The role of HMGB1 on TDI-induced NLPR3 inflammasome activation via ROS/NF-κB pathway in HBE cells.

To explore the potential role of HMGB1 on TDI-induced NLRP3 inflammasome activation, HBE cells were treated with TDI-HSA conjugate to observe the changes of HMGB1, TLR4, NF-κB, Nrf2 and NLRP3 inflammasome related proteins expressions, ROS release and MMP. NAC, TPCA-1 and Resatorvid pre-treatments were applied to explore the effects of ROS, NF-κB and TLR4 on TDI-induced NLRP3 inflammasome activation. The CRISPR/Cas9 system was used to construct HMGB1 gene knockout HBE cell line and then to explore the role of HMGB1 on TDI-HSA induced NLRP3 inflammasome activation. GL pre-treatment was applied to further confirm the role of HMGB1. Results showed that TDI increased HMGB1, TLR4, P-p65, Nrf2 proteins expressions and ROS release, decreased MMP level and activated NLRP3 inflammasome in HBE cells in a dose dependent manner. NAC, TPCA-1 and Resatorvid pre-treatments decreased the expression of P-p65 and inhibited NLRP3 inflammasome activation. Inhibition of HMGB1 decreased Nrf2 expression and ROS release, improved MMP level and reduced NLRP3 inflammasome activation. GL ameliorated NLRP3 inflammasome activation via inhibiting HMGB1 regulated ROS/NF-κB pathway. These results indicated that HMGB1 was involved in TDI-induced NLRP3 inflammasome activation as a positive regulatory mechanism. The study provided a potential target for early prevention and treatment of TDI-OA.

Hypothesis-based weight-of-evidence evaluation of the human carcinogenicity of toluene diisocyanate.

Humans are exposed to toluene diisocyanate (TDI) primarily through inhalation in workplaces where TDI is produced or used. It is classified as a possible human carcinogen, based primarily on increased tumor incidences in rodents treated with TDI by oral gavage. We used the hypothesis-based weight-of-evidence (HBWoE) method to evaluate whether the available data support the hypothesis that TDI is a human carcinogen. The epidemiology data are not sufficiently robust to support TDI as a human carcinogen; the few positive associations are more likely attributable to alternative explanations than causation. The experimental animal studies indicate that inhalation exposure to TDI does not induce tumors in rats or mice. Tumors observed after oral gavage exposure are most likely due to the conversion of approximately 5% of the administered TDI to toluene diamine (TDA), a known rodent tumorigen. This contention is supported by the observations that TDA is rapidly formed from TDI during in vitro genotoxicity assays, the spectra of responses to TDA and TDI in these assays and in oral bioassays are essentially the same, and TDI is not genotoxic in rodents or humans in vivo after inhalation exposure, when TDA is not formed to a biologically significant degree. We conclude that the weight of the evidence indicates that the conversion of TDI to TDA does not occur in mammalian species under physiological exposure conditions (i.e. inhalation), but is necessary for carcinogenesis to occur. Thus, a causal association between TDI exposure and carcinogenic effects is not plausible in humans.

Toluene diisocyanate reactivity with glutathione across a vapor/liquid interface and subsequent transcarbamoylation of human albumin.

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.

Leukocyte nicotinamide adenine dinucleotide phosphate-reduced oxidase is required for isocyanate-induced lung inflammation.

BACKGROUND: Isocyanates are low-molecular-weight compounds noted for inducing occupational and environmental asthma. Isocyanate-induced lung disease, an oxidant stress-dependent pulmonary inflammation, is the leading cause of occupational asthma. OBJECTIVES: To address the role of leukocyte-produced oxidants in airway inflammation induced by toluene diisocyanate (TDI), and to elucidate the role of leukocyte nicotinamide adenine dinucleotide phosphate-reduced (NADPH) oxidase in pathogenesis by TDI. METHODS: Wild-type mice and NADPH oxidase-deficient mice (neutrophil cytosolic factor 1 mutant, Ncf1(-/-)) were intranasally injected, challenged with inhalatory TDI, and then investigated for lung inflammation. RESULTS: Cell infiltration in lung tissue and leukocytes in bronchoalveolar lavage, airway reactivity to a methacholine challenge, and TDI-induced inflammatory cytokine expression and nuclear factor activation in the lung tissue were all markedly lower in Ncf1(-/-) mice. Wild-type mice treated with blocking antibodies against CD4 and IL-17 showed markedly lower TDI-induced airway hyperresponsiveness. CONCLUSION: Leukocyte NADPH oxidase is an essential regulator in TDI-induced airway inflammation through redox modification of immune responses.

