Memory deficits associated with sublethal cyanide poisoning relative to cyanate toxicity in rodents.

Food (cassava) linamarin is metabolized into neurotoxicants cyanide and cyanate, metabolites of which we sought to elucidate the differential toxicity effects on memory. Young 6-8 weeks old male rats were treated intraperitoneally with either 2.5 mg/kg body weight (bw) cyanide (NaCN), or 50 mg/kg bw cyanate (NaOCN), or 1 µl/g bw saline, daily for 6 weeks. Short-term and long-term memories were assessed using a radial arm maze (RAM) testing paradigm. Toxic exposures had an influence on short-term working memory with fewer correct arm entries (F(2, 19) = 4.57 p < 0.05), higher working memory errors (WME) (F(2, 19) = 5.09, p < 0.05) and longer RAM navigation time (F(2, 19) = 3.91, p < 0.05) for NaOCN relative to NaCN and saline treatments. The long-term working memory was significantly impaired by cyanide with fewer correct arm entries (F(2, 19) = 7.45, p < 0.01) and increased working memory errors (F(2, 19) = 9.35 p < 0.05) in NaCN relative to NaOCN or vehicle treated animals. Reference memory was not affected by either cyanide or cyanate. Our study findings provide an experimental evidence for the biological plausibility that cassava cyanogens may induce cognition deficits. Differential patterns of memory deficits may reflect the differences in toxicity mechanisms of NaOCN relative to NaCN. Cognition deficits associated with cassava cyanogenesis may reflect a dual toxicity effect of cyanide and cyanate.

Inhalation dosimetry of hexamethylene diisocyanate vapor in the rat and human respiratory tracts.

Hexamethylene diisocyanate (HDI) is a reactive chemical used in the commercial production of polyurethanes. Toxic effects in rodents exposed to HDI vapor primarily occur in the nasal passages, yet some individuals exposed occupationally to concentrations exceeding current regulatory limits may experience temporary reduction in lung function and asthma-like symptoms. Knowledge of interspecies differences in respiratory tract dosimetry of inhaled HDI would improve our understanding of human health risks to this compound. HDI uptake was measured in the upper respiratory tract of anesthetized Fischer-344 rats. Nasal uptake of HDI was >90% in rats at unidirectional flow rates of 150 and 300 ml/min and a target air concentration of 200 ppb. Uptake data was used to calibrate nasal and lung dosimetry models of HDI absorption in rats and humans. Computational fluid dynamics (CFD) models of the nasal passages were used to simulate inspiratory airflow and HDI absorption. Transport of HDI through lung airways was simulated using convection-diffusion based mass transport models. HDI nasal uptake of 90% and 78% was predicted using the rat and human nasal CFD models, respectively. Total respiratory tract uptake was estimated to be 99% in rats and 97% in humans under nasal breathing. Predicted human respiratory uptake decreased to 87% under oral breathing conditions. Absorption rates of inhaled HDI in human lung airways were estimated to be higher than the rat due to lower uptake in head airways. Model predictions demonstrated significant penetration of HDI to human bronchial airways, although absorption rates were sensitive to breathing style.

Hemoglobin adducts in workers exposed to 1,6-hexamethylene diisocyanate.

We investigated the utility of 1,6-hexamethylene diamine (HDA) hemoglobin adducts as biomarkers of exposure to 1,6-hexamethylene diisocyanate (HDI) monomer. Blood samples from 15 spray painters applying HDI-containing paint were analyzed for hemoglobin HDA (HDA-Hb) and N-acetyl-1,6-hexamethylene diamine (monoacetyl-HDA-Hb) by GC-MS. HDA-Hb was detected in the majority of workers (≤1.2-37 ng/g Hb), whereas monoacetyl-HDA-Hb was detected in one worker (0.06 ng/g Hb). The stronger, positive association between HDA-Hb and cumulative HDI exposure (r(2) = 0.3, p < 0.06) than same day exposure (p ≥ 0.13) indicates long-term elimination kinetics for HDA-Hb adducts. This association demonstrates the suitability of HDA-Hb adducts for further validation as a biomarker of HDI exposure.

