Development of an information-intensive structure-activity relationship model and its application to human respiratory chemical sensitizers.

Structure-activity relationship (SAR) models are recognized as powerful tools to predict the toxicologic potential of new or untested chemicals and also provide insight into possible mechanisms of toxicity. Models have been based on physicochemical attributes and structural features of chemicals. We describe herein the development of a new SAR modeling algorithm called cat-SAR that is capable of analyzing and predicting chemical activity from divergent biological response data. The cat-SAR program develops chemical fragment-based SAR models from categorical biological response data (e.g. toxicologically active and inactive compounds). The database selected for model development was a published set of chemicals documented to cause respiratory hypersensitivity in humans. Two models were generated that differed only in that one model included explicate hydrogen containing fragments. The predictive abilities of the models were tested using leave-one-out cross-validation tests. One model had a sensitivity of 0.94 and specificity of 0.87 yielding an overall correct prediction of 91%. The second model had a sensitivity of 0.89, specificity of 0.95 and overall correct prediction of 92%. The demonstrated predictive capabilities of the cat-SAR approach, together with its modeling flexibility and design transparency, suggest the potential for its widespread applicability to toxicity prediction and for deriving mechanistic insight into toxicologic effects.

Development of immunoassays for biomonitoring of hexamethylene diisocyanate exposure.

Hexamethylene diisocyanate (HDI) is used widely to manufacture polyurethanes for paints and coatings. It is an irritant and a chemical asthmagen. The U.S. Occupational Safety and Health Administration time-weighted average permissible exposure limit is 5 ppb and the ceiling limit is 20 ppb. We sought to develop a sensitive and specific immuno-bioassay to supplement workplace air monitoring and detect recent HDI exposure. For this, we produced rabbit antiserum to HDI-adducted keyhole limpet hemocyanin (HDI-KLH). The specificity of the antiserum was demonstrated by its reaction with a variety of HDI-conjugated proteins and the absence of reactions with conjugates of other diisocyanates, namely toluene diisocyanate and diphenyl methylene diisocyanate. Four immunoassays were developed and compared for their ability to detect decreasing quantities of HDI-adducted human serum albumin (HSA) containing 2 mol HDI adduct per mol HSA (HDI(2)-HSA) as determined by matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry. The sensitivities of some of the assays are within the range (0.82-45 nM) of current analytic methods. A Western analysis procedure has a sensitivity of 600 nM HDI adduct on HSA. ELISA inhibition assay, in which microtiter plates are coated with the HDI(2)-HSA antigen, has a sensitivity of 300 nM HDI adduct. An immunoblot assay has a sensitivity of 9 nM HDI adduct. The most sensitive bioassay (1.8 nM HDI adduct) is a three-antibody sandwich ELISA in which wells of microtiter plates are coated with the IgG fraction of the anti-HDI-KLH antisera. Compared with analytic methods for HDI biomonitoring, the immunoassays are faster and less costly and accommodate numerous samples simultaneously. The assays have the potential to affect industrial biomonitoring programs significantly.

Importance of inflammatory and immune components in a mouse model of airway reactivity to toluene diisocyanate (TDI).

