) Normal/QNB 1.84 ± 0.52 2.52 ± 0.56* 1.86 ± 0.24 1.17 ± 0.60 1.20 ± 0.64 PFIB/saline 0.72 ± 0.30 1.28 ± 0.87 1.40 ± 0.21 6.72 ± 5.10* 5.31 ± 3.65* PFIB/QNB 2.79 ± 1.54# 2.11 ± 1.37 1.02 ± 0.55 3.74 ± 1.29 3.38 ± 0.36

*p<0.05 versus normal saline group, #p<0.05 versus PFIB group at the same time, n=6.

Table 3. Effects of QNB (5 mg/kg i.p. at 30 min before exposure) on PFIB (130 mg/m3 for 5 min) inhalation-induced changes in mice lung non-protein sulfhydryl, protein sulfhydryl, and total sulfhydryl concentration (µmol/g)

Time (h) 24 81624

NP-SH (normal: 0.62 ± 0.13) Normal/QNB 0.76 ± 0.14 0.80 ± 0.08 0.87 ± 0.10 0.90 ± 0.12 0.85 ± 0.11 PFIB/saline 0.49 ± 0.04 0.53 ± 0.03 0.60 ± 0.12 0.38 ± 0.17 0.53 ± 0.02 PFIB/QNB 0.46 ± 0.07 0.43 ± 0.10 0.51 ± 0.03 0.63 ± 0.20 0.50 ± 0.05

P-SH (normal: 15.17 ± 0.75) Normal/QNB 15.08 ± 1.28 15.03 ± 1.82 16.12 ± 0.42 15.53 ± 0.89 17.25 ± 2.75 PFIB/saline 13.64 ± 1.07 14.52 ± 0.33 12.32 ± 1.23* 9.38 ± 1.22* 13.00 ± 0.50 PFIB/QNB 14.42 ± 0.33 13.51 ± 1.81 14.20 ± 1.38 13.15 ± 1.57# 12.65 ± 1.11

T-SH (normal:15.79 ± 0.81) Normal/QNB 15.85 ± 1.17 15.83 ± 1.87 16.99 ± 0.45 16.43 ± 0.90 18.09 ± 2.80 PFIB/saline 14.13 ± 1.10 15.05 ± 0.33 12.92 ± 1.31* 9.76 ± 1.27* 13.53 ± 0.52 PFIB/QNB 14.88 ± 0.33 13.94 ± 1.79 14.72 ± 1.36 13.78 ± 1.76# 13.15 ± 1.14

*p<0.05 versus normal saline group, #p<0.05 versus PFIB group at the same time, n=6. 282 J Occup Health, Vol. 47, 2005

Table 4. Effects of QNB (5 mg/kg i.p. at 30 min before exposure) on PFIB (140 mg/m3 for 8 min) inhalation-induced hemorheological changes in rats

Time (h) 28 16

whole blood viscosity at a shear rate of 1 sÐ1 (mPa × S) (normal: 27.91 ± 3.20) Normal/QNB 21.33 ± 2.26* 24.34 ± 6.01 27.19 ± 12.31 PFIB/saline 26.14 ± 0.35 29.67 ± 2.67 80.64 ± 9.31* PFIB/QNB 22.55 ± 2.06# 23.55 ± 1.60# 54.80 ± 10.76#

erythrocyte aggregation (normal: 22.81 ± 2.54) Normal/QNB 17.76 ± 1.88* 20.26 ± 5.00 22.64 ± 10.25 PFIB/saline 22.03 ± 2.41 26.47 ± 1.65 61.13 ± 7.60* PFIB/QNB 16.77 ± 1.30# 20.04 ± 1.82# 44.47 ± 10.13#

plasma viscosity (mPa × S) (normal: 1.22 ± 0.06) Normal/QNB 1.18 ± 0.07 1.26 ± 0.04 1.21 ± 0.13 PFIB/saline 1.23 ± 0.12 1.11 ± 0.13 1.24 ± 0.07 PFIB/QNB 1.27 ± 0.06 1.12 ± 0.12 1.32 ± 0.13

hematocrit (%) (normal: 38.6 ± 2.14) Normal/QNB 37.98 ± 2.94 35.67 ± 3.56 36.14 ± 2.90 PFIB/saline 37.80 ± 1.69 38.66 ± 0.96 39.50 ± 2.03 PFIB/QNB 37.25 ± 1.77 38.76 ± 1.85 38.90 ± 2.33

leucocyte counts in peripheral blood (103/mm3) (normal: 12.73 ± 2.30) Normal/QNB 8.63 ± 0.32* 12.91 ± 1.82 12.85 ± 4.38 PFIB/saline 7.77 ± 1.62* 9.20 ± 1.65* 8.16 ± 1.27* PFIB/QNB 8.65 ± 2.77 8.80 ± 1.90 11.96 ± 0.47#

*p<0.05 versus normal saline group, #p<0.05 versus PFIB group at the same time, n=6.

