Guinea pig respiratory response to isocyanates

TOXICOLOGY AND APPLIED PHARMACOLOGY 71, 113-l 22 (1983)

Guinea Pig Respiratory

Response

to Isocyanates’

L. S. MULLIN,~ C. K. WOOD, AND N. D. KRIVANEK Central

Research and Development Department, for Toxicology and Industrial Medicine.

Received

February

E. I. du Pont de Nemours and Company, Haskell Laborator), Elkton Road, P.O. 90s 50. Newark. Delaware 19711

19. 1983; accepted

May

27, 1983

Guinea Pig Respiratory Response to Isocyanates.MULLIN. L. S.. WOOD, C. K., AND KRIVANEK. D. (1983). Toxicol. Appl. Pharmacol. 71, 113-l 22. Exposure to some isocyanates (e.g., toluene diisocyanate) has been associated with development of respiratory sensitization. In this study. guinea pig respiratory response to protein conjugates of isocyanatoethyl methacrylate (IEM) and isocyanatoethyl propionate (IEP) was evaluated. Guinea pigs were exposed to daily induction exposures with an aerosol of bovine serum albumin (BSA) or BSA conjugated with IEM or IEP. After approximately 2 weeks significant increases in respiratory rate occurred in the guinea pigs exposed to the isocyanate conjugates. The number of animals responding was related to the degree of conjugation of isocyanate to protein. No response to unconjugated BSA was observed. The isocyanates conjugated to another carrier, guinea pig serum albumin (GSA), elicited responses. In guinea pigs responding to BSA-IEM, 0.01 ppm IEM monomer did not elicit responses; 0.1 to 0.4 ppm IEM vapor elicited responses similar to conjugates but which were delayed: 0.5 and 0.6 ppm induced irritation responses.An IEM polymer aerosol that contained <0.0@04% monomer did not elicit a response. These data suggesta response threshold. Guinea pigs developing responses to either of the isocyanate conjugates displayed cross-reactions to challenge with the other. A conjugate of BSA with hexyl isocyanate (HI) did not induce cross-responses in guinea pigs reactive to BSA-IEM. Application of BSA-IEP or IEP monomer to the scratched skin of guinea pigs that responded by inhalation to BSA-IEP resulted in immediate wheal and flare responses not seen in unexposed animals. All of these findings suggest induction of Type I hypersensitivity (asthmatic) directed toward the isocyanate portion of the conjugate and not the protein. N.

The manufacture and use of isocyanates and polyurethane enamels and foam have been associated with development of respiratory sensitization responses in certain exposed individuals (Butcher et al., 1977; Cavelier et al., 1977; Mapp et al., 1979; O’Brien et al., 1979; White et al., 1980). These asthmatic-type responses occurred either immediately or were delayed several hours after exposure. The responses have been attributed to airborne isocyanate monomer (Karol et al., 1979a). At

’ This paper was presented at the 2 1st Annual Society of Toxicology Meeting (Paper 559), Boston, Mass., February 1982. 2 To whom correspondence should be addressed.

our laboratory and elsewhere (Stevens and Palmer, 1970) early attempts to duplicate the same type of respiratory response in guinea pigs exposed to isocyanate monomer vapor produced equivocal results. However, in 1978, Karol et al.. reported the development of hypersensitivity responses in the respiratory tract of guinea pigs exposed to an aerosol of tolyl isocyanate (TI) conjugated to ovalbumin (OA). The responses were found to be induced by the hapten (TI) by demonstrating that sensitive animals did not respond to the OA carrier protein alone, but did respond to Tl conjugated to another carrier protein, bovine serum albumin (BSA). The purpose of the present study was to evaluate the respiratory responses induced by two other isocyanates. 113

MULLIN,

114

WOOD, AND KRIVANEK

isocyanatoethyl methacrylate, a material considered for use as a nolvurethane activator and _ _ an impurity in the manufacture of this material, isocyanatoethyl propionate. The responses produced by these materials were compared to those produced by hexyl isocyanate, the monoisocyanate analog of a frequently used polyurethane activator, hexamethylene diisocyanate (HDI). METHODS Test Material

