Determination of methyl isopropyl hydantoin from rat erythrocytes by gas-chromatography mass-spectrometry to determine methyl isocyanate dose following inhalation exposure.

Methyl isocyanate (MIC) is an important precursor for industrial synthesis, but it is highly toxic. MIC causes irritation and damage to the eyes, respiratory tract, and skin. While current treatment is limited to supportive care and counteracting symptoms, promising countermeasures are being evaluated. Our work focuses on understanding the inhalation toxicity of MIC to develop effective therapeutic interventions. However, in-vivo inhalation exposure studies are limited by challenges in estimating the actual respiratory dose, due to animal-to-animal variability in breathing rate, depth, etc. Therefore, a method was developed to estimate the inhaled MIC dose based on analysis of an N-terminal valine hemoglobin adduct. The method features a simple sample preparation scheme, including rapid isolation of hemoglobin, hydrolysis of the hemoglobin adduct with immediate conversion to methyl isopropyl hydantoin (MIH), rapid liquid-liquid extraction, and gas-chromatography mass-spectrometry analysis. The method produced a limit of detection of 0.05 mg MIH/kg RBC precipitate with a dynamic range from 0.05-25 mg MIH/kg. The precision, as measured by percent relative standard deviation, was <8.5%, and the accuracy was within 8% of the nominal concentration. The method was used to evaluate a potential correlation between MIH and MIC internal dose and proved promising. If successful, this method may be used to quantify the true internal dose of MIC from inhalation studies to help determine the effectiveness of MIC therapeutics.

Assessment of dermal uptake of diphenylmethane-4,4'-diisocyanate using tape stripping and biological monitoring.

Very little is known about the dermal uptake of isocyanates, and dermal exposure to isocyanates has been discussed as a factor involved in the induction of respiratory diseases. To investigate the dermal uptake of diphenylmethane-4,4'-diisocyanate (4,4'-MDI). Four volunteers were dermally exposed to 10, 25, 49 and 50 mg 4,4'-MDI, respectively, for eight hours. The exposed areas were tape stripped. Urine and blood were biologically monitored for 48 hours. Tape strips, plasma, and urine were analysed by liquid chromatography-mass spectrometry. In total, 35-70% of the applied dose of 4,4'-MDI was absorbed by the skin. Very low fractions of applied dose were found in the tape strips. The 4,4'-MDA concentration in plasma and urine was low, but peaked in urine at 10-14 hours and plasma at 8-32 hours after exposure. 4,4'-MDI is readily absorbed by human skin. Only small fractions of 4,4'-MDI remain as such in the superficial skin layers. The amounts found in blood and urine were only small fractions of the total applied doses which indicates that very small amounts of 4,4'-MDI penetrate the skin and reach the blood stream. The dermal uptake and distribution of 4,4'-MDI is much slower compared to that associated with airway uptake. Our data strongly indicate that formation of 4,4'-MDA from 4,4'-MDI upon reacting with water in the skin can only occur to a very limited extent.

Albumin adducts and urinary metabolites resulting from occupational exposure to 1,5-naphthalene diisocyanate.

OBJECTIVES: 1,5-Naphthalene diisocyanate (NDI) is used in the plastic industry as a curing agent. 1,5-Naphthalene diisocyanate is classified as a sensitizing agent. The objective of this study has been to develop biomonitoring methods for the evaluation of exposure to NDI. MATERIAL AND METHODS: We obtained blood and urine samples from a group of 20 male workers exposed to NDI. The workers answered a questionnaire about their exposure history, job description, the number of years with the company and the time spent working with NDI over the 10 days of the study. Total plasma, albumin, and urine were analyzed for the presence of 1,5-naphthalenediamine (NDA) after acid hydrolysis using gas chromatography-mass spectrometry (GC-MS). RESULTS: 1,5-Naphthalenediamine was found in about 60% of the samples obtained from the workers. 1,5-Naphthalenediamine was obtained after acid hydrolysis of plasma, albumin, and urine at levels up to 1.5 pmol NDA/mg of plasma proteins, 1.15 pmol NDA/mg of albumin, and 55.3 pmol NDA/ml of urine, respectively. CONCLUSIONS: 1,5-Naphthalenediamine found in urine correlates best with the plasma levels (r = 0.91, p < 0.01). The albumin-adduct levels did not correlate with the NDI-specific immunoglobulin E (IgE) or total IgE present in the workers. The adduct and metabolite levels correlate with the air levels of NDI. Int J Occup Med Environ Health 2017;30(4):579-591.

