New Method to Biomonitor Workers Exposed to 1,6-Hexamethylene Diisocyanate.

Isocyanates such as 1,6-hexamethylene diisocyanate (HDI), 4,4'-methylenediphenyl diisocyanate, and toluene diisocyanate are highly reactive compounds that have a variety of commercial applications, including manufacturing polyurethane foam, elastomers, paints, adhesives, coatings, insecticides, and many other products. Their primary route of occupational exposure is through inhalation. Due to their high chemical reactivity, they are toxic and have adverse effects at the cellular and subcellular levels, leading to irritative and immunological reactions associated with lung disease. High concentrations of isocyanates are strong respiratory irritants. Bronchial sensitization and asthma are among the major adverse clinical reactions associated with low-level chronic exposure to isocyanates. Albumin adducts have been linked to the mechanism of occupational asthma caused by isocyanates. Isocyanates react in vivo with albumin, which is recognized by the immune system. Albumin adducts of isocyanates trigger immune responses and are probably the antigenic basis for isocyanate asthma. Sensitization to isocyanates is the main pathway for adverse health effects. Therefore, markers for the biologically effective dose such as albumin adducts of HDI are needed. A new isocyanate adduct of HDI with lysine─Nε-[(6-amino-hexyl-amino)carbonyl]-lysine (HDI-Lys)─was synthesized and characterized by 1H-NMR, 13C-NMR, and mass spectrometry (MS). Appropriate internal standards─HDI-Lys-4,4'-5,5'-d4 (HDI-d4-Lys) and Nε-[(7-amino-heptyl-amino)carbonyl]-lysine (Hep-Lys)─were synthesized to establish a LC-MS/MS method for the analysis of HDI adducts in in vitro modified albumin and in workers. The presence of HDI-Lys was found after pronase digestion of albumin and confirmed by two independent chromatographic approaches: with a C8 reversed-phase column and with a hydrophilic interaction liquid chromatography column. Quantification was performed with positive electrospray ionization (ESI)-MS. The adduct peak found in vivo was confirmed with the less sensitive negative ESI-MS. In summary, these are new compounds and methods to determine isocyanate-specific adducts with albumin in workers exposed to HDI.

Literature review and evaluation of biomarkers, matrices and analytical methods for chemicals selected in the research program Human Biomonitoring for the European Union (HBM4EU).

Humans are potentially exposed to a large amount of chemicals present in the environment and in the workplace. In the European Human Biomonitoring initiative (Human Biomonitoring for the European Union = HBM4EU), acrylamide, mycotoxins (aflatoxin B1, deoxynivalenol, fumonisin B1), diisocyanates (4,4'-methylenediphenyl diisocyanate, 2,4- and 2,6-toluene diisocyanate), and pyrethroids were included among the prioritized chemicals of concern for human health. For the present literature review, the analytical methods used in worldwide biomonitoring studies for these compounds were collected and presented in comprehensive tables, including the following parameter: determined biomarker, matrix, sample amount, work-up procedure, available laboratory quality assurance and quality assessment information, analytical techniques, and limit of detection. Based on the data presented in these tables, the most suitable methods were recommended. According to the paradigm of biomonitoring, the information about two different biomarkers of exposure was evaluated: a) internal dose = parent compounds and metabolites in urine and blood; and b) the biologically effective = dose measured as blood protein adducts. Urine was the preferred matrix used for deoxynivalenol, fumonisin B1, and pyrethroids (biomarkers of internal dose). Markers of the biological effective dose were determined as hemoglobin adducts for diisocyanates and acrylamide, and as serum-albumin-adducts of aflatoxin B1 and diisocyanates. The analyses and quantitation of the protein adducts in blood or the metabolites in urine were mostly performed with LC-MS/MS or GC-MS in the presence of isotope-labeled internal standards. This review also addresses the critical aspects of the application, use and selection of biomarkers. For future biomonitoring studies, a more comprehensive approach is discussed to broaden the selection of compounds.

Quo vadis blood protein adductomics?

