Article

Dilysine-Methylene Diphenyl Diisocyanate (MDI), a Urine Biomarker of MDI Exposure?

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Abstract

Biomonitoring methylene dipheny diisocyanate (MDI) in urine may be useful in industrial hygiene and exposure surveillance approaches toward disease (occupational asthma) prevention, and in understanding pathways by which internalized chemical is excreted. We explored possible urine biomarkers of MDI exposure in mice after respiratory tract exposure to MDI, as glutathione (GSH) reaction products (MDI-GSH), and after skin exposure to MDI dissolved in acetone. LC-MS analyses of urine identified a unique 543.29 m/z [M+H]⁺ ion from MDI exposed mice, but not controls. The 543.29 m/z [M+H]⁺ ion was detectable within 24 hours of a single MDI skin exposure and following multiple respiratory tract exposures to MDI-GSH reaction products. The 543.29 m/z [M+H]⁺ ion possessed properties of di-lysine-MDI, including (a) an isotope distribution pattern for a molecule with the chemical formula C27H38N6O6, (b) expected collision induced dissociation (CID) fragmentation pattern upon MS/MS, and (c) a retention time in reverse phase LC-MS identical to synthetic di-lysine-MDI. Further MDI-specific western blot studies suggest albumin (which contains multiple di-lysine sites susceptible to MDI carbamylation) as a possible source for di-lysine-MDI, and the presence of MDI conjugated albumin in urine up to 6 days post respiratory tract exposure. Two additional [M+H]⁺ ions (558.17 and 863.23 m/z) were found exclusively in urine of mice exposed to MDI-GSH via the respiratory tract and possessed characteristics of previously described cyclized MDI-GSH and oxidized glutathione (GSSG)-MDI conjugates respectively. Together the data identify urinary biomarkers of MDI exposure in mice and possible guidance for future translational investigation.

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... The apparent molecular weight of the biomarker recognized by the new mAbs is consistent with that of an albumin-HDI adduct and mirrors recently published studies of mice exposed to aromatic MDI. (34) Together, the data demonstrate the potential utility of the new mAbs for biomonitoring exposure to HDI and its oligomers. ...
... The biomarker identified by the mAbs possesses an apparent mw consistent with that expected for an albumin adduct, as recently described in mouse studies with MDI. (34) Thus, 6. Western blots of urine from mice exposed to aliphatic diisocyanate. ...
... Each lane contained 20 mL of urine dialyzed against phosphate buffer and depleted of endogenous mouse Ig using protein A. Note that major band *68 kDa is consistent with an albumin adduct as recently published in analogous studies with MDI. (34) ALIPHATIC ISOCYANATE MAbS 71 the presently described urine western blot technique with the new a-aliphatic di/polyisocyanate mAbs, or a further developed high-throughput detection methodology (e.g., sandwich ELISA), could provide the foundation for a biomonitoring approach toward occupational exposure surveillance. Sequencing of the newly developed mAbs cDNA defines molecular determinants for aliphatic di-and polyisocyanate recognition, generated via recombination of germline antibody genes. ...
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Aliphatic di- and polyisocyanates are crucial chemical ingredients in many industrial processes and are a well-recognized cause of occupational asthma. Serologic detection of "chemical epitopes" in biological samples could serve as an exposure surveillance approach toward disease prevention, and thus we sought to generate aliphatic isocyanate-specific monoclonal antibodies (mAbs). Three hybridomas were generated from Balb/c mice immunized with a commercial product containing a combination of uretdione, homopolymer, and monomeric forms of hexamethylene diisocyanate (HDI). Three stable hybridomas were subcloned by limiting dilution, two secreting IgG1κ and one secreting IgMκ mAb that bind aliphatic di- and polyisocyanates (conjugated to albumin), but not aromatic toluene or methylene diphenyl diisocyanate (TDI or MDI). Each mAb demonstrates slight differences in epitope specificity, for example, recognition of hydrogenated MDI (HMDI) or different carrier proteins (transferrin, actin) reacted with vapor phase HDI, and is encoded by unique recombination of different germline antibody genes, with distinct complementary determining regions. By western blot, all three mAbs detect a molecule with characteristics of an albumin adduct uniquely in urine from mice skin exposed to a mixture of aliphatic di- and polyisocyanate. Together, the data define molecular determinants of humoral immune recognition of aliphatic di- and polyisocyanates through new mAbs, which will serve as useful research reagents and may be applicable to future exposure surveillance efforts.
... Although MDA reflects the total pool of MDI-adducts in urine, at present it is not clear yet what exactly these MDI-adducts excreted in urine are, and how their relative abundance impacts the half-life of MDA. We hypothesize that MDA in hydrolyzed urine reflects two distinct clearance phases -one fast clearance of non-protein derived adducts (possibly adducts with glutathione, Wisnewski et al., 2019), and one slow clearance of MDI adducts with proteins-or protein fragments following their recycling in the liver. Elevated levels of MDA in pre-shift urine observed in our cohort of workers likely reflect the slower clearance of MDI-protein adducts, such as those with lysine terminals in albumin, and therefore they reflect exposures on previous days, or slower MDI skin absorption from previous day exposures (Jones et al., 2017). ...
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Several studies on the prognosis of occupational asthma have shown that a significant proportion of patients continue to experience asthmatic symptoms and nonspecific bronchial hyperresponsiveness after cessation of work. The determinants of this unfavourable prognosis of asthma are: long duration of exposure before the onset of asthma; long duration of symptoms before diagnosis; baseline airway obstruction; dual response after specific challenge test; and the persistence of markers of airway inflammation in bronchoalveolar lavage fluid and bronchial biopsy. The relevance of immunological markers in the outcome of occupational asthma has not yet been assessed. Further occupational exposure in sensitized subjects leads to persistence and sometimes to progressive deterioration of asthma, irrespective of the reduction of exposure to the specific sensitizer, and only the use of particular protective devices effectively prevents the progression of the disease. A long-term follow-up study of toluene diisocyanate (TDI)-induced asthma showed that the improvement in bronchial hyperresponsiveness to methacholine occurred in a small percentage of subjects and only a long time after work cessation. Bronchial sensitivity to TDI may disappear, but non-specific bronchial hyperresponsiveness often persists unchanged, suggesting a permanent deregulation of airway tone. Steroid treatment significantly reduces nonspecific bronchial hyperresponsiveness only when started immediately after diagnosis.