Acute life-threatening extrinsic allergic alveolitis in a paint controller.

BACKGROUND: Occupational diisocyanate-induced extrinsic allergic alveolitis (EAA) is a rare and probably underestimated diagnosis. Two acute occupational EAA cases have been described in this context, but neither of them concerned hexamethylene diisocyanate (HDI) exposure. AIMS: To investigate the cause of a life-threatening EAA arising at work in a healthy 30-year-old female paint quality controller. METHODS: Occupational medical assessment, workplace evaluation, airborne and biological monitoring and immunodermatological tests. RESULTS: Diagnosis of EAA relied on congruent clinical and radiological information, confirmed occupational HDI exposure and positive IgG antibodies and patch tests. The patient worked in a small laboratory for 7 years, only occasionally using HDI-containing hardeners. While working with HDI for 6 h, she developed breathlessness, rapidly progressing to severe respiratory failure. Workplace HDI airborne exposure values ranged from undetectable levels to 4.25 p.p.b. Biological monitoring of urinary hexamethylene diamine in co-workers ranged from <1.0 to 15.4 µg/g creatinine. Patch tests 8 months later showed delayed skin reaction to HDI at 48 h. Subsequent skin biopsy showed spongiotic dermatitis with infiltration of CD4(+) and CD8(+) T cells. CONCLUSIONS: We believe this is the first reported case of acute life-threatening EAA following exposure to HDI. Low concentrations of airborne HDI and relatively high urinary hexamethylene diamine suggest significant skin absorption of HDI could have significantly contributed to the development of this acute occupational EAA.

The effect of the uremic toxin cyanate (CNO⁻) on anaerobic cysteine metabolism and oxidative processes in the rat liver: a protective effect of lipoate.

Chronic renal failure (CRF) patients have an increased plasma level of urea, which can be a source of cyanate. This compound can cause protein carbamoylation thereby changing biological activity of proteins. Therefore, in renal failure patients, cyanate can disturb metabolism and functioning of the liver. This work presents studies demonstrating that the treatment of rats with cyanate alone causes the following changes in the liver: (1) inhibition of rhodanese (TST), cystathionase (CST) and 3-mercaptopyruvate sulfotransferase (MPST) activities, (2) decrease in sulfane sulfur level (S*), (3) lowering of nonprotein sulfhydryl groups (NPSH) group level, and (4) enhancement of prooxidant processes (rise in reactive oxygen species (ROS) and malondialdehyde (MDA) level). This indicates that cyanate inhibits anaerobic cysteine metabolism and shows prooxidant action in the liver. Out of the above-mentioned changes, lipoate administered with cyanate jointly was able to correct MDA, ROS and NPSH levels, and TST activity. It had no significant effect on MPST and CST activities. It indicates that lipoate can prevent prooxidant cyanate action and cyanate-induced TST inhibition. These observations can be promising for CRF patients since lipoate can play a dual role in these patients as an efficient antioxidant defense and a protection against cyanate and cyanide toxicity.

Chronic inhalation exposures of Fischer 344 rats to 1,6-hexamethylene diisocyanate did not reveal a carcinogenic potential.

