Analysis of the reactivity of [14C]toluene diisocyanate (TDI) in an isolated, perfused lung model

F

ELSEVIER

Chemico-Biological

Interactions

98 (1995) 167-183

Analysis of the reactivity of [ “C]toluene diisocyanate (TDI) in an isolated, perfused lung model A.L. Kennedya, L. Lastbomb, G. Skarpingc, M. Dalenec, A. Ryrfeldtb, P. Moldeusb, W.E. Brown*a aDepartment of Biological Sciences, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA bDepartment of Toxicology, Karolinska Instituter, Stockholm, Sweden ‘Department of Occupational and Environmental Medicine, University Hospital, Lund, Sweden

Received 6 March 1995; revision received 1 June 1995; accepted 6 June 1995

Abstract An isolated, perfused, guinea pig lung model was used to investigate the molecular events which occur when a 14C-labeled TDI vapor reaches the airways. Exposure concentrations of 0.2 and 0.7 ppm were tested. Perfusate composition included: Krebs Ringer buffer only, as well as buffer containing either guinea pig serum albumin, human serum albumin, or diluted guinea pig plasma. Radioactivity was detected in the perfusate within minutes of exposure, and following a delay, increased linearly. The rate of uptake was dependent on TDI concentration and the composition of the perfusate. Biochemical characterization of the state of the 14C-labeled material in the perfusate was performed. The distribution between low and high molecular weight reaction products was determined by molecular sieve fractionation and varied as a function of perfusate composition but no variability was observed as a function of time during the 45 min of exposure. An increase in nucleophile concentration in the perfusate was associated with both a higher percentage of conjugated products (from 15% with buffer only to 45% with diluted guinea pig plasma) and an increase in the rate of TDX uptake (from 0.5 urn Eq/min with buffer alone to 0.1 ug Eq/min with diluted GPSA as perfusate at 0.7 ppm). GC-MS analysis of the samples for free TDA, before and after acid hydrolysis, showed that the low molecular weight product(s), which represented from 55-85% of the circulating radioactivity, was composed of hydrolyzable and non-hydrolyzable conjugates and metabolites with approximately 4% of the label

Abbreviations: BSA, bovine serum albumin; GC-MS, gas chromatography-mass spectrometry; GC-SIM, gas chromatography-selective ion monitoring: GPSA, guinea pig serum albumin; HSA, human serum albumin; IVPL, in vitro perfused lung; MIC, methyl isocyanate; PPPA, pentafluoropropionic anhydride; PNBPA, pamnitrobenzyl-Wn-propylamine; SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis; TDA, toluene diamine; TDDA, trideuterated 2,4-, 2,6-toluene diamine; TDI, toluene diisocyanate; TDX, route compound of toluene diisocyanate. *Corresponding author.

OC09-2797/95/$09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0009.2797(95)03644-2

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associated with free TDA. Although varies, this result is analogous to the fused lung (IVPL) system may be a anate-induced disease and metabolic Keywords: Toluene diisocyanate;

the distribution between high and low molecular weight species findings from in vivo studies and suggests that the isolated, peruseful tool in investigating the molecular mechanisms of isocyactivity of the lung.

Perfused lung model; Biochemical

reactivity

1. Introduction Toluene diisocyanates (TDI) are highly reactive, bifunctional isocyanates used extensively in the production of polyurethanes. Vapor state exposure to TDI results in a number of airway effects ranging from irritation to chronic lung disease [ 11. In some cases, an asthmatic response is observed following exposure. The airway reactions appear to have both pharmacologic and immunologic components [2]. This disease process is quite complex and has not been fully defined at the molecular level [3]. Biochemical reactivity of isocyanates has been demonstrated using in vitro systems [4] to include a variety of biologically relevant functional groups. This reactivity has been confirmed in vivo through the analysis of tissues and fluids from worker populations, exposed volunteers [5,6], and controlled animal studies [7,20]. Jin et al. [8] for example, have used immunological methods to identify 5 TDI-protein adducts in bronchoalveolar lavage following inhalation exposure using a guinea pig model system. The availability of high sensitivity, gas chromatography/mass spectrometry methods to detect TDA in hydrolyzed biological samples at concentrations as low as 0.05 pg/l [9,10] have enabled measurement of TDX, the root compound independent of metabolism or conjugation, in much larger populations. Several chemical reactions and cellular and molecular targets have been characterized but the etiology of the disease process is still uncertain. Entry of isocyanates to the circulatory system has been the focus of a number of studies [7,1 l-131. In general, the uptake of the root compound, as followed for example, by 14C ring labeling of TDI, into the bloodstream has been shown to be kinetically linear, dose dependent, isocyanate independent and species independent [3]. The final form, denoted as TDX, of the isocyanate in the circulation following in vivo inhalation exposure has been shown to be primarily a conjugated macromolecular species. Less than 5% of the radioactive material was found in a low molecular weight form, also denoted as TDX, (e.g. the amine (hydrolyzed isocyanate), glutathione adducts etc.) The results of these studies do not establish, however, whether the reaction takes place in the lumen of the airways or directly in the circulatory system. Neither do they establish the site of primary uptake, that is what the uptake distribution is between the nasal cavity and the lung. This paper will describe the use of a perfused lung model to provide a more sophisticated in vitro system which closely mimics the in vivo environment but eliminates the contribution from such variables as nasal uptake and systemic organ metabolism. Using radioactively labeled toluene diisocyanate, this study examines the contribution of the lung to the uptake of isocyanates, the rate of TDI uptake when delivered directly to the lung and the molecular distribution of the TDX in the lung and perfusate following vapor exposure of an isolated lung to TDI.