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The authors report an important application of (MDI) that does not seem to be widely known. The authors have been involved in a compensation dispute concerning a surface worker from a coal mine. This atopic patient had claimed compensation from the Belgian Fund for Occupational Diseases for bronchial asthma which his physician had regarded as occupational, essentially on the basis of a convincing temporal relation between the disease and periods of work. The claim was rejected, because no substantial exposure to a known asthma-causing agent has been identified. At the time of appeal, the patient has quit his work at the coal mine for 2 years. He gave a list of the products that he had frequently loaded on wagons for transportation underground. The source of exposure for the patient, was probably the handling of half-empty and leaking MDI drums coming back from the mine. No precautions seemed to have been taken and the worker's description, corroborated by others, suggested that exposure was at times considerable. On the basis of this information the occupational nature of the patient's asthma was accepted.
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The in vivo fate of globin adducts with styrene 7,8-oxide (SO), an electrophilic metabolic intermediate of styrene, was studied in male Wistar rats dosed intraperitoneally with racemic SO, 100mg/kg b.w. Regioisomeric hydroxy(phenyl)ethyl (HPE) adducts at Cys, N-terminal Val, Lys and His in globin were determined and their elimination from blood was followed during 60 days, corresponding to life span of rat erythrocytes. In the rat urine, Nα-acetylated products of hydrolytic cleavage of the HPE adducts with Cys, Lys and His were determined. On the first day post-exposure, abundant Nα-acetyl-HPE-Cys adducts (mercapturic acids) formed via direct conjugation of SO with hepatic glutathione were excreted rapidly, but then a much slower phase of elimination reflecting formation of Nα-acetyl-HPE-Cys via cleavage of the adducted globin was observed. A two-phase elimination occurred also in urinary Nα-acetyl-HPE adducts with His and Lys. While a decline by 75-85% during the first 7days post-exposure most likely reflected elimination of adducted albumin, the subsequent slow decline until day 60 corresponded to elimination kinetics of the adducted globin. Thus, the study not only provided original data on the fate of SO-globin adducts but also allowed to reveal general toxicokinetics properties of the urinary cleavage products as a novel type of chemical exposure biomarkers.
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4,4′–Methylenediphenyl diisocyanate (MDI) is the most important isocyanate used in the industry. Lung sensitization with bronchial asthma is the main disorder in exposed workers. Albumin adducts of MDI might be involved in specific immunological reactions. MDI adducts with lysine (MDI-Lys) of albumin have been found in MDI-workers and construction workers. MDI-Lys is an isocyanate-specific adduct of MDI with albumin. In the present study, we report MDI-adducts in workers undergoing diagnostic MDI challenge tests. The workers were exposed for 2 h to 5 ppb of MDI. The adduct levels increase significantly after the exposure to MDI in the challenge chamber. About 0.9% of the dose was bound to albumin. So far, only urinary metabolites of MDI were measured to monitor isocyanate workers. However, such urinary metabolites are not isocyanate specific. Therefore, we propose to measure albumin adducts for monitoring MDI exposed subjects.
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Methylene diphenyl diisocyanate (MDI) is an important industrial chemical and asthmagenic respiratory sensitizer, however its metabolism remains unclear. In this study we used LC-MS and LC-MS/MS to identify novel reaction products of MDI with oxidized glutathione (GSSG), including an 837 m/z [M + H]+ ion corresponding to GSSG bound (via one of its N-termini) to partially hydrolyzed MDI, and an 863 m/z [M + H]+ ion corresponding to GSSG cross-linked by MDI (via its two γ-glutamine N-termini). Further studies with heavy isotope labeled and native reduced glutathione (GSH) identified an [M + H]+ ion corresponding to previously described mono(GSH)-MDI, and evidence for “oligomeric” GSH-MDI conjugates. This study also investigated transformational changes in MDI after incubation with an S9 fraction prepared from murine liver. LC-MS analyses of the S9 reaction products revealed the formation of [M + H]+ ions with m/z's and retention times identical to the newly described GSSG-MDI (837 and 863) conjugates and the previously described mono(GSH)-MDI conjugate. Together the data identify novel biological transformations of MDI, which could have implications for exposure-related health effects, and may help target future in vivo studies of metabolism.
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A new experimental model was designed to study the fate of globin adducts with styrene 7,8-oxide (SO), a metabolic intermediate of styrene and a model electrophilic compound. Rat erythrocytes were incubated with SO at 7 or 22°C. Levels of specific amino acid adducts in globin were determined by LC/MS analysis of the globin hydrolysate and the erythrocytes with known adduct content were administered intravenously to recipient rats. The course of adduct elimination from the rat blood was measured over following 50 days. In the erythrocytes incubated at 22 °C, a rapid decline in the adduct levels on the first day post-transfusion followed by a slow phase of elimination was observed. In contrast, the adduct elimination in erythrocytes incubated at 7 °C was nearly linear copying elimination of intact erythrocytes. In the urine of recipient rats, regioisomeric SO adducts at cysteine, valine, lysine and histidine in the form of amino acid adducts and/or their acetylated metabolites as well as SO-dipeptide adducts were identified by LC/MS supported by synthesized reference standards. S-(2-Hydroxy-1-phenylethyl)cysteine and S-(2-hydroxy-2-phenylethyl)cysteine, the most abundant globin adducts, were excreted predominantly in the form of the corresponding urinary mercapturic acids (HPEMAs). Massive elimination of HPEMAs via urine occurred within the first day from the erythrocytes incubated at both 7 and 22 °C. However, erythrocytes incubated at 7 °C showed also a slow second phase of elimination so that HPEMAs were detected in urine up to 50 days post-transfusion. These results indicate for the first time that globin adducts can be cleaved in vivo to modified amino acids and dipeptides. The cleavage products and/or their predictable metabolites are excreted in urine over the whole life span of erythrocytes. Some of the urinary adducts may represent a new type of noninvasive biomarkers of exposure to adduct-forming chemicals.
