RESUMEN
BACKGROUND AND AIM: The submandibular salivary glands (SMG) represent a suitable model for studying epithelial cell growth and differentiation. Bisphenol A (BPA) is a xenoestrogen, synthesized to produce polymers such as polycarbonates and epoxy resins. There are concerns about the occurrence of BPA in food, water as well as its appearance in human tissues and body fluids. Lycopene (LYC) is a carotenoid compound that exerts antioxidant and anti-inflammatory properties. This work was performed to study possible protective effect of LYC against BPA toxicity in SMG. MATERIAL AND METHODS: 40 albino rats were divided into 4 groups; Group I: served as controls. Group II: rats received LYC (4 mg/kg, p.o), Group III: rats received BPA (10 mg/kg, p.o) and Group IV: rats received LYC (4 mg/kg, p.o) and BPA (10 mg/kg, p.o). All drugs were administered for 45 days then under anesthesia, rats were sacrificed. The SMG specimens were taken for histological and biochemical studies. RESULTS: BPA resulted in a significant rise of malondialdehyde, tumor necrosis factor-α and interleukine-1ß. In contrast, the tissue levels of glutathione and PPAR-γ were significantly decreased. BPA activated Wnt/ß-catenin pathway evidenced by upregulating WNT3a, ß-catenin and c-myc expression. Moreover, SMG of BPA showed degenerative changes that affected the parenchymal and stromal elements of the glands. The immunohistochemical localization of cytokeratin 5,6 and 18 of BPA rats revealed weak immunostaining of the serous secretory cells, myoepithelial cells and ductal cells. Upon treatment with LYC, glutathione and PPAR-γ were restored. CONCLUSION: LYC acted as a protective agent against BPA-induced pathological changes in SMG.
Asunto(s)
Antioxidantes , PPAR gamma , Ratas , Antioxidantes/farmacología , Antioxidantes/uso terapéutico , beta Catenina/metabolismo , Carotenoides , Resinas Epoxi/metabolismo , Glutatión/metabolismo , Queratina-5/metabolismo , Licopeno/química , Malondialdehído , PPAR gamma/metabolismo , Sustancias Protectoras/farmacología , Glándulas Salivales , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba , Agua , AnimalesRESUMEN
Bisphenol A (BPA) is an industrial synthetic chemical that is extensively used for manufacturing polycarbonate plastics and epoxy resins. However, there is limited literature on BPA-induced temporal neurobehavioral transformation and oxidative stress-mediated neurodegeneration in the subtle region of the zebrafish brain. Consequently, an investigational setup was prepared to study the temporal response to duration-dependent BPA exposure on neurobehavioral, oxidative stress, and neurodegeneration in zebrafish. Zebrafish were divided into five groups: naïve, control, 7 days (BPA7D), 14 days (BPA14D), and 21 days (BPA21D). Our findings indicated that chronic waterborne exposure to BPA substantially altered the light/dark preference and bottom-dwelling behavior of zebrafish in the BPA14D, and BPA21D groups compared with naïve and control groups. Biochemical studies revealed that there was a significant downregulation in the cellular level of small-molecule antioxidants evidenced by reduced glutathione (GSH) and activity of antioxidant enzymes of glutathione biosynthesis in a duration-dependent manner after exposure to BPA. However, exposure to BPA for 7 days did not induce substantial alteration in biochemical parameters, such as GSH level, protein carbonylation, and superoxide dismutase activity, although the neurobehavioral responses expressively differed from those of the naïve and control groups. Moreover, our histopathological observation also indicated a temporal augmentation in chromatin condensation in the periventricular gray zone (PGZ) of the zebrafish brain after chronic exposure to BPA. The overall outcomes of the present study indicated that the transformed neurobehavioral phenotypes in zebrafish are a consequence of BPA-induced oxidative stress and PGZ neurodegeneration and clearly show a temporal transformation under BPA exposure.