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2. Materials and methods 2.1. Chemicals We obtained 2,4- and 2,6-TDA (toluene diamine) from Fluka (Buchs, Switzerland); 2,4and 2,6-TDI (80:20) from Janssen Chimica (Beerse, Belgium); pentafluoropropionic anhydride (PFPA), from Pierce (Rockford, IL, USA); trideuterated 2,4- and 2,6-TDA 99.7% pure deuterated material (CD3C6H3[NH&) (TDDA) from Synthelec (Lund, Sweden); para-nitrobenzyl-N-n-propylamine (PNBPA) from Regis Chemical; diclofenac and salbutamol from Sigma Chemical Co. (St. Louis, MO, USA). Bovine serum albumin (fraction V) (BSA) was obtained from Boehringer Marmheim GmbH (Germany); and L-670 596 was kindly provided by Dr. C. Pickett, Merck-Frosst Canada Inc. (Canada). 14C-labeled TDI (80:20, 2,4-TDI:2,6-TDI; 20.6 mCi/mmol); human serum albumin (HSA) and guinea pig serum albumin (GPSA) from Sigma Chemical Co. All other chemicals were of analytical grade and obtained from a local supplier. 2.2. Isolated perfused lung system Male, Dunkin-Hartley, guinea pigs (Bio Jet Service, Uppsala, Sweden) weighing between 250-400 g were used in this study. The animals were anaesthetized by intraperitoneal injection with pentobarbital (Mebumal Vet., NordVacc, Sweden) (120 mg/kg) and the lungs were surgically removed as previously described [ 141. In brief, the chest was opened, the superior caval vein cut and about 500 U heparin injected into the right ventricle. The pulmonary artery was cannulated via an incision in the right ventricle. An outlet cannula was secured in the left atria1 appendage via an incision in the apex of the heart. Tracheotomy was performed and the lung and heart excised and suspended in a well-humidified artificial thoracic chamber. Fig. 1 illustrates the apparatus used for the lung perfusion experiments. The lungs were perfused at a constant hydrostatic pressure (about 15 cm H,O). The perfusion medium used to equilibrate the lungs was a Krebs-Ringer bicarbonate buffer (composition in mM: NaCl 118.0, KC1 4.7, CaC& 2.5, MgS04 1.2, NaHCO, 24.9, KHzPO4 1.2) with the addition of 12.5 mM HEPES, 5 mM glucose and 2% bovine serum albumin, fraction V. The pH of the perfusate solution was controlled to between 7.35-7.45 by administration of CO:! and heated to 37°C with a circulating waterbath. In order to avoid post mortem bronchoconstriction of the isolated guinea pig lungs, salbutamol was present in the perfusion buffer (50 nM), as described previously [ 151. The lung was ventilated at 55 breaths/min by creating an alternating negative pressure (-l--8 cm H,O) inside the thoracic chamber using an animal respirator (Model 680, Harvard Apparatus, USA) and a vacuum source connected to the thoracic chamber. The tracheal airflow was measured with a heated pneumotachograph (A. Fleisch, Switzerland) and a pressure transducer sensor 164pc (Honeywell, USA). The thoracic pressure changes were monitored with a pressure sensor 174pc (Honeywell, USA). The signals were transformed and amplified via an interface system (Colboume Instruments, USA) and recorded directly on a computer. The perfusion flow was measured manually. The lungs were allowed to stabilize for 30 min before starting the experiment. Only lungs with stable baseline values for perfusion flow, compliance and conductance were used. Baseline values of conductance and compliance were 84.1 f 7.8 ml/&Pa and 5.2 * 0.8 ml/kPa (mean * S.D.; n = 6), respectively. Baseline perfusion flow was 24.4 * 3.0

A.L. Kennedy et al./Chemico-Biological

Interactions 98 (I 995) 167-183

Isocyanate Atmosphere Generation System see Figure lc

A.L. Kennedy et al./Chemico-Biological

dilution

air

AirflOW

Cl-flOW

w

_

Regulators

l

.