The GSTP1 Ile105 Val polymorphism modifies the metabolism of toluene di-isocyanate.

BACKGROUND: Toluene di-isocyanate (TDI) is widely used in the production of polyurethane foams and paints. As TDI causes respiratory disease in only a fraction of exposed workers, genetic factors may play a key role in disease susceptibility. Polymorphisms in TDI metabolising genes may affect elimination kinetics, resulting in differences in body retention, and in its turn differences in adverse effects. OBJECTIVES: To analyze how genotype modifies the associations between (i) TDI in air (2,4-TDI and 2,6-TDI) and its metabolites toluene diamine (TDA; 2,4-TDA and 2,6-TDA) in hydrolyzed urine; and (ii) 2,4-TDA and 2,6-TDA in hydrolyzed plasma and 2,4-TDA and 2,6-TDA in urine. METHODS: Workers exposed to TDI were analyzed for 2,4-TDI and 2,6-TDI in air (N=70), 2,4-TDA and 2,6-TDA in hydrolyzed urine (N=124) and in plasma (N=128), and genotype: CYP1A1*2A, CYP1A1*2B, GSTA1-52, GSTM1O, GSTM3B, GSTP1 I105V, GSTP1 A114V, GSTT1O, MPO-463, NAT1*3, *4, *10, *11, *14, *15, NAT2*5, *6, *7, and SULT1A1 R213H. RESULTS: GSTP1 105 strongly modified the relationship between 2,4-TDA in plasma and in urine: ValVal carriers had about twice as steep regression slope than IleIle carriers. A similar pattern was found for 2,6-TDA. CYP1A1*2A, GSTM1, GSTP1, GSTT1, and MPO possibly influenced the relationship between TDA in plasma and urine. CONCLUSION: Our results show, for the first time, genetic modification on the human TDI metabolism. The findings suggest that GSTP1 genotype should be considered when evaluating biomarkers of TDI exposure in urine and plasma. Moreover, the results support earlier findings of GSTP1 105 Val as protective against TDI-related asthma.

[Determination and analysis of toluene diisocyanate metabolites in mice using gas chromatography-mass spectrometry].

In the present research we used gas chromatography-mass spectrometry (G C-MS) to determine metabolites of toluene diisocyanate (TDI) in mice and deduce the pathway for toluene diisocyanate metabolism in the organism. Conditions for TDI chromatography: Supelco PTETM-5 chromatographic column (30 mm x 0.25 mm x 0.25 microm); initial column temperature: 90 degrees C, which was maintained for 30 min, then the temperature was increased at a rate of 40 degrees C x min(-1) to 280 degrees C, and maintained for 5.25 min; temperature for the vaporizing chamber: 250 degrees C; carrier gas: helium flowing at 1.0 microL x min(-1). Conditions for chromatography of TDI metabolites in the organism: 94% methyl, 4% ethenyl-bonded-phase fused-silica capillary column (30 + 2 m x 0.25 + 0.02 mm); initial column temperature: 30 degrees C, which was maintained for 5 min, after and then was increased at a rate of 80 degrees C x min(-1) to 280 degrees C, and maintained for 5 min; temperature for the vaporizing chamber: 250 degrees C; carrier gas: helium flowing at 1.0 microL x min(-1). Conditions for mass spectrometry: EI for ionization; 70 eV for ionization energy; 280 degrees C for connecting tube temperature; 35-350 micro for range of scanning; and 1.0 microL for sample size. The results showed that 2 ,4-toluene diisocyanate was metabolized into 2,4-diaminotoluene. Under the conditions selected for GC-MS, TDI metabolites in the organism can be isolated and identified.