The polyisocyanates of 1,6-hexamethylene diisocyanate (HDI) find widespread commercial use as components of paints and in the formulation of light-stable polyurethane coating materials. This 2-year study assessed the oncogenicity of the diisocyanate monomer HDI in male and female Fischer-344 rats exposed 6 h/day, 5 days/week to mean analytical air concentrations of 0, 0.005, 0.025, and 0.164 ppm HDI. During the in-life phase, transient eye irritation was observed in 0.164 ppm males, and a slight body weight decrease (5%) in the 0.164 ppm females during the second year of exposure. There were no exposure-related effects on mortality. Compound-related, non-neoplastic histopathologic changes were limited to the respiratory tract and changes were characterized by epithelial tissue reaction to the acute irritant properties of HDI vapor. For tissues of the nasal cavity, the major histopathologic findings were degeneration of the olfactory epithelium characterized by destruction of the epithelial architecture often with narrowing or atrophy and occasional focal erosion or ulceration. In addition, there was variable degeneration of the respiratory epithelium with hyperkeratosis of the epithelium, epithelial and mucus secretory cell hyperplasia, squamous metaplasia, chronic-active inflammation, and errosive or ulcerative changes. These tissue effects along with a statistically significant decrease in body weight of female rats demonstrated attainment of a maximum tolerated dose. There was no evidence of progression of these changes in the nasal epithelium to neoplasia nor evidence of any compound-related neoplastic lesions for any of the other tissues examined. Therefore, it is concluded that HDI did not show a carcinogenic potential in this study.

Quantitative plasma biomarker analysis in HDI exposure assessment.

Quantification of amines in biological samples is important for evaluating occupational exposure to diisocyanates. In this study, we describe the quantification of 1,6-hexamethylene diamine (HDA) levels in hydrolyzed plasma of 46 spray painters applying 1,6-hexamethylene diisocyanate (HDI)-containing paint in vehicle repair shops collected during repeated visits to their workplace and their relationship with dermal and inhalation exposure to HDI monomer. HDA was detected in 76% of plasma samples, as heptafluorobutyryl derivatives, and the range of HDA concentrations was < or =0.02-0.92 microg l(-1). After log-transformation of the data, the correlation between plasma HDA levels and HDI inhalation exposure measured on the same workday was low (N = 108, r = 0.22, P = 0.026) compared with the correlation between plasma HDA levels and inhalation exposure occurring approximately 20 to 60 days before blood collection (N = 29, r = 0.57, P = 0.0014). The correlation between plasma HDA levels and HDI dermal exposure measured on the same workday, although statistically significant, was low (N = 108, r = 0.22, P = 0.040) while the correlation between HDA and dermal exposure occurring approximately 20 to 60 days before blood collection was slightly improved (N = 29, r = 0.36, P = 0.053). We evaluated various workplace factors and controls (i.e. location, personal protective equipment use and paint booth type) as modifiers of plasma HDA levels. Workers using a downdraft-ventilated booth had significantly lower plasma HDA levels relative to semi-downdraft and crossdraft booth types (P = 0.0108); this trend was comparable to HDI inhalation and dermal exposure levels stratified by booth type. These findings indicate that HDA concentration in hydrolyzed plasma may be used as a biomarker of cumulative inhalation and dermal exposure to HDI and for investigating the effectiveness of exposure controls in the workplace.

Contact allergy to aliphatic polyisocyanates based on hexamethylene-1,6-diisocyanate (HDI).

BACKGROUND: Aliphatic polyisocyanates based on hexamethylene 1,6-diisocyanate (HDI) are components of lacquers, coatings, and spray paints. They are mainly composed of HDI trimers, but also contain larger oligomers, and minute amounts of HDI monomers (<1%). HDI trimers occur as biuret, isocyanurate and asymmetrical types. OBJECTIVES: We report on 4 patients with allergic contact dermatitis/contact allergy resulting from HDI-based polyisocyanates in polyurethane paints. METHODS: The patients were examined at the Finnish Institute of Occupational Health in 2000-2009. The first two patients were diagnosed by testing with their own polyurethane hardeners and ingredients of the hardeners. In 2002, HDI isocyanurate (HDI-IC) trimer was added to our isocyanate series, and the last two patients were screened with the series. RESULTS: Patient no. 1 was exposed and sensitized to HDI biuret trimer, patient no. 2 to HDI-IC trimer, and patient no. 4 to HDI asymmetrical trimer. Patient no. 3 was positive with HDI-IC trimer. He had been handling several paint hardeners containing HDI-based polyisocyanates, but the subtypes of the trimers remained unidentified. All 4 patients were negative with HDI monomer. CONCLUSIONS: HDI trimers are novel contact allergens in workers who handle polyurethane paints. The allergic reactions cannot be explained by sensitization to HDI monomer.