171

Interactions 98 (1995) 167-183

To lung chamber

.: .:./ iiii!ib Heating

__-___I

C

I

block

__IIm_s

Fig. 1. Schematic of the perfused lung apparatus. (A) Complete system with monitoring devices used prior to and following each exposure. (B) System minus the monitoring device as used during exposure with isocyanate. (C) Enlarged schematic of isocyanate vapor generation system.

ml/mm (mean f S.D.; n = 6). These values are well within the control parameters for lungs in this system as reported by Lastbom et al. [ 161. The 6 perfusion experiments were each done with 70 ml recirculating medium containing variable constituents. The specific parameters for each experiment are given in Table 1. 2.3. Isocyanate vapor generation system An apparatus for the generation of TDI in air was developed and is illustrated in Fig. lc. An ampoule containing about 13 ul of 14C-labeled TDI was placed in a temperature conTable 1 Experimental

conditions

prior to exposure

Experiment no.

Perfusate composition

Tidal volume (ml)

Perfusion flow (mumin)

Conductance (mINkPa)

Compliance (mlikPa)

1 2 3 4 5 6

buffe? only buffer + 1% HSA buffer only buffer + 1% HSA buffer + I % GPSA dilutedb GP plasma

1.9 1.9 1.9 2.5 2.0 2.0

28 25.5 19 24 25 25

85.9 93.0 73.5 88.1 87.8 75.6

4.4 4.4 5.1 6.1 5.3 5.4

aKrebs-Ringer bicarbonate buffer (composition given in Materials and methods) HEPES, 5 mM glucose and 50 nM salbutamol. bFresh guinea pig plasma was diluted 1 part to 5 parts of buffer and used as perfusate.

containing

12.5 mM

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trolled (-20-70°C) heating block. Preheated air was passed across the liquid TDI generating the TDI vapor. The TDI vapor was then diluted with dry air (l-10 l/min) before introducing it to the lung. The temperature of the heating block was controlled using Peltier elements. AFC 25.00 Mass flow controllers (ASM Montpellier, France) with a Vacutec UFC 502 flow control box (Vacutec AB, Svedala, Sweden) were used for regulating and controlling the air speed. All tubing in the system was made of either glass or Teflon. The final TDI concentration was influenced by the exposed surface area of TDI in the ampoule, the temperature and the delivery air speed. The specific conditions for all experiments reported in this paper were: a total airflow rate equal to 0.4 Ymin, exposure duration of 45 min and an isocyanate vial temperature of 10°C. 2.4. Sampling and analysis of TDI vapor concentrations For the determination of TDI vapor exposure concentrations, PNBPA impregnated glass fiber filters (Whatman) were placed in line at the point of entry (Fig. lb) to the lung. Air was drawn through the filters at a speed of 2 l/min for 5 min using SKC Model 224-36 pumps (SKC Inc., Eighty Four, PA, USA). Preparation of the sample and analysis by HPLC were performed following a previously published method [17]. Following sampling, the PNBPA-TDI derivative was extracted from the filter in 1 ml of acetonitrile and analyzed by HPLC using UV detection at 254 nm. The HPLC equipment consisted of a Waters 680 gradient controller, a Waters 484 UV-detector and a Waters 740 integrator (Millipore-Waters, Milford, MA, USA). Isocratic elution was accomplished using acetonitrile-water (70:30, v/v) with 9.9 mM phosphoric acid on a Waters Cts pBondapack column. Each exposure concentration was determined by averaging 6 measurements; 3 taken prior to the exposure and 3 taken after the exposure was terminated. The lung exposure conditions and corresponding average TDI concentrations for each experiment are given in Table 2. In summary, these experiments were performed at two isocyanate concentrations, 0.2 and 0.7 ppm, for 45 min comparing variable perfusate compositions.

Table 2 Perfusate uptake of 14C during exposure Experiment no.