BACKGROUND: Nearly 9 million individuals are exposed to agents in the workplace associated with asthma, and isocyanates represent the most common cause of occupationally induced asthma. OBJECTIVES: Nonetheless, the immunological mechanisms responsible for isocyanate-induced asthma are not clear. A murine model for toluene diisocyanate (TDI) asthma is described and employed to examine inflammatory and immune components that may be involved in the disease. METHODS: Groups (n = 6) of C57BL/6J and athymic mice were sensitized by subcutaneous injection (20 microl on day 1, 5 microl on days 4 and 11), and 7 days later challenged by inhalation (100 p.p.b., days 20, 22 and 24) with TDI. Twenty-four hours following the last challenge the tracheae and lungs were examined for histological changes as well as for the expression of Th1, Th2 and pro-inflammatory cytokines. Mice were also examined for airway reactivity to methacholine challenge and for specific and total IgE and IgG antibodies. RESULTS: TDI sensitization resulted in increased reactivity to methacholine challenge as well as a significant inflammatory response in the trachea and nares of wild-type mice, but not in the athymic mice nor in the lungs of the C57BL/6J mice. Airway inflammation was characterized by inflammatory cell influx, goblet cell metaplasia and epithelial damage. Histological changes in the trachea were accompanied by increased mRNA expression of interleukin (IL)-4, tumour necrosis factor alpha, lymphotoxin beta, lymphotactin and Rantes, as well as TDI-specific IgG antibodies and elevated levels of total IgE. IgE-specific antibodies were not detected with this exposure regimen but were produced when the TDI concentrations were increased. CONCLUSIONS: These studies provide a unique murine model for occupational asthma that generates both inflammatory and immune mediators similar to those occurring in TDI-induced asthma in humans.

Subclinical immunologic and physiologic responses in hexamethylene diisocyanate-exposed auto body shop workers.

BACKGROUND: Diisocyanates are potent sensitizing agents and currently the most commonly identified cause of occupational asthma in industrialized countries. However, diisocyanate asthma is difficult to diagnose and exposure and host risk factors are unclear. Auto body shops, one of the most common hexamethylene diisocyanate (HDI) exposure settings, are particularly difficult to study due to their small size and episodic exposures. Surveillance studies of such workers are limited. OBJECTIVES: We have initiated a cross-sectional field epidemiologic study, Survey of Painters and Repairers of Auto bodies by Yale (SPRAY), to characterize the effects of diisocyanate exposures on actively employed auto body shop workers. Methods and Results We present here questionnaire, physiologic, immunologic, and exposure data on 75 subjects enrolled in the study. No overt cases of clinically apparent diisocyanate asthma were identified based on spirometry, methacholine challenge, peak flows, and symptoms. HDI-specific lymphocyte proliferation was present in 30% of HDI-exposed workers and HDI-specific IgG in 34% of HDI-exposed workers, but they were not associated. HDI-specific IgE was detected in two workers. HDI-specific lymphocyte proliferation, increased methacholine responsiveness, and symptoms of chest tightness and shortness of breath were more common in the most heavily HDI-exposed workers, the painters. More long-term follow-up of this cohort should clarify the significance of these HDI-specific immunologic responses, physiologic changes, and symptoms. CONCLUSIONS: These findings demonstrate the presence of HDI-specific immune responses in a large proportion of healthy HDI-exposed workers.

Isolated airway exposure to toluene diisocyanate results in skin sensitization.

Toluene diisocyanate (TDI), a highly reactive industrial chemical is a leading cause of occupational asthma in westernized countries. It has also been reported to be a skin sensitizer in mice and guinea pigs although instances of skin sensitivity in humans are rare. It is uncertain if skin-contact is necessary to initiate the dermal sensitization. This study sought to determine if exclusive airway exposure to TDI could result in skin sensitivity. A group of guinea pigs was administered 50 microl of 0.6% TDI intratracheally (it.), another group received intranasal (in.) application of 0.6, 1.2, or 1.8% TDI. Eighty percent (4/5) of the it.-dosed animals, and 92% (11/12) of in.-dosed animals exhibited skin sensitivity. None of 14 control animals gave a positive reaction to patch challenge with TDI. These findings indicate that exclusive exposure of the airways to TDI can result in skin sensitivity and suggest that such events may be possible in TDI workers and should be considered in all workers exposed via the airways to chemical sensitizers.

Rapid reduction of intracellular glutathione in human bronchial epithelial cells exposed to occupational levels of toluene diisocyanate.