Biological monitoring of exposure to toluene diisocyanate.

OBJECTIVES: Toluene diisocyanate (TDI) is used in the manufacture of polyurethane and is a potent inducer of diseases of the airways. In this study, 2,4- and 2,6-toluenediamine in hydrolyzed urine and plasma were evaluated as biomarkers of exposure to 2,4- and 2,6-TDI, respectively. METHODS: For 81 exposed workers from nine different plants, the personal 8-hour time-weighted-average exposure to TDI was monitored by a filter method with 1-(2-methoxyphenyl)piperazine. In parallel, urinary samples (U1) were collected during the last 4 hours of the workshift. On a different occasion, blood samples and additional urinary samples (U2) were collected from the exposed workers, and also from a reference group consisting of 121 unexposed workers. The biomarker levels were determined in urine and plasma by the use of alkaline hydrolysis. RESULTS: There were strong associations between the personal air and biomarker levels, with correlation coefficients in the range of 0.75-0.88 for the U1 samples and in the range of 0.50-0.78 for the plasma samples. By weighted linear regression, the relations were calculated between the air and biomarker levels. The slopes of the obtained regression curves ranged from 1.8 to 2.7 m3/1 for air-urine and from 2.2 to 2.9 m3/1 for air-plasma, and the intercepts were all close to the origin of the coordinates. Through the extrapolation of these regression curves, biological exposure limits were calculated. CONCLUSIONS: The biological monitoring methods and strategies presented in this report are useful for assessing exposure to TDI in practice.

2,4-toluenediisocyanate and hexamethylene-diisocyanate adducts with blood proteins: assessment of reactivity of amino acid residues in vitro.

Diisocyanates, reactive compounds used in plastics industry and potent occupational allergens, readily bind to proteins both in vitro and in vivo, however, the pattern of adducts with individual amino acids has not been investigated systematically. In this study, potential of the proteinogenic amino acid residues for carbamoylation with 2,4-toluenediisocyanate (2,4-TDI) and hexamethylenediisocyanate (HDI) was evaluated. The diisocyanates were incubated in an in vitro system (buffer pH 7.4/dioxane 50:50) with: (a) a series of Nalpha-benzyloxycarbonyl amino acids (Z-amino acids) and N-acetylcysteine (Ac-Cys), model compounds for non-N-terminal amino acids of the protein chain; (b) dipeptides Val-Phe and Asp-Phe, model compounds for N-termini of globin and albumin, respectively. Reactivity of the compounds tested, evaluated from their depletion during incubation with the diisocyanates (measured by HPLC), was in the order: Ac-Cys = Asp-Phe > Val-Phe = Nalpha-Z-Lys >> Nalpha-Z-His for 2,4-TDI, and Ac-Cys > Asp-Phe > Val-Phe = Nalpha-Z-Lys > Nalpha-Z-His > N-Z-Tyr for HDI, however, the adducts with Ac-Cys were unstable. Reactions of other amino acid residues (e.g. Ser, Thr, Met, Trp, Arg, Asn, Gln) with 2,4-TDI and HDI were not observed. Thus, N-terminal amino acids and Lys residues are likely to produce most abundant adducts with diisocyanates in proteins. Further, three amino compounds with increasing pKa values (Val-Phe, Val and Nalpha-Z-Lys) were incubated with 2,4-TDI and N-acetyl-S-[4-(2-amino)tolylcarbamoyl]cysteine, a 2,4-TDI-derived thiocarbamate with carbamoylating activity, in media with 10% and no dioxane, respectively. Here, reactivity of the amino compounds was decreasing in the order: Val-Phe > Val > Nalpha-Z-Lys, which reflects the mechanism of the amine-isocyanate reaction. The experiments also demonstrate the effect of a solvent (organic phase content) on the yield of the carbamoylation reactions.

Comparison of the binding potential of various diisocyanates on DNA in vitro.

