Laboratory Techniques for Identifying Causes of Allergic Dermatitis.

This article reviews the laboratory's role in identifying causes of chemical-induced allergic dermatitis. Several topics will be discussed. Allergen hazard identification refers to testing of chemicals for their sensitization potential. Animal-based, in silico, in chemico, and in vitro tests have been developed to identify the skin sensitization hazard of potential chemical allergens, but only a few of these are accepted by regulatory agencies. Laboratory investigations have also evaluated the stability of several commercially available allergic contact dermatitis patch tests. Such studies are considered product testing and are usually conducted in analytical chemistry laboratories.

Characterization and comparative analysis of 2,4-toluene diisocyanate and 1,6-hexamethylene diisocyanate haptenated human serum albumin and hemoglobin.

Diisocyanates (dNCOs) are low molecular weight chemical sensitizers that react with autologous proteins to produce neoantigens. dNCO-haptenated proteins have been used as immunogens for generation of dNCO-specific antibodies and as antigens to screen for dNCO-specific antibodies in exposed individuals. Detection of dNCO-specific antibodies in exposed individuals for diagnosis of dNCO asthma has been hampered by poor sensitivities of the assay methods in that specific IgE can only be detected in approximately 25% of the dNCO asthmatics. Apart from characterization of the conjugates used for these immunoassays, the choice of the carrier protein and the dNCO used are important parameters that can influence the detection of dNCO-specific antibodies. Human serum albumin (HSA) is the most common carrier protein used for detection of dNCO specific-IgE and -IgG but the immunogenicity and/or antigenicity of other proteins that may be modified by dNCO in vivo is not well documented. In the current study, 2,4-toluene diisocyanate (TDI) and 1,6-hexamethylene diisocyanate (HDI) were reacted with HSA and human hemoglobin (Hb) and the resultant adducts were characterized by (i) HPLC quantification of the diamine produced from acid hydrolysis of the adducts, (ii) 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay to assess extent of cross-linking, (iii) electrophoretic migration in polyacrylamide gels to analyze intra- and inter-molecular cross-linking, and (iv) evaluation of antigenicity using a monoclonal antibody developed previously to TDI conjugated to Keyhole limpet hemocyanin (KLH). Concentration-dependent increases in the amount of dNCO bound to HDI and TDI, cross-linking, migration in gels, and antibody-binding were observed. TDI reactivity with both HSA and Hb was significantly higher than HDI. Hb-TDI antigenicity was approximately 30% that of HSA-TDI. In conclusion, this data suggests that both, the extent of haptenation as well as the degree of cross-linking differs between the two diisocyanate species studied, which may influence their relative immunogenicity and/or antigenicity.

Reactivity measurement in estimation of benzoquinone and benzoquinone derivatives' allergenicity.

Benzoquinone (BQ) and benzoquinone derivatives (BQD) are used in the production of dyes and cosmetics. While BQ, an extreme skin sensitizer, is an electrophile known to covalently modify proteins via Michael Addition (MA) reaction whilst halogen substituted BQD undergo nucleophilic vinylic substitution (SNV) mechanism onto amine and thiol moieties on proteins, the allergenic effects of adding substituents on BQ have not been reported. The effects of inserting substituents on the BQ ring has not been studied in animal assays. However, mandated reduction/elimination of animals used in cosmetics testing in Europe has led to an increased need for alternatives for the prediction of skin sensitization potential. Electron withdrawing and electron donating substituents on BQ were assessed for effects on BQ reactivity toward nitrobenzene thiol (NBT). The NBT binding studies demonstrated that addition of EWG to BQ as exemplified by the chlorine substituted BQDs increased reactivity while addition of EDG as in the methyl substituted BQDs reduced reactivity. BQ and BQD skin allerginicity was evaluated in the murine local lymph node assay (LLNA). BQD with electron withdrawing groups had the highest chemical potency followed by unsubstituted BQ and the least potent were the BQD with electron donating groups. The BQD results demonstrate the impact of inductive effects on both BQ reactivity and allergenicity, and suggest the potential utility of chemical reactivity data for electrophilic allergen identification and potency ranking.