Perfusate composition

Average TDI atmospheric concentration”

ppm

S.D.

ug Eq/mind

R2 value

yg Eq/mld

I

buffer buffer buffer buffer buffer diluted

0.2 0.2 0.7 0.8 0.7 0.7

0.02 0.05 0.08 0.07 0.07 0.1 I

0.0235 0.0310 0.0541 0.06 19 0.1017 0.082 1

0.86 0.97 0.98 0.95 0.96 0.98

0.0109 0.0099 0.0201 0.0250 0.0337 0.0268

2 3 4 5 6

only + 1%HSA only + 17r HSA + 1% GPSA GP plasma

Rate of TDX uptake in perfusateb

%I = 6 for all determinations. bRate determined as linear regression from 2045 min of exposure. ‘Aliquot of terminal perfusate following removal of timed aliquot. dpg Eq = (dpm x 174.17 mg/mm x 1000 pg/mg)/(2.22 x IO” dpm/mCi

x

20.8 mCi/mm)

TDX cont. in terminal perfusa&

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173

2.5. The exposure protocol

With the exposure concentration set, and the initial state of the lungs measured, the equilibrating perfusate containing 2% BSA was replaced by the experimental perfusate. After establishing that no significant lung function had changed as a result of the new perfusate, the pressure transducer was removed from the experimental system, to avoid contamination with radioactivity, thus the 14C exposure system was configured as diagrammed in Fig. lb. Just prior to initiation of the exposure, a 1 ml perfusate sample was collected into a 1.5-ml microfuge tube from the tube leading to the lower perfusate reservoir. At time zero, the line from the TDI generation system was connected to a T-joint which replaced the pressure transducer at the entrance to the lung. At 5, 10, 20,25, 30, 35,40 and 45 min during the exposure, 1 ml perfusate samples were taken. The perfusate pH was maintained constant throughout each experiment. 2.6. Collection of samples at termination of exposure The TDI inlet line was removed from the system, the perfusate was drained, and the total volume was determined. A fraction was immediately titrated to pH 3 with HCl. The remainder of each sample was stored at -70°C. Lungs from experiments 3-6 were removed from the apparatus and lavaged with two, 7-ml saline washes. Duplicate aliquots were taken from each pooled lavage for scintillation analysis. These lungs were frozen for later biochemical analysis. Lungs from experiments 1 and 2 were inflated with 10% buffered formalin and stored submerged in the same fixative for future histological analysis. 2.7. Scintillation analysis of samples Duplicate, 100 pl aliquots of the terminal perfusates, kinetic time samples taken during exposure, and molecular sieve fractionation samples were dissolved in 5 ml of Ecoscint (Hintze) scintillation cocktail and quantitated for 14C using liquid scintillation analysis on a Beckman LS 180 1 scintillation counter. 2.8. Hydrolysis of perfasate To a 100 yl perfusate sample, 1.5 ml of 3 M HzSO4 was added. The sample was then hydrolyzed at 100°C for 16 h. Five ml of saturated NaOH, 2 ml of toluene and 0.5 ng TDDA (internal standard) were then added. The mixture was shaken for -10 min and then centrifuged at 1500 x g for 5 min. A 1.5 ml volume of the organic layer was transferred to a new test tube and 20 yl of PFPA was added. The mixture was immediately shaken vigorously for -10 min. The excess of the reagent and acid was removed by extraction with 2 ml of 1 phosphate buffer solution (pH 7.5). A 1 ml volume of the toluene layer containing the amide derivative and the internal standard was transferred to a 1.5-ml auto sampler vial with a Teflon seal and was then injected into the GC-MS system described below. 2.9. Electrophoretic analysis of terminal perfusate samples Aliquots of, perfusate samples were subjected to SDS polyacrylamide gel electrophoresis following the procedure of Laemmli [18], using a 12.5% resolving gel. The gel was stained with Coomassie Blue and destained in an isopropanol-acetic acid solution. The gel

