Assessment of cellular and molecular metrics for dose selection in an in vivo comet assay: A case study with MDI.

The in vivo comet assay can evaluate the genotoxic potential of a chemical in theoretically any tissue that can be processed to a single cell suspension. This flexibility enables evaluation of point-of-contact tissues using a relevant route of test material administration; however, assessing cytotoxicity is essential for the interpretation of comet results. Histopathological evaluation is routinely utilized to assess cytotoxicity, but temporal- and cell-specific considerations may compromise applicability to the comet assay. In the present study, 1,1'-methylenebis(4-isocyanatobenzene) (4,4'-MDI) was administered to rats for 6 h by nose-only inhalation, and the comet assay was conducted to evaluate genotoxicity in the site-of-contact tissue (bronchoalveolar lavage cells) and distal tissues (liver and glandular stomach). Given the reactive nature of MDI, cellular and molecular metrics at the site-of-contact- including inflammation, macrophage activation, apoptosis/necrosis, and oxidative stress- were used to set appropriate exposure concentrations, in addition to the standard systemic measures of toxicity. In the range-finding study, a concentration of 4 mg/m3 was considered the maximum noninflammatory concentration; hence target concentrations of 2, 5, and 11 mg/m3 were selected for the comet study. In the lung lavage, MDI exposure substantially increased total protein and ß-glucuronidase, along with cellular apoptosis. Although MDI did not increase the comet assay response (% tail DNA) in any of the tissues examined, the positive control (ethyl methanesulfonate, EMS) significantly increased % tail DNA in all tissues. In total, these data indicate that appropriate cellular and molecular measurements may facilitate dose selection to discern cellular status in the comet assay.

Quantitative Risk Assessment: Developing a Bayesian Approach to Dichotomous Dose-Response Uncertainty.

Model averaging for dichotomous dose-response estimation is preferred to estimate the benchmark dose (BMD) from a single model, but challenges remain regarding implementing these methods for general analyses before model averaging is feasible to use in many risk assessment applications, and there is little work on Bayesian methods that include informative prior information for both the models and the parameters of the constituent models. This article introduces a novel approach that addresses many of the challenges seen while providing a fully Bayesian framework. Furthermore, in contrast to methods that use Monte Carlo Markov Chain, we approximate the posterior density using maximum a posteriori estimation. The approximation allows for an accurate and reproducible estimate while maintaining the speed of maximum likelihood, which is crucial in many applications such as processing massive high throughput data sets. We assess this method by applying it to empirical laboratory dose-response data and measuring the coverage of confidence limits for the BMD. We compare the coverage of this method to that of other approaches using the same set of models. Through the simulation study, the method is shown to be markedly superior to the traditional approach of selecting a single preferred model (e.g., from the U.S. EPA BMD software) for the analysis of dichotomous data and is comparable or superior to the other approaches.

Intestinal Microbiome-Macrophage Crosstalk Contributes to Cholestatic Liver Disease by Promoting Intestinal Permeability in Mice.

BACKGROUND AND AIMS: Mounting evidence supports an association between cholestatic liver disease and changes in the composition of the microbiome. Still, the role of the microbiome in the pathogenesis of this condition remains largely undefined. APPROACH AND RESULTS: To address this, we have used two experimental models, administering alpha-naphtylisocyanate or feeding a 0.1% 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet, to induce cholestatic liver disease in germ-free mice and germ-free mice conventionalized with the microbiome from wild-type, specific pathogen-free animals. Next, we have inhibited macrophage activation by depleting these cells using clodronate liposomes and inhibiting the inflammasome with a specific inhibitor of NOD-, LRR-, and pyrin domain-containing protein 3. Our results demonstrate that cholestasis, the accumulation of bile acids in the liver, fails to promote liver injury in the absence of the microbiome in vivo. Additional in vitro studies supported that endotoxin sensitizes hepatocytes to bile-acid-induced cell death. We also demonstrate that during cholestasis, macrophages contribute to promoting intestinal permeability and to altered microbiome composition through activation of the inflammasome, overall leading to increased endotoxin flux into the cholestatic liver. CONCLUSIONS: We demonstrate that the intestinal microbiome contributes to cholestasis-mediated cell death and inflammation through mechanisms involving activation of the inflammasome in macrophages.