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The nasal passages are the portal of entry of the respiratory tract. Because laboratory rodents are obligate nose breathers and highly dependent on the sense of smell, normal structure and function of the upper airways are critical to the health and well-being of these animals. The rodent nose is a structurally complex organ with multiple functions. Besides housing the essential components of the olfactory system, the nasal passages filter, humidify, and warm the inspired air preparing it for the delicate gas exchange tissues in the lung. The nasal cavity has been described as an efficient “scrubbing tower” that removes most, but not all, inhaled chemicals potentially harmful to the lower respiratory airways and pulmonary parenchyma (Brain 1970).
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Diisocyanate chemicals essential to polyurethane production are a well-recognized cause of occupational asthma. The pathogenesis of diisocyanate-induced asthma, including the pathways by which the chemical is taken up and its distribution in exposed tissue, especially the lung, remain unclear. We developed an antiserum with specificity for methylene diphenyl diisocyanate (MDI) the most abundantly produced and utilized diisocyanate world-wide, and established its ability to detect MDI in situ. Polyclonal MDI-specific IgG were induced by immunizing rabbits with MDI-conjugated to keyhole limpet hemocyanin (KLH) emulsified in complete Freund's adjuvant, followed by two booster injections with incomplete Freund's adjuvant. The antiserum contains IgG that recognize a variety of different MDI conjugated proteins, but not unconjugated or mock exposed proteins by dot blot analysis. The antiserum further demonstrates specificity for proteins conjugated with MDI, but not other commonly used diisocyanates. Immunochemical studies with cytospun airway cells and formalin-fixed paraffin embedded lung tissue sections from mice intranasally exposed to MDI (as reversibly reactive glutathione conjugates, e.g. GSH-MDI) demonstrated the antiserum's ability to detect MDI in tissue samples. The data demonstrate penetration of MDI into the lower airways, localized deposition in the epithelial region surrounding airways, and uptake by alveolar macrophages. The new immunochemical reagent should be useful for further studies delineating the uptake and tissue distribution of MDI, especially as it relates to adverse health effects from exposure.
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Autoradiographic Analyses of Guinea Pig Airway Tissues Following Inhalation Exposure to 14C-Labeled Methyl Isocyanate. Kennedy, A. L., Singh, G., Alarie, Y., and Brown, W. E. (1993). Fundam. Appl. Toxicol. 20, 57-67.
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Isocyanates have been a leading chemical cause of occupational asthma since their utility for generating polyurethane was first recognized over 60 years ago, yet the mechanisms of isocyanate asthma pathogenesis remain unclear. The present study provides in vivo evidence that a GSH-mediated pathway underlies asthma-like eosinophilic inflammatory responses to respiratory tract isocyanate exposure. In naive mice, a mixture of GSH reaction products with the chemical allergen, methylene-diphenyl diisocyanate (MDI), induced innate immune responses, characterized by significantly increased airway levels of chitinase YM-1, and IL-12/IL-23β (but not α) subunit. However, in mice immunologically sensitized to MDI via prior skin exposure, identical GSH-MDI doses induced substantially greater inflammatory responses, including significantly increased airway eosinophil numbers and mucus production, along with IL-12/IL-23β, chitinases and other indicators of alternative macrophage activation. The "self"-protein albumin in mouse airway fluid was uniquely modified by GSH-MDI at position 414K, a preferred site of MDI reactivity on human albumin. The 414K-MDI conjugation appears to covalently cross-link GSH to albumin via GSH's NH2-terminus, a unique conformation possibly resulting from cyclized mono(GSH)-MDI, or "asymmetric" (S,N'-linked) bis(GSH)-MDI conjugates. Together, the data support a possible thiol-mediated transcarbamoylating mechanism linking MDI exposure to pathogenic eosinophilic inflammatory responses.
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Background Isocyanates are one of the most commonly reported causes of occupational asthma; however, the risks of developing isocyanate asthma in modern production facilities remain poorly defined. We evaluated TDI exposure and respiratory health among an inception cohort of workers during their first year of employment at a new polyurethane foam production factory.Methods Forty-nine newly hired workers were evaluated pre-employment, 6-months, and 12-months post-employment through questionnaire, spirometry, and TDI-specific serology. Airborne TDI levels were monitored by fixed-point air sampling and limited personal sampling. Qualitative surface SWYPE™ tests were performed to evaluate potential sources of skin exposure.ResultsAirborne TDI levels overall were low; over 90% of fixed-point air measurements were below the limit of detection (0.1 ppb). Over the first year of employment, 12 of the 49 original workers (24.5%) were lost to follow-up, no additional workers were enrolled, and seven of the 49 original workers (14.2%) developed either new asthma symptoms (N = 3), TDI-specific IgG (N = 1), new airflow obstruction (N = 1) and/or a decline in FEV1 ≥ 15% (N = 3), findings that could indicate TDI-related health effects. The prevalence of current asthma symptoms was significantly higher in the workers lost to follow-up compared to those who completed the 12-month follow-up (25% vs. 2.7%; P = 0.04).Conclusions The findings suggest possible early TDI-related health effects in a modern polyurethane production plant. These findings also highlight the need for further longitudinal evaluation of these workers and the challenges of studying workers at risk for isocyanate asthma. Am. J. Ind. Med. © 2014 Wiley Periodicals, Inc.