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was dried using BioGel Wrap and exposed to Kodak X-Omat film at -70°C for autoradiographic analysis. The film was developed following manufacturer’s specifications. 2.10. Molecular sieve fractionation of terminal perjksates The relative distribution of high and low molecular weight components in the perfusate samples was determined by scintillation analysis following molecular sieve fractionation using pre-washed Centricon 10 microconcentrators (Amicon). 100 pl aliquots were diluted to 1 ml with PBS and spun at 5000 x g for 40 min. Retentates were repeatedly washed with 1 ml volumes. Filtrate fractions (~10 kDa) were removed and retentates (>lO kDa) were recovered by centrifugation at 746 x g for 4 min. 100 pl aliquots of the filtrates and retentates were subjected to scintillation analysis. TDX concentration calculations were made for total perfusate, retentate and filtrate fractions using the scintillation analysis results and the specific activity of the original TDI. 2.1 I. Determination of total TDX concentration in per&ate fractions TDA quantitation using GC-SIM was performed directly on the perfusate fraction ~10 kDa without hydrolysis and on the fraction >lO kDa after hydrolysis with sulfuric acid. The TDA was monitored as the PFPA derivative. GC-SIM analysis was performed as previously described [lo]. In brief, a Trio 1000 mass spectrometer (Fisons Instruments, VG-Biotech, Altrincham, Cheshire, England) connected to a Carlo-Erba Mega gas chromatograph equipped with an A200S autosampler (Fisons Instruments, Milan, Italy) was employed. The injector temperature was 290°C and the column oven temperature was 110°C isothermal for 1 min, then raised lS’C/min to 280°C where it was kept for 2 min. The temperature of the ion source was 230°C and the GC-MS interface temperature was 280°C. The capillary inlet pressure of helium was 0.8 kg/m3. The instrument was used in the chemical ionization mode with negative ion monitoring using ammonia as the reagent gas. The source read out pressure was 2 x 10T4 mbar, which was not the actual pressure inside the ionization cavity. The ions monitored were m/z = 394 and m/z = 374 of TDAPFPA and m/z = 397 and m/z = 377 of TDDA-PFPAcorresponding to the M-20 and M-40 ions. TDDA is trideuterated 2,4-, 2,6-TDA used as an internal standard. Fused silica capillary columns with chemically bounded stationary phases, DB-5 J & W Scientific (Folsom, CA, USA) 30 m x 0.24 mm I.D., with a film thickness of 0.25 pm were used for the GC-MS determinations. 3. Results A series of experiments were completed using an isolated, perfused guinea pig lung model to investigate the molecular events which occur when radiolabeled isocyanate is introduced into the lower airway. 3.1. Lung function prior to exposure Over the course of 45 min of exposure, there is a weak effect on lung function as a result of exposure to TDI at 0.6 ppm. Flow rate and conductance compared to control are unchanged while an effect on compliance was observed [ 141. The data in Table 1 shows that the initial state of each lung used in this set of experiments was within the control para-

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meters [16] characteristic of lungs used in this experimental design. These initial parameters were not affected by the variety of perfusate compositions used in these experiments. 3.2. Uptake of 14C into perfusates For each exposure, samples of the perfusates

0

10

20 TIME

were collected at periodic intervals

and

-I 30

40

50

40

50

(minutes)

10000

0

0

10

20

30

TIME (minutes) 30000

20000

10000

0 0

10

20

30

40

50

TIME (minutes) Fig. 2. Uptake of 14C into the perfusate during exposure to “C-labeled TDI. (A) 0, 0.2 ppm TDI using buffer only perfusate; QO.2 ppm TDI using 1%HSA perfusate; (B) l ,0.8 ppm TDI using buffer only perfusate; U. 0.7 ppm TDI using 1% HSA perfusate; (C) l,0.7 ppm TDI using 1%GPSA perfusate; QO.7 ppm TDI using diluted guinea pig plasma perfusate. In all cases, the lines represent linear regression fit of data from 20-45 min of exposure