Indoor air pollution evaluation with emphasize on HDI and biological assessment of HDA in the polyurethane factories.

Today, many raw materials used in factories may have a dangerous effect on the physiological system of workers. One of them which is widely used in the polyurethane factories is diisocyanates. These compounds are widely used in surface coatings, polyurethane foams, adhesives, resins, elastomers, binders, and sealants. Exposure to diisocyanates causes irritation to the skin, mucous membranes, eyes, and respiratory tract. Hexamethylene diamine (HDA) is metabolite of hexamethylene diisocyanate (HDI). It is an excretory material by worker's urine who is exposed to HDI. Around 100 air samples were collected from five defined factories by midget impinger which contained dimethyl sulfoxide absorbent as a solvent and tryptamine as reagent. Samples were analyzed by high-performance liquid chromatography with EC\UV detector using NIOSH 5522 method of sampling. Also, 50 urine samples collected from workers were also analyzed using William's biological analysis method. The concentration of HDI into all air samples were more than 88 microg/m(3), and they have shown high concentration of pollutant in the workplaces in comparison with NIOSH standard, and all of the workers' urine were contaminated by HDA. The correlation and regression test were used to obtain statistical model for HDI and HDA, which is useful for the prediction of diisocyanates pollution situation in the polyurethane factories.

Near-infrared light via light-emitting diode treatment is therapeutic against rotenone- and 1-methyl-4-phenylpyridinium ion-induced neurotoxicity.

Parkinson's disease is a common progressive neurodegenerative disorder characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta. Mitochondrial dysfunction has been strongly implicated in the pathogenesis of Parkinson's disease. Thus, therapeutic approaches that improve mitochondrial function may prove to be beneficial. Previously, we have documented that near-infrared light via light-emitting diode (LED) treatment was therapeutic to neurons functionally inactivated by tetrodotoxin, potassium cyanide (KCN), or methanol intoxication, and LED pretreatment rescued neurons from KCN-induced apoptotic cell death. The current study tested our hypothesis that LED treatment can protect neurons from both rotenone- and MPP(+)-induced neurotoxicity. Primary cultures of postnatal rat striatal and cortical neurons served as models, and the optimal frequency of LED treatment per day was also determined. Results indicated that LED treatments twice a day significantly increased cellular adenosine triphosphate content, decreased the number of neurons undergoing cell death, and significantly reduced the expressions of reactive oxygen species and reactive nitrogen species in rotenone- or MPP(+)-exposed neurons as compared with untreated ones. These results strongly suggest that LED treatment may be therapeutic to neurons damaged by neurotoxins linked to Parkinson's disease by energizing the cells and increasing their viability.

Comparative assessment of early acute lung injury in mice and rats exposed to 1,6-hexamethylene diisocyanate-polyisocyanate aerosols.