Chemicals are measured regularly in air, food, the environment, and the workplace. Biomonitoring of chemicals in biological fluids is a tool to determine the individual exposure. Blood protein adducts of xenobiotics are a marker of both exposure and the biologically effective dose. Urinary metabolites and blood metabolites are short term exposure markers. Stable hemoglobin adducts are exposure markers of up to 120 days. Blood protein adducts are formed with many xenobiotics at different sites of the blood proteins. Newer methods apply the techniques developed in the field of proteomics. Larger adducted peptides with 20 amino acids are used for quantitation. Unfortunately, at present the methods do not reach the limits of detection obtained with the methods looking at single amino acid adducts or at chemically cleaved adducts. Therefore, to progress in the field new approaches are needed.

Biomarkers, matrices and analytical methods targeting human exposure to chemicals selected for a European human biomonitoring initiative.

The major purpose of human biomonitoring is the mapping and assessment of human exposure to chemicals. The European initiative HBM4EU has prioritized seven substance groups and two metals relevant for human exposure: Phthalates and substitutes (1,2-cyclohexane dicarboxylic acid diisononyl ester, DINCH), bisphenols, per- and polyfluoroalkyl substances (PFASs), halogenated and organophosphorous flame retardants (HFRs and OPFRs), polycyclic aromatic hydrocarbons (PAHs), arylamines, cadmium and chromium. As a first step towards comparable European-wide data, the most suitable biomarkers, human matrices and analytical methods for each substance group or metal were selected from the scientific literature, based on a set of selection criteria. The biomarkers included parent compounds of PFASs and HFRs in serum, of bisphenols and arylamines in urine, metabolites of phthalates, DINCH, OPFRs and PAHs in urine as well as metals in blood and urine, with a preference to measure Cr in erythrocytes representing Cr (VI) exposure. High performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was the method of choice for bisphenols, PFASs, the HFR hexabromocyclododecane (HBCDD), phenolic HFRs as well as the metabolites of phthalates, DINCH, OPFRs and PAHs in urine. Gas chromatographic (GC) methods were selected for the remaining compounds, e.g. GC-low resolution MS with electron capture negative ionization (ECNI) for HFRs. Both GC-MS and LC-MS/MS were suitable for arylamines. New developments towards increased applications of GC-MS/MS may offer alternatives to GC-MS or LC-MS/MS approaches, e.g. for bisphenols. The metals were best determined by inductively coupled plasma (ICP)-MS, with the particular challenge of avoiding interferences in the Cd determination in urine. The evaluation process revealed research needs towards higher sensitivity and non-invasive sampling as well as a need for more stringent quality assurance/quality control applications and assessments.

Is It Realistic to Propose Determination of a Lifetime Internal Exposome?

Biomonitoring of xenobiotics has been performed for many years in occupational and environmental medicine. It has revealed hidden exposures and the exposure of workers could be reduced. Although most of the toxic effects of chemicals on humans were discovered in workers, the scientific community has more recently focused on environmental samples. In several countries, urinary and blood samples have been collected and analyzed for xenobiotics. Health, biochemical, and clinical parameters were measured in the biomonitoring program of the Unites States. The data were collected and evaluated as group values, comparing races, ages, and gender. The term exposome was created in order to relate chemical exposure to health effects together with the terms genome, proteome, and transcriptome. Internal exposures were mostly established with snapshot measurements, which can lead to an obvious misclassification of the individual exposures. Albumin and hemoglobin adducts of xenobiotics reflect the exposure of a larger time frame, up to 120 days. It is likely that only a small fraction of xenobiotics form such adducts. In addition, adduct analyses are more work intensive than the measurement of xenobiotics and metabolites in urine and/or blood. New technology, such as high-resolution mass spectrometry, will enable the discovery of new compounds that have been overlooked in the past, since over 300,000 chemicals are commercially available and most likely also present in the environment. Yet, quantification will be challenging, as it was for the older methods. At this stage, determination of a lifetime internal exposome is very unrealistic. Instead of an experimental approach with a large number of people, which is economically and scientifically not feasible, in silico methods should be developed further to predict exposure, toxicity, and potential health effects of mixtures. The computer models will help to focus internal exposure investigations on smaller groups of people and smaller number of chemicals.

