inhibition of cholinesterase activity by isocyanates

TOXICOLOGY AND APPLIED PHARMACOLOGY 63,45-52 (1982)

Inhibition of Choline&erase Activity by lsocyanates

WILLIAM E. BROWN, *J ALLEN H. GREEN,* MERYL H. KAROL,?

AND YVES c. E. ALARrEt

*Department of Biological Sciences, Carnegie-Mellon University, Pittsburgh, Pennsylvania 15213, and tDepartment of Industrial Environmental Health Sciences, Graduate School of Public Health,

University of Pittsburgh, Pittsburgh, Pennsylvania 15261

Received July 27, 1981; accepted November 23, 1981

Inhibition of Cholinesterase Activity by Isocyanates. BROWN, W. E., GREEN, A. H., KAROL,

M. H.. AND ALARIE, Y. C. E. (1982). Toxicol. Appl. Pharmacol. 63, 45-52. Exposure of workers to isocyanates may result in irritation and/or sensitization of the respiratory tract. An immunologic mechanism for sensitization has been presented previously. This investigation explored whether, as a possible mechanism for the irritation reaction, the toxic respiratory effect of isocyanates might be due to their ability to inhibit cholinesterases. Hexamethylene diisocyanate (HDI), hexyl isocyanate (HI), and 2,6-toluene diisocyanate (2,6-TDI) were found to completely inhibit purified human serum cholinesterase when added at molar ratios of 4: 1 to 8: 1 (isocyanate:enzyme). By contrast, molar ratios of 50: 1 or greater were required for 50% enzyme inhibition by 2,4-toluene diisocyanate (2,4-TDI), phenyl isocyanate, or o-tolyl isocyanate. Enzyme inhibition was also achieved by exposure of purified cholinesterase to at- mospheres containing 1 ppm isocyanates. Under these conditions, HDI and HI were again the most potent enzyme inhibitors with much less reactivity shown by 2,4-TDI and 2,6-TDI. Under more physiologic conditions, when whole human plasma was the source of cholinesterase, HDI and HI were still potent enzyme inhibitors. However, with the latter two isocyanates, the molar concentrations needed to effect 50% enzyme inhibition suggested affinity labeling by these reagents. The potent cholinesterase inhibition shown by HDI and HI may offer explanation for observed respiratory symptomatology noted upon exposure to these isocyanates.

Several diisocyanates are currently used in the production of polyurethanes. Among these compounds, toluene diisocyanate (TDI), hexamethylene diisocyanate (HDI), and diphenylmethane diisocyanate (MDI) have been reported to cause pulmonary hypersensitivity in exposed workers (Avery et al., 1969; Pepys et al., 1972; O’Brien et al., 1979; Zeiss et al., 1980). Specific antibodies have been associated with TDI sensitivity in humans and animals (Karol et al., 1978; Karol and Alarie, 1980; Karol, 1980; Karol et al., 1980) and with MD1 sensitivity in humans (Zeiss et al., 1980). Evidence is

I To whom correspondence and reprint requests should be addressed.

therefore accumulating that hypersensitivity responses to diisocyanates in exposed workers may be due to an immunologic mechanism.

Diisocyanates also have been associated with toxicologic reactions. High concentrations of TDI produced inflammatory reactions on the nasal mucosa of mice (Sangha and Alarie, 1979) and on the human cornea (Luckenbach and Kielar, 1980). A direct pharmacologic action of TDI was reported on the tracheobronchial tree (Van Ert and Battigelli, 1975) and on circulating lympho- cytes in in vitro preparations (Davies et al.. 1977).

Both TDI and HDI are potent sensory ir-

45 0041-008X/82/040045-08$02.00/0 Copyright 0 1982 by Academic Press. Inc All rights of reproduction in any form reserved

46 BROWN ET AL.

ritants of the nasal mucosa in mice (Sangha their solubility and dissolution in the aqueous reaction

and Alarie, 1979; Sangha et al., 198 1). This mixture. Previous studies (Brown and Weld, 1973b)

irritation response had a slow onset, and re- found that isocyanate reactions with serine proteases

covery following exposure was also ex- were very rapid. Therefore, in the current study, reac-

tremely slow. These findings suggested co- tions were allowed to proceed at 25°C for 10 min at which time a S-p1 sample of the reaction mixture was

valent interaction of diisocyanates with molecules associated with cholinergic nerve endings in the nasal mucosa. Since isocyanates are active-site-directed reagents for serine proteases (Brown and Wold, 1973a), a study was initiated to determine if isocyanates reacted specifically with active sites on cholinesterase. Inhibition of this enzyme could explain the activity of isocyanates as sensory irritants, and provide a pharmacologic basis for pulmonary reactions observed in workers upon first encounter with isocyanates and in the absence of specific antibodies to isocyanates.