The aliphatic diisocyanate monomer 1,6-hexamethylene diisocyanate (HDI) is used as a building block for non-volatile polycondensation products, such as HDI-isocyanurate (HDI-IC) and HDI-biuret (HDI-BT). This paper describes the results from acute inhalation studies with these types of polyisocyanate aerosols in OF1 and C57BL/6J mice and in Wistar rats. The modifying role of different concentrations of residual HDI in HDI-BT on pulmonary irritation was also addressed. These data close data gaps for acute mouse inhalation studies in direct comparison with rats. The sensory irritant potency was examined in OF1 mice during a 3h nose-only exposure to the polyisocyanate aerosols. Concurrent with exposure, breathing patterns suitable to distinguish upper/lower respiratory tract irritation where examined. Functional measurements in barometric plethysmographs (Penh) addressed changes in respiratory function in C57BL/6J mice exposed for 6h up to 16h postexposure. These measurements revealed that these polyisocyanates elicit changes slow in onset suggestive of pulmonary irritation rather than upper airway irritation. This conclusion was supported by similarly exposed OF1 mice exposed to non-irritant, surface active respirable particles of amorphous silica. In C57BL/6J mice and Wistar rats, nose-only exposed for 6h to 10mg/m(3) of aerosolized HDI-BT HDI (0.1% or 2% residual HDI), the pulmonary irritation potency was comparatively assessed by bronchoalveolar lavage (BAL) on postexposure day 1. Similarly air-exposed animals served as concurrent controls. Most changes in BAL suggestive of acute pulmonary irritation were more pronounced in Wistar rats than in C57BL/6J mice. A conclusive dependence of BAL endpoints on the residual content of residual HDI monomer in the polyisocyanate was not found. The results of this study show that mice may be particularly suitable to functionally analyze at which location of the respiratory tract predominant irritation may occur. However, with regard to analysis of lower respiratory tract irritation, rats were demonstrated to be more susceptible than mice. In summary, this study supports the conclusion that data from rat inhalation studies with these types of isocyanates appear to be more conservative and less variable than the respective data from mice.

Alpha-ketoglutarate and N-acetyl cysteine protect PC12 cells from cyanide-induced cytotoxicity and altered energy metabolism.

Cyanide is a rapidly acting neurotoxin that inhibits cellular respiration and energy metabolism leading to histotoxic hypoxia. This results in the dissipation of mitochondrial membrane potential (MMP) accompanied by decreased cellular ATP content which in turn is responsible for increased levels of intracellular calcium ions ([Ca(2+)](i)) and total lactic acid content of the cells. Rat pheochromocytoma (PC12) cells possess much of the biochemical machinery associated with synaptic neurons. In the present study, we evaluated the cytoprotective effects of alpha-ketoglutarate (A-KG) and N-acetylcysteine (NAC) against cyanide-induced cytotoxicity and altered energy metabolism in PC12 cells. Cyanide-antagonism by A-KG is attributed to cyanohydrin formation whereas NAC is known for its antioxidant properties. Data on leakage of intracellular lactate dehydrogenase and mitochondrial function (MTT assay) revealed that simultaneous treatment of A-KG (0.5 mM) and NAC (0.25 mM) significantly prevented the cytotoxicity of cyanide. Also, cellular ATP content was found to improve, followed by restoration of MMP, intracellular calcium [Ca(2+)](i) and lactic acid levels. Treatment with A-KG and NAC also attenuated the levels of peroxides generated by cyanide. The study indicates that combined administration of A-KG and NAC protected the cyanide-challenged PC12 cells by resolving the altered energy metabolism. The results have implications in the development of new treatment regimen for cyanide poisoning.

Gene expression profiling of in vitro cultured macrophages after exposure to the respiratory sensitizer hexamethylene diisocyanate.

Occupational exposure to chemicals is one of the main causes of respiratory allergy and asthma. Identification of chemicals that trigger allergic asthma is difficult as underlying processes and specific markers have not yet been clearly defined. Moreover, adequate classification of the respiratory toxicity of chemicals is hampered due to the lack of validated in vivo and in vitro test methods. The study of differential gene expression profiles in appropriate human in vitro cell systems is a promising approach to identify selective markers for respiratory allergy. As alveolar macrophages display important immunological and inflammatory properties in response to foreign substances in the lung, we aimed at gaining more insight in changes of human macrophages transcriptome and to identify selective genetic markers for respiratory sensitization in response to hexamethylene diisocyanate (HDI). In vitro cultures of human THP-1 cells were differentiated into macrophages and exposed to 55 microg/ml HDI for 6 and 10h. Using human oligonucleotide microarrays, changes were observed in the expression of genes that are involved in diverse biological and molecular processes, including detoxification, oxidative stress, cytokine signaling, and apoptosis, which can lead to the development of asthma. These genes are possible markers for respiratory sensitization caused by isocyanates.