Hemoglobin Adducts and Urinary Metabolites of Arylamines and Nitroarenes.

Arylamines and nitroarenes are intermediates in the production of pharmaceuticals, dyes, pesticides, and plastics and are important environmental and occupational pollutants. N-Hydroxyarylamines are the toxic common intermediates of arylamines and nitroarenes. N-Hydroxyarylamines and their derivatives can form adducts with hemoglobin (Hb-adducts), albumin, DNA, and tissue proteins in a dose-dependent manner. Most of the arylamine Hb-adducts are labile and undergo hydrolysis in vitro, by mild acid or base, to form the arylamines. According to current knowledge of arylamine adduct-formation, the hydrolyzable fraction is derived from the reaction products of the arylnitroso derivatives that yield arylsulfinamide adducts with cysteine. Hb-adducts are markers for the bioavailability of N-hydroxyarylamines. Hb-adducts of arylamines and nitroarenes have been used for many biomonitoring studies for over 30 years. Hb-adducts reflect the exposure history of the last four months. Biomonitoring of urinary metabolites is a less invasive process than biomonitoring blood protein adducts, and urinary metabolites have served as short-lived biomarkers of exposure to these hazardous chemicals. However, in case of intermittent exposure, urinary metabolites may not be detected, and subjects may be misclassified as nonexposed. Arylamines and nitroarenes and/or their metabolites have been measured in urine, especially to monitor the exposure of workers. This review summarizes the results of human biomonitoring studies involving urinary metabolites and Hb-adducts of arylamines and nitroarenes. In addition, studies about the relationship between Hb-adducts and diseases are summarized.

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.

Biomonitoring Human Albumin Adducts: The Past, the Present, and the Future.

Serum albumin (Alb) is the most abundant protein in blood plasma. Alb reacts with many carcinogens and/or their electrophilic metabolites. Studies conducted over 20 years ago showed that Alb forms adducts with the human carcinogens aflatoxin B1 and benzene, which were successfully used as biomarkers in molecular epidemiology studies designed to address the role of these chemicals in cancer risk. Alb forms adducts with many therapeutic drugs or their reactive metabolites such as ß-lactam antibiotics, acetylsalicylic acid, acetaminophen, nonsteroidal anti-inflammatory drugs, chemotherapeutic agents, and antiretroviral therapy drugs. The identification and characterization of the adduct structures formed with Alb have served to understand the generation of reactive metabolites and to predict idiosyncratic drug reactions and toxicities. The reaction of candidate drugs with Alb is now exploited as part of the battery of screening tools to assess the potential toxicities of drugs. The use of gas chromatography-mass spectrometry, liquid chromatography, or liquid chromatography-mass spectrometry (LC-MS) enabled the identification and quantification of multiple types of Alb xenobiotic adducts in animals and humans during the past three decades. In this perspective, we highlight the history of Alb as a target protein for adduction to environmental and dietary genotoxicants, pesticides, and herbicides, common classes of medicinal drugs, and endogenous electrophiles, and the emerging analytical mass spectrometry technologies to identify Alb-toxicant adducts in humans.

Comparison of biological effects with albumin adducts of 4,4'-methylenediphenyl diisocyanate in workers.

Lung sensitization and asthma are the main health effects of 4,4'-methylenediphenyl diisocyanate (MDI). Albumin adducts (isocyanate-specific adducts) of MDI might be involved in the etiology of sensitization reactions. Albumin adducts of MDI were analyzed in sera of diisocyanate-exposed worker with and without diisocyanate occupational asthma (DA), as well as in exposed workers with and without diisocyanate-specific IgG antibodies. In DA-positive workers and IgG-positive workers, albumin adducts were significantly higher versus workers without DA and those who were specific IgG negative. The odds ratio to be DA-positive was 57 times larger for workers with adduct levels above 230 fmol/mg. The odds ratio to be IgG-positive was 10 times larger for workers with adduct levels above 113 fmol/mg. Therefore, albumin adducts appear to be a good predictor of the biological effects. The albumin-adduct levels in workers without biological effects were in the range of the adduct levels found in previous studies of healthy MDI-factory and construction site workers.