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Antioxidantes , Pez Cebra , Animales , Pez Cebra/metabolismo , Antioxidantes/farmacología , Cromatina , Resinas Epoxi/metabolismo , Compuestos de Bencidrilo/toxicidad , Estrés Oxidativo , Glutatión/metabolismo , Encéfalo/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
Age-associated changes leading to a decline in cardiac structure and function contribute to the increased susceptibility and incidence of cardiovascular diseases (CVD) in elderly individuals. Indeed, age is considered a risk factor for heart failure and serves as an important predictor for poor prognosis in elderly individuals. Effects stemming from chronic, low-grade inflammation, inflammaging, are considered important determinants in cardiac health; however, our understanding of the mechanisms involved remains unresolved. A steady decline in mitochondrial function is recognized as an important biological consequence found in the aging heart which contributes to the development of heart failure. Dysfunctional mitochondria contribute to increased cellular stress and an innate immune response by activating the NLRP-3 inflammasomes, which have a role in inflammaging and age-related CVD pathogenesis. Emerging evidence suggests a protective role for CYP450 epoxygenase metabolites of N-3 and N-6 polyunsaturated fatty acids (PUFA), epoxylipids, which modulate various aspects of the immune system and protect mitochondria. In this article, we provide insight into the potential roles N-3 and N-6 PUFA have modulating mitochondria, inflammaging and heart failure.
Asunto(s)
Insuficiencia Cardíaca/inmunología , Insuficiencia Cardíaca/metabolismo , Animales , Enfermedades Cardiovasculares/sangre , Enfermedades Cardiovasculares/inmunología , Enfermedades Cardiovasculares/metabolismo , Resinas Epoxi/metabolismo , Ácidos Grasos Insaturados/sangre , Insuficiencia Cardíaca/sangre , Humanos , Inmunidad Innata/fisiología , Inflamasomas/metabolismo , Inflamasomas/fisiología , Inflamación/sangre , Inflamación/inmunología , Inflamación/metabolismo , Mitocondrias/metabolismo , Mitocondrias/fisiologíaRESUMEN
During the 20th century, synthetic polymers were greatly used in the field of art. In particular, the epoxy resins were used for both conservation and for creating sculptures. The biodeterioration of these polymers has not been adequately studied. The aim of this investigation was to examine the microflora responsible for the deterioration of an epoxy statue exposed to outdoor conditions. Fungal and bacterial microflora were isolated from the art object, clustered by fluorescence-ITS (internal transcribed spacer), identified by ITS and 16S rRNA sequencing and tested for their lipolytic abilities by three agar assays. Different algal, bacterial, cyanobacterial and fungal clone libraries were constructed. The surrounding airborne microflora was analyzed using culture-dependent and culture-independent approaches. The results indicated the presence, on the statue surface, of an interesting and differentiate microbial community composed of rock-inhabiting members, algal photobionts (Trebouxia spp., Chloroidium ellipsoideum and Chlorella angustoellipsoidea), Cyanobacteria (Leptolyngbya sp., Phormidium sp., Cylindrospermum stagnale, Hassallia byssoidea and Geitlerinema sp.), black yeasts related to the species Friedmanniomyces endolithicus, Pseudotaeniolina globosa, Phaeococcomyces catenatus and Catenulostroma germanicum and several plant-associated fungi. This investigation provides new information on the potential microfloral inhabitants of epoxy resin discovering a new ecological niche, occupied mainly by several members of rock-colonizing microbial species.