Preparation

and Analysis

The test materials were 2-isocyanatoethyl methacrylate WM CH,=C;OCHrCHzN=C=O;

6 99.4% pure) and 2-isocyanatoethyl propionate (IEP; 0 CH,CH&HZCHIN=C=O; 99+% pure) both obtained from Dow Chemical Company and hexyl isocyanate (HI; CHs(CH,)sN=C=O, 99+% pure) obtained from E. I. du Pont de Nemours and Company. Isocyanate-protein conjugates were prepared by the slow addition of 0.25 ml isocyanate monomer to 50 ml of aqueous borate buffer solution (pH 9.4) that contained 1% w/v fraction V bovine serum albumin (BSA), guinea pig serum albumin (GSA), or in a preliminary series of exposures ovalbumin (OA) (Sigma Chemical Co., St. Louis, MO.). After vigorous stirring at room temperature for approximately 1 hr, the conjugate solution was dialyzed over a 72-hr period against three changes of distilled water, filtered through a sterile 0.45 @cmFalcon filter, and frozen. The degree of conjugation was increased by increasing reaction time and/or temperature (from 0 to 25°C). Conjugate solutions were stored frozen until used. Protein concentration in conjugate solutions was determined spectrophotometrically according to the method of Lowry et al. (1951). The degree of isocyanate conjugation to protein was determined by fluorescence spectrophotometry. Serial dilutions (25-100 pg protein/ml) of 1% (w/v) aqueous unreacted protein solutions and of conjugate solutions were prepared with 0.4 M potassium borate buffer (pH 10.4). Fluoropa reagent solution was prepared according to the supplier’s specifications.3 Equal volumes of protein or 3 50 mg Fluoropa crystals (Pierce Chemical Co., Rockford, Ill.) were dissolved in I ml methanol, 0.2 ml of 2-

conjugate dilutions and Fluoropa reagent were mixed, and fluorescence of the mixture was measured on a PerkinElmer MTF3 fluorescence spectrophotometer (X ex = 348 nm, X em = 428 nm). The relative difference in lluorescence between unreacted and conjugated proteins (adjusted for concentration) was considered to reIlect depletion of BSA, GSA, and OA primary amine groups due to reaction with isocyanate and, therefore, was considered a measure of the degree of isocyanate conjugation to protein. This measure was expressed as percentage conjugation. Exposure

Procedure

Male Hartley strain (Charles River Breeding Laboratories, Wilmington, Mass.) guinea pigs weighing 350 to 400 g on arrival were quarantined, observed for 10 days prior to exposure, and determined to be in good health. For exposure, each guniea pig was placed in a Lucite body plethysmograph with a rubber dam neck piece. Each plethysmograph was connected to a Gould Model PM6 air pressure transducer and respiration rate was recorded on a Gould Model 2800 recorder. Four guinea pigs per group were exposed by head only to house-line air for a 5-min control period, followed by a IO-min exposure to an aerosol of protein conjugate, and a 5-min recovery period on house-line air. During the induction period, guinea pigs were exposed once daily, 5 days per week. Their chamber positions were rotated every other day to ensure that all animals received the same aerosol&stribution, although no distributional differences were apparent. Exposures were conducted under positive pressure in a 10-l glass cylinder which contained side ports for placement of the plethysmographs as illustrated in Fig. 1. Protein solutions were aerosolized from a DeVilbiss Model 40 nebulizer by house-line air delivered from a precalibrated flow meter. This nebulizer generates particles in the 3- to 4-pm mass median diameter range (Dennis, 1976). Aerosolized protein conjugate passed directly into the test chamber and was dispersed by a funnel-shaped baffle. A separate air line was used to supply air during control and recovery periods and during exposure, if necessary, for diluting air. Chamber atmosphere samples were collected by drawing a portion of chamber air (2 liters/min for 10 min) by vacuum through a train of three fiitted-end midget impingers each containing 10 ml of distilled water. The amount of trapping solution per impinger was measured. and concentration of protein in solution was determined by the Lowry et al. (195 1) method. Chamber concentration of protein was determined based on airflow through the impinger and expressed as micrograms protein per liter air. The degree of isocyanate protein conjugation was determined prior to testing.

mercaptoethanol, 0.3 ml of 30% Brij 35 solution, and 100 ml of pH 10.4 borate buffer.