during the observation period (Table 3). epithelium cells (Fig. 4D). These pathological alterations Compared with the normal group, the blood viscosity were noticeably less after QNB (5 mg/kg i.p.) at shear rate of 1 sÐ1 and erythrocyte aggregation were pretreatment, and was characterized by maintenance of significantly raised in rats in parallel with the severity of the alveolar structure (Fig. 4E), although vacuoles and pulmonary edema. The hemorheological effects of blebs in type I pneumocytes, and fluid leakage into the pretreatment with QNB (5 mg/kg i.p. 30 min before the alveolar space could still be seen (Fig. 4F). PFIB exposure) were shown at 2 h post PFIB exposure. On the other hand, hematocrit and plasma viscosity were Discussion not altered significantly. Leukopenia also quickly PFIB is a most potent toxic gas that can induce appeared at 2 h after PFIB inhalation. The effect of QNB pulmonary edema after inhalation, the main pathological (5 mg/kg i.p.) pretreatment on leucocyte counts in changes of which are a destroyed lung gas-blood barrier peripheral blood was a decrease in normal animals and and the leakage of protein and water including red blood an increase at 16 h post PFIB inhalation (Table 4). cells from the circulation system into the alveolar space, as indexed by increased wet lung weight and dry lung 4. Ultrastructure study weight, as well as high protein concentration in BALF. No pathological change was found with types I and II Cholinolytics are reported to have wide pharmacological pneumocytes and alveolar vasculature in the normal/ effects besides blocking cholinergic receptors. In the saline and normal/QNB groups (Fig. 4A, 4B). The lungs current paper we hypothesized that QNB and a high of all mice sacrificed at 12 h after exposure to PFIB (130 dosage of anisodamine could attenuate pulmonary edema mg/m3) for 5 min showed severe and generalized induced by PFIB inhalation and confirmed this by the disruption and slough, in the form of excessive lysosomes observation of a significant decrease of wet lung/body and lamination swelling in type II epithelium cells (Fig. weight ratio, dry lung/body weight ratio, total protein 4C), as well as increased vesicle vacuolation in type I concentration in BALF and mortality. Tianhong ZHANG, et al.: Intervention of PFIB-Induced ALI by Cholinolytics 283

Fig. 4. Representative electron photomicrographs of lungs from mice × 30,000. (A): normal group, type II pneumocyte ( ). (B): normal/QNB group. (C)Ð(D): PFIB group sacrificed at 12 h post exposure to PFIB (130 mg/m3 × 5 min), excessive lysosomes ( ) and lamination swelling ( ) in type II epithelium cells, increased vacuolation ( ) in type I epithelium cells. (E)Ð(F): pretreatment group with QNB (5 mg/kg) i.p. at 30 min before exposure. Fluid leakage ( FL ) into the alveolar space from the type I epithelium cell (I) junction, which appears widened.

It is speculated that the primary (direct) injury of PFIB consumption of protein sulfhydryl, which could be inhalation is caused by PFIB binding to nucleophilic partially blocked by QNB administration, clearly supports groups of amino acid in the alveolar membrane, the large the above speculation regarding the role of the sulfhydryl area of alveoli providing support for this interaction. redox state, although no change of nonprotein sufhydryl Among the nucleophilic groups of amino acid, sulfhydryl (e.g. glutathione) was observed and further studies are is possibly one of the most sensitive groups to active needed to clarify this exception. electrophilic chemicals such as PFIB and phosgene. The secondary (indirect) injury of PFIB may be caused Additionally, the maintenance of the cytoskeleton, protein by multiple factors including excessive inflammation and molecular activity, and cell metabolism might be related blood coagulation abnormality, both of which are to the regulation of the intracellular sulfhydryl redox state. involved in the pathogenesis of various kinds of ALI. The current results that PFIB inhalation induced The lung MPO study and leucocyte counts in peripheral 284 J Occup Health, Vol. 47, 2005 blood showed neutrophil sequestration and accumulation 12, 843Ð851(1992) in the lung may be one of the key factors in PFIB 4) J Patocka and J Bajgar: Toxicology of inhalation-induced ALI. Perfluoroisobutene. ASA Newsletter 22, 69 (1998) Blood viscosity is the intrinsic resistance to blood flow 5) CM Arroyo: The Chemistry of Perfluoroisobutylene in the blood vessels and may a key factor involved in (PFIB) with Nitrone and Nitroso Spin Traps: an EPR/ Spin Trapping Study. Chem Biol Interact 105, 119Ð many pathophysiological processes; e.g. shear stress 129 (1997) damage at the blood-endothelial interface, facilitation of 6) AF Lailey, L Hill, IW Lawston, D Stanton and DG plasma protein interaction with the endothelium, increased Upshall: Protection by cysteine esters against propensity for thrombosis, impaired microcirculatory flow chemically induced pulmonary edema. Biochem and blood infusion to an organ. 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