New specific and sensitive biomonitoring methods for chemicals of emerging health relevance.

In this publication the challenges to cope for the aim to obtain innovative biomonitoring methods in our laboratory are visualized for di(2-propylheptyl)phthalate, 2-mercaptobenzothiazole, 3,5-di-tert-butyl-4-hydroxytoluene, 4-nonylphenol, 4-tert-octylphenol, 3-(4-methylbenzylidene)camphor, 4,4'-methylene diphenyl diisocyanate, and Hexabromocyclododecane. For these substances new specific markers were explored based on animal or human kinetic data with urine being the preferred matrix compared to blood. The determination of these markers was complex in all cases, because the sample preparation as well as the detection by high performance liquid chromatography, capillary gas chromatography coupled to tandem mass spectrometers or high resolution mass spectrometry should enable the lowest possible detection limit by use of minimal biological sample volumes. To get a first hint of a possible background level, the analytical methods were applied to urine samples of about 40 persons for each chemical. For Di(2-propylheptyl)phthalate and 2-Mercaptobenzothiazole first results are presented from population biomonitoring.

Determination of albumin adducts of 4,4'-methylenediphenyl diisocyanate after specific inhalative challenge tests in workers.

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 2h to 5ppb of MDI. The adduct levels increase significantly after the exposure to MDI in the challenge chamber. About 0.6% 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.

Biomarkers of exposure in Monday morning urine samples as a long-term measure of exposure to aromatic diisocyanates.

PURPOSE: Exposure to diisocyanates is a known occupational hazard. One method for monitoring occupational exposure is by analyzing biomarkers in hydrolyzed urine and plasma. The half-life of the biomarkers in plasma is about 3 weeks, and the urinary elimination is divided into one fast (hours) and one slow phases (weeks). Polymorphism in glutathione S-transferase enzymes (GST) is earlier shown to modify the metabolism. The aim of the study was to assess whether biomarkers of exposure in urine collected after two non-exposed days correlate with levels in plasma and whether they can be used as a measure for long-term exposure to aromatic diisocyanates and further whether polymorphisms in GST influenced the correlations. METHODS: Biomarkers of exposure was analyzed in urine and blood samples collected from 24 workers, exposed to at least one of toluene-, methylenediphenyl- or naphthalene diisocyanate, on a Monday morning after at least two unexposed days. Moreover, genotype was determined for 19 of the workers. RESULTS: The corresponding specific gravity-adjusted biomarkers in urine and plasma levels for the different diisocyanates correlated well (r between 0.689 and 0.988). When taking all samples together, the correlation coefficient was 0.926. Polymorphism in the GSTM1 genotype seemed to modify the association. CONCLUSION: Urine collected after two unexposed days can possibly be used as long-term biomarker of exposure for aromatic diisocyanates.

Analytical determination of specific 4,4'-methylene diphenyl diisocyanate hemoglobin adducts in human blood.

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.

Determination of isocyanate specific albumin-adducts in workers exposed to toluene diisocyanates.

Toluene diisocyanates (2,4-TDI and 2,6-TDI) are important intermediates in the chemical industry. Among the main damages after low levels of TDI exposure are lung sensitization and asthma. It is therefore necessary to have sensitive and specific methods to monitor isocyanate exposure of workers. Urinary metabolites or protein adducts have been used as biomarkers in workers exposed to TDI. However, with these methods it was not possible to determine if the biomarkers result from exposure to TDI or to the corresponding toluene diamines (TDA). This work presents a new procedure for the determination of isocyanate-specific albumin adducts. Isotope dilution mass spectrometry was used to measure the adducts in albumin present in workers exposed to TDI. 2,4-TDI and 2,6-TDI formed adducts with lysine: N(ϵ)-[({3-amino-4-methylphenyl}amino)carbonyl]-lysine, N(ϵ)-[({5-amino-2-methylphenyl}amino)carbonyl]-lysine, and N(ϵ)- [({3-amino-2-methylphenyl}amino)carbonyl]-lysine. In future studies, this new method can be applied to measure TDI-exposures in workers.