RESPONSE

TO

ISOCYANATES

Tubmg To Pressure Transducers

Nebulizer With Test Material

L-l Te& Meter

Impingers

FIG.

I. Schematic

diagram

of guinea

Animals were exposed daily during the induction period to a protein-isocyanate conjugate until a positive respiratory response occurred as determined by the respiratory index (see Data Analysis). Exposure continued during a positive response except in the case ofrespiratory collapse, at which time the guinea pig was removed from exposure. After a positive response, the guinea pig was challenged the following day with an exposure to BSA. This exposure was followed approximately I hr later by an exposure to GSA-isocyanate conjugate. If the GSA-isocyanate conjugate produced no response, the guinea pig was reexposed to the original BSA-isocyanate conjugate to determine whether the original conjugate continued to elicit a response. Once an isocyanate conjugate induced a respiratory response, animals were challenged on subsequent days with conjugates of other isocyanates to test for cross-response. Some guinea pigs responding to BSA-IEM were also challenged with IEM vapors to determine whether the monomer alone could elicit a response. IEM vapor was generated by passing clean filtered air over liquid monomer in a midget impinger. Chamber IEM concentration was determined by bubbling a portion of chamber air through toluene in a midget impinger and analyzing the solution for IEM content. The analytical method was a modification of the method of Schanche and Hermann (1974) with a gas chromatograph equipped with a nitrogen detector for analysis. Finally, one group of guinea pigs responding to BSAIEM conjugate was exposed to an isocyanate copolymer solution to determine whether that solution, either copolymer or residual monomer, was capable of eliciting a

pig aerosol

exposures

response. The copolymer solution consisted of 20% styrene/IEM/dodecyl mercaptan polymer (44/46/10) in dimethyl phthalate (DMP) and was delivered into the exposure chamber as an aerosol from a DeVilbiss nebulizer. Nominal chamber aerosol concentration was determined from the weight of sample used per volume of air delivered.

Duta A nalysjs A positive respiratory response in guinea pigs was characterized by a marked increase in respiration rate. This response was followed in some cases by gasping respiration (slow irregular rate with increased tidal volume) and “respiratory collapse.” Examples are presented under Results. Both the frequency of respiration rate increase and the time to onset of respiratory collapse were included in the formula developed by Karol et al. (1978) to calculate the respiratory index (RI).4 Each animal served as its own control. The criterion for a positive response was an increase of greater than three standard deviations from its average respiratory index during the induction period. A negative respiratory index resulted if respiration rate during or following exposure never exceeded the control respiration rate. If no respiratory responses occurred by the

4 RI = 20 (&)“* [In(Rr/&)] [(210 - 13T)/lS], where respiration rate (average rate during 5-min control period), RI = highest respiration rate occurring for 3 consecutive min during or following aerosol exposure, and T = time in minutes to respiratory coiIapse: if collapse does not occur. T = 15.

R, = starting

MULLIN,

116

WOOD, AND KBIVANEK

fourth week ofexposure, it was concluded that the response for that animal to the test material was negative. Scratch Test A scratch test for detection of immediate hypersensitivity was employed. The scratch test differsfrom standard guinea pig skin tests for delayed hypersensitivity which is a Tcell mediated response that takes about 24 hr to develop. A wheal and Ilare response in the scratch test occurs within minutes. Vaso-active substances released from skin mast cells result in vasodilation (erythema) and vascular permeability (wheal) (Fudenberg et al., 1978). This test shows the presence of a factor in the circulation capable of inducing a Type I hypersensitivity reaction. Several antigens were applied to the scratched skin of control and BSAIEP-exposed guinea pigs. Two weeks after respiratory responses first occurred in guinea pigs exposed to a 62% BSA-IEP conjugate, the back and neck of these four guinea pigs and two control (untreated) guinea pigs of the same age were shaved and 0.5 ml each of acetone, 0.5% IEP in acetone, 0.5% IEM in acetone, 0.76% aqueous BSA, or 0.74% aqueous BSA-IEP was applied to scratched areas of skin. Alter 1 hr skin was evaluated for erythema and wheal response and scored as negative, mild, moderate, or severe (Fudenberg et al., 1978).

