Last data update: Apr 22, 2024. (Total: 46599 publications since 2009)
Records 1-30 (of 53 Records) |
Query Trace: Siegel PD [original query] |
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Laboratory Techniques for Identifying Causes of Allergic Dermatitis
Chipinda I , Anderson SE , Siegel PD . Immunol Allergy Clin North Am 2021 41 (3) 423-438 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. |
Isothiazolinone detection in dish soap and personal care products: Comparison of lovibond isothiazolinone test kit and ultrahigh-performance liquid chromatography-tandem mass spectrometry
Kimyon RS , Zang LY , Siegel PD , Voller LM , Schlarbaum JP , Warshaw EM . Dermatitis 2020 32 (4) 245-250 BACKGROUND: Isothiazolinones are commonly used preservatives, which may cause allergic contact dermatitis. The Lovibond Isothiazolinone Test Kit (LITK) has been reported to successfully identify clinically relevant, occult isothiazolinones in patient personal care products. OBJECTIVE: The aim of the study was to analyze dish soaps and personal care products that do not declare isothiazolinones ("no-ISO") for the presence of isothiazolinones via 2 methods: LITK and ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). METHODS: No-ISO dish soaps (n = 9), a convenience sample of patient products (n = 6), and controls (positive [isothiazolinone declared], n = 5; negative, n = 2) were tested with LITK (X3) and UHPLC-MS/MS. RESULTS: Several no-ISO dish soaps and personal products were positive for isothiazolinones (LITK, n = 12; UHPLC-MS/MS, n = 3). Ultrahigh-performance liquid chromatography-tandem mass spectrometry specifically identified methylisothiazolinone alone in 1 no-ISO dish soap, methylchloroisothiazolinone in another, and both in a third. Using UHPLC-MS/MS as the criterion standard, we observed the accuracy of LITK for 9 dish soaps was poor (sensitivity, 66.7%; specificity, 20%) and very poor for 6 personal care products (sensitivity, 0%; specificity, 0%). CONCLUSIONS: Personal products may contain undeclared isothiazolinones. The current study found that LITK had poor accuracy for testing dish soap and personal care products. Clinicians should be aware of these factors when managing patients with contact allergy to isothiazolinones. |
Formaldehyde release from predispersed tattoo inks: Analysis using the chromotropic acid method
Liou YL , Voller LM , Liszewski W , Ericson ME , Siegel PD , Warshaw EM . Dermatitis 2020 32 (5) 327-332 BACKGROUND: Allergic contact dermatitis to tattoo ink may last from weeks to years. Formaldehyde is a strong sensitizer that may be present in predispersed tattoo inks. OBJECTIVES: The aim of this study was to evaluate the presence of formaldehyde in predispersed tattoo inks using the chromotropic acid method. METHODS: Tattoo inks from 39 companies were evaluated. Inclusion criteria included availability to purchase inks online through US tattoo product wholesalers or individual Web sites. Brands were grouped based on prevalence of use: common, uncommon, or rare. For common brands, 8 colors (primary colors, secondary colors, black, and white) were purchased. For uncommon and rare brands, 5 colors (primary colors, black, and white) were purchased. Each ink was tested with standard chromotropic acid method procedures; concentration of formaldehyde released was quantified using spectrophotometry. RESULTS: In total, 127 tattoo inks were purchased and tested. Ninety-three (73%) tested positive for formaldehyde release; 34 (27%) tested negative. Formaldehyde release did not correlate with color or brand. At least 1 ink from all brands (except 1) was positive for formaldehyde release. CONCLUSION: Approximately three-quarters of selected US tattoo inks tested positive for formaldehyde release. Clinicians should be aware of tattoo ink as a potential source of formaldehyde. |
Resolution of pulmonary inflammation induced by carbon nanotubes and fullerenes in mice: Role of macrophage polarization
Lim CS , Porter DW , Orandle MS , Green BJ , Barnes MA , Croston TL , Wolfarth MG , Battelli LA , Andrew ME , Beezhold DH , Siegel PD , Ma Q . Front Immunol 2020 11 1186 Pulmonary exposure to certain engineered nanomaterials (ENMs) causes chronic lesions like fibrosis and cancer in animal models as a result of unresolved inflammation. Resolution of inflammation involves the time-dependent biosynthesis of lipid mediators (LMs)-in particular, specialized pro-resolving mediators (SPMs). To understand how ENM-induced pulmonary inflammation is resolved, we analyzed the inflammatory and pro-resolving responses to fibrogenic multi-walled carbon nanotubes (MWCNTs, Mitsui-7) and low-toxicity fullerenes (fullerene C60, C60F). Pharyngeal aspiration of MWCNTs at 40 mug/mouse or C60F at a dose above 640 mug/mouse elicited pulmonary effects in B6C3F1 mice. Both ENMs stimulated acute inflammation, predominated by neutrophils, in the lung at day 1, which transitioned to histiocytic inflammation by day 7. By day 28, the lesion in MWCNT-exposed mice progressed to fibrotic granulomas, whereas it remained as alveolar histiocytosis in C60F-exposed mice. Flow cytometric profiling of whole lung lavage (WLL) cells revealed that neutrophil recruitment was the greatest at day 1 and declined to 36.6% of that level in MWCNT- and 16.8% in C60F-treated mice by day 7, and to basal levels by day 28, suggesting a rapid initiation phase and an extended resolution phase. Both ENMs induced high levels of proinflammatory leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) with peaks at day 1, and high levels of SPMs resolvin D1 (RvD1) and E1 (RvE1) with peaks at day 7. MWCNTs and C60F induced time-dependent polarization of M1 macrophages with a peak at day 1 and subsequently of M2 macrophages with a peak at day 7 in the lung, accompanied by elevated levels of type 1 or type 2 cytokines, respectively. M1 macrophages exhibited preferential induction of arachidonate 5-lipoxygenase activating protein (ALOX5AP), whereas M2 macrophages had a high level expression of arachidonate 15-lipoxygenase (ALOX15). Polarization of macrophages in vitro differentially induced ALOX5AP in M1 macrophages or ALOX15 in M2 macrophages resulting in increased preferential biosynthesis of proinflammatory LMs or SPMs. MWCNTs increased the M1- or M2-specific production of LMs accordingly. These findings support a mechanism by which persistent ENM-induced neutrophilic inflammation is actively resolved through time-dependent polarization of macrophages and enhanced biosynthesis of specialized LMs via distinct ALOX pathways. |
Urushiol compounds detected in Toxicodendron-labeled consumer products using mass spectrometry
Zhang AJ , Aschenbeck KA , Law BF , B'Hymer C , Siegel PD , Hylwa SA . Dermatitis 2020 31 (2) 134-139 BACKGROUND: Urushiol, the culprit allergen in Toxicodendron plants such as poison ivy, is an oily mixture of 15 and 17 carbon side chain alk-(en)-yl catechols. Recently, consumer products have been identified that contain Toxicodendron as an ingredient on their label; however, no studies have assessed whether urushiol is indeed present within these products. OBJECTIVE: The aim of the study was to determine whether urushiol compounds are present in consumer products labeled as containing Toxicodendron species. METHODS: Gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry were performed on 9 consumer products labeled as containing Toxicodendron species, including topical homeopathic remedies. Single ion monitoring gas chromatography-mass spectrometry was programmed in selective ion mode to detect 3-methylcatechol characteristic fragment ions of alk-(en)-yl catechols after silanization. Similarly, single ion monitoring liquid chromatography-tandem mass spectrometry was programmed to detect 4 urushiol pentadecylcatechols and 5 urushiol heptadecylcatechols using previously reported mass-to-charge ratios. RESULTS: Gas chromatography-mass spectrometry detected alk-(en)-yl catechols in 67% (6/9) of the products tested. Liquid chromatography-tandem mass spectrometry detected multiple urushiol pentadecylcatechols and heptadecylcatechols in 44% (4/9) of the products tested. CONCLUSIONS: Alk-(en)-yl catechols and multiple urushiols were detected in consumer products listing Toxicodendron species as an ingredient. Clinicians should be aware of these known allergenic ingredients in consumer products. |
Etiological contact allergen chemical identification and confirmation
Siegel PD , Law BF , Warshaw EM . Dermatitis 2019 31 (2) 99-105 Identification of the etiological chemical agent(s) associated with a case(s) of allergic contact dermatitis (ACD) is important for both patient management and public health surveillance. Traditional patch testing can identify chemical allergens to which the patient is allergic. Confirmation of allergen presence in the causative ACD-associated material is presently dependent on labeling information, which may not list the allergenic chemical on the product label or safety data sheet. Dermatologists have expressed concern over the lack of laboratory support for chemical allergen identification and possibly quantification from patients' ACD-associated products. The aim of the study was to provide the clinician a primer to better understand the analytical chemistry of contact allergen confirmation and unknown identification, including types of analyses, required instrumentation, identification levels of confidence decision tree, limitations, and costs. |
Isothiazolinone content of US consumer adhesives: Ultrahigh-performance liquid chromatographic mass spectrometry analysis
Goodier MC , Zang LY , Siegel PD , Warshaw EM . Dermatitis 2019 30 (2) 129-134 BACKGROUND: There are limited data regarding the prevalence and concentration of isothiazolinone preservatives in consumer adhesives. OBJECTIVES: The aim of this study was to determine the prevalence and concentration of 5 specific isothiazolinones (methylisothiazolinone [MI], methylchloroisothiazolinone [MCI], benzisothiazolinone [BIT], butyl BIT, and octylisothiazolinone) in US adhesives. METHODS: Thirty-eight consumer adhesives were analyzed using ultrahigh-performance liquid chromatographic-mass spectrometry. Fisher exact tests were used to test for isothiazolinone content and: 1) glue format (2) application purpose and 3) extraction method. RESULTS: Nineteen adhesives (50%) had at least 1 isothiazolinone, and 15 contained 2 isothiazolinones. Frequencies and concentrations were as follows: MI (44.7%; 4-133 ppm), MCI (31.6%; 7-27 ppm), BIT (15.8%; 10-86 ppm), and octylisothiazolinone (2.6%; 1 ppm). Butyl BIT was not detected in any of the adhesives. Format (stick vs liquid) was not statistically associated with isothiazolinone presence. At least half of adhesives in the following application purposes had at least 1 isothiazolinone: shoe, craft, fabric, and school. All-purpose glues had a statistically significant lower concentration of MI and MCI, whereas craft glues were associated with higher concentrations of MI and MCI. Compared with other glues, fabric adhesives were associated with a higher risk of containing BIT. CONCLUSIONS: Half of the tested adhesives contained at least 1 isothiazolinone. Methylisothiazolinone and MCI were the most common. Consumers and dermatologists should be aware of adhesives as a source of isothiazolinones. |