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.

Determination of albumin adducts of 4,4'-methylenediphenyl diisocyanate in workers of a 4,4'-methylenedianiline factory.

Lung sensitization and asthma are the main health effects of 4,4'-methylenediphenyl diisocyanate (MDI). Albumin adducts (isocyanate specific adducts) of MDI might be involved in the etiology of sensitization reactions. Albumin adducts of MDI have been found in subjects classified as 4,4'-methylenedianiline (MDA) workers. The mean adduct levels in these MDA-workers were 1.5 times higher than in MDI-workers of the same company. MDA-specific hemoglobin adducts, were present ten times more in the MDA-workers than in the MDI-workers. MDA-workers with specific work task had significantly higher albumin adduct levels.

Biomarkers of exposure, effect, and susceptibility in workers exposed to chloronitrobenzenes.

Biomonitoring methods were applied to workers exposed to high levels of chloronitrobenzenes. The external dose, internal dose, biologically effective dose, and biological effects were determined. Individual susceptibility was assessed by analyzing genetic polymorphisms of glutathione S-transferases M1, P1 and T1, and N-acetyltransferases 1 and 2. When the markers of exposure and susceptibility were compared with the frequency of chromosomal aberrations, clinical blood and urine parameters, and health effects typical of chloronitrobenzenes exposure, only a few of the comparisons were statistically significant. A statistically significantly higher frequency of chromosomal aberrations was detected in workers with a high level of hemoglobin-adducts.

Use of genetically manipulated Salmonella typhimurium strains to evaluate the role of human sulfotransferases in the bioactivation of nitro- and aminotoluenes.

Various nitro- and aminotoluenes demonstrated carcinogenic activity in rodent studies, but were inactive or weakly active in conventional in vitro mutagenicity assays. Standard in vitro tests do not take into account activation by certain classes of enzymes. This is true in particular for sulfotransferases (SULTs). These enzymes may convert aromatic hydroxylamines and benzylic alcohols, two major classes of phase-I metabolites of nitro- and aminotoluenes, to reactive esters. Here it is shown that expression of certain human SULTs in Salmonella typhimurium TA1538 or TA100 strongly enhanced the mutagenicity of various nitrotoluenes and nitro- and amino-substituted benzyl alcohols. Human SULT1A1, SULT1A2, and SULT1C2 showed the strongest activation. The observation that some nitrotoluenes as well as some aminobenzyl alcohols were activated by SULTs in the absence of cytochromes P450 implies that mutagenic sulfuric esters were formed at both the exocyclic nitrogen and the benzylic carbon, respectively. Nitroreductase deficiency (using strain YG7131 instead of TA1538 for SULT1A1 expression) did not affect the SULT-dependent mutagenicity of 1-hydroxymethylpyrene (containing no nitro group), moderately enhanced that of 2-amino-4-nitrobenzyl alcohol, and drastically attenuated the effects of nitrobenzyl alcohols without other substituents. The last finding suggests that either activation occurred at the hydroxylamino group formed by nitroreductase or the nitro group (having a strong -M effect) had to be reduced to an electron-donating substituent to enhance the reactivity of the benzylic sulfuric esters. The results pointed to an important role of SULTs in the genotoxicity of nitrotoluenes and alkylated anilines. Activation occurs at nitrogen functions as well as benzylic positions.

Activation of aryl hydrocarbon receptor signaling by extracts of teak and other wood dusts.

Wood dusts, as a group, are categorized as known human carcinogens, but the risks of exposure to specific types of wood dusts and the carcinogenic chemicals they contain are not well studied. Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is linked to the carcinogenic action of specific classes of chemicals. Here we examined whether chemicals in various wood dusts had the potential to activate AhR signaling as a potential toxic mechanism of action. We found that methanol extracts of teak, walnut, mahogany, and poplar dusts contained a wide range of AhR ligand activity, whereas extracts of oak, pine, and other softwoods did not contain appreciable activity. Teak dust extract, being particularly potent, was subjected to chemical analysis. The 2-methylanthraquinone (2-MAQ) accounted for the AhR ligand activity and was present at an average concentration of 0.27 parts per hundred in teak dust. Pure 2-MAQ potently induced AhR signaling (EC50 115 nM), confirming that this was the active ligand. Aqueous extracts of teak dust made using yeast or mammalian cell culture medium also contained robust AhR activity, suggesting the 2-MAQ ligand is soluble at bioactive concentrations in physiologically relevant fluids. The high concentration and potency of 2-MAQ in teak wood suggest it may mediate toxic effects through activation of AhR signaling in exposed wood workers.