RESULTS Response to Conjugates An initial exposure was conducted with ovalbumin (OA) as a carrier protein with a low degree of isocyanate conjugation (16%). Four guinea pigs exposed to an average concentration of 34 &liter OA-IEM daily developed positive respiratory responses during the second week of exposure. One died of respiratory collapse during exposure. All surviving animals reacted strongly to challenge with OA alone, while none responded to challenge with BSA-IEM, indicating the response was elicited by the carrier protein rather than the isocyanate component of the conjugate. Lack of response to the isocyanate was probably due to its low degree of conjugation on the carrier. Since the strong reactions to OA alone could override potential responses to an isocyanate conjugate, its use as a carrier protein was discontinued in favor of BSA which, by itself, did not induce positive respiratory responses in this test system.

Six groups of guinea pigs were exposed to BSA-IEM. Due to the large amount of data generated during these studies, respiratory responses of every animal tested are not presented. However, a representative example of positive responses in a test animal is presented in Table 1. Only animals which had an increase of three standard deviations in respiratory index over their index during induction exposures were considered to have a positive response. Responses to conjugate generally occurred after several minutes of exposure and subsided during the 5-min recovery period following exposure. For BSA-IEM exposures, the degree of conjugation ranged from 24 to 98%. The concentration of protein in chamber air was generally similar for all exposure groups (see Table 2). The number of animals developing respiratory responses was related to the degree of isocyanate conjugation on protein. Two of four guinea pigs exposed to the 24% conjugate, three of four exposed to the 72% conjugate, and all animals exposed to the 89% and greater conjugates developed positive responses during the second or third week of exposure. Often the responses were transient and lasted only several days. However, the responses could be evoked again if animals were rechallenged after a l-week rest period. Due to the short duration of the response, not every animal was challenged with GSA-IEM. However, when animals responsive to the original material were challenged, 80% responded positively to GSAIEM conjugate, while none responded to BSA alone (see Table 1). Those challenged with GSA alone also responded negatively. This result is considered strong evidence that the isocyanate portion of the molecule was critical for eliciting the response. All guinea pigs exposed to the 90% BSAIEM conjugate developed positive respiratory responses within 8 to 15 days after initiation of exposures. They were treated with disodium cromoglycate, DSCG, a bronchial asthma prophylactic agent. DSCG was administered as an aerosol (60 pg/liter for 10 min) r/z hr prior to exposure on test Days 10 and 16.

RESPONSE

TO TABLE

RESPIRATORY

RESPONSE

OF GUINEA

PIG No.

Respiratory (breath/min)

Control W

Day No.

Test material

2 3 4 5 8 9 IO II 12 12 15-19 22 23 23

BSA-IEM BSA-IEM BSA-IEM BSA-IEM BSA-IEM BSA-IEM BSA-IEM BSA-IEM BSA-IEM BSA GSA-IEM Not exposed BSA-IEM BSA BSA-IEP

a Percentage b Calculation ( Statistically d Exposure ’ Exposure ’ Exposure x Exposure

I

48020

EXPOSED

7c

94 86

20.3 10.5 6.4 2.8 9.6 3.3 14.0 8.6 94.2 8.6 100.0 59.5 6.4 67.4

reaction of protein primary amine with isocyanate. of respiratory index (RI) is detailed in text. significant response (+3 SD of previous Rls). Minute 3 to postexposure Minute 3. Minute 2 followed by “respiratory collapse” at Minute Minute 2 through 10. Minute 3 through 7.