Article
Diisocyanates are industrially important chemicals that serve as polymerizing agents in a variety of polyurethane products. In addition to their many industrial uses, diisocyanates have been implicated as causative agents of occupational allergic respiratory disease, although the specific mechanism(s) by which these diseases occur remains unknown. In this study the sites of conjugation of the two most industrially important monomeric diisocyanates, methylene diphenyl diisocyanate (MDI) and toluene diisocyanate (TDI) on human serum albumin are identified utilizing multiplexed tandem mass spectrometry on a quadrupole time-of-flight mass spectrometer. Analysis of human albumin reacted with MDI and TDI over the range of 1:1–40:1 (isocyanate:protein) mol ratio reveals that MDI and TDI react with a maximum of 20 and 37 residues, respectively. Conjugation of diisocyanates to albumin proceeds in a concentration-dependant manner with MDI and TDI reacting at a preferred subset of 5 and 10 residues, respectively, in the limiting case of a 1:1mol ratio. MDI reacts at fewer residues than does TDI, and is not observed to react with any residues exclusive of TDI. These results cannot be explained on the basis of simple sterics or hydrophobicity, but rather on the basis of increased reactivity of one TDI isocyanate moiety due to electron withdrawing character of the second isocyanate moiety. Furthermore, reaction of diisocyanates with albumin in a phosphate buffered saline (PBS) solution provides three additional reactive sites that are not observed in ammonium bicarbonate buffer. Two lysine residues, Lys199 and Lys525, are observed to be reactive to both diisocyanates at all concentrations and in all solvent systems employed in this study. This study presents a comprehensive conjugation map of MDI and TDI on human albumin. The results suggest that several albumin residues are reactive toward both MDI and TDI and may be useful for biomonitoring of diisocyanate exposures.
Article
4,4'-Methylene diphenyl diisocyanate (MDI) is one of the most important isocyanates in the industrial production of polyurethane and other MDI-based synthetics. Because of its high reactivity, it is known as a sensitizing agent, caused by protein adducts. Analysis of MDI is routinely done by determination of the nonspecific 4,4'-methylenedianiline as a marker for MDI exposure in urine and blood. Since several publications have reported specific adducts of MDI and albumin or hemoglobin, more information about their existence in humans is necessary. Specific adducts of MDI and hemoglobin were only reported in rats after high-dose MDI inhalation. The aim of this investigation was to detect the hemoglobin adduct 5-isopropyl-3-[4-(4-aminobenzyl)phenyl]hydantoin (ABP-Val-Hyd) in human blood for the first time. We found values up to 5.2 ng ABP-Val-Hyd/g globin (16 pmol/g) in blood samples of workers exposed to MDI. Because there was no information available about possible amounts of this specific MDI marker, the analytical method focused on optimal sensitivity and selectivity. Using gas chromatography-high-resolution mass spectrometry with negative chemical ionization, we achieved a detection limit of 0.02 ng ABP-Val-Hyd/g globin (0.062 pmol/g). The robustness of the method was confirmed by relative standard deviations between 3.0 and 9.8 %. Combined with a linear detection range up to 10 ng ABP-Val-Hyd/g globin (31 pmol/g), the enhanced precision parameter demonstrates that the method described is optimized for screening studies of the human population.
Article
Methylene diphenyl diisocyanate (MDI) is among the leading chemical causes of occupational asthma world-wide, however, the mechanisms of disease pathogenesis remain unclear. This study tests the hypothesis that glutathione (GSH) reacts with MDI to form quasi-stable conjugates, capable of mediating the formation of MDI-conjugated "self" protein antigens, which may participate in pathogenic inflammatory responses. To test this hypothesis, an occupationally relevant dose of MDI (0.1% w/v) was reacted with varying concentrations of GSH (10 μM-10 mM), and the reaction products were characterized with regard to mass/structure, and ability to carbamoylate human albumin, a major carrier protein for MDI in vivo. LC-MS/MS analysis of GSH-MDI reaction products identified products possessing the exact mass of previously described S-linked bis(GSH)-MDI and its partial hydrolysis product, as well as novel cyclized GSH-MDI structures. Upon co-incubation of GSH-MDI reaction products with human albumin, MDI was rapidly transferred to specific lysines of albumin, and the protein's native conformation/charge was altered, based on electrophoretic mobility. Three types of modification were observed, intra-molecular MDI cross-linking, addition of partially hydrolyzed MDI, and addition of "MDI-GSH", where MDI's 2(nd) NCO had reacted with GSH's "N-terminus". Importantly, human albumin carbamoylated by GSH-MDI was specifically recognized by serum IgG from MDI exposed workers, with binding dependent upon the starting GSH concentration, pH, and NaCl levels. Together, the data define a non-enzymatic, thiol-mediated transcarbamoylating mechanism by which GSH may promote immune responses to MDI exposure, and identify specific factors that might further modulate this process.
Article
Abstract Hexamethylene diisocyanate (HDI) is a reactive chemical used in the commercial production of polyurethanes. Toxic effects in rodents exposed to HDI vapor primarily occur in the nasal passages, yet some individuals exposed occupationally to concentrations exceeding current regulatory limits may experience temporary reduction in lung function and asthma-like symptoms. Knowledge of interspecies differences in respiratory tract dosimetry of inhaled HDI would improve our understanding of human health risks to this compound. HDI uptake was measured in the upper respiratory tract of anesthetized Fischer-344 rats. Nasal uptake of HDI was >90% in rats at unidirectional flow rates of 150 and 300 ml/min and a target air concentration of 200 ppb. Uptake data was used to calibrate nasal and lung dosimetry models of HDI absorption in rats and humans. Computational fluid dynamics (CFD) models of the nasal passages were used to simulate inspiratory airflow and HDI absorption. Transport of HDI through lung airways was simulated using convection-diffusion based mass transport models. HDI nasal uptake of 90% and 78% was predicted using the rat and human nasal CFD models, respectively. Total respiratory tract uptake was estimated to be 99% in rats and 97% in humans under nasal breathing. Predicted human respiratory uptake decreased to 87% under oral breathing conditions. Absorption rates of inhaled HDI in human lung airways were estimated to be higher than the rat due to lower uptake in head airways. Model predictions demonstrated significant penetration of HDI to human bronchial airways, although absorption rates were sensitive to breathing style.