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Bacterias/genética , Plásticos Biodegradables/metabolismo , Cianobacterias/genética , Resinas Epoxi/metabolismo , Hongos/genética , Agar , Bacterias/clasificación , Biodegradación Ambiental , Biodiversidad , Cianobacterias/clasificación , Hongos/clasificación , Consorcios Microbianos/genética , ARN Ribosómico 16S/genéticaRESUMEN
Bisphenol A (BPA), a monomer of polycarbonate plastics and epoxide resin, acts as an endocrine-active compound and has been shown to enhance the inflammatory response to allergen challenge. Previous reports in rodents have demonstrated that perinatal BPA exposure alters airway inflammation following sensitization and challenge to ovalbumin in juvenile and adult offspring. Additionally, a high concentration of BPA has been shown to enhance mediator release in mast cell lines. This study aimed to determine if short-term BPA exposure, at levels relevant to human exposure, enhances mast cell release of histamine and cysteinyl leukotrienes (CysLTs). Primary murine bone marrow-derived mast cells (BMMC) produced from the femurs of female C57BL/6 mice were stimulated with BPA or estradiol (E2) in vitro. It was observed that both BPA and E2 increased BMMC histamine release over a range of nanomolar concentrations (1-1000 nM). The estrogen receptor (ER) antagonist ICI 182,780 partially blocked the ability of E2, but not BPA, to elevate histamine release. BPA also increased CysLT release, which was not abrogated by ER inhibition. It was also observed that the ability of BPA to enhance histamine and CysLT release was inhibited by blocking the extracellular signal-regulated kinase (ERK) pathway with U0126 or by chelating extracellular calcium (Ca(2+)) using EGTA. In summary, these experiments are the first to demonstrate that acute BPA exposure enhances mast cell histamine and CysLT release in vitro--an effect that is not dependent on an ER-mediated mechanism. Instead, BPA-induced mast cell histamine and CysLT release may be mediated, in part, by the ERK pathway and extracellular Ca(2+) concentrations. These data suggest that exposure to BPA at levels relevant to human exposure may provoke an acute inflammatory response in atopic individuals via enhanced mast cell activation.
Asunto(s)
Compuestos de Bencidrilo/metabolismo , Resinas Epoxi/metabolismo , Mastocitos/metabolismo , Fenoles/metabolismo , Animales , Compuestos de Bencidrilo/inmunología , Células de la Médula Ósea/citología , Butadienos/farmacología , Calcio/metabolismo , Células Cultivadas , Estradiol/análogos & derivados , Estradiol/inmunología , Estradiol/farmacología , Receptor alfa de Estrógeno/antagonistas & inhibidores , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Femenino , Fulvestrant , Histamina/metabolismo , Humanos , Leucotrieno D4/metabolismo , Mastocitos/citología , Mastocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Nitrilos/farmacología , Fenoles/inmunologíaRESUMEN
Assembly, release and maturation of HIV-1 particles comprise a highly dynamic sequence of events, characterized by a series of dramatic rearrangements of the viral structural proteins and overall virion architecture. HIV-1 morphogenesis is a relatively rapid and asynchronous process, showing high variability between cells and individual virions. Therefore, bulk biochemical methods are not ideally suited to study specific aspects of this process in detail. In contrast, imaging represents a direct approach to analyze individual particles and events. While live-cell imaging can reveal the dynamics of intracellular events with high temporal resolution, it falls short in revealing ultra-structural details. Thus, live-cell fluorescence microscopy and electron microscopy (EM) can complement each other to gain insight into both the dynamics of assembly and the structures detected at HIV-1 assembly sites. In this chapter we describe microscopic setups, tools, and methods for live-cell fluorescence microscopy as well as for different EM techniques, which have been successfully used by us and others to study HIV-1 assembly at the host cell plasma membrane. These methods can be used in a complementary manner to investigate the effects of cellular factors, mutations in the viral genome or antiviral drugs on dynamic and structural aspects of HIV-1 morphogenesis.