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

Biomonitoring of methylene diphenyl 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 the 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 m/ z 543.29 [M + H]+ ion from MDI-exposed mice but not from controls. The m/ z 543.29 [M + H]+ ion was detectable within 24 h of a single MDI skin exposure and following multiple respiratory tract exposures to MDI-GSH reaction products. The m/ z 543.29 [M + H]+ ion possessed properties of dilysine-MDI, including (a) an isotope distribution pattern for a molecule with the chemical formula C27H38N6O6, (b) the expected collision-induced dissociation (CID) fragmentation pattern upon MS/MS, and (c) a retention time in reversed-phase LC-MS identical to that of synthetic dilysine-MDI. Further MDI-specific Western blot studies suggested albumin (which contains multiple dilysine sites susceptible to MDI carbamylation) as a possible source for dilysine-MDI and the presence of MDI-conjugated albumin in urine up to 6 days after respiratory tract exposure. Two additional [M + H]+ ions ( m/ z 558.17 and 863.23) 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.

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.

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.

Immunochemical detection of the occupational allergen, methylene diphenyl diisocyanate (MDI), in situ.

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, remains 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 was 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.

Role of dermal exposure in systemic intake of methylenediphenyl diisocyanate (MDI) among construction and boat building workers.

The causal relationship between inhalation exposure to methylenediphenyl diisocyanate (MDI) and the risk of occupational asthma is well known, but the role of dermal exposure and dermal uptake of MDI in this process is still unclear. The aims of this study were to measure dermal exposure to and the dermal uptake of MDI among workers (n=24) who regularly handle MDI-urethanes. Dermal exposure was measured by the tape-strip technique from four sites on the dominant hand and arm. The workers with the highest exposure (n=5) were biomonitored immediately after their work shift, in the evening and the next morning, using urinary 4,4´methylenedianiline (MDA) as a marker. Dermal uptake was evaluated by comparing workers' MDA excretions both when they were equipped with respiratory protective devices (RPDs) and when they did not use them. The measured amounts of MDI on their hands varied from below 0.1 to 17 µg/10 cm(2) during the test. MDI concentrations were in the range of 0.08 to 27 µg m(-3) in the breathing zone outside the RPDs. MDA concentrations varied from 0.1 to 0.2 µmol mol(-1) creatinine during the test period. The decreasing effect of RPDs on inhalation exposure was absent in the next morning urine samples; this excretion pattern might be an indication of dermal uptake of MDI.

Micronuclei, hemoglobin adducts and respiratory tract irritation in mice after inhalation of toluene diisocyanate (TDI) and 4,4'-methylenediphenyl diisocyanate (MDI).

Toluene diisocyanate (TDI) and 4,4'-methylenediphenyl diisocyanate (MDI), used in the production of polyurethane foam, are well known for their irritating and sensitizing properties. Contradictory results have been obtained on their genotoxicity. We investigated the genotoxicity and protein binding of inhaled TDI and MDI in mice by examining micronucleated polychromatic erythrocytes (PCEs) in bone marrow and peripheral blood and TDI- and MDI-derived adducts in hemoglobin. Male C57Bl/6J mice (8 per group) were exposed head-only to TDI vapour (mean concentrations 1.1, 1.5, and 2.4mg/m(3); the mixture of isomers contained, on the average, 63% 2,4-TDI and 37% 2,6-TDI) or MDI aerosol (mean concentrations 10.7, 20.9 and 23.3mg/m(3)), during 1h/day for 5 consecutive days. Bone marrow and peripheral blood were collected 24h after the last exposure. Inhalation of TDI caused sensory irritation (SI) in the upper respiratory tract, and cumulative effects were observed at the highest exposure level. Inhalation of MDI produced SI and airflow limitation, and influx of inflammatory cells into the lungs. Hemoglobin adducts detected in the exposed mice resulted from direct binding to globin of 2,4- and 2,6-TDI and MDI, and dose-dependent increases were observed especially for 2,4-TDI-derived adducts. Adducts originating from the diamines of TDI (toluene diamine) or MDI (methylene dianiline) were not observed. No significant increase in the frequency of micronucleated PCEs was detected in the bone marrow or peripheral blood of the mice exposed to TDI or MDI. The ratio of PCEs and normochromatic erythrocytes (NCEs) was reduced at the highest concentration of MDI, and a slight reduction of the PCE/NCE ratio, dependent on cumulative inhaled dose, was also seen with TDI. Our results indicate that inhalation of TDI or MDI (1h/day for 5 days), at levels that induce toxic effects and formation of TDI- or MDI-specific adducts in hemoglobin, does not have detectable genotoxic effects in mice, as studied with the micronucleus assay.