Toluene diisocyanate (TDI) is a recognized chemical asthmogen, yet the mechanism of this toxicity and the molecular reactions involved have not been elucidated. We have previously shown that TDI vapor forms adducts with the apical surface of the respiratory epithelium, and that it colocalizes with ciliary tubulin. In vitro, we have shown rapid reaction of TDI with glutathione (GSH) and transfer of the bisGS-TDI adduct to a sulfhydryl-containing major histocompatibility complex peptide. This study sought to determine if intracellular GSH is altered following exposure to TDI. We used the dye CellTracker Green (chloromethylfluorescein, CMFDA) for detection of glutathione. One-day and 6-day air-liquid cultures of human bronchoepithelial cells (HBE) were exposed to 20-100 ppb TDI vapor for 5, 15, or 30 min. Cells were subsequently imaged using a confocal microscope. Both 1- and 6-day cultures showed a decrease in intensity of the thiol staining as a function of the TDI exposure dose. Doses as low as 20 ppb, the current permissible exposure limit (PEL) to TDI, resulted in rapid (within 5 min) decreases in fluorescence. The decreased fluorescence was not due to cytotoxicity or decrease in either esterase or glutathione-S-transferase activity, enzymes necessary for activation of the fluorescence of CMFDA. The decrease in glutathione levels was verified using another fluorescent label, ThioGlo(TM) 1, and cell extracts. In addition, the mucus produced by 6-day air-liquid interface HBE cells in response to TDI exposure appeared to be protective, as HBE cells underlying mucus retained more fluorescence than did cells in the same cultures that were not covered with mucus. These results, along with previous data, strongly suggest that TDI enters pulmonary cells and reacts rapidly with intracellular GSH, and that this can occur at the current PEL of 20 ppb. This rapid reaction suggests the importance of cellular thiols in TDI-induced pulmonary disease.

Cell and molecular biology of chemical allergy.

OBJECTIVE: The objective of this review is to provide current approaches to gain increased understanding of the molecular basis of chemical allergenicity. Chemical allergy refers to an allergic reaction to a low molecular weight agent (ie, <1 kD). The symptoms and pathology of chemical asthma resemble those of allergy to larger sized agents, such as pollens, weeds, and danders. The differences relate to mechanisms of disease. To stimulate an immune response, low molecular weight chemicals function as haptens and bind to carrier macromolecules. This article focuses on the chemical reactions and physicochemical characteristics of chemical allergens. DATA SOURCES: Data were obtained from published clinical reports and from the Documentation of Threshold Limit Values (1998) published by the American Congress of Governmental Industrial Hygienists. RESULTS: In vitro studies indicate the stoichiometric reaction of some chemical allergens with glutathione and the subsequent transfer of the allergen from glutathione to other nucleophiles. Computer-generated structure-activity relationship models have been developed for chemicals that induce respiratory allergy. The models, based on physicochemical properties of the agents, have high sensitivity and specificity. CONCLUSIONS: The structure-activity relationship model suggests that chemical binding is the essential feature of chemical allergens. Their in vivo reactions with thiols may result in glutathione deficiency with consequent alteration in cellular reduction-oxidation (redox) status, release of cytokines, and promotion of the T helper cell 2 phenotype. Prevention of permanent disease is dependent on periodic medical surveillance of affected workers. When detected early, the disease can frequently be reversed.

Identification of human lung and skin proteins conjugated with hexamethylene diisocyanate in vitro and in vivo.

Diisocyanates are asthma-causing chemicals used in the commercial production of polyurethane. We have previously shown that human lung epithelial cell proteins can become conjugated with hexamethylene diisocyanate (HDI) and may be biologically important in diisocyanate-induced asthma. The objective of this study was to identify specific human lung and skin proteins that become conjugated with diisocyanate after in vitro and in vivo exposure. Following in vitro exposure of human airway epithelial cells (A549), keratin 18, the 78-kD glucose-regulated protein, trans-1, 2-dihyrobenzene-1,2-diol dehydrogenase, and actin were identified as prominent diisocyanate-conjugated proteins through use of a combination of immunocytochemical and mass spectrometric techniques. Following in vivo inhalation of an HDI aerosol, keratin 18 was also identified as the predominant diisocyanate-conjugated protein in human endobronchial biopsy samples, whereas albumin was the predominant diisocyanate-conjugated protein in bronchoalveolar lavage fluid. Keratin was also identified as a predominant diisocyanate-conjugated protein in human skin biopsy samples after epicutaneous exposure to liquid-phase HDI, although the major skin diisocyanate-conjugated protein (56-kD) differed from the predominant lung diisocyanate-conjugated keratin (47-kD). The data from this study identify keratin and other proteins as potential "carriers" for diisocyanates in vivo, and suggest that HDI conjugation of these proteins may play a role in the pathogenesis of diisocyanate-induced asthma.