Determination of pulmonary irritant threshold concentrations of hexamethylene-1,6-diisocyanate (HDI) prepolymers by bronchoalveolar lavage in acute rat inhalation studies according to TRGS 430.

Pulmonary irritant threshold concentrations of two hexamethylene-1,6-diisocyanate (HDI)-based prepolymers (I: polymeric emulsfier modified and II: oligomeric allophanate modified) were determined in acute inhalation studies according to TRGS 430 (Dangerous Substances Technical Rule, isocyanates, Germany), based on benchmark extrapolation of bronchoalveolar lavage fluid (BALF) total protein. It was also investigated if the method is robust enough to be transferred to an independent laboratory. Five male Wistar rats per group were exposed nose-only to the test substances as liquid aerosols to concentrations of 0, 0.5, 3, 15 mg/m(3) for both test substances with an additional test group at 50 mg/m(3) for test substance I. The duration of the exposure was 6h, followed by serial sacrifices 1 day, 3 days and 7 days post exposure. BALF was analyzed for biochemical and cytological markers indicative for injury of the bronchoalveolar region. The exposure of rats to test substance I and II caused dose depended lung irritation with BALF total protein concentration being the most sensitive indicator of pulmonary effects. The extrapolated no observed adverse effect level of test substance I was 1.1 mg/m(3) and that of test substance II 2.3 mg/m(3). The acute pulmonary irritant threshold concentrations were found to be similar to those reported by [Pauluhn, J., 2004. Pulmonary irritant potency of polyisocyanate aerosols in rats: comparative assessment of irritant threshold concentrations by bronchoalveolar lavage. J. Appl. Toxicol. 24, 231-247] for HDI-homopolymers and other HDI-based polyisocyanates, and were at least 30 times higher than the MAK (occupational exposure limit) value for the HDI monomer (0.035 mg/m(3)). Thus the EBW (exposure assessment value) for these two HDI-based prepolymers can be established at 10x MAK, i.e. at 0.35 mg/m(3).

A critical review of the effects of gold cyanide-bearing tailings solutions on wildlife.

Wildlife deaths associated with cyanide-bearing mine waste solutions have plagued the gold mining industries for many years, yet there is little published data showing the relationship between wildlife mortality and cyanide toxicity. A gap of knowledge exists in monitoring, understanding the causal relationships and managing risks to wildlife from cyanide-bearing waste solutions and tailings. There is a need for the gold industry to address this issue and to meet the International Cyanide Management Code (ICMC) guidelines. The perceived extent of the issue varies, with one study finding the issue inadequately monitored and wildlife deaths grossly underestimated. In Nevada, USA during 1990 and 1991, 9512 carcasses were reported of over 100 species, although there was underestimation due to reporting being voluntary. Of these, birds comprised 80-91% of vertebrate carcasses reported annually. At Northparkes, Australia in 1995, it was initially estimated that 100 bird carcasses were present by mine staff following a tailings incident; when a thorough count was conducted, 1583 bird carcasses were recorded. Eventually, 2700 bird deaths were documented over a four-month period. It is identified that avian deaths are usually undetected and significantly underestimated, leading to a perception that a risk does not exist. Few guidelines and information are available to manage the risks of cyanide to wildlife, although detoxification, habitat modification and denying wildlife access have been used effectively. Hazing techniques have proven ineffective. Apparently no literature exists that documents accurate wildlife monitoring protocols on potentially toxic cyanide-bearing mine waste solutions or any understanding on the analysis of any derived dataset. This places the onus on mining operations to document that no risk to wildlife exists. Cyanide-bearing tailings storage facilities are environmental control structures to contain tailings, a standard practice in the mining industry. Cyanide concentrations below 50 mg/L weak-acid-dissociable (WAD) are deemed safe to wildlife but are considered an interim benchmark for discharge into tailings storage facilities (TSFs). Cyanide is a fast acting poison, and its toxicity is related to the types of cyanide complexes that are present. Cyanide in biota binds to iron, copper and sulfur-containing enzymes and proteins required for oxygen transportation to cells. The accurate determination of cyanide concentrations in the field is difficult to achieve due to sampling techniques and analytical error associated with loss and interferences following collection. The main WAD cyanide complexes in gold mine tailings are stable in the TSF environment but can release cyanide ions under varying environmental conditions including ingestion and absorption by wildlife. Therefore distinction between free, WAD and total cyanide forms in tailings water for regulatory purposes is justified. From an environmental perspective, there is a distinction between ore bodies on the basis of their copper content. For example, wildlife deaths are more likely to occur at mines possessing copper-gold ores due to the formation of copper-cyanide complexes which is toxic to birds and bats. The formation of copper-cyanide complex occurs preferentially to gold cyanide complex indicating the relative importance of economic vs. environmental considerations in the tailings water. Management of cyanide to a perceived threshold has inherent risks since cyanide has a steep toxicity response curve; is difficult to accurately measure in the field; and is likely to vary due to variable copper content of ore bodies and ore blending. Consequently, wildlife interaction needs to be limited to further reduce the risks. A gap in knowledge exists to design or manage cyanide-bearing mine waste solutions to render such facilities unattractive to at-risk wildlife species. This gap may be overcome by understanding the wildlife behaviour and habitat usage of cyanide-bearing solutions.