Isothiazolinone content of US residential interior wall paint: A high-performance liquid chromatographic-mass spectrometry analysis
Goodier MC , Siegel PD , Zang LY , Warshaw EM . Dermatitis 2018 29 (6) 332-338 BACKGROUND: There is limited information regarding isothiazolinone content in residential wall paints in the United States. OBJECTIVE: The aim of this study was to evaluate the prevalence of 5 isothiazolinones-methylisothiazolinone (MI), methylchloroisothiazolinone, benzisothiazolinone (BIT), butyl BIT, and octylisothiazolinone-in US residential wall paints. METHODS: Forty-seven paints were obtained from retailers in Minneapolis/St Paul, Minnesota. Paint samples were assessed for the presence of the 5 isothiazolinones using high-performance liquid chromatographic-mass spectrometry. RESULTS: At least 1 isothiazolinone was detected in all 47 paints. However, no paint contained butyl BIT, and only 1 paint had octylisothiazolinone. The MI and BIT were found in 96% and 98% of the paints, respectively. Methylisothiazolinone ranged in concentration from 17 to 358 ppm, whereas BIT varied from 29 to 1111 ppm. Methylchloroisothiazolinone was found solely in oil-based paints. Isothiazolinones were declared in 15% of Safety Data Sheets but did not correlate with high-performance liquid chromatographic-mass spectrometry. One "preservative-free" paint had BIT at 71.5 ppm. Paint sheen was not statistically associated with BIT or MI concentrations. Unpigmented paints and paints with volatile organic compound claims had significantly lower concentrations of MI, but not BIT. CONCLUSIONS: All paints contained at least 1 isothiazolinone. Methylisothiazolinone and BIT were the most common. Safety Data Sheets are insufficient for ascertaining isothiazolinone content in US paints. |
Circulating miRs-183-5p, -206-3p and -381-3p may serve as novel biomarkers for 4,4'-methylene diphenyl diisocyanate exposure
Lin CC , Law BF , Siegel PD , Hettick JM . Biomarkers 2018 24 (1) 1-45 BACKGROUND: Occupational exposure to the most widely used diisocyanate, 4,4'-methylene diphenyl diisocyanate (MDI), is a cause of occupational asthma (OA). Early recognition of MDI exposure and sensitization is essential for the prevention of MDI-OA. OBJECTIVE: Identify circulating microRNAs (miRs) as novel biomarkers for early detection of MDI exposure and prevention of MDI-OA. MATERIALS AND METHODS: Female BALB/c mice were exposed to one of three exposure regimens: dermal exposure to 1% MDI in acetone; nose-only exposure to 4580 +/- 1497 mug/m(3) MDI-aerosol for 60 minutes; or MDI dermal exposure/sensitization followed by MDI-aerosol inhalation challenge. Blood was collected and miRCURY miRs qPCR Pro fi ling Service was used to profile circulating miRs from dermally exposed mice. Candidate miRs were identified and verified from mice exposed to three MDI-exposure regimens by TaqMan(R) miR assays. RESULTS: Up/down-regulation patterns of circulating mmu-miRs-183-5p, -206-3p and -381-3p were identified and verified. Circulating mmu-miR-183-5p was upregulated whereas mmu-miRs-206-3p and -381-3p were downregulated in mice exposed via all three MDI exposure regimens. DISCUSSION AND CONCLUSION: Upregulation of circulating miR-183-5p along with downregulation of circulating miRs-206-3p and -381-3p may serve as putative biomarkers of MDI exposure and may be considered as potential candidates for validation in exposed human worker populations. |
Possible role of regional variation in allergic contact dermatitis: case report
Jiang A , Harrison JC , Siegel PD , Maibach H . Contact Dermatitis 2018 78 (3) 228-229 A 27-year-old male presented to our dermatitis clinic with 6 months' duration of red oedematous lesions on his ankles. He was previously treated for this suspected allergic contact dermatitis with prednisone (30 mg daily) for 2 weeks, during which time these lesions cleared. However, upon prednisone discontinuation the lesions recurred within several days. |
Mass spectrometry-based analysis of murine bronchoalveolar lavage fluid following respiratory exposure to 4,4'-methylene diphenyl diisocyanate aerosol
Hettick JM , Law BF , Lin CC , Wisnewski AV , Siegel PD . Xenobiotica 2017 48 (6) 1-32 Diisocyanates are highly reactive electrophiles utilized in the manufacture of a wide range of polyurethane products, and have been identified as causative agents of occupational allergic respiratory disease. However, in spite of the significant occupational health burden associated with diisocyanate-induced asthma, the mechanism of disease pathogenesis remains largely unknown. To better understand the fate of inhaled diisocyanates, a nose-only aerosol exposure system was constructed and utilized to expose a BALB/c mouse model to aerosol generated from 4,4'-methylene diphenyl diisocyanate (MDI). Tissue and bronchoalveolar lavage samples were evaluated 4 hours and 24 hours post-exposure for evidence of diisocyanate-protein haptenation, and a label-free quantitative proteomics strategy was employed to evaluate relative changes to protein content of the cellular fraction of the lavage fluid. Following MDI aerosol exposure, expression of a number of proteins with immunological or xenobiotic metabolism relevance is increased, including endoplasmin, cytochrome P450 and argininosuccinate synthase. Western blot analysis indicated MDI-conjugated protein in the lavage fluid, which was identified as serum albumin. Tandem mass spectrometry analysis of MDI-albumin revealed MDI conjugation occurs at a dilysine motif at Lys525, as well as at a glutamine-lysine motif at Lys414, in good agreement with previously published in vitro data on diisocyanate-conjugated serum albumin. |