removed for assay of remaining cholinesterase activity. This stepwise sequence was continued to complete the titration. The concentration of the stock isocyanates was increased during the titration to avoid excess acetone in the final reaction mixture. A typical plasma titration would involve steps of two consecutive ~-PI additions of a 2.6 mM isocyanate solution followed by two consecutive 5-pl additions of a 10 mM isocyanate solution followed by individual consecutive 5-~1 additions of 21,41, 83, and 166 mM isocyanate solutions. For 0.5 ml of plasma, these increments correspond to cumulative ratios (nmol/mg total protein) of 0.17, 0.34, 1.07, 1.80, 3.35, 6.36, 12.5, and 24.7, respectively, of isocyanate to plasma protein. The final concentration of acetone never exceeded 10% by volume. Parallel control titrations of enzyme (plasma) with aliquots of acetone alone indicated no significant effect of acetone on enzymatic activity. Results are expressed as percentage of the enzyme

was purified from plasma obtained from the Central Blood Bank of Pittsburgh, Pennsylvania. Purification to

METHODS

a specific activity of 150 units/mg was achieved by a combined purification scheme of Main et al. (1974) and Lockridge et al. (1979).

Enzyme assays. Horse serum and human plasma cho-

Cholinesterases. Horse serum cholinesterase (12.9

linesterase were assayed according to the procedure of

units/mg), bovine erythrocyte cholinesterase (3.34 units/

Ellman et al. (1961) with butyrylthiocholine (Sigma) as substrate. For assay of bovine erythrocyte and eel cholinesterases, acetylthiocholine (Sigma) was the sub-

mg), and eel cholinesterase (355 units/mg) were pur-

strate. One unit of activity was defined as the hydrolysis of 1 pmol butyrylthiocholine (or acetylthiocholine) per

chased from Sigma Chemical Company, and used with-

minute at 25°C.

out further purification. Human serum cholinesterase plied by Mobay Chemical Corporation, were: tbluene 2,4-diisocyanate (2,4-TDI, Mondur TDS, 99.7+%), toluene 2,6-diisocyanate (2,6-TDI, 99.1%) and hexame-

activity remaining calculated as the ratio of activity re-

thylene diisocyanate (HDI, Mondur HX, 1 98%). All

maining in the reaction vial containing isocyanate to

monoisocyanates were purchased from Eastman Or-

activity in the control vial containing an equivalent

ganic Chemicals and have a purity of 98% or greater.

amount of acetone. For titration of purified cholinesterases, enzymes

were dissolved in 0.02 M potassium phosphate buffer, pH 6.9, containing 1 mM EDTA at an initial concentration of 1 mg protein/ml. Diisocyanates, kindly sup-

Exposure of cholinesterase to isocyanate vapor. At- mospheres containing 1 ppm isocyanate were generated by bubbling dried air through an impinger containing isocyanate solution as described previously (Sangha et al., 1981). Vapors from the impinger were drawn into a lo-liter Plexiglass exposure chamber which was op- erated under a constant air flow of 20 liters/min. Con- centrations of isocyanates in the atmosphere were de- termined by sampling the chamber and analyzing the samples by gas chromatography as described by Sangha et al. (1981). For exposure, 1 ml of an enzyme solution containing 1 mg horse serum cholinesterase in 0.03 M

sodium phosphate buffer, pH 7.3, was placed in a watch glass positioned in the center of the chamber. The solution occupied a surface area of 707 mm’ (15-mm ra- dius). At designated times, aliquots were withdrawn from the unstirred enzyme solution and assayed for cholinesterase activity.

Protein determination. Protein concentration was de- termined by the microbiuret method of Goa (1953).

Cholinesrerase titration with isocyanates. Titrations of cholinesterases with isocyanates were performed by the following procedure detailed here for human plasma cholinesterase. Five hundred microliters of human plasma (68 mg protein, 2.1 units/ml) were placed in a conical reaction vial. Each increment of the titration involved addition of 5 ~1 of a stock solution of isocyanate, pre- pared in acetone, to the rapidly mixing protein solution. The isocyanates were dissolved in acetone to facilitate