176

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aliquots were subjected to scintillation analysis to monitor the uptake of the radiolabeled material into the perfusate. Fig. 2 illustrates the uptake of 14C into the perfusate. Panel A includes the curves for experiments I (closed symbols) and 2 (open symbols) at 0.2 ppm. Panel B corresponds to experiments 3 (closed symbols) and 4 (open symbols) at 0.7 ppm, and panel C shows the rate plots for experiments 5 (closed symbols) and 6 (open symbols) at 0.7 ppm. In each case, the line represents a linear regression fit to the data from 20-45 min. The curvature of the initial data suggests that there is a delay of - 10 min (extrapolation of the linear fit to the X-axis) before the level of radioactivity increases linearly over the remainder of the exposure period. Using this linear approximation for the uptake, a rate of 14C uptake was derived for each experimental condition. Table 2 summarizes these rates. At both TDI concentrations tested, the uptake of label into the perfusate is slower in the buffer-only perfusate samples (Fig. 2; panel A closed symbol and panel B closed symbol). Uptake is enhanced by the presence of guinea pig components (Fig. 2; panel C), while the presence of albumin from another species does not appear to significantly enhance the uptake rate (Fig. 2; panel A open symbol and panel B open symbol). This pattern is also noted in the specific activity of the label in the collected terminal perfusate as given in Table 2. 3.3. Analysis of petfusate samples Following exposure the perfusate was removed from the perfusion system and biochemical analyses were performed. 3.3.1. Molecular weightfractionation of perfusate components. Perfusates were subjected to Centricon 10 molecular sieve fractionation to separate high molecular weight (>lO kDa) from low molecular weight ( 10 kDa) to low (< 10 kDa) components in the uptake samples remained constant over the entire exposure time. 3.3.2. GC-MS analysis of perfusate fractions. Terminal perfusate fractions were analyzed for free TDA by GC-SIM both prior to and following sulfuric acid hydrolysis. Free TDA was derivatized, extracted and quantitated. For comparison, aliquots were also counted and the total concentration of TDX in each sample was calculated. The comparison of the results of these analyses is given in Table 3. Analysis of the high molecular weight (>lO kDa) perfusate fraction after hydrolysis by GC-SIM shows that TDX is formed and for most of the samples, only a portion (26-77%) of the material is recovered as the free diamine upon hydrolysis. The low molecular weight (
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178

Fig. 3. Distribution of “C-label “C-label

following

(“C]TDl

expowre of lung in perfked

lung system. Distribution of

in the lung (filled bars), lavage (cross-hatched bars) and perfusate (gray bars) expressed as percent of

total recovered label. Distributions are given as a function ofTD1 concentration and perfusate composition.

labeled material in the perfusate pound.

is either metabolites

or conjugates

of the original com-

3.3.3. SDS pol_yacrylamide gel electrophoresis of perjkate samples. Terminal perfusate samples were also analyzed by SDS-PAGE relative to an unmodified albumin standard. Coomassie Blue staining patterns show no significant structural modifications of the proteins which would be indicative of intra or intermolecular diisocyanate crosslinking. Such modifications are normally found in diisocyanate conjugates prepared in vitro. An increase in low molecular weight protein components was found when HSA was in the perfusate. The radioactive distribution of the gel components was determined through autoradiography and illustrated that all detectable label was associated with the 68 kDa band on the gel. This was observed for all samples including the perfusate containing guinea pig plasma where multiple protein bands were observed but only one was labeled as determined at this level of detection. Low molecular weight components (<5 kDa) are generally lost from the gel during the staining and destaining procedures. 3.4. Analysis of lung and lavage samples 3.4. I. Digestion and scintillation analysis. Lung samples were weighed and minced. Two tissue samples were placed in tarred glass scintillation vials and fragment weight was determined. The remainder of the tissue was stored frozen at -70°C. Each fragment was solubilized with hyamine hydroxide (ICN) and then t4C content was quantitated by liquid scintillation analysis. Results were corrected for background and converted to pg Eq/g tissue. Fig. 3 is a bar graph comparing the total equivalents of radioactivity recovered from the lung and corresponding lavage for each experiment. Variability between samples from

179

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Table 4 Calculation

of estimated dose

Experiment “0.

TDI cow. (mg/ml)

Time (min)

VT (ml)

f (breaths/ min)

Estimated dose (mg)

Estimated dose (cpm)

Total retained (cpm)

% of estimated dose

3 4 5 6

5.1 5.0 5.7 4.3

45 45 45 4.5

1.6 2.4 1.8 1.8

55 55 55 55

2.3 3.0 2.5 1.9

5.8 7.6 6.3 4.9

1.9x 106 2.0x lo6 1.7x lo6 1.7x106

33 26 27 35

x x x x

10-6 10-6 10-6 10-6

x x x x

10.’ 10-2 1O-2 10-2

the same lung may be due to incomplete homogeneity aliquots were also subjected to scintillation analysis.

x x x x

lo6 lo6 lo6 106

of the tissue samples.