Pyridoxylamine reactivity kinetics as an amine based nucleophile for screening electrophilic dermal sensitizers.

Chemical allergens bind directly, or after metabolic or abiotic activation, to endogenous proteins to become allergenic. Assessment of this initial binding has been suggested as a target for development of assays to screen chemicals for their allergenic potential. Recently we reported a nitrobenzenethiol (NBT) based method for screening thiol reactive skin sensitizers, however, amine selective sensitizers are not detected by this assay. In the present study we describe an amine (pyridoxylamine (PDA)) based kinetic assay to complement the NBT assay for identification of amine-selective and non-selective skin sensitizers. UV-Vis spectrophotometry and fluorescence were used to measure PDA reactivity for 57 chemicals including anhydrides, aldehydes, and quinones where reaction rates ranged from 116 to 6.2 × 10(-6) M(-1) s(-1) for extreme to weak sensitizers, respectively. No reactivity towards PDA was observed with the thiol-selective sensitizers, non-sensitizers and prohaptens. The PDA rate constants correlated significantly with their respective murine local lymph node assay (LLNA) threshold EC3 values (R(2) = 0.76). The use of PDA serves as a simple, inexpensive amine based method that shows promise as a preliminary screening tool for electrophilic, amine-selective skin sensitizers.

Characterization of methylene diphenyl diisocyanate-haptenated human serum albumin and hemoglobin.

Protein haptenation by polyurethane industrial intermediate 4,4'-methylene diphenyl diisocyanate (MDI) is thought to be an important step in the development of diisocyanate (dNCO)-specific allergic sensitization; however, MDI-haptenated albumins used to screen specific antibody are often poorly characterized. Recently, the need to develop standardized immunoassays using a consistent, well-characterized dNCO-haptenated protein to screen for the presence of MDI-specific IgE and IgG from workers' sera has been emphasized and recognized. This has been challenging to achieve due to the bivalent electrophilic nature of dNCOs, leading to the capability to produce multiple cross-linked protein species and polymeric additions to proteins. In the current study, MDI was reacted with human serum albumin (HSA) and hemoglobin (Hb) at molar ratios ranging from 1:1 to 40:1 MDI/protein. Adducts were characterized by (i) loss of available 2,4,6-trinitrobenzene sulfonic acid (TNBS) binding to primary amines, (ii) electrophoretic migration in polyacrylamide gels, (iii) quantification of methylene diphenyl diamine following acid hydrolysis, and (iv) immunoassay. Concentration-dependent changes in all of the above noted parameters were observed, demonstrating increases in both number and complexity of conjugates formed with increasing MDI concentrations. In conclusion, a series of bioanalytical assays should be performed to standardize MDI-antigen preparations across lots and laboratories for measurement of specific antibody in exposed workers that in total indicate degree of intra- and intermolecular cross-linking, number of dNCOs bound, number of different specific binding sites on the protein, and degree of immunoreactivity.

Substituent effects on the reactivity of benzoquinone derivatives with thiols.

Benzoquinone (BQ) is an extremely potent electrophilic contact allergen that haptenates endogenous proteins through Michael addition (MA). It is also hypothesized that BQ may haptenate proteins via free radical formation. The objective of this study was to assess the inductive effects (activating and deactivating) of substituents on BQ reactivity and the mechanistic pathway of covalent binding to a nucleophilic thiol. The BQ binding of Cys34 on human serum albumin was studied, and for reactivity studies, nitrobenzenethiol (NBT) was used as a surrogate for protein binding of the BQ and benzoquinone derivatives (BQD). Stopped flow techniques were used to determine pseudofirst order rate constants (k) of methyl-, t-butyl-, and chlorine-substituted BQD reactions with NBT, whereas electron pair resonance (EPR) studies were performed to investigate the presence of the free radical mediated binding mechanism of BQD. Characterization of adducts was performed using mass spectrometry and nuclear magnetic resonance spectroscopy (NMR). The rate constant values demonstrated the chlorine-substituted (activated) BQD to be more reactive toward NBT than the methyl and t-butyl-substituted (deactivated) BQD, and this correlated with the respective EPR intensities. The EPR signal, however, was quenched in the presence of NBT suggesting MA as the dominant reaction pathway. MS and NMR results confirmed adduct formation to be a result of MA onto the BQ ring with vinylic substitution also occurring for chlorine-substituted derivatives. The binding positions on BQ and NBT/BQ(D) stoichiometric ratios were affected by whether the inductive effects of the substituents on the ring were positive or negative. The reactivity of BQ and BQD is discussed in terms of the potential relationship to potential allergenic potency.