ISOCYANATE CHOLINESTERASE REACTIONS 47

RESULTS

Isocyanate Inhibition of Cholinesterase Ac- tivity

As an example of a typical titration of cholinesterase with the isocyanates, the results of the titration of human serum cholinesterase with hexamethylene diisocyanate are shown in Fig. 1. Titration by successive additions of HDI to a cholinesterase solution resulted in the incremental loss of cholinesterase activity. Fifty percent inhibition of activity was achieved at a molar ratio of 3.2: 1 (isocyanate:cholinesterase) following five successive, cumulative additions of stock HDI solutions to a 1 mg/ml solution of cholinesterase. Assuming a molecular weight of 340,000 daltons (four subunits of 85,000 daltons each) (Muensch et al., 1976), the stoichiometry of the inhibition was calculated. Linear extrapolation of the early phase of the reaction to 100% enzyme inhibition (dashed line in Fig. 1) yielded a

0

\

molar ratio of approximately 6 mol HDI per mole cholinesterase (1.50 mol HDI/subunit). This high degree of specificity is in- dicative of a site-specific affinity label (Baker, 1967) whose inhibition reaction successfully competes with the self-hydrolysis rate of alkyl isocyanates under similar conditions (Brown and Wold, 1973b). Increased curvature of the titration curve was noted above a molar ratio of 3. This result was inter- preted as an increased contribution from the competing hydrolysis reaction as the number of effective reactive sites on the protein de- creases. This interpretation was supported by the finding that curvature was more pro- nounced when titrations were performed at lower protein concentrations as shown by the second curve (circles) in Fig. 1.

A comparison of the inhibitory ability of other alkyl as well as aryl isocyanates toward cholinesterases is presented in Table 1. Of the alkyl isocyanates, HDI was the most potent inhibitor of human serum cholinesterase with 50% inhibition achieved with 3.2

\ q Y q

1 \, , I I 1 1 I , I I I I 3 ,

4 8 12 16 20 24 28 32 MOLAR RATIO ( HDI/CHOLINESTERASE)

FIG. 1. Titration of purified human serum cholinesterases with HDI. Human serum cholinestetase (150 units/mg) in 0.02 M potassium phosphate buffer, pH 6.9, at protein concentrations of 0.33 mg/ml (squares) and 0.16 mg/ml (circles) were titrated with HDI. The dashed line is a linear extrapolation of the initial phase of the high protein concentration titration. Molar ratios were calculated on the basis of total protein present and a molecular weight of 340 K daltons for human serum cholinesterase.

48 BROWN ET AL.

TABLE 1

MOLAR RATIO ISOCYANATE~PROTEIN” REQUIRED FOR 50% ENZYME INHIBITION

Enzyme source

Bovine Human Isocyanate erythrocyteb serumC Eeld

Hexamethylene diisocyanate 0.24 3.20 1.75 Hexyl isocyanate 0.8 4.00 55.00 Butyl isocyanate 1.25 ND’ 108.7

2,6-Toluene diisocyanate 0.55 0.80 202.5 2,4-Toluene diisocyanate ND 67.5 700 o-Tolyl isocyanate 15.2 910 ND p-Tolyl isocyanate 15.9 ND ND Phenyl isocyanate 37.5 ND ND

’ Calculations of molar ratios assumed that all protein was cholinesterase. ’ Bovine erythrocyte cholinesterase (Sigma). Specific activity of 3.34 units/mg protein when assayed with ace-

tylthiocholine substrate. ’ Human serum cholinesterase chromatographically purified from human plasma. Specific activity of 150 units/

mg protein when assayed with butyrylthiocholine substrate. d Eel cholinesterase (Sigma). Specific activity of 355 units/mg protein when assayed with acetylthiocholine

substrate. ’ Experiment not performed.

mol isocyanate per mole enzyme. Hexyl isocyanate had slightly less potency as an inhibitor. Of the aryl isocyanates, only pure 2,6-TDI was an effective inhibitor of human cholinesterase. The remaining aryl isocyanates showed much lower specificity for the cholinesterase when compared with that shown by the alkyl isocyanates. 2,6-TDI showed a higher specificity than any of the alkyl isocyanates. The specificity shown by 2,6-TDI may reflect a difference in the hydrolysis rates of the alkyl and aryl isocyanates under these reaction conditions.

The isocyanates were tested also for their ability to inhibit cholinesterases obtained from bovine erythrocyte, horse serum, and electric eel. Results of the titration of the bovine erythrocyte enzyme yielded results similar to those obtained with the human serum enzyme (see Table 1). However, differences in inhibition stoichiometry were apparent. This finding may reflect differences in enzyme purities. The erythrocyte preparation was 3.34 units/mg, whereas the serum preparation was 150 units/mg. All molar ratios were calculated assuming both preparations were 100% cholinesterase.