Article
Methylene diphenyl diisocyanate (MDI), a low molecular weight chemical important for producing polyurethane foam, coatings, and elastomers is a major cause of occupational asthma, however, mechanisms of disease pathogenesis remain poorly understood. This study characterizes the rearranged germline and hypervariable region cDNA of new anti-MDI secreting hybridomas derived from mice immunized with MDI-conjugated to autologous serum proteins. Six IgG1 secreting clones were identified in initial screening ELISAs, based on differential binding to MDI conjugated human albumin vs. mock exposed albumin. The mAbs secreted by the hybridomas also recognized MDI conjugated to other model proteins (e.g. ovalbumin, transferrin), but did not bind unconjugated proteins, or protein conjugates prepared with other isocyanates (e.g. TDI, HDI). The mAbs displayed MDI-dose dependent binding in ELISA and Western blot, and exhibited varying degrees of cross-competition, suggesting differences in epitope specificity. The cDNA encoding the monoclonal antibodies reveal clonal differences in the CDR3 regions, germline gene usage, and patterns of somatic hypermutation related to epitope specificity. Together, the data provide new insight into the molecular determinants of humoral MDI specificity, and characterize anti-MDI IgG1 mAbs that may be developed into useful diagnostic reagents.
Article
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 (100μM) 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 H2PO4(-)/HPO4(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, H2PO4(-)/HPO4(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.
Article
Five male subjects were exposed to 1,6-hexamethylene diisocyanate (HDI) atmospheres for 7.5 h. The exposures were performed in an 8 m3 stainless steel test chamber, and the HDI atmospheres were generated by a gas-phase permeation method. HDI in air was determined by an HPLC method utilizing the 9-(N-methylaminomethyl)-anthracene reagent, and by a continuous monitoring device (MDA 7100). The average air concentration was ca 25 g/m3, and the inhaled dose of HDI for the different subjects was estimated at ca 100 g. The related amine 1,6-hexamethylene diamine (HDA) was, after acid hydrolysis of urine and plasma, determined as a heptafluorobutyric derivative, by glass capillary gas-chromatography and selected ion monitoring (SIM), in a chemical ionization mode using ammonia as reagent gas. The cumulated urinary excretion of HDA during 28 h was 8.0 to 14 g, which corresponds to ca 11 to 21% of the inhaled dose of HDI. The urinary level of HDA, in samples collected immediately after the end of the exposures, was on average 0.02 mmol/mol creatinine (range 0.01–0.03 mmol/mol creatinine). The urinary elimination was rapid, and half-time (t1/2), for the concentration of HDA in urine, showed an average of 1.2 h (range 1.1–1.4 h). No specific IgE and IgG antibodies to HDI were detected before and after provocation; nor were spirometry or bronchial reactivity changed immediately and 15 h after provocation. Analysis of HDA in hydrolysed urine, as a marker of short-time exposure to HDI, is proposed.
Article
Five men were exposed to toluene diisocyanate (TDI) atmospheres for 7.5 h. The TDI atmospheres were generated by a gas-phase permeation method, and the exposures were performed in an 8-m3 stainless-steel test chamber. The mean air concentration of TDI was ca. 40 g/m3, which corresponds to the threshold limit value (TLV) of Sweden. The inhaled doses of 2,4- and 2,6-TDI were ca. 120 g. TDI in the test chamber air was determined by an HPLC method using the 9-(N-methyl-aminomethyl)-anthracene reagent and by a continuous-monitoring filter-tape instrument. After hydrolysis of plasma and urine, the related amines, 2,4- and 2,6-toluenediamine 2,4-, and 2,6-TDA), were determined as pentafluoropropionic anhydride (PFPA) derivatives by capillary gas-chromatography using selected ion monitoring (SIM) in the electron-impact mode. The urinary elimination of the TDAs showed a possible biphasic pattern, with rapid first phases for 2,4-TDA (mean t 1/2 for the concentration in urine, 1.9 h) and for 2,6-TDA (mean t 1/2 for the concentration in urine, 1.6 h). The cumulative amount of 2,4-TDA excreted in urine within 28 h ranged from 8% to 14% of the estimated dose of 2,4-TDI, and the cumulative amount of 2,6-TDA in urine ranged from 14% to 18% of the 2,6-TDI dose. The average urinary level of 2,4-TDA was 5 g/l in the 6 to 8-h sample (range 2.8–9.6 g/l), and the corresponding value for 2,6-TDA was 8.6 g/l (range, 5.6–16.6 g/l). Biological monitoring of exposure to 2,4- and 2,6-TDI by analysis of 2,4- and 2,6-TDA in urine is feasible.
Article
This article summarizes a large body of industry air sampling data (8134 samples) in which airborne MDI concentrations were measured in a wide variety of manufacturing processes that use either polymeric MDI (PMDI) or monomeric (pure) MDI. Data were collected during the period 1984 through 1999. A total of 606 surveys were conducted for 251 companies at 317 facilities. The database includes 3583 personal (breathing zone) samples and 4551 area samples. Data demonstrate that workplace airborne MDI concentrations are extremely low in a majority of the manufacturing operations. Most (74.6%) of the airborne MDI concentrations measured in the personal samples were nondetectable, i.e., below the limits of quantification (LOQs). A variety of validated industrial hygiene sampling/analytical methods were used for data collection; most are modifications of OSHA Method 47. The LOQs for these methods ranged from 0.1-0.5 microg/sample. The very low vapor pressures of both monomeric MDI and PMDI largely explain the low airborne concentrations found in most operations. However, processes or applications in which the chemical is sprayed or heated may result in higher airborne concentrations and higher exposure potentials if appropriate control measures are not implemented. Data presented in this article will be a useful reference for employers in helping them to manage their health and safety program as it relates to respiratory protection during MDI/PMDI applications.