Contact sensitization to 4,4'-diaminodiphenylmethane and to isocyanates among general dermatology patients.

BACKGROUND: Diisocyanates and 4,4'-diaminodiphenylmethane (MDA) are industrial sensitizers. Occupational asthma is a risk among workers exposed to diisocyanates. Exposure may also lead to contact sensitization and allergic contact dermatitis. OBJECTIVE: The aim of this study was to determine the occurrence of contact sensitization to MDA and to diisocyanates among general dermatology patients. PATIENTS AND METHODS: Patch testing with MDA was carried out in 1595 patients. Diphenylmethane-4,4'-diisocyanate (MDI) and toluene-2,4-diisocyanate (TDI) were tested in 1023 patients and isophorone diisocyanate (IPDI) and 1,6-hexamethylene diisocyanate (HDI) in 433 patients. The clinical data and sources of exposure are analysed. RESULTS: MDA reactions were seen in 17 (1.1%) patients and MDI reactions in 4 patients. Six MDA-positive patients reacted to p-phenylenediamine and two to epoxy chemicals. 5/10 of the TDI reactions were seen concurrently with reactions to MDI, MDA, HDI, or to IPDI. IPDI reactions were seen in eight patients and HDI reactions in two patients. Possible sources of exposure were traced in most patients, although the association with the current dermatitis was not apparent in all cases. CONCLUSION: (Di)isocyanates may induce contact sensitization with or without allergic contact dermatitis.

Brown Norway rat asthma model of diphenylmethane-4,4'-diisocyanate (MDI): analysis of the elicitation dose-response relationship.

The known human asthmagen polymeric diphenylmethane-diisocyanate (MDI) was investigated in the Brown Norway rat skin asthma model. Two types of dose-response relationships are addressed with the following focus: (1) does sensitization dose and surface area influence the subsequent elicitation response and (2) is the elicitation response more dependent on previous elicitation doses or more on skin sensitizing dose? These two aims are investigated in two elaborated experiments, using inflammatory (bronchoalveolar lavage, BAL) and physiologic (Penh) endpoints to characterize asthma-like responses in rats. Postchallenge measurements of Penh focused on responses delayed in onset. Inflammatory endpoints in BAL were performed one day after the fourth challenge. Both protocols utilized a dermal sensitization phase with two administrations on days 0 and 7 followed by four inhalation challenges with approximately 38 mg MDI/m(3) in intervals of 2 weeks. In the first protocol three groups of rats were topically dosed with 40, 10, and 2.5 mul of MDI per rat. Each dose group consisted of three subgroups with dosed surface areas of 3.1-12.6 cm(2), 0.8-3.1 cm(2), and 0.4-0.8 cm(2), respectively. In the second protocol groups of rats were topically dosed with 40 microl of MDI per rat followed by three challenges with 37 mg MDI/m(3). At the fourth challenge subgroups of rats were either challenged with 8, 18, or 39 mg MDI/m(3). Independent of the protocol used, response was characterized by increased influx of neutrophilic granulocytes in BAL and delayed respiratory response. All groups from the first study sensitized to and challenged with MDI elicited a distinct response relative to similarly challenged naive rats. A sensitization dose dependence of the elicitation response was not found. The second protocol revealed that the elicitation dose correlates with increased neutrophils in BAL and delayed-onset respiratory responses. In summary, these data suggest that the vigor of asthma-like responses appear to be more dependent on the inhalation elicitation dose of previously challenged rats rather than the dermal induction dose.

Synthesis, mechanical properties, biocompatibility, and biodegradation of polyurethane networks from lysine polyisocyanates.