With the erythrocyte enzyme, a greater similarity between HDI and 2,6-TDI in stoichiometry for 50% enzyme inhibition was seen. This result may be a consequence of the protein concentration at which the titrations of the erythrocyte (1 .O mg/ml) and the serum (0.33 mg/ml) enzymes were performed. The higher the protein concentration, the less the difference in isocyanate hydrolysis rates will contribute to the stoichiometry (see Fig. 1). To test these as- sumptions, samples of commercially avail- able bovine erythrocyte and horse serum cholinesterases were purified on the procain- amide affinity column of Main et al. (1974) and were titrated with HDI at the same protein concentration (0.4 mg/ml). The molar ratio required for 50% inhibition was 0.93 for the erythrocyte enzyme and 1.19 for the serum enzyme.

Addition of isocyanates to preparations of purified horse serum cholinesterase resulted in inhibition parallel to that seen with purified human serum cholinesterase. By contrast, titration of cholinesterase from the electric eel yielded very different results. Only HDI was effective in inhibiting the eel


enzyme. The other alkyl isocyanates, as well as 2,6-TDI, were at least an order of mag- nitude less effective in their ability to inhibit enzyme activity.

Inhibition of Cholinesterase by Vapors of Isocyanates

Exposure of horse serum cholinesterase to isocyanate vapors resulted in enzyme inhibition as shown in Fig. 2. Under these conditions, HDI and HI were again found to be potent cholinesterase inhibitors. Within 5 min of exposure, HDI inactivated more than 80% of the cholinesterase activity and HI inactivated more than 50% of the activity. By comparison, inhibition by the aryl isocyanates required longer exposure. Of interest was the finding that 2,6-TDI and 2,4- TDI were of equivalent potency in this assay whereas when these isocyanates were added directly to purified serum cholinesterase, 2,6-TDI was considerably more potent than the 2,4-TDI isomer (Table 1).

Vapors of o-tolyl isocyanate showed very

little reactivity toward the horse serum enzyme. It is not clear whether the differences in specificity between vapor and liquid states of the isocyanates reflect differences in isocyanate solubility in the unstirred solution. or differences in hydrolysis rates of the var- ious isocyanates. However, the rapid and complete inhibition of cholinesterase activity by HDI remains the striking feature of this assay.

Isocyanate Inhibition of Plasma Cholincs- terase

In an effort to approach a more physiologic situation for interaction of isocyanates with cholinesterases, whole plasma was titrated with a variety of isocyanates. The results, shown in Fig. 3, indicated that HDI and HI retained their high degree of specificity for cholinesterase whereas 2,6-TDI showed a significant loss in specificity toward the enzyme in plasma when compared to purified enzyme. Of interest was the finding that o-tolyl isocyanate showed an increased specificity for the enzyme in plasma.

10 20 30 40 50 60 70 80 90 MINUTES OF EXPOSURE

FIG. 2. Exposure of cholinesterase to isocyanate vapors. One-milliliter aliquots of horse serum cholinesterase (12.7 units/mg, 1 mg/ml) in 0.02 M potassium phosphate buffer, pH 6.9, were exposed to I ppm atm of isocyanate vapor. The curves represent exposures to HDI (open squares), HI (filled squares), 2,6-TDI (open triangles), 2,4-TDI (filled triangles), and o-tolyl isocyanate (diamonds).

50 BROWN ET AL.

II 11 11 1 I II

10 20 30 40 50 60 70 80 NANOMOLES ISOCYANATE/MILLIGRAM TOTAL PROTEIN

10 20 30 40 50 60 MOLAR RATIO (ISOCYANATE/CHOLlNESTERASE) X 10-4

FIG. 3. Titration of plasma with isocyanates. One-milliliter aliquots of human plasma were titrated with HDI (open squares), HI (filled squares), 2,6-TDI (open triangles), 2,4-TDI (filled triangles), and o-tolyl isocyanate (diamonds). Molar ratios were calculated assuming a specific activity of 760 units/ mg and a molecular weight of 340 K daltons for pure serum cholinesterase.

Increased addition of isocyanates to plasma caused reduction in cholinesterase activity but complete enzyme inhibition was not obtained. This result may reflect hydrolysis of isocyanates, reaction of isocyanates with other plasma proteins, as well as the pres- ence of other plasma proteins capable of hydrolyzing the butyrylthiocholine substrate but not affected by isocyanate reaction. The persistent demonstration of cholinesterase specificity by HDI and HI further empha- sizes the affinity-labeling character of these reagents.

DISCUSSION

Pharmacologic effects of isocyanates have been observed in both experimental animals and humans. Exposure to high concentrations of TDI (0.2 ppm or greater) has re-

sulted in irritation of the eye, skin, and respiratory tract, resulting in chest tightness, coughing, and reduced pulmonary function (NIOSH, 1978). Reports of stoichiometric isocyanate inactivation of serine proteases, together with carbamate inhibition of cholinesterase activity and location of cholinergic nerve endings in respiratory tract tis- sue, prompted investigation of effects of several industrial isocyanates on cholinesterases.