None of the animals had positive respiratory responses on the days DSCG was preadministered. One guinea pig responded on the day

PIG RESPIRATORY

RESPONSES

BSA BSA-IEM BSA-IEM BSA-IEM BSA-IEM BSA-IEM BSA-IEM a Number

0 24 72 89 90 92 98 positive

responses/number

11 mind 5 min.

65.0’ 4.9 66.5’

+ collapse”

9 min’ 5 minY

in rate to 54 breaths/min

SERUM

ALBUMIN-ISXYANATOETHYL

EXPOSURES

38~ 37 f 54 -+ 31-+ 49 + 47t 42f tested.

23.4 12.3 7.4 3.2 6.5 4.0 15.9 9.7 97.2’ 9.7 583.0’

7-decrease

Average chamber concentration

70

Conjugation

Duration ot response

2

TO BOVINE

METHACRYLATE

Material

CONJUGATE’)

immediately after DSCG preadministration and others responded several days later when challenged with BSA-IEM aerosol. Thus. it

TABLE GUINEA

(89%

Respiratory index ’

Increase

136 126 116 I10 110 124 122 114 202 114 200 177 100 144

Ill

TO BSA-IEM

rate

During/after challenge (Fastest 3 mitt)

113 114 109 107 104 120 107 105 104 105 100 -

II;

ISOCYANATES

8 10 14 9 24 7 8

protein (&I)

Number positive

developing responses” O/4 214 3/4 414 4/4 414 414

118

MULLIN,

WOOD,

AND

KRIVANEK

guinea pigs responsive to BSA-IEP reacted when challenged with BSA-IEM. One of these also reacted when challenged with GSA-IEM. Cross-response to a related isocyanate conjugate was similar in time of onset and duration to that induced by the original conjugate. The intensity of response as measured by the respiratory index was equal to or slightly less than that to the original material. In no caseswere the cross-reactions more severe than that to original conjugate.

appeared that DSCG diminished the respiratory response to BSA-IEM. The four guinea pigs exposed to the 90% BSA-IEM conjugate were killed immediately after exposure during the third exposure week and the lungs examined. One of these animals was killed immediately after a positive response. No gross or histopathologic abnormalities that could be attributed to BSA-IEM exposure were observed in any of these animals. Several groups of guinea pigs were exposed to BSA conjugates of other isocyanates (Table 3). All guinea pigs exposed to a 62% conjugate of BSA-IEP developed positive responses by the third week of exposure. Guinea pigs responsive to BSA-IEP also responded to challenge with GSA-IEP but not to BSA or GSA alone. Two groups of guinea pigs were exposed to BSA-HI, 71 and 84% conjugated. One animal in the 7 1% group had a slight increase in respiration rate. The response was equivocal and the animal was not challenged. The maximum degree of conjugation of BSA-HI (84%), achieved by reacting HI with BSA twice, induced no respiratory responses.

Response to Monomer

Vapor and Copolymer

Two groups of guinea pigs responsive to BSA-IEM (92 and 98% conjugated) were challenged with IEM monomer. These animals were exposed for 10 min but were monitored for a 6-hr recovery period rather than the 5-min recovery used with conjugates, since delayed reactions have been reported in humans. Combined results of the two groups are given in Table 5. No immediate or delayed responses occurred in guinea pigs challenged with 0.01 ppm. Exposure to 0.1 to 0.4 ppm IEM vapor elicited respiratory responses in some of the BSA-IEM responsive guinea pigs. The responses were qualitatively identical to those induced by conjugate, i.e., similar increases in respiratory rate. However, the responses to monomer were delayed, occurring 1 to 5 hr postexposure and generally lasted longer than those induced by conjugates. An example of the response is given in Table 6

Cross-Reactions

Some guinea pigs exposed to BSA-IEM and BSA-IEP were tested for cross reactions. Results are in Table 4. Guinea pigs responsive to BSA-IEM cross-reacted to challenge with BSA-IEP but not to BSA-HI. Similarly, two

TABLE

3

GUINEAPIGRESPIRATORYRESPONSESTOBOVINESERUMALBUMIN-ISOC(ANATOETHYLPROPIONATEAND BOVINESERLJMALBUMIN-HEXYLISOCYANATEEXPOSURES