Bone defects, such as compressive fractures in the vertebral bodies, are frequently treated with acrylic bone cements (e.g., PMMA). Although these biomaterials have sufficient mechanical properties for fixing the fracture, they are non-degradable and do not remodel or integrate with host tissue. In an alternative approach, biodegradable polyurethane (PUR) networks have been synthesized that are designed to integrate with host tissue and degrade to non-cytotoxic decomposition products. PUR networks have been prepared by two-component reactive liquid molding of low-viscosity quasi-prepolymers derived from lysine polyisocyanates and poly(epsilon-caprolactone-co-DL-lactide-co-glycolide) triols. The composition, thermal transitions, and mechanical properties of the biomaterials were measured. The values of Young's modulus ranged from 1.20-1.43 GPa, and the compressive yield strength varied from 82 to 111 MPa, which is comparable to the strength of PMMA bone cements. In vitro, the materials underwent controlled biodegradation to non-cytotoxic decomposition products, and supported the attachment and proliferation of MC3T3 cells. When cultured in osteogenic medium on the PUR networks, MC3T3 cells deposited mineralized extracellular matrix, as evidenced by von Kossa staining and tetracycline labeling. Considering the favorable mechanical and biological properties, as well as the low-viscosity of the reactive intermediates used to prepare the PUR networks, these biomaterials are potentially useful as injectable, biodegradable bone cements for fracture healing.

Brown Norway rat asthma model of diphenylmethane-4,4'-diisocyanate (MDI): impact of vehicle for topical induction.

The development of this Brown Norway (BN) rat asthma model was focused on the duplication of at least some hallmarks of human diisocyanate asthma using the skin as the initial priming route of exposure. Equal total doses of polymeric diphenylmethane-diisocyanate (MDI) were applied to similar surface areas either dissolved in di-n-octyl sebacic acid ester (20%) (SEBA), in acetone:olive oil (20%) (AOO) or undiluted. The elicitation of respiratory allergy utilized four repeated nose-only inhalation challenges of 30 min with 39 mg/m(3) MDI-aerosol approximately every 2 weeks. Emphasis was directed towards the analysis of respiratory responses delayed in onset. Endpoints suggestive of an allergic inflammatory response were examined by bronchoalveolar lavage (BAL) 1 day after the last inhalation challenge and comprised protein, LDH, cytodifferentiation of BAL cells, MCP-1, and some Th1 and Th2 cytokines. MCP-1 and cytokines were comparatively determined in three compartments: BAL fluid, BAL cells, and lung-associated lymph nodes (LALN). In all groups sensitized topically to MDI typical delayed-onset respiratory responses occurred. The lung and LALN weights, BAL-protein and -LDH were significantly increased as compared to the naïve control group challenged identically. There was compelling evidence of a neutrophilic rather than an eosinophilic inflammatory response. The patterns of interleukin (IL) IL-1alpha, TNF-alpha, IFN-gamma, GM-CSF, and IL-4 differed appreciably from one compartment to another and were essentially maximal in BAL cells. In contrast, MCP-1 was increased to the same extent in all compartments measured. Collectively, changes were slightly, although consistently more pronounced when using SEBA as vehicle when compared with the vehicle AOO or undiluted MDI. Notable was a discordance of cytokine profiles and respiratory responses. In conclusion, the priming potency of topically administered MDI and subsequent asthma-like responses following repeated inhalation exposures appear to be dependent on multiple factors, one of them appears to be associated with the type of matrix used to dissolve MDI. This animal model provides a versatile and robust experimental tool to evaluate and assess at least some features of MDI-related asthma.

Th2 Cytokines in Skin Draining Lymph Nodes and Serum IgE Do Not Predict Airway Hypersensitivity to Intranasal Isocyanate Exposure in Mice.