Asunto(s)
VIH-1/fisiología , Imagen Molecular/métodos , Ensamble de Virus , Liberación del Virus , Línea Celular , Resinas Epoxi/metabolismo , Humanos , Metilcelulosa/química , Microscopía Electrónica , Coloración y EtiquetadoRESUMEN
In this study, two halophilic bacteria were isolated from activated sludge in the epoxy wastewater treatment system. The strains were identified, and the growth and degradation characteristics were investigated. Strain J1 and J2 was identified respectively by morphological observation and 16S rDNA sequence alignment analysis. It was found that both strains belong to the Bacillus genus (Bacillus sp.) and branch Bacillus (Virgibacillus sp.). The optimized growth condition of strain J1 and J2 in the high salt CM culture medium was as follows: solution temperature 30 degrees C, pH 7.0 and 5-50 g x L(-1) of NaCl. Furthermore, the best degradation condition of the organic epoxy wastewater was: temperature 30 degrees C, pH 7.0 and NaCl concentration 30 g x L(-1). When the volume ratio of bacterial suspension mixture of J1 and J2 was 2:1 and the inoculum size of the composite strains was 10%, the highest COD removal efficiency was achieved in the epoxy wastewater treatment.
Asunto(s)
Bacillus/metabolismo , Resinas Epoxi/aislamiento & purificación , Cloruro de Sodio/análisis , Eliminación de Residuos Líquidos/métodos , Bacillus/aislamiento & purificación , Biodegradación Ambiental , China , Resinas Epoxi/metabolismo , Aguas del Alcantarillado/microbiología , Virgibacillus/aislamiento & purificación , Virgibacillus/metabolismo , Aguas Residuales/químicaRESUMEN
The presence of residual endodontic sealer in the pulp chamber may cause discoloration of the dental crown and interfere with the adhesion of restorative materials. The aim of this study was to compare the efficacy of different solvents in removing residues of an epoxy resin-based sealer (AH Plus) from the dentin walls of the pulp chamber, by scanning electron microscopy (SEM). Forty-four bovine incisor dental crown fragments were treated with 17% EDTA and 2.5% NaOCl. Specimens received a coating of AH Plus and were left undisturbed for 5 min. Then, specimens were divided in four groups (n = 10) and cleaned with one of the following solutions: isopropyl alcohol, 95% ethanol, acetone solution, or amyl acetate solution. Negative controls (n = 2) did not receive AH Plus, while in positive controls (n = 2) the sealer was not removed. AH Plus removal was evaluated by SEM, and a score system was applied. Data were analyzed by Kruskal-Wallis and Dunn tests. None of the solutions tested was able to completely remove AH Plus from the dentin of the pulp chamber. Amyl acetate performed better than 95% ethanol and isopropyl alcohol (p < 0.05), but not better than acetone (p > 0.05) in removing the sealer from dentin. No significant differences were observed between acetone, 95% ethanol, and isopropyl alcohol (p > 0.05). It was concluded that amyl acetate and acetone may be good options for cleaning the pulp chamber after obturation with AH Plus.
Asunto(s)
Dentina/efectos de los fármacos , Dentina/ultraestructura , Resinas Epoxi/análisis , Resinas Epoxi/metabolismo , Compuestos Orgánicos/farmacología , Materiales de Obturación del Conducto Radicular/análisis , Materiales de Obturación del Conducto Radicular/metabolismo , Solventes/farmacología , Animales , Bovinos , Procesamiento de Imagen Asistido por Computador , Incisivo/efectos de los fármacos , Incisivo/ultraestructura , Microscopía Electrónica de Rastreo , Compuestos Orgánicos/metabolismo , Solventes/metabolismoRESUMEN
The aim of the present study was to investigate the influence of a decellularization protocol on the structure and the mechanical behavior of small-diameter (<6 mm) tibial calf arteries and veins. Calf vessels were decellularized by a detergent-enzymatic method (DEM), partially hydrolyzed with trypsin and subsequently cross-linked using poly(ethylene glycol) diglycidyl ether. Our results showed that i) the DEM can be considered a simple and valuable procedure for the preparation of complete acellular arteries and veins able to preserve a high degree of collagen and elastic fibers, and ii) poly(ethylene glycol) diglycidyl ether cross-linking treatment provides appropriate mechanical reinforcement of blood vessels. Histologically, the decellularized vessels were obtained employing the detergent-enzymatic procedure and their native extracellular matrix histoarchitecture and components remained well preserved. Moreover, the decellularization protocol can be considered an effective method to remove HLA class I antigen expression from small-diameter tibial calf arteries and veins. Cytocompatibility of decellularized cross-linked vessels was evaluated by endothelial and smooth muscle cell seeding on luminal and adventitial vessel surfaces, respectively.