Inhalation of diisocyanate vapors is associated with immediate-type hypersensitivity reactions and direct toxic responses. The genotoxic effects of diisocyanates have not been clarified. The aim of this study was to examine the changes in DNA following in vitro exposure to three most commonly used diisocyanates (toluene diisocyanate, TDI; methylenediphenyl-4,4'-diisocyanate, MDI; and hexamethylene diisocyanate, HDI) and to compare their binding potential using melting behavior of DNA and electrophoresis studies in DNA. Following incubation of DNA with MDI (pure and mix) and HDI we found no differences in the melting behavior compared to the control calf thymus DNA. However, DNA treated with TDI showed differences in the shape of the native DNA curves due to changes in hyperchromicity and exhibited 14% more DNA reconstitution after renaturation. The small changes in the melting behavior of native DNA do not suggest the formation of DNA intrastrand cross-links but rather conformational changes of single- and double-stranded DNA. These conformational changes were further explored by agarose electrophoresis of native and denatured calf thymus DNA. Control and all diisocyanate-exposed DNA showed no differences in the size of native DNA fragments. Conversely, electrophoresis of TDI mix-incubated DNA, following denaturation, showed a distinct reduction in the double-stranded DNA fragment size compared to the control, MDI-denatured (pure and mix), and HDI-denatured DNA. These findings may help to better understand the mechanisms of the genotoxic effect of TDI.

Workers exposed to thermal degradation products of TDI- and MDI-based polyurethane: biomonitoring of 2,4-TDA, 2,6-TDA, and 4,4'-MDA in hydrolyzed urine and plasma.

The aim of the study was to investigate biomarkers of exposure to thermal degradation products of 2,4- and 2,6-toluene diisocyanate (TDI)- and 4,4'-methylenediphenyl diisocyanate (MDI)-based polyurethane and the toxicokinetics of these products. Blood and urine were collected from 15 factory workers exposed to thermal degradation products of MDI-based polyurethane glue and TDI-based flexible foam. Four of these workers were also studied during an exposure-free period. Urine and plasma were analyzed after acidic hydrolysis and the concentrations of the isocyanates' corresponding amines, 2,4-, 2,6-toluenediamine (TDA), and 4,4'-methylenedianiline (MDA), were determined as derivatives of pentafluoropropionic anhydride by gas chromatography using chemical ionization mass spectrometry monitoring negative ions. Urinary elimination rates were in the range of < 0.01-5.7 micrograms of 2,4-TDA per hour, < 0.01-3.5 micrograms of 2,6-TDA per hour, and < 0.01-1.6 micrograms of 4,4'-MDA per hour. Plasma levels were in the range of < 0.1-5.5 ng of 2,4-TDA per mL, < 0.1-2.3 ng of 2,6-TDA per mL, and < 0.1-45 ng of 4,4'-MDA per mL. The urinary half-lives of 4,4'-MDA for four of the workers were found to be 59, 61, 73, and 82 hours. The half-lives of 4,4'-MDA in plasma were 10, 14, 16, and 22 days. Elimination rate peaks of 2,4-TDA, 2,6-TDA, and 4,4'-MDA in urine varied during and between workdays. The individual variation in plasma concentrations of 2,4-TDA, 2,6-TDA, and 4,4'-MDA with time was small, but between individuals the variation was great.

Inhalation of toluene diisocyanate is associated with increased production of nitric oxide by rat bronchoalveolar lavage cells.