Isocyanate exposure in the workplace has been linked to asthma and allergic rhinitis. Recently, investigators have proposed that Th2 cytokine responses in lymph nodes draining the site of dermal application of chemicals including isocyanates may be used to identify sensitizers that cause asthma-like responses. The purpose of this study was to determine if the cytokine profile induced after dermal sensitization with isocyanates and serum IgE predict immediate (IHS) and methacholine-induced late (LHS) respiratory hypersensitivity responses after intranasal challenge. Dermal application of hexylmethane diisocyanate (HMDI), toluene diisocyanate (TDI), or methylene diisocyanate (MDI) significantly increased interleukin-4 (IL-4), IL-5, and IL-13 secretion in parotid lymph node cells. Isophorone diisocyanate (IPDI) increased IL-4 and IL-13, but not IL-5. Tolyl(mono)isocyanate (TMI), tetramethylene xylene diisocyanate (TMXDI), or the contact sensitizer dinitrochlorobenzene (DNCB), only induced minor increases in some of the Th2 cytokines. HMDI, TDI, MDI, and IPDI elicited greater increases in total serum IgE than DNCB, TMI, and TMXDI. All chemicals except TMXDI caused IHS after intranasal challenge of sensitized female BALB/c mice. Only HMDI-, TMI-, or TMXDI-sensitized and challenged mice had increases in LHS. All chemicals elicited epithelial cytotoxicity indicative of nasal airway irritation. The discordance between dermal cytokine profiles and respiratory responses suggests that dermal responses do not necessarily predict respiratory responses. Serum IgE also was not predictive of the respiratory responses to the isocyanates, suggesting that other unknown mechanisms may be involved.

Partial C-fiber ablation modulates diphenylmethane-4,4'-diisocyanate (MDI)-induced respiratory allergy in Brown Norway rats.

Brown Norway (BN) rats were topically sensitized to polymeric diphenylmethane-diisocyanate (MDI) and challenged with MDI-aerosol approximately every 2 weeks over a time period of 2 months. Half of the sensitized animals were pretreated with capsaicin for partial C-fiber defunctionalization. After the fourth challenge inflammatory and pro-inflammatory factors in bronchoalveolar lavage (BAL) fluid and cells and physiological delayed-onset breathing patterns were analyzed. The latter endpoint was examined in the capsaicin pretreated group before and after each challenge. Findings were compared against naïve but repeatedly MDI-challenged BN rats. BAL-neutrophils, -protein, and -LDH as well as lung weights were significantly increased in the MDI-sensitized and challenged rats relative to the naïve, challenged control rats. With regard to these endpoints, capsaicin pretreatment did not affect the responsiveness to MDI-aerosol. In contrast, pro-inflammatory cytokines, the Th2 cell cytokine IL-4, and the CC-chemokine MCP-1 were significantly increased in BAL-cells of capsaicin pretreated and MDI-sensitized rats, whilst in the normal MDI-sensitized rats markedly less pronounced changes (if any) occurred. In the former group, IL-4 and MCP-1 were also significantly increased in the lung draining lymph nodes. Time-related increased frequencies of delayed-onset responses were observed in MDI-sensitized rats after subsequent MDI-challenges, however, differences between capsaicin pretreated and normal rats were not found. Despite the remarkable differences between normal and capsaicin pre-treated rats in the concentrations of pro-inflammatory and Th1-/Th2-cell specific cytokines, the inflammatory endpoints in BAL as well as the physiological measurements did not identify appreciable differences amongst these groups. This study included an ancillary study addressing the analysis of the modulating effect of capsaicin pre-treatment of naïve Wistar rats exposed for single 6h to MDI-aerosol. The results indicated more pronounced changes on endpoints in the BAL-fluid of capsaicin-pretreated rats as compared to rats with intact C-fibers. This complex picture appears to suggest that C-fibers may modulate the allergic inflammatory response elicited by MDI-challenge. It appears that tachykinergic sensory C-fibers modulate the protective pathways against irritant-related lung inflammation and, similarly, also pro-inflammatory immunological factors modulating allergic inflammation. Although difficult to disentangle unequivocally the mechanisms involved, neuro-immunological factors may be important in triggering and maintaining this complex disease and cytokine/chemokine patterns may not necessarily predict the functional outcome of test.

Inconsistencies between cytokine profiles, antibody responses, and respiratory hyperresponsiveness following dermal exposure to isocyanates.