Detailed mechanistic investigation into the S-nitrosation of cysteamine.

The nitrosation of cysteamine (H2NCH2CH2SH) to produce cysteamine-S-nitrosothiol (CANO) was studied in slightly acidic medium by using nitrous acid prepared in situ. The stoichiometry of the reaction was H2NCH2CH2SH + HNO2 → H2NCH2CH2SNO + H2O. On prolonged standing, the nitrosothiol decomposed quantitatively to yield the disulfide, cystamine: 2H2NCH2CH2SNO → H2NCH2CH2S-SCH2CH2NH2 + 2NO. NO2 and N2O3 are not the primary nitrosating agents, since their precursor (NO) was not detected during the nitrosation process. The reaction is first order in nitrous acid, thus implicating it as the major nitrosating agent in mildly acidic pH conditions. Acid catalyzes nitrosation after nitrous acid has saturated, implicating the protonated nitrous acid species, the nitrosonium cation (NO+) as a contributing nitrosating species in highly acidic environments. The acid catalysis at constant nitrous acid concentrations suggests that the nitrosonium cation nitrosates at a much higher rate than nitrous acid. Bimolecular rate constants for the nitrosation of cysteamine by nitrous acid and by the nitrosonium cation were deduced to be 17.9 ± 1.5 (mol/L)-1 s-1 and 6.7 × 104 (mol/L)-1 s-1, respectively. Both Cu(I) and Cu(II) ions were effective catalysts for the formation and decomposition of the cysteamine nitrosothiol. Cu(II) ions could catalyze the nitrosation of cysteamine in neutral conditions, whereas Cu(I) could only catalyze in acidic conditions. Transnitrosation kinetics of CANO with glutathione showed the formation of cystamine and the mixed disulfide with no formation of oxidized glutathione (GSSG). The nitrosation reaction was satisfactorily simulated by a simple reaction scheme involving eight reactions.

Haptenation: chemical reactivity and protein binding.

Low molecular weight chemical (LMW) allergens are commonly referred to as haptens. Haptens must complex with proteins to be recognized by the immune system. The majority of occupationally related haptens are reactive, electrophilic chemicals, or are metabolized to reactive metabolites that form covalent bonds with nucleophilic centers on proteins. Nonelectrophilic protein binding may occur through disulfide exchange, coordinate covalent binding onto metal ions on metalloproteins or of metal allergens, themselves, to the major histocompatibility complex. Recent chemical reactivity kinetic studies suggest that the rate of protein binding is a major determinant of allergenic potency; however, electrophilic strength does not seem to predict the ability of a hapten to skew the response between Th1 and Th2. Modern proteomic mass spectrometry methods that allow detailed delineation of potential differences in protein binding sites may be valuable in predicting if a chemical will stimulate an immediate or delayed hypersensitivity. Chemical aspects related to both reactivity and protein-specific binding are discussed.

Discrimination of haptens from prohaptens using the metabolically deficient Cpr(low/low) mouse.