Isocyanates are used commercially, particularly in the manufacture of polyurethane coatings and foam. These compounds can pose an occupational health hazard since there is a risk of respiratory disease following isocyanate exposure. The purpose of the present study was to investigate whether a single, sublethal isocyanate inhalation is associated with increased production of the free radical nitric oxide (NO). Mature male Sprague-Dawley rats were exposed to air or toluene diisocyanate (TDI; 2 ppm) for 4 hr. Indices of pulmonary function were assessed before and after exposure to TDI fumes. At 20 hr postexposure, bronchoalveolar lavage cells (BALC) and fluid were harvested. NO synthase (NOS)-dependent reactive species production by alveolar macrophages was assessed by determining N(omega)-nitro-L-arginine methyl ester-inhibitable chemiluminescence following stimulation with unopsonized zymosan. Northern blot analysis was used to index inducible NOS mRNA levels in BALC, while nitrite and nitrate (NOx) levels were measured to determine NOx levels in the lavage fluid and the production of NO by cultured adherent BALC was indexed by measuring nitrite levels. Exposure to aerosolized TDI was associated with an increase in the number of alveolar macrophages, lymphocytes, and polymorphonuclear leukocytes harvested by bronchoalveolar lavage, relative to that from air-exposed rats. NOx levels in the lavage fluid and NOS-dependent production of reactive species by alveolar macrophages were increased following TDI exposure. In addition, inducible NO production by BALC (i.e., mRNA levels and nitrite levels in BALC conditioned media) was elevated following TDI treatment. These findings indicate that pulmonary inflammatory responses induced by TDI exposure are associated with increases in inducible NO production. Therefore, the potential role of NO in the initial pulmonary response to TDI exposure warrants further investigation.

Biodegradation of toluene diamine (TDA) in activated sludge acclimated with aniline and TDA.

The biodegradability of toluene diamine (TDA) which has been regarded as a "recalcitrant compound" was examined in activated sludges. In this study, a microorganic-enzyme system which metabolized TDA was obtained by acclimating the activated sludge with aniline and TDA. In the sludge subject to be 200 days' acclimation, the considerable increase in respiration rate with the addition of TDA, accompanied the sharp decrease in its concentration. This indicated that TDA was metabolized fortuitously. The rate of biodegradation of TDA in the absence of aniline was first order with respect to its concentration when the initial TDA concentration was less than about 5 mg/l. The rate constant in this relation was proportional to mixed liquor suspended solid (MLSS). However, when the initial TDA concentration exceeded 5 mg/l, the plots were deviated from a first order rate equation.

Route-dependent comparative metabolism of [14C]toluene 2,4-diisocyanate and [14C]toluene 2,4-diamine in Fischer 344 rats.

This study was initiated to evaluate the pharmacokinetics/metabolism of 14C-labeled toluene 2,4-diisocyanate (2,4-[14C]-TDI) following oral and inhalation exposure in the rat. For comparison, the pharmacokinetics/metabolism of toluene 2,4-diamine (2,4-[14C]TDA) was also evaluated. Groups of 3 or 4 male rats were given either a single 60 mg/kg oral dose of 2,4-[14C]-TDI or were exposed to 2,4-[14C]TDI vapors at a target concentration of 2 ppm for a 4-hr period. Additional groups of male rats were given single 3 or 60 mg/kg oral doses or a single 3 mg/kg intravenous (iv) dose of 2,4-[14C]TDA. All rats were euthanized by 48 hr postexposure. Following oral administration of 2,4-[14C]TDI, > 93% of the administered radioactivity was recovered in the urine, feces, cage wash, and tissues. Approximately 8% of the oral dose was excreted in the urine while 81% was eliminated in the feces. It is estimated that during inhalation exposure, essentially all of the inhaled 2,4-[14C]TDI was retained by the animal. At 48 hr post-inhalation exposure approximately 15 and 47% of the recovered radioactivity was in the urine and feces, respectively. Following oral or inhalation exposure to 2,4-[14C]TDI, no radioactivity was eliminated as either expired 14C organics or 14CO2. Comparison of the 2,4-[14C]TDI inhalation group with the oral 2,4-[14C]TDI and 2,4-[14C]TDA treatment groups indicated that a larger percentage of the inhaled radioactivity was in the tissues/carcass (34% vs 2-4%) and the excretion of radioactivity into the urine was slower (t1/2 = 20 hr vs 5-8 hr) following TDI inhalation. The total amount of free+acetylated TDA metabolites detected in the urine specimens (0-12 hr) following oral and inhalation exposure to 2,4-[14C]TDI was 15 and 0.26 microgram eq 2,4-TDA, respectively. No free 2,4-TDA was detected in the urine specimen from the inhalation group. In comparison, 638 and 20 micrograms eq 2,4-TDA was detected in the urine specimen after oral administration of 60 and 3 mg/kg 2,4-[14C]TDA, respectively. Following 2,4-[14C]TDI inhalation and oral exposure approximately 90 and 65% of the quantitated urinary metabolites existed as acid-labile conjugates, respectively. In contrast, only 16-39% of the quantitated urinary metabolites existed as acid-labile conjugates following oral administration of 2,4-[14C]TDA. Inhalation exposure to 2,4-TDI primarily results in the formation of acid-labile conjugates with little or no 2,4-TDA being formed.(ABSTRACT TRUNCATED AT 400 WORDS)