Vapor pressure and predicted stability of American Contact Dermatitis Society Core Allergens
Jou PC , Siegel PD , Warshaw EM . Dermatitis 2016 27 (4) 193-201 BACKGROUND: Accurate patch testing is reliant on proper preparation of patch test allergens. The stability of patch test allergens is dependent on several factors including vapor pressure (VP). OBJECTIVE: This investigation reviews the VP of American Contact Dermatitis Society Core Allergens and compares stability predictions based on VP with those established through clinical testing. METHODS: Standard references were accessed for determining VP in millimeters of mercury and associated temperature in degrees celsius. If multiple values were listed, VP at temperatures that most approximate indoor storage conditions (20 degrees C and 25 degrees C) were chosen. For mixes, the individual component with the highest VP was chosen as the overall VP, assuming that the most volatile substance would evaporate first. Antigens were grouped into low (≤0.001 mm Hg), moderate (<1 to >0.001 mm Hg), and high (≥1 mm Hg) volatility using arbitrary cutoff values. CONCLUSIONS: This review is consistent with previously reported data on formaldehyde, acrylates, and fragrance material instability. Given lack of testing data, VP can be useful in predicting patch test compound stability. Measures such as air-tight multidose reagent containers, sealed single-application dispensers, preparation of patches immediately before application, and storage at lower temperatures may remedy some of these issues. |
The Influence of Diisocyanate Antigen Preparation Methodology on Monoclonal and Serum Antibody Recognition
Hagerman LM , Law BF , Bledsoe TA , Hettick JM , Kashon ML , Lemons AR , Wisnewski AV , Siegel PD . J Occup Environ Hyg 2016 13 (11) 829-39 Exposure to diisocyanates (dNCOs), such as methylene diphenyl diisocyanate (MDI) can cause occupational asthma (OA). Currently, lab tests for dNCO specific IgE are specific, but not sensitive, which limits their utility in diagnosing dNCO asthma. This may be due to variable preparation and poor characterization of the standard antigens utilized in these assays. The aim of this study was to produce and characterize a panel of antigens prepared using three different commonly employed methods and one novel method. The conjugates were examined for recognition by anti-MDI monoclonal antibodies (mAbs) in varying enzyme linked immunosorbant assay (ELISA) formats, extent of crosslinking, total amount of MDI, the sites of MDI conjugation, relative shape/charge, and reactivity with human serum with antibodies from sensitized, exposed workers. Results indicate that while there are minimal differences in the total amount of MDI conjugated, the extent of crosslinking, and the conjugation sites, there are significant differences in the recognition of differently prepared conjugates by mAbs. Native and denaturing polyacrylamide gel electrophoresis demonstrate differences in the mobility of different conjugates, indicative of structural changes that are likely important for antigenicity. While mAbs exhibited differential binding to different conjugates, polyclonal serum antibodies from MDI exposed workers exhibited equivalent binding to different conjugates by ELISA. While differences in the recognition of the different conjugates exist by mAb detection, differences in antigenicity could not be detected using human serum from MDI-sensitized individuals. Thus, although dNCO conjugate preparation can, depending on the immunoassay platform, influence binding of specific antibody clones, serologic detection of the dNCO-exposure-induced polyclonal antibody response may be less sensitive to these differences. |
Characterization and comparative analysis of 2,4-toluene diisocyanate and 1,6-hexamethylene diisocyanate haptenated human serum albumin and hemoglobin
Mhike M , Hettick JM , Chipinda I , Law BF , Bledsoe TA , Lemons AR , Nayak AP , Green BJ , Beezhold DH , Simoyi RH , Siegel PD . J Immunol Methods 2016 431 38-44 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
Mbiya W , Chipinda I , Simoyi RH , Siegel PD . Toxicology 2015 339 34-39 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. 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. BQ and BQD skin allergenicity, was evaluated in the murine local lymph node assay (LLNA). 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. |
Setting occupational exposure limits for chemical allergens - understanding the challenges
Dotson GS , Maier A , Siegel PD , Anderson SE , Green BJ , Stefaniak AB , Codispoti CD , Kimber I . J Occup Environ Hyg 2015 12 Suppl 1 S82-98 Chemical allergens represent a significant health burden in the workplace. Exposures to such chemicals can cause the onset of a diverse group of adverse health effects triggered by immune-mediated responses. Common responses associated with workplace exposures to low molecular weight (LMW) chemical allergens range from allergic contact dermatitis to life-threatening cases of asthma. Establishing occupational exposure limits (OELs) for chemical allergens presents numerous difficulties for occupational hygiene professionals. Few OELs have been developed for LMW allergens because of the unique biological mechanisms that govern the immune-mediated responses. The purpose of this article is to explore the primary challenges confronting the establishment of OELs for LMW allergens. Specific topics include: (1) understanding the biology of LMW chemical allergies as it applies to setting OELs; (2) selecting the appropriate immune-mediated response (i.e., sensitization versus elicitation); (3) characterizing the dose (concentration)-response relationship of immune-mediated responses; (4) determining the impact of temporal exposure patterns (i.e., cumulative versus acute exposures); and (5) understanding the role of individual susceptibility and exposure route. Additional information is presented on the importance of using alternative exposure recommendations and risk management practices, including medical surveillance, to aid in protecting workers from exposures to LMW allergens when OELs cannot be established. |