The murine local lymph node assay (LLNA) is a validated, well accepted method for identification of chemical contact allergens. Both direct acting haptens and prohaptens (requiring metabolic activation) can be identified, but not differentiated by this assay. This study was used to assess the utility of a pan microsomal metabolic deficient mouse to distinguish between direct acting haptens and prohaptens in the LLNA. Hapten and prohapten induced cell proliferation was compared in C57BL/6J (B6) wild type (WT) versus homozygous (HO) knockout mice with a hypomorphic NADPH-Cytochrome P450 Reductase (CPR) gene (termed Cpr(low/low)) resulting in low CPR enzyme activity. Mice were dosed with known prohaptens; benzo(a)pyrene (BaP), carvone oxime (COx) and paracetamol (PCM) and haptens; oxazolone (OX), 4-ethoxymethylene-2-phenyl-2-oxazolin-5-one (EtOX), and N-acetylbenzoquinoneimine (NABQI) in this study. Skin microsomes from the WT, HO and heterozygous (HT) Cpr(low/low) mice were compared and evaluated for CPR activity. Lymphocyte proliferative responses to BaP, COx and PCM were significantly abrogated by 36.4%, 45.2% and 50.8%, respectively; in Cpr(low/low) knock out (KO) mice versus WT mice; while the lymphocyte proliferative responses to the direct acting haptens OX, EtOX and NABQI were comparable. CPR activity, determined as Units/mg protein, was determined to be significantly lower in the Cpr(low/low) mice compared to the WT. Results of the present study suggest potential utility of the Cpr(low/low) mice in the LLNA to differentiate prohaptens from direct acting haptens.

Use of the human monocytic leukemia THP-1 cell line and co-incubation with microsomes to identify and differentiate hapten and prohapten sensitizers.

Consumer and medical products can contain leachable chemical allergens which can cause skin sensitization. Recent efforts have been directed at the development of non-animal based tests such as in vitro cell activation assays for the identification of skin sensitizers. Prohapten identification by in vitro assays is still problematic due to the lack of prohapten bioactivation. The present study evaluated the effect of hapten and prohapten exposure on cell surface markers expression (CD86, CD54 and CD40) in the human monocytic leukemia, THP-1, cell line. Upregulation of activation and costimulatory markers are key events in the allergic sensitization process and have been reported to serve as indicators of skin sensitization. Cells were exposed to the prohaptens benzo(a)pyrene (BaP), 7,12-dimethylbenz(a)anthracene (DMBA), carvone oxime (COx), cinnamic alcohol (CA) and isoeugenol (IEG) at concentrations ranging from 1 to 10 µM for 24 and 48 h. The direct-binding haptens dinitrochlorobenzene (DNCB), benzoquinone (BQ), hydroxylethyl acrylate (HEA) and benzylbromide (BB) were used as positive controls. Cells were also exposed to the irritants sodium dodecyl sulfate (SDS) and sulfanilamide (SFA). Bioactivation of prohaptens was achieved by adding aroclor-induced rat liver microsomes (S9) to the cell cultures. Consistent upregulation of surface expressions of CD86, CD54 (ICAM-1) and CD40 was observed in THP-1 cells treated with direct-acting haptens (±S9) or prohapten (+S9). Upregulation of these markers was not observed after exposure to skin irritants or prohaptens in the absence of exogenously added S9. In conclusion, modification of in vitro cell culture assays to include co-incubation with microsomes enhances identification of prohaptens and allows them to be clearly distinguished from direct-binding haptens.

Fungal pigments inhibit the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of darkly pigmented fungi.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used to discriminate moniliaceous fungal species; however, darkly pigmented fungi yield poor fingerprint mass spectra that contain few peaks of low relative abundance. In this study, the effect of dark fungal pigments on the observed MALDI mass spectra was investigated. Peptide and protein samples containing varying concentrations of synthetic melanin or fungal pigments extracted from Aspergillus niger were analyzed by MALDI-TOF and MALDI-qTOF (quadrupole TOF) MS. Signal suppression was observed in samples containing greater than 250ng/µl pigment. Microscopic examination of the MALDI sample deposit was usually heterogeneous, with regions of high pigment concentration appearing as black. Acquisition of MALDI mass spectra from these darkly pigmented regions of the sample deposit yielded poor or no [M+H](+) ion signal. In contrast, nonpigmented regions within the sample deposit and hyphal negative control extracts of A. niger were not inhibited. This study demonstrated that dark fungal pigments inhibited the desorption/ionization process during MALDI-MS; however, these fungi may be successfully analyzed by MALDI-TOF MS when culture methods that suppress pigment expression are used. The addition of tricyclazole to the fungal growth media blocks fungal melanin synthesis and results in less melanized fungi that may be analyzed by MALDI-TOF MS.