A robust structure-activity relationship (SAR) model for esters that cause skin irritation in humans.

A structure-activity relationship (SAR) model has been developed to discriminate skin irritant from nonirritant esters. The model is based on the physicochemical properties of 42 esters that were tested in humans for skin irritation. Nineteen physicochemical parameters that represent transport, electronic, and steric properties were calculated for each chemical. Best subsets regression analysis indicated candidate models for further analysis. Regression analyses identified significant models (p < 0.05) that had variables that were also significant (p < 0.05). These candidate models were evaluated using linear discriminant analysis to determine if the irritant esters could be discriminated from nonirritant esters. The stability of the model was evident from the consistency of parameters among ten submodels generated using multiple random sampling of the database. The sensitivity of the ten models, evaluated by "leave-one-out" cross-validation, ranged from 0. 846 to 0.923, with a mean of 0.885 +/- 0.025 (95% CI). The specificity ranged from 0.615 to 0.923, with a mean of 0.738 +/- 0.06 (CI). Compared with nonirritant esters, irritant esters had lower density, lower water solubility, lower sum of partial positive charges, higher Hansen hydrogen bonding parameter, and higher Hansen dispersion parameter. The results indicate that physicochemical features of esters contribute to their ability to cause skin irritation in humans, and that chemical partitioning into the epidermis and intermolecular reactions are likely important components of the response. This model is applicable for prediction of human irritation of esters yet untested.

Intracellular S-glutathionyl adducts in murine lung and human bronchoepithelial cells after exposure to diisocyanatotoluene.

Diisocyanatotoluene (toluene diisocyanate, TDI), a 4:1 mixture of 2, 4- and 2,6-isomers used in the preparation of polyurethanes, causes occupational asthma by an as yet unknown mechanism. We previously showed that it forms adducts with the apical surface of the bronchoepithelium in vivo, and with ciliary microtubules in cultured human bronchoepithelial (HBE) cells. These results suggested that TDI may not enter HBE cells. In vitro studies, however, showed that TDI avidly forms bis adducts with glutathione (GSH) and that these adducts transfer monoisocyanato-monoglutathionyl-TDI to a sulfhydryl-containing peptide. This study sought to elucidate intracellular reactions of TDI. Using an electron paramagnetic resonance spectrometric (EPR) method, we established that the level of thiol-dependent quenching of phenoxyl radicals of etoposide was decreased >40% in pulmonary tissue of mice that received TDI intrabronchially. Similarly, HBE cells exposed to 100 ppb TDI vapor experienced a >30% reduction in thiol levels as determined with a thiol-specific fluorescent probe (ThioGlo 1). HPLC/UV analysis of lysates from HBE cells exposed to 200 and 500 ppb TDI vapor suggested a dose-related formation of S-glutathionyl adducts. Data from the 500 ppb TDI-treated HBE cells verified the identity of the 2-monoglutathionyl-4-monoisocyanato adduct. The results provide firm evidence that TDI enters pulmonary cells and reacts with GSH. This rapid reaction leading to formation of S-glutathionyl adducts of TDI suggests the importance of cellular thiols in TDI-induced pulmonary disease.

Isocyanate-conjugated human lung epithelial cell proteins: A link between exposure and asthma?