90 Material BSA-IEP BSA-HI BSA-HI 0 Questionable reaction. min on Day 12.

Average chamber concentration

Conjugation 62 71 84 RI statistically

protein (&I)

Number positive

24-r4 41 r2s 36+ 5 significant

(27.4)

but respiratory

developing responses 414 1 “j4 014

rate increased

from

100 to only 132 breaths/

RESPONSE

TO

ISOCYANATES

TABLE GUINEA

PIG RESPIRATORY

II9

4

CHALLENGES

RESPONSES TO CROSS-REACTION

Challenge Induction

%

90

Material

Conjugation

Material

BSA-IEM BSA-IEM

12 89 62

BSA-HI BSA-IEP BSA-IEM GSA-IEM

BSA-IEP

Conjugation

(Days 12 and 23). These responseswere not seen when control animals were exposed to monomer. Challenge with 0.5 and 0.6 ppm IEM produced severe upper respiratory tract irritation, as indicated by a 30 and 50% decreasein respiration during exposure, respectively, in both control animals and those previously exposed to conjugates. No delayed responseswere detected at these concentrations, but recovery from irritation was slow and may have masked a positive respiratory response. Four guinea pigsresponsiveto BSA-IEM were

Chamber protein concentration bg/U

Positive responses to challenges

71

68

O/3

62

21

212

89

?-I

2/L!

90

34

112

challenged with an aerosol solution of 20% styrene/IEM/dodecyl mercaptan copolymer in DMP. This sample contained less than 0.0004% total residual monomer (IEM and IEP). None of the animals displayed a positive responsewhen monitored during or for 6 hr following exposure.

Scratch Test

The scratch test results are given in Table 7. Control guinea pigs did not react to any of the materials which were applied to the skin (BSA, BSA-IEP, acetone, IEP, or IEM). No TABLE 5 guinea pigs that had been exposed to BSAGUINEA PIG RESPIRATORY RESFQNSESTO ISOCYAN- IEP aerosol reacted to acetone. Three of the ATOETHYL METHACR~LATE MONOMER AND COFQL~MER four guinea pigs which had been exposed by CHALLENGE AFTER INDUCTION WITH BOVINE SERUM ALinhalation to BSA-IEP had mild skin reactions BUMIN-ISOCYANATOETHYL METHACRYLATE to IEP in acetone and one also had a mild skin reaction to IEM in acetone. One BSAPositive Challenge Chamber responses to IEP-exposed guinea pig reacted moderately to material concentration challenge skin application of BSA-IEP and mildly to BSA while two others reacted mildly to BSAIEM 0.01 ppm O/3 IEP and not at all to BSA. Responseto both IEM 0.1 ppm I”/3 BSA-IEP conjugate and IEP monomer inIEM 0.3 ppm I ‘14 dicated the responsewas directed to the isoIEM 0.3 ppm 2”/3 IEM 0.4 ppm 2”/2 cyanate rather than the protein. IEM IEM IEM copolymer’

0.5 ppm 0.6 ppm 0.5 mg/l

Obj2 Ob/4 o/4

’ Asthmatic-type respiratory responses. bAnimaIs had respiratory irritation response. but no asthmatic reaction. ’ Containing less than 0.0004% total monomer.

DISCUSSION The technique of using protein conjugates of haptens to induce a sensitization response is not new. In 1920. Landsteiner and Lamp1

MULLIN,

120

WOOD, AND KRIVANEK TABLE 6

RESPIRATORY

RESPONSES OF GUINEA

PIG No. 48857

EXPOSED TO BSA-IEM

(97.6%

CONJUGATE)

Respiratory rate (breath/min) Day No. 1 2 3 4 5 8 9 10 11 12 16 17 23

Test material

Control (a4

During/afier challenge (Fastest 3 min)