Carbamylation and oxidation of proteins lead to apoptotic death of lymphocytes.

The apoptotic/necrotic changes in isolated human peripheral blood mononuclear cells (MNCs) subjected to hydrogen peroxide (H2O2), cyanate (NaOCN) and their combination were examined. The mitochondrial potential (ΔΨm), the activities of caspases (-2, -3, -6, -8 and -9) and the level of carbonyls and amino groups in proteins were determined and DNA fragmentation. Apoptotic or necrotic cells were identified by fluorescence microscopy using double staining with Hoechst 33258/propidium iodide. Treatment of MNCs with NaOCN (1 mmol/L and 2 mmol/L), alone and in combination with H2O2 (100 µmol/L), led to a significant decrease in the content of amine groups and a significant increase in the carbonyl level of MNCs in comparison with the control. Measurements taken at three time points (30, 60 and 150 min) showed a significant decrease in ΔΨm in MNCs incubated with H2O2, cyanate and their combination. The highest decrease in ΔΨm was observed after 150 min, when a combination of NaOCN and H2O2 was applied. We observed significant increases in the activities of caspases-2 and -3 in cells exposed to H2O2 and the combination of NaOCN and H2O2. An increase in caspase-2 but not in caspase-3 activity was noted in cells incubated with cyanate. A significant increase in caspase-9 activity in MNCs was observed in all arrangements of tested compounds in comparison with the control. In H2O2-treated cells, a higher level of necrotic cells was noted in comparison to apoptotic cells, whereas carbamylation led mainly to apoptotic cell death. The combination of cyanate and H2O2 increased the population of necrotic cells.

Gene therapy in the nervous system with superoxide dismutase.

Neuronal death following necrotic insults involves the generation of reactive oxygen species (ROS). We investigated the effects of antioxidant gene therapy on ROS accumulation after exposure to either sodium cyanide, kainic acid or oxygen glucose deprivation (OGD). Specifically, we generated herpes simplex virus-1 amplicon vector expressing the gene for the antioxidant enzyme CuZnSOD. Overexpression of this gene in primary hippocampal cultures resulted in increased enzymatic activity of the corresponding protein. CuZnSOD significantly protected hippocampal neurons against sodium cyanide insult and the subsequent lipid peroxidation. However, it did not protect against OGD- or kainic-acid-induced toxicity. Moreover, CuZnSOD significantly worsened the toxicity, hydrogen peroxide accumulation and lipid peroxidation induced by kainic acid. As a possible explanation for this surprising worsening, CuZnSOD overexpression increased glutathione peroxidase activity in the presence of sodium cyanide but had no effect on catalase or glutathione peroxidase activity in the presence of kainic acid. Thus, cells were unlikely to be able to detoxify the excess hydrogen peroxide produced as a result of the CuZnSOD overexpression. These studies can be viewed as a cautionary note concerning gene therapy intervention against necrotic insults.