BACKGROUND: Isocyanates are a group of highly reactive cross-linking chemicals that cause airway inflammation and asthma in exposed individuals. Isocyanates have been detected along the airway epithelia of exposed workers and animals, prompting the hypothesis that isocyanates can directly bind to epithelial cell proteins. OBJECTIVE: We tested the hypothesis that hexamethylene diisocyanate (HDI) binds directly to lung epithelial cell proteins and initiated studies to evaluate the immunostimulatory potential of HDI-conjugated lung epithelial cell proteins. METHODS: Human lung epithelial cell lines were exposed to vapor- and liquid-phase HDI, and the cellular proteins were analyzed for HDI conjugation by Western blotting and tested for the ability to induce lymphocyte proliferation in vitro. RESULTS: A number of epithelial cell polypeptides, ranging from 25 to 110 kd in apparent molecular weight, were conjugated with HDI after exposure of the human lung epithelial cell lines (A549 and NCI-H292) to HDI concentrations greater than 0.005% (vol/vol) in the liquid phase. Vapor-phase HDI exposure resulted in a more restricted HDI conjugation pattern, with major HDI-conjugated polypeptides migrating at 47, 71, and 91 kd. HDI-conjugated epithelial cell proteins specifically stimulated proliferation of PBMCs from subjects with isocyanate-induced asthma but not HDI-exposed nonasthmatic individuals or atopic subjects with nonisocyanate-related asthma. CONCLUSIONS: The data demonstrate that epithelial cell proteins readily react with HDI and that HDI-conjugated epithelial cell proteins can stimulate lymphocyte proliferation. Further characterization and evaluation of HDI-conjugated epithelial cell proteins will elucidate their potential role in the pathogenesis of isocyanate-induced asthma.

Toluene diisocyanate colocalizes with tubulin on cilia of differentiated human airway epithelial cells.

Toluene diisocyanate (TDI), a highly reactive industrial chemical with widespread use in the manufacture of polyurethane and plastics, is the leading cause of occupational asthma associated with chemical exposure. We report the effects of TDI vapor (20, 100, 500, 1000 ppb) in vitro on differentiated human bronchial epithelial cells. Increased mucus was observed by electron microscopy at all TDI concentrations. Cytotoxicity, as evidenced by cell pyknosis and DNA fragmentation, was detected following a 30-min exposure to TDI concentrations of 100 ppb or higher. At 1000 ppb, transepithelial resistance was lost. Using confocal microscopy and double staining, TDI was found colocalized with ciliary tubulin in cultures that had been exposed to 20 and 100 ppb. These findings are the first to identify TDI binding to human pulmonary epithelial cells and indicate extensive binding to the cilia of differentiated epithelial cells. The in vivo implications of these findings include decreased ciliary movement and longer retention of TDI and hence increased exposure. Altered cytoskeletal-derived signal transduction may be a consequence of tubulin involvement. The effects of such changes on respiratory sensitization remain to be explored.

The importance of the diluent for airway transport of toluene diisocyanate following intranasal dosing of mice.

Uncertainty of the transport of reactive chemicals to the lung is a major concern when using intranasal dosing of animals. In a preliminary study using mice, intranasal instillation of the dyes methylene blue (in water) and Sudan black B (in 1:4 ethyl acetate:olive oil), indicated that the following conditions were necessary to achieve transport to the lung: (1) aqueous diluent, (2) light anesthesia prior to dosing, (3) holding the animal in a supine position during chemical application, and (4) maintaining the animal in the same position postdosing. Using these conditions, we investigated the distribution of toluene diisocyanate (TDI), a major industrial asthmogen, to the lung following intranasal administration. Female C57BL/6 mice received 20 microl of 1% TDI in ethyl acetate:olive oil (1:4). Group 1 received a single application on day 1; group 2, single applications on 2 consecutive days; group 3, single applications on 4 consecutive days; and group 4, a single application of the vehicle on 2 consecutive days. All mice were necropsied 24 h after the final application. The nasal passages, upper pharynx, trachea, lungs, and olfactory bulbs of each animal were examined with hematoxylin-eosin and immunohistochemical staining, the latter using a rabbit anti-TDI antiserum. Histopathology revealed desquamation of ciliated epithelial cells as well as inflammatory cell debris in the nasal cavity and upper pharynx of animals in groups 1-3. The intensity of these changes was dependent on the number of applications. No inflammation was observed in the trachea, lungs, or olfactory bulbs in any of the groups. Immunohistochemical examination revealed positive staining for the TDI moiety in epithelial cells of the nasal cavity and upper pharynx in animals of groups 1-3. No staining was observed in the trachea, lungs, or olfactory bulbs of any animal. These results suggest that TDI, when dissolved in olive oil:ethyl acetate and applied intranasally, does not reach the trachea and/or lower airways.