% Increase

BSA-IEM BSA-IEM BSA-IEM BSA-IEM BSA-IEM BSA-IEM BSA-IEM BSA-IEM BSA-IEM 0.4 ppm IEM BSA-IEM BSA-IEM 0.3 ppm IEM

131 122 126 135 117 119 118 118 117 118 110 101 109

144 140 138 146 122 120 132 134 164 237 170 174 192

9.9 14.8 9.5 8.1 4.3 0.8 11.9 13.6 40.2 100.8 54.5 72.3 76.1

Respiratory index” 12.2 17.6 11.5 10.2 5.1 1.0 14.0 16.0 45.7b 108.4 b 59.7b 75.3b 81.3’

Duration of response

9 min’ 1 hrd 9 min’ 11 min’ 30 min8

’ Calculation of respiratory index (RI) is described in text. b Statistically significant response (+3 SD of previous RIs). ’ Exposure Minute 3 to postexposure Minute 1. d One hour postexposure to two hours postexposure (during exposure there was a 16% respiratory rate decrease). e Exposure Minute 2 to exposure Minute 10. ’ Exposure Minute 2 to postexposure Minute 2. BFour and one-half to five hour postexposure (during exposure there was a 23% respiratory rate decrease).

demonstrated antibody reaction against paminobenzenearsenate conjugates in rabbits injected with p-aminobenzenearsenate globulin (Landsteiner, 1962). Karol et al. (1978) and the present study have modified the tech-

nique by using aerosolized conjugates to induce hypersensitivity in the guinea pig respiratory tract. Protein conjugates were used in this study rather than monomers because it is likely that isocyanates act as haptens, in-

TABLE 7 SCRATCH

TEST RESPONSES OF GUINEA PIGS EXPOSED BY INHALATION ALBUMIN-ISXYANATOETHYL PROPIONATE

TO BOVINE

SERUM

BSA-IEP exposed guinea pigs Material

Control guinea pigs (number of skin responses”)

Number of skin responses’

Severity of response b

BSA-IEP Acetone 0.5% IEP in Acetone 0.5% IEM in Acetone

o/2 o/2 012 012 012

l/4 314 O/4 314 l/4

1+ 2+, 1++ 3+ 1+

BSA

a Wheal and flare reaction. b +, mild; ++, moderate (Fudenberg et al., 1978).

RESPONSE

TO

ducing an immune response after conjugation to a larger molecule. Also use of conjugates allowed delivery of greater amounts of antigen without inducing toxic effects such as sensory irritation. Respiratory responses observed in guinea pigs on this study suggest that the test materials induced an immediate (Type I) hypersensitivity resembling human isocyanate-induced asthma (Karol et al., 1979a). Evidence includes the type of response observed (increase in respiratory rate followed in some cases by gasping respiration), the time course until onset of response (lo-14 days), and the immediate wheal and flare reaction observed in the scratch test. Further support consists of inhibition of the responses by disodium cromoglycate (DSCG). DSCG is a drug which prevents allergic asthmatic attack by inhibiting the release of the mediators of anaphylaxis initiated by the interaction of antigens with reagenic (&E-type) antibodies (Cox, 1967). In guinea pigs infected with Trichinella spiralis, maximum bronchoconstriction occurred on Day 12 at which time DSCG significantly reduced the severity of effect (Carney, 1976). Further study should be directed toward determining which, if any, types of antibodies are involved in the guinea pig respiratory response. This finding would help in establishing the guinea pig response as a valid model for human respiratory sensitization reactions. In humans, respiratory sensitization is mediated by IgE immunoglobulins. Later IgG-type (blocking) antibodies, which bind circulating antigen without initiating a sensitization response, may result in hyposensitization (Fudenberg et al., 1978). In guinea pigs both IgEand IgG-type antibodies have been found (Dobson et al., 1971). Karol et al. (1978, 1979b) found hapten-specific precipitating (IgG-type) antibodies by double diffusion and immune electrophoretic techniques and reagenic &E-type) antibodies by passive cutaneous anaphylaxis were produced during exposure to isocyanate-protein conjugates. In the present study the respiratory responses lasted for only a few days. This finding might correspond to a build-up first of reagenic (IgE-