Article
Exposure to toluene diisocyanate (TDI), an industrially important crosslinking agent used in the production of polyurethane products, can cause asthma in sensitive workers. Albumin has been identified as a major reaction target for TDI in vivo, and TDI-albumin reaction products have been proposed to serve as exposure biomarkers and to act as asthmagens, yet they remain incompletely characterized. In the current study, we used a multiplexed tandem mass spectrometry (MS/MS) approach to identify the sites of albumin conjugation by TDI vapors, modeling the air/liquid interface of the lung. Vapor phase TDI was found to react with human albumin in a dose-dependent manner, with up to 18 potential sites of conjugation, the most susceptible being Lys351 and the dilysine site Lys413-414. Sites of vapor TDI conjugation to albumin were quantitatively limited compared with those recently described for liquid phase TDI, especially in domains IIA and IIIB of albumin. We hypothesize that the orientation of albumin at the air/liquid interface plays an important role in vapor TDI conjugation and, thus, could influence biological responses to exposure and the development of in vitro assays for exposure and immune sensitivity.
Article
Glutathione has previously been identified as a reaction target for toluene diisocyanate (TDI) in vitro and in vivo, and has been suggested to contribute to toxic and allergic reactions to exposure. In this study, the reactivity of reduced glutathione (GSH) with TDI in vitro was further investigated using a mixed phase (vapor/liquid) exposure system to model the in vivo biophysics of exposure in the lower respiratory tract. HPLC/MS/MS was used to characterize the observed reaction products. Under the conditions tested, the major reaction products between TDI vapor and GSH were S-linked bis(GSH)-TDI and to a lesser extent mono(GSH)-TDI conjugates (with one N═C═O hydrolyzed). The vapor-phase-generated GSH-TDI conjugates were capable of transcarbamoylating human albumin in a pH-dependent manner, resulting in changes in the self-protein's conformation/charge, on the basis of electrophoretic mobility under native conditions. Specific sites of human albumin-TDI conjugation, mediated by GSH-TDI, were identified (Lys(73), Lys(159), Lys(190), Lys(199), Lys(212), Lys(351), Lys(136/137), Lys(413/414), and Lys(524/525)) along with overlap with those susceptible to direct conjugation by TDI. Together, the data extend the proof-of-principle for GSH to act as a "shuttle" for a reactive form of TDI, which could contribute to clinical responses to exposure.
Article
Isocyanates, low-molecular weight chemicals essential to polyurethane production, are one of the most common causes of occupational asthma, yet the mechanisms by which exposure leads to disease remain unclear. While isocyanate asthma closely mirrors other Type I Immune Hypersensitivity (Allergic) disorders, one important characteristic of hypersensitivity (‘allergen’-specific IgE) is reportedly absent in a large portion of affected individuals. This variation from common environmental asthma (which typically is induced by high-molecular weight allergens) is important for two reasons. (1) Allergen-specific IgE is an important mediator of many of the symptoms of bronchial hyper-reactivity in ‘allergic asthma’. Lack of allergen-specific IgE in isocyanate hypersensitive individuals suggests differences in pathogenic mechanisms, with potentially unique targets for prevention and therapy. (2) Allergen-specific IgE forms the basis of the most commonly used diagnostic tests for hypersensitivity (skin prick and RAST). Without allergen-specific IgE, isocyanates may go unrecognized as the cause of asthma. In hypersensitive individuals, chronic exposure can lead to bronchial hyperreactivity that persists years after exposure ceases. Thus, the question of whether or not isocyanate asthma is an IgE-mediated disease, has important implications for disease screening/surveillance, diagnosis, treatment and prevention. The present Pro/Con Debate, addresses contemporary, controversial issues regarding IgE in isocyanate asthma. Cite this as: A. V. Wisnewski and M. Jones, Clinical & Experimental Allergy, 2010 (40) 1155–1162.
Article
Diphenylmethane diisocyanate (MDI), the chemical commonly used as a cross-linking agent in commercial polyurethane production, is a well-recognized cause of asthma. Reaction products between MDI and "self" proteins are hypothesized to act as antigens capable of inducing airway inflammation and asthma; however, such MDI antigens remain incompletely understood. We used a variety of analytical methods to characterize the range of MDI-albumin reaction products that form under physiological conditions. Sites of MDI conjugation on antigenic MDI-albumin products, as defined by serum immunoglobulin G (IgG) from MDI-exposed workers, were determined by high-performance liquid chromatography (HPLC) followed by tandem mass spectrometry (MS/MS). The data identified 14 MDI conjugation sites (12 lysines and 2 asparagines) on human albumin and highlight reaction specificity for the second lysine in dilysine (KK) motifs, and this may be a common characteristic of "immune-sensitizing" chemicals. Several of the MDI conjugation sites are not conserved in albumin from other species, and this may suggest species differences in epitope specificity for self protein (albumin)-isocyanate conjugates. The study also describes new applications of contemporary proteomic methodology for characterizing and standardizing MDI-albumin conjugates destined for use in clinical research.