Lavage

3.4.2. CuZcuZution of estimated dose. Total dose for each in vitro perfused lung (IVPL) experiment was calculated using a standard formula as follows: Concentration (mg/ml) x Time (min) x Tidal Volume (ml) x Frequency (breaths/min) = Dose (mg) Using the specific activity of the labeled TDI, the counting efficiency, the lung volume, the ventilation rates as well as the molecular weight of TDI, total estimated dose in mg was converted to total dpms. These calculations are summarized in Table 4. 3.4.3. Determination of retained dose. One of the advantages of the use of radiolabeled compounds is the ability to quantitate the actual label uptake as a percentage of the estimated dose. Total dpms recovered from the tissue and fluids derived from a single lung exposure were determined and the percentage of the total estimated dose this represented was calculated as shown in Table 4. 4. Discussion

This study was performed to investigate the transport and airway reactions of inhaled toluene diisocyanate. To demonstrate the isolated effects of isocyanates delivered directly to the lung, the perfused lung model was used as a method to evaluate the airway specific reactions of isocyanates as well as the uptake (transport) of the compound or its metabolites from the lumen of the lung into the circulatory system [ 15,161. The isolated perfused and ventilated lung model has the advantage over in vivo studies in that the effects of compounds of interest can be studied on the lung itself, without interference from other organs. The isolated lung can be exposed to controlled amounts of a variety of agents via the pulmonary circulation or by inhalation, and their uptake and metabolism can be measured. Acute changes in airway conductance, lung compliance and vascular resistance or blood/ buffer flow can be monitored. It is possible to relate functional effects to the release of mediators or to changes in the biochemistry of the lung. Also, the mode of perfusion, the composition of the perfusate, its pH, gas tensions and temperature, breathing parameters and compositions of inhaled air can all be easily manipulated. Compromises of the system include the lack of functional innervation of the lung. Thus centrally-mediated neural reflexes induced by irritants cannot occur. However, local reflex pathways are still intact. Also, the

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bronchiole circulation is not cannulated, causing perfusion buffer to flow in a retrograde fashion from the pulmonary circulation into the bronchial circulation. The isolated lung model represents an integrated in vitro model system, whereby all lung cells are present in their normal position with respect to each other. It is however not always easy to attribute an effect of a studied compound to a particular cell type [ 191. Due to the reactive nature of the isocyanate functional group and the aqueous nature of the environment to which it is introduced (the airway lumen), hydrolysis to the diamine product would be predicted and therefore, free TDA could be present in the perfusate. However, competing reactions with nucleophilic groups on airway molecules and metabolism of both the isocyanate and its diamine hydrolysis product in the lung could result in only low levels of free diamine to be observed in the perfusate. In vivo TDI, inhalation studies with guinea pigs and rats have shown that conjugated forms of the isocyanate appear to predominate in the circulation of these species and extremely low levels of free diamine are observed following exposure. It is not possible from whole animal studies to determine the specific contribution of lung to the distribution of molecules in the circulation since the contribution of nasal cavity absorption and/or the metabolic activity of systemic organs can not be determined. The use of the IVPL system in this study allowed the direct evaluation of the reactivity of TDI vapors in the airway. Macromolecular conjugation assessment and quantitation of the formation of free diamine were used as initial reaction endpoints. Hydrolysis of the high molecular weight radioactive adducts was performed to assess the stability and nature of the functional group modifications by TDI vapors. Comparison of the GC-SIM data with total scintillation values suggests that variable functional group modifications occurred, only some of which are susceptible to hydrolytic cleavage, under the conditions tested. We have previously shown that the perfused lung model can be used successfully to follow the effect of isocyanates on lung function [16]. A slight reduction in compliance values was found at a concentration of 0.6 ppm. Using mass spectroscopy, it was possible to monitor the uptake of TDX after hydrolysis and it was found that some form of the isocyanate is taken into the circulation. To facilitate a molecular analysis of these reactions, radioactively labeled TDI was used in the present study. System adaptations were required and included development of a miniaturized vapor generation system (Fig. lc) and minimization of recirculation volume of the perfusate. In addition, several proteins and diluted plasma were used to test the effect of variable perfusate components on uptake and distribution (Table 1). To balance between a sufficiently low concentration to minimize lung damage and a high enough concentration to provide a measurable level of label in the perfusate, the 14C labeled material was used at the highest possible specific activity (20.6 mCi/ mmol) which meant that the total volume of TDI available was 13 pl. Thus the miniaturized delivery system described in Fig. lc was developed to deliver a constant concentration of isocyanate from a minimum volume of TDI. As seen by the standard deviation on the average concentrations, that represents a combined average of triplicate sampling taken immediately before and after each exposure (Table 2), the atmospheric concentration was maintained at a constant level during each exposure series. The chosen concentrations also gave measurable levels of radioactive label in the perfusate even at the low concentration. These concentrations (0.2 ppm and 0.7 ppm) also represented values that bracketed the concentration (0.6 ppm) where lung function measurements had been made [16] and the