Quantitation of 4,4′-methylene diphenyl diisocyanate human serum albumin adducts
Luna LG , Green BJ , Zhang F , Arnold SM , Siegel PD , Bartels MJ . Toxicol Rep 2014 1 743-751 4,4′-Methylene diphenyl diisocyanate (herein 4,4′-MDI) is used in the production of polyurethane foams, elastomers, coatings, adhesives and the like for a wide range of commercial products. Occupational exposure to MDI levels above current airborne exposure limits can elicit immune mediated hypersensitivity reactions such as occupational asthma in sensitive individuals. To accurately determine exposure, there has been increasing interest in developing analytical methods to measure internal biomarkers of exposure to MDI. Previous investigators have reported methodologies for measuring MDI diamine metabolites and MDI-Lysine (4,4′-MDI-Lys) adducts. The purpose of this study was to develop and validate an ultra performance liquid chromatography isotope dilution tandem mass spectrometry (UPLC-ID/MS/MS) quantitation method via a signature peptide approach to enable biomonitoring of 4,4′-MDI adducted to human serum albumin (HSA) in plasma. A murine, anti-4,4′-MDI monoclonal IgM antibody was bound to magnetic beads and utilized for enrichment of the MDI adducted HSA. Following enrichment, trypsin digestion was performed to generate the expected 414 site (primary site of adduction) 4,4′-MDI-adducted HSA signature peptide that was quantified by UPLC-ID/MS/MS. An Agilent 6530 UPLC/quadrupole time of flight MS (QTOF) system was utilized for intact adducted protein analysis and an Agilent 6490 UPLC/MS/MS system operated in multiple reaction monitoring (MRM) mode was utilized for quantification of the adducted signature peptide biomarker both for in chemico and worker serum samples. Worker serum samples were initially screened utilizing the previously developed 4,4′-MDI-Lys amino acid method and results showed that 12 samples were identified as quantifiable for 4,4′-MDI-Lys adducts. The signature peptide adduct approach was applied to the 12 worker samples identified as quantifiable for 4,4′-MDI-Lys adducts. Results indicated no positive results were obtained above the quantification limit by the signature peptide approach. If the 414 site of lysine adduction accounted for 100% of the 4,4′-MDI adductions in the signature peptide adduct approach, the three highest quantifiable samples by the 4,4′-MDI-Lys method should have at least been detectable by the signature peptide method. Results show that although the 4,4′-MDI signature peptide approach is more selective, it is 18 times less sensitive than the 4,4′-MDI-Lys method, thus limiting the ability to detect adduct levels relative to the 4,4′-MDI-Lys amino acid method. |
Exposures and cross-shift lung function declines in wildland firefighters
Gaughan DM , Piacitelli CA , Chen BT , Law BF , Virji MA , Edwards NT , Enright PL , Schwegler-Berry DE , Leonard SS , Wagner GR , Kobzik L , Kales SN , Hughes MD , Christiani DC , Siegel PD , Cox-Ganser JM , Hoover MD . J Occup Environ Hyg 2014 11 (9) 591-603 Respiratory problems are common among wildland firefighters. However, there are few studies directly linking occupational exposures to respiratory effects in this population. Our objective was to characterize wildland fire fighting occupational exposures and assess their associations with cross-shift changes in lung function. We studied 17 members of the Alpine Interagency Hotshot Crew with environmental sampling and pulmonary function testing during a large wildfire. We characterized particles by examining size distribution and mass concentration, and conducting elemental and morphological analyses. We examined associations between cross-shift lung function change and various analytes, including levoglucosan, an indicator of wood smoke from burning biomass. The levoglucosan component of the wildfire aerosol showed a predominantly bimodal size distribution: a coarse particle mode with a mass median aerodynamic diameter about 12 mum and a fine particle mode with a mass median aerodynamic diameter < 0.5 mum. Levoglucosan was found mainly in the respirable fraction and its concentration was higher for fire line construction operations than for mop-up operations. Larger cross-shift declines in forced expiratory volume in one second were associated with exposure to higher concentrations of respirable levoglucosan (p < 0.05). Paired analyses of real-time personal air sampling measurements indicated that higher carbon monoxide (CO) concentrations were correlated with higher particulate concentrations when examined by mean values, but not by individual data points. However, low CO concentrations did not provide reliable assurance of concomitantly low particulate concentrations. We conclude that inhalation of fine smoke particles is associated with acute lung function decline in some wildland firefighters. Based on short-term findings, it appears important to address possible long-term respiratory health issues for wildland firefighters. [Supplementary materials are available for this article. Go to the publisher's online edition of Journal of Occupational and Environmental Hygiene for the following free supplemental resources: a file containing additional information on historical studies of wildland fire exposures, a file containing the daily-exposure-severity questionnaire completed by wildland firefighter participants at the end of each day, and a file containing additional details of the investigation of correlations between carbon monoxide concentrations and other measured exposure factors in the current study.]. |