Biological monitoring of exposure to 1,5-naphthalene diisocyanate and 4,4'-methylenediphenyl diisocyanate.

OBJECTIVES: Biological monitoring of occupational sensitizers, such as 1,5-naphthalene diisocyanate (NDI) and 4,4'-methylenediphenyl diisocyanate (MDI) is of high importance. In this study, 1,5-naphthalenediamine (NDA) and 4,4'-methylenedianiline (MDA) in hydrolysed urine and plasma were evaluated as biomarkers of exposure to NDI and MDI, respectively. METHODS: The air exposure to NDI and MDI was monitored for 30 exposed workers at four different plants. In parallel, urinary as well as blood plasma samples were collected. Biomarker levels were determined in hydrolysed urine and plasma by means of gas chromatography-mass spectrometry. RESULTS: Air exposure to both MDI and NDI was correlated to their corresponding urinary and plasma biomarkers. The correlation coefficients for the associations between air and biomarker levels were in the range of 0.51-0.65 and 0.53-0.96 for MDI and NDI, respectively. For NDI, but not for MDI, the significance and correlation coefficients were increased by adjusting the urinary biomarker levels for creatinine content or density. CONCLUSIONS: Biomarker and air levels of MDI and NDI were correlated, but there was a large individual variation.

Upper reference limits for biomarkers of exposure to aromatic diisocyanates.

OBJECTIVES: The objectives of this study were to determine the levels of 2,4-toluenediamine, 2,6-toluenediamine, 1,5-naphthtalenediamine and 4,4'-methylenediphenyldianiline in hydrolyzed urine and plasma for occupationally unexposed workers and to calculate upper reference limits (URLs). These analytes are biomarkers of exposure to 2,4-toluene diisocyanate and 2,6-toluene diisocyanate (2,4-TDI and 2,6-TDI), 1,5-naphtalene diisocyanate (NDI) and 4,4'-methylenediphenyl diisocyanate (MDI), respectively. METHODS: The biomarker levels were determined in urinary and plasma samples obtained from 121 occupationally unexposed workers. Based on these biomarkers levels and the biomarker levels in an occupationally exposed group of workers, URLs were calculated. The method used for these calculations was based on the receiver operator characteristic (ROC) curve method technique, and the URLs were set at the optimum of sum of sensitivity and specificity. RESULTS: The URLs for the different diisocyanates were calculated to be in the range of 0.1-0.5 microg/L. Occupationally unexposed workers had detectable biomarker levels of the diisocyanates investigated. Especially abundant was the biomarkers of MDI which were found in 97% of both urinary and plasma samples. For the other biomarkers, 0-15% of the unexposed workers had detectable levels. The detected levels were mostly close to the limit of detection (LOD), but urinary levels of biomarkers of MDI up to 60 times the LOD were found. The sensitivities and specificities for classification of the workers as occupationally exposed or not, were in the range of 88-100% and 97-100%, respectively. CONCLUSIONS: The URLs were calculated that may be applicable when screening for occupational exposure. A worker with a biomarker level above the URL will be classified as occupationally exposed. Biomarkers of aromatic diisocyanates, especially biomarkers of MDI, were present among occupationally unexposed workers, but the source and nature of the exposure is unknown.

Identification and characterisation of adducts between serum albumin and 4,4'-methylenediphenyl diisocyanate (MDI) in human plasma.

Diisocyanates are potent inducers of airways disease. Methylenediphenyl diisocyanate (MDI) is a widely used diisocyanate in the chemical industry. The aim of this study was to identify major and also immunologically relevant protein conjugates of MDI in plasma. Plasma was obtained from an MDI-exposed worker. The plasma was dialysed and then fractionated using ion exchange chromatography (IEC) and gel filtration. These fractions and also aliquots of unfractioned plasma were hydrolysed, derivatised and analysed for isocyanate adduct content using gas chromatography-mass spectrometry. In addition, immunologically relevant proteins were identified through specific IgG immunoblotting using pooled sera from two exposed workers. It was shown by dialysis that 96% of the hydrolysed MDI derivatives were protein bound and that 95% of the MDI adducts co-eluted with serum albumin in plasma using IEC. All MDI-protein adducts co-eluted with serum albumin using gel filtration. IgG immunoblotting showed a major 66 kDa protein and also some intermolecular reactions in serum albumin. This study shows serum albumin to be the major protein in plasma that forms adducts in vivo with MDI. Thus, a quick and simple quantitative method for biological monitoring may be developed for MDI exposure. The results also showed that MDI-specific IgG antibodies preferentially bind to the serum albumin in in-vitro-synthesised MDI-plasma protein conjugates.

