Phenyl isocyanate is a potent chemical sensitizer.

Diisocyanates, as a chemical class, are recognized for their ability to cause respiratory and dermal sensitization. By contrast, monofunctional isocyanates have not been associated with cases of clinical sensitization. We investigated the immunologic activity of phenyl isocyanate (PI), an aromatic monoisocyanate, which is a trace constituent in commercial diphenyl methane diisocyanate (MDI) products, since it is has been reported to cause an "asthma-like' syndrome in rats. The potency of PI as a contact sensitizer was assessed using the mouse ear swelling test. PI was found to be the most potent isocyanate tested yielding an SD50 (dose predicted to sensitize 50% of the mice) of 0.04 mumol/kg, compared with SD50 values of 0.5, 2.1, and 30.4 mumol/kg, for the diisocyanate sensitizers hexamethylene diisocyanate, MDI, and toluene diisocyanate (TDI), respectively. When tested for ability to stimulate humoral immune responses, antibody titers to PI were more than ten-fold greater than those induced by TDI. The mean hapten-specific IgG antibody titer to PI was 1.4 x 10(4), compared with 1.3 x 10(3) for TDI. The anti-PI IgG1 anaphylactic antibody titer (1.2 x 10(4) was significantly greater than the anti-TDI IgG1 titer of 6.4 x 10(2). Hapten-specific IgE was not detected to either isocyanate. These results indicate that phenyl isocyanate is a potent inducer of both cellular and humoral immune responses. This activity may be a significant contribution to the sensitization potential of commercial products in which it is a trace contaminant.

A murine model for assessing the respiratory hypersensitivity potential of chemical allergens.

Using equimolar quantities of 2 chemical allergens, toluene diisocyanate (TDI), noted for its ability to cause respiratory hypersensitivity, and dinitrochlorobenzene (DNCB), noted for its dermal sensitizing activity, the mouse was evaluated as a possible model to indicate respiratory hypersensitivity. A previously published procedure (Garssen et al. (1989) Immunology 68, 51-58) was followed whereby chemicals were applied epicutaneously to the shaved flank of BALB/c mice. Eight days later, animals were challenged by intranasal application of the chemical. The lungs were evaluated at 48 h. Both TDI and DNCB elicited mild mononuclear inflammatory cuffing around pulmonary vasculature. No reaction was noted around pulmonary airways. Sera, drawn 48 h following the intranasal challenge with chemical allergen, were evaluated for total IgE, hapten-specific IgE and IgG, and for IL-2, IL-4, IL-5, IL-6, and interferon gamma. Animals exposed to TDI demonstrated decreased total IgE and the presence of TDI-specific IgG. Cytokine levels were unchanged in both groups. These results indicate that in this mouse model, total serum IgE and the production of hapten-specific IgG antibodies distinguished a respiratory from a contact sensitizing chemical. Further comparison of the serologic response of mice to these two classes of chemicals is required to determine if the murine model can be used to predict dermal versus respiratory sensitizing activity of chemical allergens.

Methods to assess RAST results in patients exposed to chemical allergens.

RAST is routinely used to assess the presence of IgE antibodies against environmental allergens in the sera of symptomatic patients. With exposure to low-mol.-mass (chemical) allergens, RAST is also done, but the interpretation of results is more difficult. A study was undertaken to assess methods which have been employed to determine the positivity of sera and to develop an objective, statistically based procedure for determination of RAST positivity to chemical allergens. Sera were obtained from 14 nonsensitized (control) subjects and examined for total IgE by radioimmunoassay, and for cyanuric chloride (CyCl)-specific IgE by RAST. Linear regression analysis revealed the correlation between the tests to be 0.94. Equations for the 95% and 99% predicted intervals were obtained. Sera from 19 cyanuric chloride workers were tested for total and specific IgE. Positivity of RAST results was determined by three methods: RAST inhibition, RAST ratio (binding to CyCl-human serum albumin (HSA) disks/HSA disks), and the regression equation where values outside the 99% predicted interval were considered to be positive and those > 95% < 99% were considered to be borderline values. Determination of CyCl RAST positivity by the regression method gave results comparable to those obtained from RAST inhibition, whereas the RAST ratio resulted in many more false positive conclusions. This statistical approach to RAST analysis was also used to assess sera from patients exposed to the unrelated chemical allergens, toluene diisocyanate and formaldehyde. Conclusions based on the regression method were in good agreement with those from RAST inhibition assay. Moreover, use of one standard curve may be sufficient for interpretation of RAST results for diverse haptenic allergens. These results indicate that the regression method provides a statistical basis from which to determine positivity of RAST analyses while eliminating the need for RAST inhibition assays.