Arterial stiffness, oxidative stress, and smoke exposure in wildland firefighters
Gaughan DM , Siegel PD , Hughes MD , Chang CY , Law BF , Campbell CR , Richards JC , Kales SF , Chertok M , Kobzik L , Nguyen PS , O'Donnell CR , Kiefer M , Wagner GR , Christiani DC . Am J Ind Med 2014 57 (7) 748-56 OBJECTIVES: To assess the association between exposure, oxidative stress, symptoms, and cardiorespiratory function in wildland firefighters. METHODS: We studied two Interagency Hotshot Crews with questionnaires, pulse wave analysis for arterial stiffness, spirometry, urinary 8-iso-prostaglandin F2alpha (8-isoprostane) and 8-hydroxy-2'-deoxyguanosine (8-OHdG), and the smoke exposure marker (urinary levoglucosan). Arterial stiffness was assessed by examining levels of the aortic augmentation index, expressed as a percentage. An oxidative stress score comprising the average of z-scores created for 8-OHdG and 8-isoprostane was calculated. RESULTS: Mean augmentation index % was higher for participants with higher oxidative stress scores after adjusting for smoking status. Specifically for every one unit increase in oxidative stress score the augmentation index % increased 10.5% (95% CI: 2.5, 18.5%). Higher mean lower respiratory symptom score was associated with lower percent predicted forced expiratory volume in one second/forced vital capacity. CONCLUSIONS: Biomarkers of oxidative stress may serve as indicators of arterial stiffness in wildland firefighters. |
Toluene diisocyanate (TDI) disposition and co-localization of immune cells in hair follicles
Nayak AP , Hettick JM , Siegel PD , Anderson SE , Long CM , Green BJ , Beezhold DH . Toxicol Sci 2014 140 (2) 327-37 Diisocyanates (dNCOs) are potent chemical allergens utilized in various industries. It has been proposed that skin exposure to dNCOs produces immune sensitization leading to work-related asthma and allergic disease. We examined dNCOs sensitization by using a dermal murine model of toluene diisocyanate (TDI) exposure to characterize the disposition of TDI in the skin, identify the predominant haptenated proteins, and discern the associated antigen uptake by dendritic cells. Ears of BALB/c mice were dosed once with TDI (0.1% or 4% v/v acetone). Ears and draining lymph nodes (DLNs) were excised at selected time points between 1 h and 15 days post-exposure and were processed for histological, immunohistochemical, and proteomic analyses. Monoclonal antibodies specific for TDI-haptenated protein (TDI-hp) and antibodies to various cell markers were utilized with confocal microscopy to determine co-localization patterns. Histopathological changes were observed following exposure in ear tissue of mice dosed with 4% TDI/acetone. Immunohistochemical staining demonstrated TDI-hp localization in the stratum corneum, hair follicles, and sebaceous glands. TDI-hp were co-localized with CD11b+ (integrin alphaM/Mac-1), CD207+ (langerin), and CD103+ (integrin alphaE) cells in the hair follicles and in sebaceous glands. TDI-hp were also identified in the DLN 1 h post-exposure. Cytoskeletal and cuticular keratins along with mouse serum albumin were identified as major haptenated species in the skin. The results of this study demonstrate that the stratum corneum, hair follicles, and associated sebaceous glands in mice are dendritic cell accessible reservoirs for TDI-hp and thus identify a mechanism for immune recognition following epicutaneous exposure to TDI. |
Concentrations and stability of methyl methacrylate, glutaraldehyde, formaldehyde and nickel sulfate in commercial patch test allergen preparations
Siegel PD , Fowler JF , Law BF , Warshaw EM , Taylor JS . Contact Dermatitis 2014 70 (5) 309-15 BACKGROUND: Epicutaneous patch tests are used to reproduce allergy and diagnose allergic contact dermatitis. Reliable allergen test preparations are required. OBJECTIVES: The purpose of the present study was to measure the actual concentrations of nickel(II) sulfate hexahydrate (NiSO4 ), methyl methacrylate, formaldehyde, and glutaraldehyde, and to compare them with the labelled concentrations, in commercial patch test allergen preparations found in dermatology clinics where patch testing is routinely performed. MATERIALS AND METHODS: The commercial in-date and out-of-date patch test allergen preparations concentrations of NiSO4 , methyl methacrylate, formaldehyde and glutaraldehyde from one to three participating clinics were analysed with chromatographic or wet chemical techniques. RESULTS: NiSO4 and formaldehyde concentrations were at or above the labelled concentrations; however, formaldehyde loss occurred with storage. NiSO4 particulate was uniformly distributed throughout the petrolatum. 'In-use' methyl methacrylate reagent syringes all contained ≤ 56% of the 2% label concentration, with no observable relationship with expiration date. Lower methyl methacrylate cocentrations were consistently measured at the syringe tip end, suggesting loss resulting from methyl methacrylate's volatility. The concentrations of glutaraldehyde patch test allergen preparations ranged from 27% to 45% of the labelled (1% in pet.) concentration, independently of expiration date. CONCLUSIONS: Some false-negative methyl methacrylate, formaldehyde or glutaraldehyde patch test results may be attributable to instability of the test preparations. |