Asunto(s)
Prótesis Vascular , Vasos Sanguíneos/trasplante , Ingeniería de Tejidos/métodos , Animales , Vasos Sanguíneos/citología , Bovinos , Adhesión Celular , Proliferación Celular , Células Cultivadas , Colágeno/metabolismo , Reactivos de Enlaces Cruzados/metabolismo , Células Endoteliales/citología , Resinas Epoxi/metabolismo , Glicina/metabolismo , Humanos , Inmunohistoquímica , Microscopía Electrónica de Rastreo , Miocitos del Músculo Liso/citología , Tripsina/metabolismoRESUMEN
INTRODUCTION: Patch testing is performed to diagnose allergic contact dermatitis to avoid exposure to the revealed contact allergens. Nevertheless, patch testing is not routinely performed in the evaluation of patients with chronic idiopathic (spontaneous) urticaria (CIU). The objective of this study was to determine the frequency of sensitization to patch test allergens in severe CIU and assess a role of the allergen avoidance in CIU remission. METHODS: The cases of patients with severe CIU were retrospectively studied. The patients were patch tested using T.R.U.E. TEST. Three groups were studied: CIU with positive patch test group; CIU with negative patch test group and control group, which included the cases of not patch-tested patients with CIU. The groups were followed up monthly to assess changes in Chronic Urticaria Severity Score (CUSS) after allergen avoidance. RESULTS: Forty-three subjects with severe CIU were patch tested. Nickel sulphate was positive in 4 (9.3%) cases, potassium dichromate in 2 (4.7%) cases, cobalt, balsam of Peru, paraphenylene diamine, fragrance mix and epoxy resin were positive in 1 (2.3%) case. Their baseline CUSS (5.4 ± 0.5) improved significantly after 1 month of allergen avoidance (3.2 ± 1.1; P < 0.001); but similar improvement of CUSS (5.3 ± 0.5) was seen in 34 patients with CIU with negative patch test (3.2 ± 1.3; P < 0.001) and in 49 patients with CIU of control group (5.2 ± 0.4 to 3.4 ± 1.3; P < 0 < 0.001) after 1 month. CONCLUSIONS: There is no relationship between avoidance of contact allergens and the course of CIU.
Asunto(s)
Alérgenos/inmunología , Dermatitis Alérgica por Contacto/diagnóstico , Pruebas del Parche , Urticaria/diagnóstico , Adulto , Bálsamos/metabolismo , Enfermedad Crónica , Cobalto/inmunología , Dermatitis Alérgica por Contacto/inmunología , Resinas Epoxi/metabolismo , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Níquel/inmunología , Pruebas del Parche/métodos , Fenilendiaminas/inmunología , Dicromato de Potasio/inmunología , Estudios Retrospectivos , Índice de Severidad de la Enfermedad , Urticaria/inmunologíaRESUMEN
Ascorbate oxidase purified from Lagenaria siceraria fruit was immobilized onto epoxy resin "Araldite" membrane with 79.4% retention of initial activity of free enzyme. The biosensor showed optimum response within 15s at pH 5.8 and 35°C, which was directly proportional to ascorbate concentration ranging from 1-100µM. There was a good correlation (R(2) = 0.99) between serum ascorbic acid values by standard enzymic colorimetric method and the present method. The enzyme electrode was used for 200 times without considerable loss of activity during the span of 90 days when stored at 4°C.