ISOCYANATES

I?1

type) antibodies followed within days by the production of blocking (IgG) antibodies. However it may be that the loss of response corresponded to a depletion of mediator (reagenic antibodies) due to frequency of challenge. Therefore a rest period allowed the mediator to again build up and responses could be reinduced. The exact function of the various immunoglobulin in the guinea pig asthmatic response has not been fully established. In addition, further work should be done to define the nature of the antigenic determinant of the conjugates. The positive response of the test animals to challenge with isocyanates conjugated to GSA (a different carrier and a guinea pig self-protein) and to isocyanate (IEM) monomer was evidence that the haptenic groups rather than the carrier protein were responsible for eliciting the asthmatic response. This isocyanate-directed response was seen with respiratory challenge by aerosol conjugate and monomer vapor as well as with dermal challenge by the scratch test. The delayed response to challenge with monomer supports the likelihood that a protein-isocyanate conjugation reaction occurred in vivo prior to respiratory reaction. In the present study HI appeared less antigenic than IEM and IEP. No cross-reactions to HI conjugates were observed. Based on the small number of animals tested, the degree of reliability attributed to the lack of cross-reaction to HI is not known. It appears, however, that in this limited study, the antigen had to be not only an isocyanate, but an isocyanate which was structurally similar to the original material. This finding is in contrast to a clinical study by O’Brien et al. ( 1979). Those investigators found that workers exposed only to TDI, an aromatic isocyanate, cross-reacted to diphenylmethane diisocyanate and also to HDI, an aliphatic isocyanate, upon bronchial challenge. The guinea pig respiratory test has not been evaluated for detecting other known human respiratory sensitizers. Materials such as trimellitic anhydride (Zeiss et al.. 1977) and phthalic anhydride (Chester et al., 1977) have been reported to cause respiratory sensitization

122

MULLIN,

WOOD,

in humans. These materials are difficult to conjugate to protein homogeneously without cross-linking and potential loss of hapten specificity due to their multiple reactive sites. Antigenicity of haptens in the present assay may depend on the carrier protein and the nature of the conjugate formed. The results of this test are not quantitatively predictive of the amount of isocyanate monomer which will sensitize humans nor does the test predict a dose which will elicit a response in a sensitized human. However, it appears that respiratory response in sensitized guinea pigs is to some extent dose related. In the BSAIEM study, the number of animals which became responsive was related to the degree of conjugation of the carrier protein. There was no correlation, however, between degree of conjugation of BSA-IEM and time to onset of response (minutes after exposure), response severity, or duration of response. The response to challenge with IEM monomer was concentration related at levels below 0.5 ppm in both the number of animals responding and the duration and severity of response. Higher concentrations resulted in sensory irritation and respiratory slowing which may have obscured any potential asthmatic responses. Lack of response to polymer also suggested that a critical level of monomer was needed to trigger a response. Further study of the nature of respiratory sensitization and the types of materials which are antigenic should be conducted. ACKNOWLEDGMENTS The

authors

wish

to thank

for conducting guinea typing manuscript.

G. Bessicks

pig exposures

and

and

for

B. T., JONES, H. W., DIEM,

R. N., O’NEIL, J. E., DHARMAJAN,

C. E., GLINDV., WEILL,

choconstriction in the guinea pig and the effect of disodium cromoglycate. Int. Archs. Allergy Appl. Im-

50, 322-328.

C., PHAM,

ROMACH, L. (1977).

Notes Dot.

P., MUR,

A., AND respiratory

BUI

DINH function.

J. M., LONG, Cub.

88, 315-327.

E. H.,

SCHWARTZ,

GREENSTEIN,

H. J., PAYNE,

S. (1977).

Clin. Allergy COX,

Q. T., MEREAU,

F., CICOLLELLA, Isocyanates and

CHESTER,

Phthalic

C. B., AND

anhydride

asthma.

7, 1 S-20.

J. S. G. (1967).

inhibitor

of

Nature

(London)

Disodium

reagenic

cromoglycate:

A specific

antibody-antigen

mechanisms.

216, 1328-1329. Handbook on Aerosols.

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