Formation, solvolysis, and transcarbamoylation reactions of bis(S-glutathionyl) adducts of 2,4- and 2,6-diisocyanatotoluene.

During our ongoing studies of the reactions of toluene diisocyanate (2,4- and 2,6-diisocyanatotoluene, TDI) in vivo, it became apparent that reactive form(s) of these diisocyanates reach(es) the circulatory system after passage through the respiratory system. Based on recent work by others regarding the transcarbamoylation reactions of monoisocyanates, we hypothesized that the reactive form could be masked as an S-thiocarbamoylglutathione adduct of one or more of the isocyanato moieties. In this study, the glutathione adducts of 2,4- and 2,6-diisocyanatotoluene were synthesized under physiological conditions. Bis adducts were the major products when near-equimolar amounts of glutathione and the individual diisocyanato compounds were mixed at physiological pH, and were formed in high yield. Little to no mono adducts formed under these reaction conditions. The masses of the bis adducts were confirmed by electrospray mass spectrometry (MS), and 1H NMR analysis strongly suggested that the thiol of the cysteine residue of glutathione was the nucleophile in each case. The rates of solvolysis of the two bis adducts in aqueous buffer under conditions of physiological temperature and pH were determined, and electrospray MS analysis showed that the corresponding mono(glutathionyl)-TDIs were formed in these reactions. Incubation in vitro of each of the bis(glutathionyl)-TDI adducts with a 12 amino acid peptide (Thr-Cys-Val-Glu-Trp-Leu-Arg-Arg-Tyr-Leu-Lys-Asn) at pH 7.5 resulted in transfer of one mono(glutathionyl)-toluylisocyanato moiety to the peptide as detected by HPLC and on-line electrospray MS analyses. In both the solvolysis and transfer experiments, the 2,4-TDI-derived bis(glutathionyl) adduct reacted most quickly, while both the bis(glutathionyl)-2,6-TDI adduct and its transfer product with the peptide were more stable than their 2,4-TDI-derived counterparts. The results indicate high stoichiometry in formation and ready transfer to nucleophilic sites of protein, and suggest that the isocyanato moiety of both 2,4- and 2,6-TDI may be regenerated in vivo from their bis(glutathionyl) adducts. As a consequence, the thiol status of particular tissues may be a contributing factor to individual TDI toxicity susceptibility, and a mechanism by which toxicity at sites distant to the initial point of contact may be proposed.

Use of anti-TNF-alpha antiserum to investigate toxic alveolitis arising from cotton dust exposure.

Cotton dust has been associated with byssinosis and toxic alveolitis. A murine animal model has been developed with which to investigate the pathogenesis of these disorders. Studies with the model have reproduced the neutrophilic inflammation characteristic of the alveolitis, and have shown the presence of tumor necrosis factor-alpha (TNF-alpha) in the bronchoalveolar lavage (BAL) fluid. The current study investigated the role of TNF-alpha in the inflammatory response by use of a polyclonal antiserum to recombinant murine TNF-alpha. Following a 4-h exposure to cotton dust, experimental animals showed a 40-fold increase in BAL cells with 92% neutrophils. There was a 24-fold increase in TNF-alpha in the BAL fluid. Up regulation of TNF-alpha mRNA expression was detected in BAL cells. Mice pretreated with anti-TNA-alpha antiserum displayed a marked attenuation of the neutrophilic inflammation; however, the level of TNF-alpha mRNA expression was not reduced in these mice. These studies support a major role of TNF-alpha in the toxic alveolitis induced by cotton dust inhalation.