Development, validation and characterization of an analytical method for the quantification of hydrolysable urinary metabolites and plasma protein adducts of 2,4- and 2,6-toluene diisocyanate, 1,5-naphthalene diisocyanate and 4,4'-methylenediphenyl diisocyanate.

Occupational exposure to diisocyanates within the plastic industry causes irritation and disorders in the airway. The aim of this study was to develop, validate and characterize a method for the determination of 2,4-toluenediamine (2,4-TDA), 2,6-toluenediamine (2,6-TDA), 1,5-diaminonaphthalene (1,5-NDA) and 4,4'-methylenedianiline (4,4'-MDA) in hydrolysed urine and plasma, and to study the correlation between the plasma and urinary levels of these potential biomarkers of 2,4-toluene diisocyanate (2,4-TDI), 2,6-toluene diisocyanate (2,6-TDI), 1,5-naphthalene diisocyanate (1,5-NDI) and 4,4'-methylenediphenyl diisocyanate (4,4'-MDI), respectively. Samples were hydrolysed with 0.3 M NaOH at 100 degrees C for 24 h. The diamines were extracted, derivatized with pentafluoropropionic acid anhydride, and quantified by selected ion monitoring on gas chromatography-mass spectrometry. The repeatability and reproducibility of the method were 7-18% and 7-19%, respectively. Dialysis experiments showed that the metabolites of 2,4-TDI, 2,6-TDI, 1,5-NDI and 4,4'-MDI in plasma were exclusively protein adducts. No free diamines were found in urine, indicating that all diisocyanate-related metabolites were in a conjugated form. For each diisocyanate-related biomarker, there were strongly significant correlations (p<0.001) between individual levels of metabolites in plasma and urine, with Spearman's rank correlation coefficient (rs) values of 0.74-0.90. The methods presented here will be valuable for the development of biological monitoring methods for diisocyanates.

The lessons of Bhopal [toxic] MIC gas disaster scope for expanding global biomonitoring and environmental specimen banking.

Bhopal Toxic gas tragedy represents one of the worst chemical accidents of the world. Autopsy and toxicological studies, apart from presenting evidence of acute and even chronic cyanide toxicity, provided a unique example of the incriminated chemical being traced to the bodies of the victims. The entry of methyl isocyanate (MIC) into the blood stream was established by the presence of carbamoylated end-terminal amino acids of haemoglobin and other tissue proteins. The presence of MIC trimer and a few other identified as well as unidentified tank residue constituents in the blood and viscera further established a close nexus of the products of pyrolysis of MIC in the aerosol inhaled by the victims. The Bhopal studies exemplify the scope for biological monitoring (BM) and environmental specimen banking (ESB) in chemical accidents as part of the global efforts.

Albumin adducts, hemoglobin adducts and urinary metabolites in workers exposed to 4,4'-methylenediphenyl diisocyanate.

4,4'-Methylenediphenyl diisocyanate (MDI) is the most widely used isocyanate in the manufacture of polyurethanes. MDI has been implicated as one of the major causes of occupational asthma. Hydrolysis of MDI can yield 4,4'-methylenedianiline (MDA), which is a suspected human carcinogen. Thus the need to monitor occupational exposure to MDI is of great significance. The use of air monitors alone has been found to be insufficient and there is a need for sensitive markers of recent and long-term exposure. We obtained biological samples from a group of 20 workers exposed to MDI vapor during the manufacture of polyurethane products. The air levels of MDI in the factory were measured using personal, work room and work station monitors. In most cases the levels were below detection limits. The blood and urine samples were analyzed for the presence of adducts and metabolites using GC-MS methods. Urinary base-extractable metabolites were found above control levels in 15 of the 20 workers and ranged from 0.035 to 0.83 pmol MDA/ml. The level of the acetylated metabolite N'-acetyl-4,4'-methylenedianiline (AcMDA) ranged from 0.13 to 7.61 pmol/ml. The amount of MDA released after acid hydrolysis was on average 6.5 times higher than the amount of free MDA and AcMDA present in urine. MDA was detected as a hemoglobin (Hb) adduct in all of the 20 subjects. The level ranged from 70 to 710 fmol/g Hb. In one individual the Hb adduct of AcMDA was detected. This is the first time a Hb adduct of AcMDA has been detected after occupational exposure to MDI. This is a further piece of evidence for the biological availability of the suspected human carcinogen MDA from in vivo hydrolysis of MDI. Plasma albumin conjugates of MDI can cause the onset of respiratory disorders in both man and animal models. Thus we investigated the presence of plasma protein adducts. The plasma MDA levels ranged from 0.25 to 5.4 pmol/ml. Up to 120 fmol/mg were found to be covalently bound to albumin.