Rapid and simple kinetics screening assay for electrophilic dermal sensitizers using nitrobenzenethiol.

The need for alternatives to animal-based skin sensitization testing has spurred research on the use of in vitro, in silico, and in chemico methods. Glutathione and other select peptides have been used to determine the reactivity of electrophilic allergens to nucleophiles, but these methods are inadequate to accurately measure rapid kinetics observed with many chemical sensitizers. A kinetic spectrophotometric assay involving the reactivity of electrophilic sensitizers to nitrobenzenethiol was evaluated. Stopped-flow techniques and conventional UV spectrophotometric measurements enabled the determination of reaction rates with half-lives ranging from 0.4 ms (benzoquinone) to 46.2 s (ethyl acrylate). Rate constants were measured for seven extreme, five strong, seven moderate, and four weak/nonsensitizers. Seventeen out of the 23 tested chemicals were pseudo-first order, and three were second order. In three out of the 23 chemicals, deviations from first and second order were apparent where the chemicals exhibited complex kinetics whose rates are mixed order. The reaction rates of the electrophiles correlated positively with their EC3 values within the same mechanistic domain. Nonsensitizers such as benzaldehyde, sodium lauryl sulfate, and benzocaine did not react with nitrobenzenethiol. Cyclic anhydrides, select diones, and aromatic aldehydes proved to be false negatives in this assay. The findings from this simple and rapid absorbance model show that for the same mechanistic domain, skin sensitization is driven mainly by electrophilic reactivity. This simple, rapid, and inexpensive absorbance-based method has great potential for use as a preliminary screening tool for skin allergens.

Increased cell proliferation in spleen and lymph nodes peripheral to contact allergen application site.

The local lymph node assay (LLNA) is widely used to identify chemicals that are contact sensitizers. The assay involves dosing mice with the chemical on both ears and pooling the superficial parotid lymph nodes for assessment of lymphocyte proliferation as a marker of sensitization. The present study explored potential reduction in animal usage by dosing one ear with the allergen and the other with vehicle-only. The respective draining lymph nodes were processed separately for tritiated thymidine ((3)H-TdR) incorporation. Cell proliferation in proper axillary and renal nodes, as well as in the spleen was also assessed. Cross-contamination of the chemicals from the dosed ears to other parts of the body via preening was prevented by dosing restrained animals and washing off the residual chemical with saline after 4h. Dosing the left ear with 0.02% oxazolone (OX) on unrestrained animals resulted in marked cell proliferation in its draining lymph node (stimulation index, SI=12.8) and in the lymph node draining the contra-lateral vehicle-dosed ear (SI=6), as well as the proper axillary lymph nodes (SI=3.3). Increased (3)H-TdR incorporation was not observed in the renal lymph nodes (SI=1.1). Similar stimulation of cells was observed in the lymph node draining the ear contra-lateral to the 30% hexylcinnamaldehyde (HCA)-dosed ear. Increased proliferative activity was observed in contra-lateral draining lymph nodes of restrained mice demonstrating that these results cannot be attributed to cross-contamination of adjacent skin. A significant increase in proliferation of splenocytes was also observed. It is concluded that dermal application of a contact allergen, as exemplified by OX and HCA, may induce cell proliferation in the neighboring lymph nodes and spleen indicative of hapten and/or haptenated proteins diffusing through the skin to peripheral nodes and the blood to produce systemic sensitization. It is also possible that lymphatic capillaries may communicate between the left and right side of the mouse head. Thus the contra-lateral draining superficial parotid node cannot be used as a control for application of contact allergen to a single ear in a modified LLNA.