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Ascorbato Oxidasa/metabolismo , Técnicas Biosensibles , Enzimas Inmovilizadas/metabolismo , Resinas Epoxi/metabolismo , Anhídridos Ftálicos/metabolismo , Proteínas de Plantas/metabolismo , Ascorbato Oxidasa/aislamiento & purificación , Ácido Ascórbico/sangre , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Técnicas Biosensibles/estadística & datos numéricos , Colorimetría , Cucurbitaceae/enzimología , Enzimas Inmovilizadas/aislamiento & purificación , Frutas , Humanos , Proteínas de Plantas/aislamiento & purificaciónRESUMEN
The disposition of [(14)C]-labeled n-butyl glycidyl ether (BGE, 3-butoxy-1,2-epoxypropane) was studied in rats and mice. The majority of a single p.o. dose (2-200 mg/kg) was excreted in urine (rats, 84-92%; mice, 64-73%) within 24 h. The rest of the dose was excreted in feces (rats, 2.6-7.7%; mice, 5.3-12%) and in expired air as (14)CO(2) (rats, 1.5%; mice, 10-18%), or remained in the tissues (rats, 2.7-4.4%; mice, 1.5-1.7%). No parent BGE was detected in rat or mouse urine. Fifteen urinary metabolites were identified, including 3-butoxy-2-hydroxy-1-propanol and its monosulfate or monoglucuronide conjugates, 3-butoxy-2-hydroxypropionic acid, O-butyl-N-acetylserine, butoxyacetic acid, 2-butoxyethanol, and 3-butoxy-1-(N-acetylcystein-S-yl)-2-propanol, the mercapturic acid metabolite derived from conjugation of glutathione (GSH) with BGE at the C-1 position. Some of these metabolites underwent further omega-1 oxidation to form a 3'-hydroxybutoxy substitution. One urinary metabolite was from omega-oxidation of 3-butoxy-1-(N-acetylcystein-S-yl)-2-propanol to yield the corresponding carboxylic acid. Oxidative deamination of 3-butoxy-1-(cystein-S-yl)-2-propanol gave the corresponding alpha-keto acid and alpha-hydroxy acid metabolites that were present in mouse urine but not in rat urine. An in vitro incubation of BGE with GSH showed that the conjugation occurred only at the C-1 position with or without the addition of GSH S-transferase.
Asunto(s)
Compuestos Epoxi/metabolismo , Resinas Epoxi/metabolismo , Administración Oral , Animales , Biotransformación , Radioisótopos de Carbono , Desaminación , Relación Dosis-Respuesta a Droga , Compuestos Epoxi/administración & dosificación , Compuestos Epoxi/orina , Resinas Epoxi/administración & dosificación , Heces/química , Femenino , Glucurónidos/orina , Glutatión/análogos & derivados , Glutatión/orina , Masculino , Ratones , Oxidación-Reducción , Ratas , Ratas Endogámicas F344 , Especificidad de la Especie , Sulfatos/orina , Distribución TisularRESUMEN
Cyracure UVR 6105 is a cycloaliphatic epoxy monomer and has both carboxylate and epoxy groups, with the potential for rapid polymerization. It is widely used in industry for the preparation of inks, resins, coatings, and was proposed for incorporation into dental composites. The objective of this study was to determine the mutagenic potential of this chemical related to its metabolite products. Several doses of Cyracure UVR 6105 were dissolved in DMSO and subjected to the Ames Salmonella mutagenicity assay. A metabolic activation system (S9-mix) was used consisting of Arochlor-induced liver S9 homogenate enriched with NADP and glucose-6-phosphate cofactors. In contrast to studies without S9-mix, Cyracure UVR 6105 exhibited enhanced genotoxic activities with strains TA100 and TA1535 in the presence of liver S9-mix. From in vitro metabolism of Cyracure UVR 6105 with S9-mix, as used in the Ames assay, several metabolites were identified. The alcohol metabolite, 3,4-epoxycyclohexylmethanol, containing intact epoxy group was identified in the organic solvent extract. This metabolite was synthesized and proved to be mutagenic against TA100 when assayed in the presence and absence of S9-mix. Results showed that the increased mutagenicity of Cyracure UVR-6105 in the presence of liver enzymes is due to the formation of the mutagenic metabolite 3,4-epoxycyclohexylmethanol.