Article
4,4'-Methylenediphenyl diisocyanate (MDI) is the most important of the isocyanates used as intermediates in the chemical industry. Among the main types of damage after exposure to low levels of MDI are lung sensitization and asthma. Albumin adducts of MDI might be involved in the etiology of sensitization reactions. It is, therefore, necessary to have sensitive and specific methods for monitoring the isocyanate exposure of workers. To date, urinary metabolites or protein adducts have been used as biomarkers in workers exposed to MDI. However, with these methods it is not possible to determine whether the biomarkers result from exposure to MDI or to the parent aromatic amine 4,4'-methylenedianiline (MDA). This work presents a procedure for the determination of isocyanate-specific albumin adducts. In a long-term experiment, designed to determine the carcinogenic and toxic effects of MDI, rats were exposed chronically for 3 months, to 0.0 (control), 0.26, 0.70, and 2.06 mg MDI/m(3) as aerosols. Albumin was isolated from plasma, digested with Pronase E, and analyzed by LC-MS/MS. MDI formed adducts with lysine: N(6)-[({4-[4-aminobenzyl]phenyl}amino)carbonyl]lysine (MDI-Lys) and N(6)-[({4-[4-(acetylamino)benzyl]phenyl}amino)carbonyl] lysine (AcMDI-Lys). For the quantitation of the adducts in vivo, isotope dilution mass spectrometry was used to measure the adducts in 2 mg of albumin. The adducts found in vivo (MDI-Lys and AcMDI-Lys) and the corresponding isotope labeled compounds (MDI-[(13)C(6)(15)N(2)]Lys and Ac[(2)H(4)]MDI-Lys) were synthesized and used for quantitation. The MDI-Lys levels increased from 0-24.8 pmol/mg albumin, and the AcMDI-Lys levels increased from 0-1.85 pmol/mg albumin. The mean ratio of MDI-Lys/AcMDI-Lys for each dose level was greater than >20. The albumin adducts correlate with other biomarkers measured in the same rats in the past: urinary metabolites and hemoglobin adducts released after mild base hydrolysis. This method will enable one to measure isocyanate-specific albumin adducts in workers. This new biomonitoring procedure will allow for the assessment of suspected exposure sources and may contribute to the identification of individuals who are particularly vulnerable for developing bronchial asthma and other respiratory diseases after exposure to isocyanates. In addition, it will help to improve the production of antigens for the analysis of antibodies in exposed workers.
Article
Naphthalene is a nasal toxicant and carcinogen in the rat. Upper respiratory tract (URT) uptake of naphthalene was measured in the male and female F344 rat at exposure concentrations of 1, 4, 10, or 30 ppm at inspiratory flow rates of 150 or 300 ml/min. To assess the potential importance of nasal cytochrome P450 (CYP) metabolism, groups of rats were pretreated with the CYP inhibitor 5-phenyl-1-pentyne (PP) (100 mg/kg, ip). In vitro metabolism of naphthalene was similar in nasal tissues from both genders and was reduced by 80% by the inhibitor. URT uptake in female rats was concentration dependent with uptake efficiencies (flow 150 ml/min) of 56, 40, 34, and 28% being observed at inspired concentrations of 1, 4, 10, and 30 ppm, respectively. A similar effect was observed in male rats (flow 150 ml/min) with uptake efficiencies of 57, 49, 37, and 36% being observed. Uptake was more efficient in the male than female rat, likely due to their larger size (226 vs. 144 g). Uptake of naphthalene was significantly reduced by inhibitor pretreatment with the effect being greater at the lower inspired concentrations. Specifically, in pretreated female rats (150 ml/min), URT uptake averaged 25, 29, and 26% at inspired concentrations of 4, 10, and 30 ppm, respectively. Thus, the concentration dependence of uptake was virtually abolished by PP pretreatment. These results provide evidence that nasal CYP metabolism of naphthalene contributes to URT scrubbing of this vapor and is also involved in the concentration dependence of uptake that is observed.
Article
Five male subjects were exposed to 1,6-hexamethylene diisocyanate (HDI) atmospheres for 7.5 h. The exposures were performed in an 8 m3 stainless steel test chamber, and the HDI atmospheres were generated by a gas-phase permeation method. HDI in air was determined by an HPLC method utilizing the 9-(N-methylaminomethyl)-anthracene reagent, and by a continuous monitoring device (MDA 7100). The average air concentration was ca 25 micrograms/m3, and the inhaled dose of HDI for the different subjects was estimated at ca 100 micrograms. The related amine 1,6-hexamethylene diamine (HDA) was after acid hydrolysis of urine and plasma, determined as a heptafluorobutyric derivative, by glass capillary gas-chromatography and selected ion monitoring (SIM), in a chemical ionization mode using ammonia as reagent gas. The cumulated urinary excretion of HDA during 28 h was 8.0 to 14 micrograms, which corresponds to ca 11 to 21% of the inhaled dose of HDI. The urinary level of HDA, in samples collected immediately after the end of the exposures, was on average 0.02 mmol/mol creatinine (range 0.01-0.03 mmol/mol creatinine). The urinary elimination was rapid, and half-time (t 1/2), for the concentration of HDA in urine, showed an average of 1.2 h (range 1.1-1.4 h). No specific IgE and IgG antibodies to HDI were detected before and after provocation; nor were spirometry or bronchial reactivity changed immediately and 15 h after provocation. Analysis of HDA in hydrolysed urine, as a marker of short-time exposure to HDI, is proposed.
Article
Hexane-1,6-diamine was determined in the urine of car painters exposed to paint aerosols based on 1,6-bis(carbonylamino)hexane. The concentration of inhaled functional NCO groups during a 15-min exposure averaged 2.8 ± 0.8 µmol m–3(standard deviation, n= 5). The peak diamine concentration in urine of 63 ± 33 nmol (mmol creatinine)–1(standard deviation, n= 5) occurred 30 min after the end of exposure. The diamine was extracted from the urine with Sep-Pak silica gel cartridges and determined as the perfluoroamide derivative by capillary gas chromatography-mass fragmentography. The determination of diisocyanate-derived diamines in urine offers a selective and sensitive means of biological monitoring of occupational isocyanate exposure.
Article
Through the use of radioactively labeled methyl isocyanate (MIC), the deposition, penetration, and clearance of this highly reactive compound in the airway at the tissue and cellular levels have been directly examined. Guinea pigs were exposed to 14C-MIC vapors at concentrations ranging from 0.38 to 15.2 ppm for periods of 1-6 hr. Solubilization of tissues from these animals showed the airway tissues to have the highest level of radioactivity. In the nasal region, 14C deposition, as monitored by histoautoradiography, was limited to the epithelial layer, was related to dose, and was dependent on the specific epithelial cell type. The squamous epithelium was minimally labeled on the surface and the label did not penetrate the cell layer. However, radioactivity was detected throughout the entire nasal respiratory epithelial layer. The lack of nasal deposition in tracheotomized animals demonstrated that the 14C accumulation at this site was due to the scrubbing action of the nasal region with no contribution from blood recirculation. Cellular localization in the tracheobronchial region showed epithelial and subepithelial deposition in a dose-dependent manner with accumulation of the label at the subepithelial region. Radioactivity penetrated to the level of the terminal bronchiole but was not detected in the alveolar region. The persistence of airway radioactivity over the 48-hr postexposure period monitored suggests the covalent modification of airway macromolecules. Despite its broad specificity and high reactivity, MIC undergoes selective reactions in the airways which are dependent on respiratory region and cell type.