Dermal, inhalation, and internal exposure to 1,6-HDI and its oligomers in car body repair shop workers and industrial spray painters.

OBJECTIVES: To study inhalation and dermal exposure to hexamethylene diisocyanate (HDI) and its oligomers as well as personal protection equipment (PPE) use during task performance in conjunction with urinary hexamethylene diamine (HDA) in car body repair shop workers and industrial spray painters. METHODS: Personal task based inhalation samples (n = 95) were collected from six car body repair shops and five industrial painting companies using impingers with di-n-butylamine (DBA) in toluene. In parallel, dermal exposure was assessed using nitril rubber gloves. Gloves were submerged into DBA in toluene after sampling. Analysis for HDI and its oligomers was performed by LC-MS/MS. Urine samples were collected from 55 workers (n = 291) and analysed for HDA by GC-MS. RESULTS: Inhalation exposure was strongly associated with tasks during which aerosolisation occurs. Dermal exposure occurred during tasks that involve direct handling of paint. In car body repair shops associations were found between detectable dermal exposure and glove use (odds ratio (OR) 0.22, 95% confidence interval (CI) 0.09 to 0.57) and inhalation exposure level (OR 1.34, 95% CI 0.97 to 1.84 for a 10-fold increase). HDA in urine could be demonstrated in 36% and 10% of car body repair shop workers and industrial painting company workers respectively. In car body repair shops, the frequency of detectable HDA was significantly elevated at the end of the working day (OR 2.13, 95% CI 1.07 to 4.22 for 3-6 pm v 0-8 am). In both branches HDA was detected in urine of approximately 25% of the spray painters. In addition HDA was detected in urine of a large proportion of non-spray painters in car body repair shops. CONCLUSION: Although (spray) painting with lacquers containing isocyanate hardeners results in the highest external exposures to HDI and oligomers, workers that do not perform paint related tasks may also receive a considerable internal dose.

Tape-strip sampling for measuring dermal exposure to 1,6-hexamethylene diisocyanate.

OBJECTIVES: This study describes the development and evaluation of a method for sampling layers of the stratum corneum for the quantitation of dermal exposure to 1,6-hexamethylene diisocyanate (HDI). METHODS: HDI deposited on skin was collected by the removal of stratum corneum with adhesive tape, derivatized with 1-(2-methoxyphenyl)piperazine, and quantitated as the urea derivative (HDIU) by liquid chromatography-mass spectrometry (LC-MS). This LC-MS method was tested by analyzing tape spiked with HDI-containing products, then applied to tape samples collected from the skin of an auto-body shop worker exposed to polyurethane paint aerosols. RESULTS: The limits of detection and quantitation were 20 and 50 fmol per injection, respectively. The recovery of HDI from the tape was 99.3% [95% confidence interval (95% CI) 97.1-102]. HDIU was stable at -40 degrees C, degrading by 0.28% (95% CI 0.10-0.46) per day. Quantifiable amounts of HDI were observed in 42.6% of the first three successive tape-strip samples collected from 36 different sites on the skin of the worker. The amount of HDI recovered from the collection sites on skin, measured by summing the levels collected with three successive tape-strips, ranged from nondetectable to 1874 pmol. CONCLUSIONS: This study demonstrates that HDI on skin can be collected with tape-strips and quantified at occupational levels using LC-MS.