Zinc diethyldithiocarbamate allergenicity: potential haptenation mechanisms.

BACKGROUND: Zinc diethyldithiocarbamate (ZDEC) and its disulfide, tetraethylthiuram disulfide (TETD), are rubber accelerators and contact allergens that cross-react in some individuals. OBJECTIVE: This study explored potential protein haptenation mechanisms of ZDEC and its oxidation products. METHODS: ZDEC oxidation/reduction products and sites of protein binding were assessed using high-performance liquid chromatography and mass spectrometry. The murine local lymph node assay (LLNA) was employed to probe haptenation mechanisms of ZDEC by examining its allergenicity along with its oxidation products and through elimination of oxidation and chelation mechanisms by substituting cobalt for zinc [cobalt (II) dithiocarbamate, CoDEC]. RESULTS: Oxidation of ZDEC by hypochlorous acid (bleach, HOCl), iodine, or hydrogen peroxide resulted in production of TETD, tetraethylthiocarbamoyl disulfide, and tetraethyldicarbamoyl disulfide (TEDCD). Albumin thiols reduced TETD with subsequent mixed disulfide formation/haptenation. ZDEC directly chelated the copper ion on the active site of the superoxide dismutase, whereas CoDEC did not bind to Cu proteins or form mixed disulfides with free thiols. ZDEC, sodium diethyldithiocarbamate, TEDCD, and TETD were all positive in the LLNA except CoDEC, which was non-allergenic. CONCLUSION: The thiol is the critical functional group in ZDEC's allergenicity, and haptenation is predominantly through chelation of metalloproteins and formation of mixed disulfides.

Mercaptobenzothiazole allergenicity-role of the thiol group.

The rubber accelerator, 2-mercaptobenzothiazole (MBT), is known to cause allergic contact dermatitis (ACD), but the mechanism is unknown. The role of the thiol group in MBT's allergenicity was investigated in the present study. Guinea pigs were sensitized to MBT using a modified guinea pig maximization test (GPMT) and reactivity was assessed toward 2-mercaptobenzothiazole disulfide (MBTS), 2-hydroxybenzothiazole (HBT; thiol-substituted), 2-(methylthio)benzothiazole (MTBT; thiol-blocked), and benzothiazole (BT; thiol-lacking). MBT and MBTS, but not BT, HBT, or MTBT, elicited ACD in MBT-sensitized animals, demonstrating that the thiol group is critical to MBT's allergenicity. In addition, both MBT and MBTS were shown to inhibit both glutathione reductase and thioredoxin reductase, and thus contribute to the stability of MBT-protein mixed disulfides. It is concluded that the probable haptenation mechanism of MBT is through initial oxidation to MBTS with subsequent reduction to form mixed disulfides with proteins.

Oxidation of 2-mercaptobenzothiazole in latex gloves and its possible haptenation pathway.

The rubber accelerator, 2-mercaptobenzothiazole (MBT), has been reported to cause allergic contact dermatitis from gloves and other rubber products, but its chemical fate when exposed to occupational oxidants and the mechanism of its pathogenesis are not known. It was hypothesized that the thiol group is critical to MBT's (its oxidation products or metabolites) covalent binding and/or haptenation to nucleophilic protein residues. Oxidative transformation of MBT to the disulfide 2,2'-dithiobis(benzothiazole) (MBTS) was observed within the glove matrix when hypochlorous acid, iodine, and hydrogen peroxide were used as oxidants. Cysteine reduced MBTS to MBT with subsequent formation of the mixed disulfide 2-amino-3-(benzothiazol-2-yl disulfanyl)propionic acid which was identified and characterized. Spectrophotometry and mass spectrometry experiments demonstrated the simultaneous reduction of MBTS and disulfide formation with Cys34 on bovine serum albumin, suggesting a potential route of protein haptenation through covalent bonding between protein cysteinyl residues and the MBT/MBTS thiol moiety. Metabolism of MBT using isoniazid and dexamethasone-induced rat liver microsomes, to give a protein reactive epoxide intermediate and provide an alternative protein haptenation mechanism, was not observed. The data suggest that the critical functional group on MBT is the thiol, and haptenation is via the formation of mixed disulfides between the thiol group on MBT and a protein sulfhydryl group.