In this study, initial enzyme inhibition experiments were conducted by direct addition of isocyanates to purified human serum or bovine erythrocyte cholinesterase. Under these conditions, the alkyl mono- and diisocyanates were potent enzyme inhibitors. By comparison, the aryl isocyanates, with the exception of pure 2,6-TDI, were much poorer enzyme inhibitors. When the electric eel was the source of cholinesterase, HDI


was the only isocyanate capable of effective enzyme inhibition. Both HI and butyl isocyanate were at least 10 times less potent as inhibitors. The different specificities of isocyanates for cholinesterases from different sources are probably a reflection of the differences in substrate specificities of each enzyme (Augustinsson, 197 1) and in molecular structure of the different enzymes (Muensch et al., 1976; Rosenberry and Richardson, 1977). This discrimination of isocyanates for cholinesterases from different sources resem- bles results seen with insecticides (carbamates and organophosphates) (Metcalf, 1971).

Exposure of purified horse serum cholinesterase to two alkyl isocyanates (HDI and HI), either as vapors or liquid, resulted in effective enzyme inhibition. In the studies with isocyanate vapors, a small volume of enzyme solution was introduced into the watch glass to assure a large surface area for exposure to isocyanate. Under these conditions, both the solubility of isocyanate and the hydrolysis rate of isocyanate vapors be- came important. Results confirmed the affinity of HDI for cholinesterase. HDI inactivated more than 80% of the enzyme activity after less than 5 min of exposure. At 1 ppm, the amount of HDI in air would be equivalent to 4.1 X lo-’ M. The inhibition of cholinesterase by exposure to this concentration should be compared to the 10m3 to lo-’ M concentration of TDI required to demon- strate an effect on cyclic AMP levels in in vitro studies with leukocytes (Davies et al., 1977; Van Ert and Battigelli, 1975).

Experiments were conducted with human plasma cholinesterase to better simulate the physiologic situation. Under these conditions, HDI and HI were potent cholinesterase inhibitors. On a volume basis, 10 ~1 of a 10.5 mM HDI solution inhibited 60% of the cholinesterase activity in 1 ml of plasma. Calculation of the ratio of moles isocyanate: mole cholinesterase to effect this inhibition yielded a ratio of 7.7 X 103. This high ratio reflects the small amount of enzyme present in plasma (approximately 0.05 to 0.07% by

weight) (Main et al., 1974; Das and Liddell, 1970). Of the aryl isocyanates tested with human plasma, o-tolyl isocyanate showed significant reactivity toward the enzyme. This result contrasts with that found using purified enzyme where only 2,6-TDI was found to be a specific enzyme inhibitor. The latter diisocyanate appears to be less potent in plasma, possibly due to the potential for competing reactions with the many other plasma proteins. In recent studies with calf brain cholinesterase, it was found that factors present in crude cholinesterase preparations increased the binding of cholinergic ligands to the enzyme (Hollunger and Nik- lasson, 198 1). Purification of the enzyme and the concomitant removal of these factors from solution resulted in less binding of cer- tain ligands to cholinesterase. Such an oc- currence might offer an explanation for the increased affinity of o-tolyl isocyanate toward cholinesterase in plasma when compared to the lower affinity of this ligand for the purified enzyme.

The studies reported here have attempted to investigate a possible mechanism for the irritation response observed following exposure to isocyanates. The striking finding was that HDI was consistently a potent inhibitor of cholinesterase activity regardless of the source or purity of enzyme, the vapor or liquid state of added isocyanate, and the apparent self-hydrolysis of isocyanate. Cal- culation of the molar ratio of HDI to purified enzyme needed for complete inhibition indicated stoichiometry and affinity labeling by HDI.

In view of the industrial use of HDI in polyurethane applications, the finding of stoichiometric inactivation of the cholinesterase in whole plasma by HDI is of consid- erable importance. The biological imph- cations of this isocyanate-cholinesterase interaction remain to be elucidated.

ACKNOWLEDGMENTS

This investigation was supported by the Winters Foundation, Grant ES01 532 from the National Institute

52 BROWN ET AL.

of Environmental Health Sciences, and Grant OH00865 from the National Institute of Occupational Safety and Health. The authors thank Wei-Duan Lin Liao and Charlene Yen for development of serum cholinesterase purification protocol and helpful discussion of results.

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inhibition of cholinesterase activity by isocyanates

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