Relationship between allergic contact dermatitis and electrophilicity.

To evaluate the role of electrophilicity in the induction of allergic contact dermatitis (ACD) in humans, we compared the structure-activity relationship (SAR) model of ACD with those of electrophilic and nonelectrophilic subsets of chemicals in the ACD database. For these analyses, electrophilicity was defined as the potential of a chemical to induce mutations in Salmonella. It was found that electrophilicity accounted for approximately 30-40% of ACD-inducing ability, and the remainder was associated with nonelectrophilic structures. The identification of these moieties opens the possibility for studying their role in ACD.

Chemical carcinogenicity: can it be predicted from knowledge of mutagenicity and allergic contact dermatitis?

We investigated the suggestion [R.E. Albert, Environ. Health Perspect. 105 (1997) 940-948.] that results of mutagenicity testing in Salmonella combined with allergic contact dermatitis (ACD) testing in humans would be predictive of carcinogenicity in rodents. Using the cancer bioassay results of the US National Toxicology Program (NTP), Salmonella mutagenicity tests and a highly predictive structure-activity relational model of ACD, we conclude that the combination is not more predictive than the results of the Salmonella mutagenicity assay alone.

Immunologic cross-reactivity between respiratory chemical sensitizers: reactive dyes and cyanuric chloride.

BACKGROUND: CyCl is a low molecular weight reactive chemical used as an intermediate in the production of plastics, herbicides, pharmaceuticals, and fiber-reactive dyes. It is a potent inducer of specific IgE antibody. The CyCl functionality is a structural component of monochlorotriazine and dichlorotriazine dyes. OBJECTIVE: We have investigated the immunologic cross-reactivity between cyanuric chloride (CyCl) and reactive dyes and it was hypothesized that this moiety might be a dye epitope and that it might stimulate an allergic antibody response in dye-exposed workers. METHODS: To test this hypothesis, we have used sera with IgE antibodies to CyCl and also sera from dye-exposed workers who have IgE antibodies to Procion Orange MX2R, an azo dye containing the dichlorotriazine group. As a control group we have used dye-exposed workers with IgE antibody to Remazol Black B, a diazo dye containing the vinyl sulfone-reactive group. RESULTS: Using RAST and RAST inhibitions, we identified negligible cross-reactivity between CyCl and dichlorotriazine dye. CONCLUSION: The results of this study imply that the allergenic moiety on the dye residue resides in the chromophore rather than in the common structural component of CyCl and dichlorotriazine dyes.

Target organs and systems: methodologies to assess immune system function.

Immunotoxicity encompasses both reduced and heightened immune function. Diverse chemicals can impair functioning of the immune system. Both monographs and books have been devoted to detailed descriptions of immunotoxicity. This paper gives a brief overview of the methods currently used to assess the immunotoxic potential of chemicals. It also discusses the trend toward the use of alternative methods to mammalian models, such as feral species, in vitro assays, and computational models. The strategy of using a tier approach to screen chemicals for immunotoxicity is described, together with the rationale for, and limitations of, this approach. Interpretation of data with regard to clinical disease and human health is addressed. The immune system poses substantial complexities in this regard as the system has functional reserve and functional redundancy.