Article
Surveillance programs established around the world have determined that diisocyanate chemicals are the most common cause of occupational asthma. In the United States approximately 100,000 workers are exposed to these chemical compounds in the workplace each year and 5-10% of these workers will develop occupational asthma. There are no known reliable risk factors or biomarkers which can be used to predict which exposed worker will develop diisocyanate-occupational asthma. Diisocyanate-occupational asthma workers manifest characteristic physiologic responses after specific bronchoprovocation which correlate with pathologic changes in their airways. However, the mechanism(s) for diisocyanate-occupational asthma remains unclear. Specific IgE antibody production to diisocyanates is found in only 10-30% of these workers. Bronchial biopsies and bronchoalveolar lavage have confirmed the presence of T-lymphocytes and eosinophils in the airways of these workers suggesting that T-cell mediated immune responses are more likely to play a central role in this disease. It is essential to diagnose diisocyanate-occupational asthma as early as possible in order to prevent or reduce the significant asthma morbidity associated with continuous long term exposure to these chemicals. Treatment of choice is removal of the worker from further exposure. Prospective studies evaluating larger populations of diisocyanate-exposed workers is essential for a better understanding of the pathogenesis and natural course of diisocyanate-occupational asthma.
Article
4,4'-Methylenediphenyl diisocyanate (MDI) is the most important of the isocyanates used as intermediates in the chemical industry. Among the main types of damage after exposure to low levels of MDI are lung sensitization and asthma. Protein adducts of MDI might be involved in the etiology of sensitization reactions. It is therefore necessary to have sensitive and specific methods for monitoring the isocyanate exposure of workers. To date, urine metabolites or protein adducts have been used as biomarkers in workers exposed to MDI. However, with these methods it is not possible to determine if the biomarkers result from exposure to MDI or to the parent aromatic amine 4,4'-methylenedianiline (MDA). This work presents a procedure for quantitating isocyanate-specific hemoglobin adducts. Blood proteins are used as markers of exposure and possibly as markers of dose size for the modifications of macromolecules in the target organs where the disease develops. For the quantitation of hemoglobin adducts, N(1)-[4-(4-isocyanatobenzyl)phenyl]acetamide (AcMDI) was reacted with the tripeptide valyl-glycyl-glycine and with valine yielding N-[4-(4-acetylaminobenzyl)phenyl]carbamoyl]valyl-glycyl-glycine and N-[4-[4-(acetylaminobenzyl)phenyl]carbamoyl]valine, respectively. N-[4-[4-(Acetylamino-3,5-dideuteriobenzyl)-2, 6-dideuteriophenyl]carbamoyl]valine was synthesized from valine, as was N(1)-[4-(4-isocyanato-3,5-dideuteriobenzyl)-2, 6-dideuteriophenyl]acetamide, for use as an internal standard. These adducts were cleaved in 2 M HCl to yield the corresponding hydantoins, 3-[4-(4-aminobenzyl)phenyl]-5-isopropyl-1, 3-imidazoline-2,4-dione (MDA-Val-Hyd) and 3-[4-(4-amino-3, 5-dideuteriobenzyl)-2,6-dideuteriophenyl]-5-isopropyl-1, 3-imidazoline-2,4-dione, respectively. In globin of rats exposed to MDI, MDA-Val-Hyd could be found in a dose-dependent manner. The adduct was identified by HPLC/MS/MS and quantified by GC/MS after derivatization with heptafluorobutyric anhydride. The amount of MDA-Val-Hyd found after acid hydrolysis of globin at 100 degrees C is about 12 times larger than the sum of N-acetyl-4, 4'-methylenedianiline (AcMDA) and MDA obtained from mild base hydrolysis of hemoglobin. The MDA-Val-Hyd is an isocyanate-specific adduct. MDA and AcMDA released after mild base hydrolyses result most likely from a sulfinamide adduct which is a typical adduct of arylamines. According to these results, higher amounts of isocyanate adducts than arylamine adducts should be expected in workers exposed to isocyanates.
Article
In 1976-1992 245 new cases of asthma induced by diisocyanates were diagnosed, caused by hexamethylene diisocyanate (HDI) in 39%, diphenylmethane diisocyanate (MDI) in 39%, and toluene diisocyanate (TDI) in 17% of the cases. Our aim was to study the clinical outcome of diisocyanate-induced asthma. A questionnaire was sent to the 235 patients alive in 1995, and validated by reexamining clinically 91 of them. The study was carried out on average 10 () yr after the diagnosis. Of the patients 82% experienced symptoms of asthma, 34% used no medication, and 35% were on regular medication. The patients having displayed immunoglobulin E (IgE) antibodies to isocyanates used less medication (OR 0.273; CI 0.098, 0.758) and had fewer symptoms of asthma (OR 0.329; CI 0.124, 0.875) than the IgE-negative ones. They also had a significantly shorter duration of symptoms (p = 0.0025), latency period (p = 0.0249), and duration of exposure (p = 0.0008) than the IgE-negative patients. This did not, however, entirely explain the more favourable outcome of the IgE-positive patients. Patients with HDI-induced asthma used less medication (OR 0.412; CI 0.229, 0.739) than patients with MDI- and TDI-induced asthma. The results confirm the generally rather poor medical outcome of diisocyanate-induced asthma; the persistence of symptoms and unspecific bronchial reactivity were pronounced in TDI-induced asthma. A more favourable outcome was associated with IgE mediation and HDI inducement.