Kinetics and mechanistic studies of the hydrolysis of diisocyanate-derived bis-thiocarbamates of cysteine methyl ester.

Diisocyanates (dNCOs) are the most commonly reported cause of chemically induced occupational asthma, but the ultimate antigenic form is unknown. Reactions of the three most common monomeric dNCOs, hexamethylene dNCO (HDI), methylene diphenylisocyanate (MDI), and toluene dNCO (TDI), with cysteine methyl ester (CME) gave the corresponding bis-dithiocarbamates (HDI-CME, TDI-CME, and MDI-CME). The dissociation kinetics of these bis-thiocarbamates, in aqueous conditions, was followed spectrophotometrically under varying pH and temperature conditions. Reaction of the adducts with methylamine or human serum albumin (HSA) produced diurea, monourea, and diamine products, and this was consistent with the base-catalyzed elimination reaction (E1cB) pathway being the dominant, but not exclusive, dissociation mechanism. The hydrolysis of the adducts was first-order with respect to OH(-) concentration and overall second-order (HDI-CME, k = 3.36 x 10(2) M(-)(1) min(-)(1); TDI-CME, k = 2.49 x 10(4) M(-)(1) min(-)(1); and MDI-CME, k = 5.78 x 10(4) M(-)(1) min(-)(1) at pH 7.4) with deviation from second-order when the dNCO had an aromatic functional group. Arrhenius plots gave activation energies (HDI-CME, E(a) = 70.6 kJ/mol; TDI-CME, E(a) = 46.1 kJ/mol; and MDI-CME, E(a) = 44.5 kJ/mol) that were consistent with the following order of stability: HDI-CME > TDI-CME > MDI-CME. Therefore, the stability of different dNCO-derived thiocarbamates in aqueous environments can vary greatly. Thiocarbamate dissociation rates and type of products formed may potentially influence antigenicity and subsequent hypersensitivity/toxic reactions following dNCO exposures.

Formation and stability of a nitric oxide donor: S-nitroso-N-acetylpenicillamine.

The formation, reaction dynamics, and detailed kinetics and mechanism of the reaction between nitrous acid and N-acetylpenicillamine (NAP) to produce S-nitroso-N-acetylpenicillamine (SNAP) was studied in acidic medium. The nitrous acid was prepared in situ by the rapid reaction between sodium nitrite and hydrochloric acid. The reaction is first order in nitrite and NAP. It is also first order in acid in pH conditions at or slightly higher than the pK(a) of nitrous acid. In lower pH conditions, the catalytic effect of acid quickly saturates. Higher acid concentrations also induce a faster decomposition rate of the SNAP, thus precluding the quantitative formation of SNAP from HNO2 and NAP. Both HPLC and quadrupole time-of-flight mass spectrometry techniques proved that SNAP was the sole product produced. No nitrosation occurred on the secondary amine center in NAP, and only the thiol group reacted to form the nitrosothiol. Cu(I) ions were found to be effective SNAP-decomposition catalysts. Cu(II) ions had no effect on the stability of SNAP. Ambient oxygen in reaction solutions was found to have no effect on initial rates of formation of SNAP, products obtained, and stability of SNAP. The formation of SNAP occurs through two distinct pathways. One involves the direct reaction of NAP and HNO2 to form SNAP and eliminate water, and the second pathway involved the initial formation of the nitrosyl cation, NO+, which then nitrosates the thiol. The bimolecular rate constant for the reaction of NAP and HNO2 was derived as 2.69 M(-1) s(-1), while that of direct nitrosation by the nitrosyl cation was 3.00 x 10(4) M(-1) s(-1). A simple reaction network made up of four reactions was found to be sufficient in simulating the formation kinetics and acid-induced decomposition of SNAP.