Synthetic approaches to obtain amino acid adducts of 4,4'-methylenediphenyl diisocyanate.

4,4'-Methylenediphenyl diisocyanate (MDI) is the most important isocyanate used in the chemical industry. Lung sensitization and asthma are the main types of damage after exposure to MDI. Albumin adducts of MDI might be involved in the etiology of sensitization reactions. It is therefore necessary to have sensitive and specific biomarkers such as blood protein adducts to monitor people exposed to isocyanates. For the discovery of new isocyanate adducts with blood proteins present in vivo, new synthetic standards are needed. To achieve this, we developed five methods to obtain amino acid adducts of MDI. We synthesized and isolated MDI adducts of aspartic acid, glutamic acid, cysteine, and valine. The new adducts were characterized by LC-MS/MS and NMR. We synthesized the corresponding isotope-labeled MDI adducts to develop analytical methods using LC-MS/MS. Glutathione adducts of isocyanates are an important way of transportation of the reactive isocyanates to distant sites from the original site of exposure. Therefore, we used N-acetyl-cysteine adducts of MDI as reactants: N-acetyl-S-[[4-(4-aminobenzyl)phenyl]carbamoyl]-cysteine (MDI-AcCys) and N-acetyl-S-[[4-(4-acetylaminobenzyl)phenyl]carbamoyl]-cysteine (AcMDI-AcCys). MDI-AcCys or AcMDI-AcCys formed adducts with albumin, N(α)-acetyl lysine, and valine. Isotope-labeled albumin adducts (= d(4)-MDI-albumin) were synthesized from d(4)-MDI-AcCys and albumin. d(4)-MDI-albumin can be used as an internal standard to analyze biological samples. Such an internal standard will not correct only for the extraction recovery of the adducts but also for the potential variation of the enzymatic digestions used in the procedure to analyze albumin adducts of MDI. The synthetic procedures described in this manuscript will be applicable to the synthesis of amino acid adducts from other isocyanates.

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.

Determination of new biomarkers to monitor the dietary consumption of isothiocyanates.

Isothiocyanates (ITCs) found in cruciferous vegetables have been associated with a reduced cancer risk in humans. We determined serum albumin adducts of allyl isothiocyanate (AITC), benzylisothiocyanate (BITC), phenylethylisothiocyanate (PEITC) and sulforaphane (SFN) in 85 healthy men from a dietary, randomized, controlled trial. After enzymatic digestion of albumin we determined the adducts of the ITCs with lysine (Lys) using liquid chromatography-tandem mass spectrometry. At the beginning of the study (and after 4 weeks) 4.7% (2.4%), 48.2% (35.3%), 5.9% (10.6%), and 24.7% (23.5%) of the samples were found positive for AITC-Lys, BITC-Lys, PEITC-Lys and SFN-Lys, respectively. This method enables the quantification of ITC adducts in albumin from large, prospective studies on diet and cancer.

Determination of a new biomarker in subjects exposed to 4,4'-methylenediphenyl diisocyanate.

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. This work presents a liquid chromatography (LC)-mass spectrometry (MS/MS) procedure for determination of isocyanate-specific albumin adducts in humans. MDI formed adducts with lysine of albumin: MDI-Lys and AcMDI-Lys. The MDI-Lys levels, 25th, 50th, 75th, 90th percentile, were 0, 65.2, 134, 244 fmol mg(-1) and 0, 30.5, 57.4, 95.8 fmol mg(-1) in the exposed construction and factory workers, respectively. This new biomonitoring procedure will allow 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.