Biomonitoring of workers exposed to 4,4'-methylenedianiline or 4,4'-methylenediphenyl diisocyanate.

4,4'-Methylenedianiline (MDA) and 4,4'-methylenediphenyl diisocyanate (MDI) are important intermediates in the production of polyurethanes. In order to biomonitor people exposed to low levels of MDA or MDI we have developed sensitive methods to measure hemoglobin (Hb) adducts and urine metabolites. Adducts and metabolites from 33 workers exposed to MDA and 27 workers exposed to MDI were analyzed by gas chromatography-mass spectrometry after hydrolysis, extraction and derivatization with heptafluorobutyric anhydride. Hb adducts of MDA were detected in 31 out of the 33 MDA workers and both MDA and N-acetyl-MDA (AcMDA) were found in 20 of these individuals. The detection limit for MDA was 20 fmol and for AcMDA 100 fmol/sample, which correspond to an absolute detection limit of approximately 1 fmol MDA and 5 fmol AcMDA, respectively. In the urine of workers exposed to MDA both MDA and AcMDA were found in all samples, with the exception of five where only MDA was detected. Acid hydrolysis of the urine samples yielded an approximately 3-fold higher concentration of MDA than the sum of MDA and AcMDA found after base hydrolysis. MDA but not AcMDA found in urine and in Hb correlate well, except for three outliers. In one workers the Hb adduct level of MDA was very low compared to the urine levels. Two workers had very high levels of MDA as Hb adducts but very low levels as urine metabolites. The former case indicates that the workers were recently exposed to higher levels of MDA. The latter case suggests a relatively low recent exposure. The air levels of MDA, monitored using personal air monitors, were below the detection limit. It was possible, however, to determine exposure to MDA for all workers with the methods presented in this publication. Workers exposed exclusively to MDI were studied. Exposure levels, as monitored using personal air samplers, were below the detection limit of 3 micrograms/m3, with the exception of three individuals. In 10 of the MDI workers, hydrolyzable Hb adducts of MDA (57-219 fmol/g Hb) were found. Except for four subjects, the presence of MDA (0.007-0.14 nmol/l) and AcMDA (0.08-3 nmol/l) was detected in all urine samples after base treatment. Following acid hydrolysis of the urine, higher levels of MDA (0.7-10 nmol/l) were found than the sum of free MDA and AcMDA. According to the present data, it was possible to detect exposure to MDI in a greater number of individuals by analyzing urinary metabolites than by measuring Hb adducts or air monitoring.

Determination of 4,4'-methylenediphenyldianiline (MDA) and identification of isomers in technical-grade MDA in hydrolysed plasma and urine from workers exposed to methylene diphenyldiisocyanate by gas chromatography-mass spectrometry.

Gas chromatography-mass spectrometry using chemical ionization with ammonia as reagent gas monitoring both positive and negative ions was applied. Negative-ion monitoring using ammonia and the pentafluoropropionic anhydride (PFPA) derivatives were chosen owing to low detection limits and good separation for the isomers studied. Technical-grade methylenediphenyldiioscyanate (MDI) was analysed and three isomers, 4,4'-, 2.4'- and 2,2'-methylenediphenyldianiline (MDA), were determined in addition to methylated MDA. Plasma and urine from an exposed worker were hydrolysed and analysed and the MDA isomers were identified in the biological samples.