Cytokine profiling of local lymph node responses has been proposed as a simple test to identify chemicals, such as low molecular weight diisocyanates, that pose a significant risk of occupational asthma. Previously, we reported cytokine messenger RNA (mRNA) profiles for dinitrochlorobenzene (DNCB) and six isocyanates: toluene diisocyanate, diphenylmethane-4,4'-diisocyanate, dicyclohexylmethane-4,4'-diisocyanate, isophorone diisocyanate, p-tolyl(mono)isocyanate, and meta-tetramethylene xylene diisocyanate. The present study was conducted to test the hypothesis that relative differences in the cytokine profile are predictive of relative differences in total serum immunoglobulin (Ig) E and respiratory responses to methacholine (Mch) following dermal exposure to the chemicals. After a preliminary experiment to determine an exposure regimen sufficient to achieve responses to Mch following dermal diisocyanate exposure, BALB/c mice received nine dermal exposures over a period of 28 days to one of six isocyanates, DNCB, or vehicle. Mice were then challenged with increasing doses of Mch and responsiveness was assessed using whole-body plethysmography. Serum antibody responses and cytokine mRNA profiles in the draining lymph node were also assessed. In separate experiments, cytokine protein assays were performed after five dermal exposures over a 14-day period. The response pattern for interleukin (IL)-4, IL-10, and IL-13 for the different isocyanates was highly reproducible as determined by RNAse protection assay, reverse transcription-PCR, or cytokine protein levels. However, the relative differences in T-helper cytokine profiles were not predictive of relative differences in either total serum IgE or respiratory responses to Mch following dermal exposure. The data suggest that the cytokine profiling approach needs to be further developed and refined before adoption and that other approaches to hazard identification should be pursued as well. Based on the weight of evidence from all the assays performed, it appears that all six isocyanates tested have some potential to cause respiratory hypersensitivity following dermal exposure.

Assessing isocyanate exposures in polyurethane industry sectors using biological and air monitoring methods.

Isocyanates, as a chemical group, are considered to be the biggest cause of occupational asthma in the UK. Monitoring of airborne exposures to total isocyanate is costly, requiring considerable expertise, both in terms of sample collection and chemical analysis and cannot be used to assess the effectiveness of protection from wearing respiratory protective equipment (RPE). Biological monitoring by analysis of metabolites in urine can be a relatively simple and inexpensive way to assess exposure to isocyanates. It may also be a useful way to evaluate the effectiveness of control measures in place. In this study biological and inhalation monitoring were undertaken to assess exposure in a variety of workplaces in the non-motor vehicle repair sector. Companies selected to participate in the survey included only those judged to be using good working practices when using isocyanate formulations. This included companies that used isocyanates to produce moulded polyurethane products, insulation material and those involved in industrial painting. Air samples were collected by personal monitoring and were analysed for total isocyanate content. Urine samples were collected soon after exposure and analysed for the metabolites of different isocyanate species, allowing calculation of the total metabolite concentration. Details of the control measures used and observed contamination of exposed skin were also recorded. A total of 21 companies agreed to participate in the study, with exposure measurements being collected from 22 sites. The airborne isocyanate concentrations were generally very low (range 0.0005-0.066 mg m(-3)). A total of 50 of the 70 samples were <0.001 mg m(-3), the limit of quantification (LOQ), therefore samples below the LOQ were assigned a value of 1/2 LOQ (0.0005 mg m(-3)). Of the 70 samples, 67 were below the current workplace exposure limit of 0.02 mg m(-3). The highest inhalation exposures occurred during spray painting activities in a truck manufacturing company (0.066 mg m(-3)) and also during spray application of polyurethane foam insulation (0.023 mg m(-3)). The most commonly detected isocyanate in the urine was hexamethylene diisocyanate, which was detected in 21 instances. The geometric mean total isocyanate metabolite concentration for the dataset was 0.29 micromol mol(-1) creatinine (range 0.05-12.64 micromol mol(-1) creatinine). A total of 23 samples collected were above the agreed biological monitoring guidance value of 1.0 micromol mol(-1) creatinine. Activities that resulted in the highest biological monitoring results of the dataset included mixing and casting of polyurethane products (12.64 micromol mol(-1) creatinine), semi-automatic moulding (4.80 micromol mol(-1) creatinine) and resin application (3.91 micromol mol(-1) creatinine). The biological monitoring results show that despite low airborne isocyanate concentrations, it was possible to demonstrate biological uptake. This tends to suggest high sensitivity of the biological monitoring method and/or that in some instances the RPE being used by operators was not effective or that absorption may have occurred via dermal or other routes of exposure. This study demonstrates that biological monitoring is a useful tool when assessing worker exposure to isocyanates, providing a more complete picture on the efficacy of control measures in place than is possible by air monitoring alone. The results also demonstrated that where control measures were judged to be adequate, most biological samples were close to or < 1 micromol mol(-1) creatinine, the agreed biological monitoring benchmark.