Pyridoxylamine reactivity kinetics as an amine based nucleophile for screening electrophilic dermal sensitizers
Chipinda I , Mbiya W , Adigun RA , Morakinyo MK , Law BF , Simoyi RH , Siegel PD . Toxicology 2013 315C 102-109 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 6.2x10-6M-1s-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 (R2=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. |
A murine monoclonal antibody with broad specificity for occupationally relevant diisocyanates
Lemons AR , Siegel PD , Mhike M , Law BF , Hettick JM , Bledsoe TA , Nayak AP , Beezhold DH , Green BJ . J Occup Environ Hyg 2014 11 (2) 101-10 Diisocyanates (dNCOs) used in industrial applications are well known low molecular weight allergens. Occupational exposure is associated with adverse health outcomes including allergic sensitization and occupational asthma. In this study, we report the production and initial characterization of a dNCO-hapten specific murine IgM monoclonal antibody (mAb). Female BALB/c mice were immunized intraperitoneally with 25 mug of 4,4'-methylene diphenyl diisocyanate (MDI)-keyhole limpet hemocyanin. Following six biweekly booster immunizations, splenocytes were recovered and fused to Sp2/0-Ag14 murine myeloma cell line for hybridoma production. Hybridomas were then screened in a solid-phase indirect enzyme-linked immunosorbent assay (ELISA) against 40:1 4,4'-MDI- human serum albumin (HSA). mAb reactivity to dNCO-HSA conjugates and dNCO-HSA spiked human serum were characterized using a sandwich ELISA. One hybridoma produced a multimeric IgM mAb (15D4) that reacted with 4,4'-MDI-HSA. Sandwich ELISA analysis demonstrated comparable reactivity with other occupationally relevant dNCO-HSA adducts, including 2,4-toluene diisocyanate (TDI)-HSA, 2,6-TDI-HSA, and 1,6-hexamethylene diisocyanate (HDI)-HSA, but not other electrophilic chemical HSA conjugates. The limit of quantification (LOQ) of 4,4'-MDI-HSA, 2,4-TDI-HSA, 2,6-TDI-HSA, and 1,6-HDI-HSA sandwich ELISAs were 567.2, 172.7, 184.2, and 403.5 ng/mL (8.67, 2.60, 2.77, and 6.07 pmol/mL), respectively. In contrast, experiments using dNCO-supplemented human sera showed an increase in the detectable limit of the assay. A mAb has been produced that has potential utility for detecting mixed diisocyanate exposures in occupational environments. The mAb may have additional utility in the standardization of specific IgE detection immunoassays as well as chromatographic-mass spectrometric methods to enrich dNCO adducted HSA in the plasma of occupationally exposed workers. |
Development of sandwich ELISAs for the detection of aromatic diisocyanate adducts
Lemons AR , Bledsoe TA , Siegel PD , Beezhold DH , Green BJ . J Immunol Methods 2013 397 66-70 Diisocyanates (dNCOs) are highly reactive low molecular weight chemicals commonly used in the manufacturing industry. Occupational exposures to dNCOs have been shown to elicit allergic sensitization and occupational asthma. Among the most commonly used dNCOs in industry are the aromatic dNCOs, toluene diisocyanate (TDI) and methylene diphenyl diisocyanate (MDI). This study aimed to develop enzyme linked immunosorbent assays (ELISA) utilizing aromatic dNCO-specific monoclonal antibodies (mAbs) for the detection of aromatic dNCO adducts. Two sandwich ELISAs were developed. The first sandwich ELISA utilized mAb 60G2 along with an anti-human serum albumin (HSA) polyclonal antibody. This assay detected MDI-, 2,4- and 2,6-TDI-HSA adducts with limits of detection (LOD) of 2.67, <0.10, and 1.70ng/mL, respectively. When spiked into human serum, the LOD of this ELISA increased to 34.37, 7.64 and 24.06ng/mL, respectively. The second ELISA utilized mAbs 62G5 and 60G2 for capture and detection. This assay was capable of detecting 2,4- and 2,6-TDI-HSA adducts with LODs of <4.90 and 26.92ng/mL, respectively, and when spiked in human serum, <4.90 and 95.93ng/mL, respectively. This 62G5-60G2 sandwich assay was also able to detect dNCO adducted transferrin, hemoglobin, keratin and actin, but with less sensitivity than dNCO-HSA. The results of this study demonstrate potential application of these ELISAs in the identification and characterization of aromatic dNCO adducts as well as in biomonitoring occupational and environmental dNCO exposures. |
Characterization of methylene diphenyl diisocyanate haptenated human serum albumin and hemoglobin
Mhike M , Chipinda I , Hettick JM , Simoyi RH , Lemons A , Green BJ , Siegel PD . Anal Biochem 2013 440 (2) 197-204 Protein haptenation by polyurethane industrial intermediate 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 dNCO leading to the capability to produce multiple cross-linked protein species and polymeric additions to proteins. In the present 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 (1) loss of available trinitrobenzene sulfonic acid (TNBS) binding to primary amines, (2) electrophoretic migration in polyacrylamide gels, (3) quantification of methylene diphenyl diamine following acid hydrolysis and (4) immunoassay. Concentration dependent changes in all the above noted parameters were observed demonstrating increase in both number and complexity of conjugates formed with increasing MDI concentration. In conclusion, a series of bio-analytical assays should be performed to standardize MDI-antigen preparations across lots and laboratories for measurement of specific antibody in exposed workers which in total indicate degree of intra- and inter-molecular cross-linking, number of dNCO bound, number of different specific binding sites on the protein and degree of immuno-reactivity. |