Gas chromatographic/tandem mass spectrometric identification and quantitation of metabolic 4-acetyltoluene-2,4-diamine from the F344 rat.

2,4-Toluenediamine (TDA) and 2,4-toluenediisocyanate (TDI) are metabolized in the Fischer 344 rat to monoacetyl-2,4-toluenediamine (Ac-TDA) and diacetyl-2,4-toluenediamine (Ac2-TDA). A gas chromatographic/tandem mass spectrometric (GC/MS/MS) method was developed to characterize the structure of the Ac-TDA metabolite (2-acetyl versus 4-acetyl), as a D3-diacetyl-TDA derivative. This method was also shown to be useful in the measurement of urinary levels of TDA, Ac-TDA and Ac2-TDA. Urine samples (1.0 g) were adjusted to pH 6.5-7.0, fortified with the internal standard D9-Ac2-TDA (D3-ring + D3-acetyl x 2) and extracted with ethyl acetate (2 x 2 ml). The extract residues were then derivatized with D6-acetic anhydride and analyzed via electron impact GC/MS/MS. MS/MS analysis of the D3-Ac2-TDA derivative of the two Ac-TDA isomers yielded different daughter ion spectra from the common parent ion (m/z 209). Analysis of urine samples from rats administered TDA (p.o., i.v.) and TDI (p.o., inhalation) indicated that all of the metabolic Ac-TDA from these test materials was the 4-acetyl-TDA isomer. Subsequent GC/MS analysis of the heptafluorobutyric acid (HFBA) derivative of this metabolite confirmed the MS/MS results. Selected ion monitoring of the M-acetyl daughter ions from the derivatized TDA, Ac-TDA and Ac2-TDA was shown to be a useful technique for quantitation of urinary levels of these compounds, with a detection limit of 35 ng g-1 urine for TDA and 10 ng g-1 urine for Ac-TDA and Ac2-TDA.

Protective effect of nifedipine upon specific bronchial responses to isocyanates.

In order to evaluate the effect of nifedipine, a calcium channel blocker, upon specific bronchial responses to isocyanates, 5 subjects with occupational asthma due to TDI were examined. Each subject was exposed to an atmosphere containing 0.01 ppm of TDI in an inhalation chamber for 15 minutes; the test was repeated on a different day 45 minutes after administration of placebo or nifedipine (20 mg sublingually) in a random fashion. Our data show an effectiveness of nifedipine at the above mentioned dose in preventing the responses arising immediately or within one hour after the exposure (cases n. 1, 2, 3, 4, 5). For preventing late responses it was necessary to repeat the administration of nifedipine (10 mg sublingually) 2 hours after the exposure (cases n. 4 and n. 5). The mechanism by which nifedipine prevents bronchial responses to isocyanates has not been elucidated.

Correlation of bacterial mutagenicity and hamster cell transformation with tumorigenicity induced by 2,4-toluenediamine.

In the presence of rat liver microsome enzymes, 2,4-toluenediamine (TDA) was mutagenic for several tester strains of Salmonella typhimurium. TDA induced morphological transformation in an in vitro carcinogenesis system using secondary culture target cells prepared from cryopreserved primary Syrian hamster embryo cells. These results now correlate bacterial mutagenicity and in vitro morphological transformation with the reported tumorigenicity of this compound.