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low concentration (0.2 ppm for 45 min (0.15 ppm.h)) is comparable to that already reported for inhalation exposure to the same labeled compound in vivo (0.145 ppm for 1 h (0.145 PPd)) [71. In a previously reported in vivo study of the uptake of TDX in guinea pigs [7], it was found that there was a linear dose response relationship between the concentration of TDX in terminal blood and the total dose of TDI expressed as ppm.h. Based on that study, the rate of uptake of TDX at 0.146 ppm is calculated to be 4.17 x 10e4 ug Eq/ml.min (for a 40 ml blood volume the rate would be 0.0167 ug Eq/min) while from this study where the TDI is inhaled directly by the lung without the scrubbing of the nasal passage, the uptake rate at 0.2 ppm is calculated to be 4.43 x 10m4ug Eq/ml.min (for the 70 ml perfusate, the rate would be 0.0310 ug Eq/min). Thus the rate of uptake directly by the lung in the perfused lung model is approximately equivalent when the rate per ml of circulation is considered. Although the concentration of TDX in terminal perfusate and the rates of uptake of TDX into perfusate do increase with higher TDI exposure concentration, the relationships do not extrapolate to zero and therefore do not follow a linear dose response relationship. However, one major factor indicates that substantially different mechanisms of uptake may be at work between the in vivo case and the IVPL system. First, although the rate of uptake of TDX into the circulatory system is approximately equivalent at the low dose (0.2 ppm) for both the in vivo and IVPL systems, the molecular weight distribution of the molecules in each circulation system after the exposure are very different. In the in vivo system >95% of the isocyanate is found conjugated to molecules >lO kDa while, at a comparable TDI exposure concentration, the IVPL system yielded >60% of the labeled material ~10 kDa molecular weight. In both cases, the TDI was found either conjugated to macromolecules or as metabolites. The nonlinearity of the uptake can be explained by a saturation response which may occur at high isocyanate concentrations. If it were assumed that the same linear dose response relationship, as found in vivo [7] (39 200 dpm/ml/ppm.h), exists at low doses in the IVPL system, then an exposure concentration of 0.15 ppm.h would have been predicted to yield a terminal perfusate TDX concentration of 5880 dpm/ml and 3000 dpm/ml was found, while at 0.6 ppm.h a terminal concentration of 23 520 dpm/ml would have been expected and 8000 dpm/ml was found. These differences which diverge significantly at higher exposure concentrations suggest that there may be a saturation phenomenon at work in the transport of TDX molecules to the circulation. This is supported by the fact that at the higher exposure concentration (0.7 ppm), only 19.2% (range 14.5-27.6) of the total retained (recovered) dose is transported to the perfusate while the remainder is associated with the lung tissue. This is to be compared with the examination of the lung and blood from the in vivo exposure of guinea pigs at an equivalent dose. In this case, only 7.8% of the dose recovered from the lung and circulation is associated with the lung [7]. The results of this study indicate that, coupled with the use of radioactively labeled compounds, the IVPL system represents an excellent model system for measuring the contribution of the lung to the uptake, distribution and fate of reactive gases. In the preliminary results reported here for toluene diisocyanate several observations were made which correlate well with in vivo studies. First, after a delay, there is a linear uptake of label into the circulation during inhalation similar to that seen for MIC [lo] and TDI [7]. Though there are similar profiles of uptake kinetics using the two systems, there are significant differences in the distribution of products between the in vivo and IVPL systems. However, the

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distribution found for the perfused lung system may reflect the fate of isocyanate which reaches the lung during in vivo exposure. More low molecular weight products are found using the IVPL system; however, free TDA, the hydrolysis product of TDI, is still not an endpoint for the exposure and thus active reaction/metabolism would seem to take place in the lung. This parallels the results reported by Jin et al. which indicate numerous protein adducts are formed in the lung upon inhalation exposure of guinea pigs to TDI vapors [8]. In addition, two general observations from the current experiments were made which impact future experimental design using the IVPL system. The first was the observation that the composition of the perfusate significantly affected the rate of transport of the TDX to the circulation. In particular, the apparent species specificity of the albumin used in the perfusate would suggest the importance of recognition of specific albumins in possible exchange or transport mechanisms. And finally, the low dose retention (20-30%) for the reactive vapor may reflect incomplete gas exchange in the lung with each theoretical breath. Together, these observations suggest that the perfused lung model is very useful for studying the direct effects of molecules entering the physiological system through either inhalation or direct entry to the circulation.

Acknowledgements This work was supported by the Swedish Work Environmental Fund. Travel support for Amy L. Kennedy and William E. Brown was provided by the International Isocyanate Institute.

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