Substituent effects on the reactivity of benzoquinone derivatives with thiols
Mbiya W , Chipinda I , Siegel PD , Mhike M , Simoyi RH . Chem Res Toxicol 2013 26 (1) 112-123 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. |
Thiurams in shoe contact dermatitis - a case series
Munk R , Sasseville D , Siegel PD , Law BF , Moreau L . Contact Dermatitis 2013 68 (3) 185-7 We present four patients who developed allergic contact dermatitis on their feet after wearing Keds® Canvas sneakers. All patients underwent patch testing with the North American Contact Dermatitis Group (NACDG) Baseline series (Chemotechnique Diagnostics AB, Vellinge, Sweden), and various other allergen trays, depending on the clinical scenario, including a glues and adhesives series (Chemotechnique Diagnostics AB), a shoe series (Chemotechnique Diagnostics AB), and a textile tray (Chemotechnique Diagnostics AB). All 4 patients developed positive reactions to the thiuram mix, as well as to pieces of their shoes (Fig. 1). We initially believed that thiuram accelerators were used in this type of rubber-based canvas shoe. However, subsequent chemical analysis failed to identify thiurams in two different pairs of shoes. Table 1 summarizes the individual characteristics and patch test results of each patient. |
Markers of upper airway inflammation associated with microbial exposure and symptoms in occupants of a water-damaged building
Akpinar-Elci M , White SK , Siegel PD , Park JH , Visotcky A , Kreiss K , Cox-Ganser JM . Am J Ind Med 2013 56 (5) 522-30 BACKGROUND: Water damage in buildings has been associated with reports of upper airway inflammation among occupants. METHODS: This survey included a questionnaire, allergen skin testing, nasal nitric oxide, and nasal lavage on 153 participants. We conducted exposure assessments of 297 workstations and analyzed collected dust for fungi, endotoxin, and (1 --> 3)-beta-D-glucan to create floor-specific averages. RESULTS: Males had higher levels of nasal inflammatory markers, and females reported more symptoms. ECP, IL-8, and MPO were significantly associated with nasal symptoms, flu-like achiness, or chills. Fungi and glucan were positively associated with blowing out thick mucus. Endotoxin was significantly associated with ECP in overall models, and with ECP, IL-8, MPO, and neutrophils among non-atopic females. CONCLUSIONS: In this study, we documented an association between endotoxin and nasal inflammatory markers among office workers. The results of our study suggest that a non-allergic response may contribute to symptoms occurring among occupants in this water-damaged building. (Am. J. Ind. Med. (c) 2013 Wiley Periodicals, Inc.) |
Hexamethylene diisocyanate (HDI) vapor reactivity with glutathione and subsequent transfer to human albumin
Wisnewski AV , Mhike M , Hettick JM , Liu J , Siegel PD . Toxicol In Vitro 2012 27 (2) 662-71 INTRODUCTION: Airway fluid glutathione (GSH) reactivity with inhaled vapors of diisocyanate, a common occupational allergen, is postulated to be a key step in exposure-induced asthma pathogenesis. METHODS: A mixed (vapor/liquid) phase exposure system was used to model the in vivo reactivity of inhaled HDI vapor with GSH in the airway fluid. HDI-GSH reaction products, and their capacity to transfer HDI to human albumin, were characterized through mass spectrometry and serologic assays, using HDI-specific polyclonal rabbit serum. RESULTS: HDI vapor exposure of 10mM GSH solutions resulted in primarily S-linked, bis(GSH)-HDI reaction products. In contrast, lower GSH concentrations (100muM) resulted in mainly mono(GSH)-HDI conjugates, with varying degrees of HDI hydrolysis, dimerization and/or intra-molecular cyclization, depending upon the presence/absence of H(2)PO(4)(-)/HPO(4)(2-) and Na(+)/Cl(-) ions. The ion composition and GSH concentration of the fluid phase, during HDI vapor exposure, strongly influenced the transfer of HDI from GSH to albumin, as did the pH and duration of the carbamoylating reaction. When carbamoylation was performed overnight at pH 7, 25 of albumin's lysines were identified as potential sites of conjugation with partially hydrolyzed HDI. When carbamoylation was performed at pH 9, more rapid (within 3h) and extensive modification was observed, including additional lysine sites, intra-molecular cross-linkage with HDI, and novel HDI-GSH conjugation. CONCLUSIONS: The data define potential mechanisms by which the levels of GSH, H(2)PO(4)(-)/HPO(4)(2-), and/or other ions (e.g. H(+)/OH(-), Na(+), Cl(-)) affect the reactivity of HDI vapor with self-molecules in solution (e.g. airway fluid), and thus, might influence the clinical response to HDI respiratory tract exposure. |
Detailed mechanistic investigation into the S-nitrosation of cysteamine
Morakinyo MK , Chipinda I , Hettick J , Siegel PD , Abramson J , Strongin R , Martincigh BS , Simoyi RH . Can J Chem 2012 90 (9) 724-738 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. |
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