Asunto(s)
Resinas Epoxi/metabolismo , Resinas Epoxi/toxicidad , Mutágenos/metabolismo , Mutágenos/toxicidad , Animales , Resinas Epoxi/química , Técnicas In Vitro , Extractos Hepáticos/química , Extractos Hepáticos/metabolismo , Espectroscopía de Resonancia Magnética , Metanol/análogos & derivados , Microsomas Hepáticos/química , Microsomas Hepáticos/metabolismo , Estructura Molecular , Pruebas de Mutagenicidad , Ratas , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/metabolismo , PorcinosRESUMEN
Organic acid anhydrides (OAAs) are low molecular weight, reactive compounds extensively used in industry. Exposure to these compounds may lead to allergic symptoms such as rhinitis and asthma. It is important to develop better and more informative methods for assessment of exposure to OAAs. The aim of this study was to develop a method for analysis of specific hexahydrophthalic anhydride (HHPA)-adducted tryptic peptides of human serum albumin (HSA) in nasal lavage (NAL). Furthermore, these peptides were evaluated as biomarkers of exposure. The proteins in the NAL samples were reduced, alkylated and digested with trypsin and the obtained peptides were analyzed using liquid chromatography/tandem mass spectrometry. The total amount of hydrolyzable HHPA in an HHPA-HSA conjugate was used for calibration. A deuterium-labeled HHPA-HSA conjugate was used as internal standard. Five volunteers were exposed to 10, 40 and 80 microg/m3 of HHPA in an exposure chamber and NAL samples were collected before and after exposure. Acceptable precisions of the assay at 13-14% were found for three adducted peptides. The mean levels of these three peptides for the five subjects ranged between 5-22, 15-75 and 33-125 pmol/mL NAL for the exposures at 10, 40 and 80 microg/m3, respectively. High correlations between air levels and the measured peptides were found on an individual basis but there were large inter-individual differences ranging between 63 and 110% for the three peptides. The large differences remained after protein adjustments. It was possible to detect exposures below 10 microg/m3 with the method. Thus, these adducted peptides may be used as biomarkers of exposure, which may better estimate the risk than previous biomarkers developed for OAAs.
Asunto(s)
Contaminantes Ocupacionales del Aire/análisis , Aire/análisis , Monitoreo del Ambiente/métodos , Resinas Epoxi/análisis , Líquido del Lavado Nasal/química , Anhídridos Ftálicos/análisis , Albúmina Sérica/química , Adulto , Contaminantes Ocupacionales del Aire/metabolismo , Contaminantes Ocupacionales del Aire/farmacología , Relación Dosis-Respuesta a Droga , Resinas Epoxi/metabolismo , Resinas Epoxi/farmacología , Humanos , Exposición por Inhalación , Masculino , Persona de Mediana Edad , Fragmentos de Péptidos/química , Anhídridos Ftálicos/metabolismo , Anhídridos Ftálicos/farmacología , Albúmina Sérica/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Tripsina/metabolismoRESUMEN
Hexahydrophthalic anhydride (HHPA), an industrially important chemical, is a highly allergenic compound. The aim of this work was to identify proteins in nasal lavage fluid (NLF) that form adducts with HHPA. Such bindings may induce production of specific immunoglobulin E (IgE) or affect physiological mechanisms of the proteins. NLF was obtained from HHPA-exposed volunteers, workers and exposed guinea pigs. HHPA-binding proteins were visualized with immunoblotting using a polyclonal antiserum against HHPA. The proteins were excised from sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, digested with trypsin and identified by tandem mass spectrometry (MS/MS) and database searches. The antiserum was found to be specific for HHPA-bound proteins. In vivo formed HHPA-binding proteins in humans were identified as antileukoproteinase, immunoglobulin G (IgG), immunoglobulin A (IgA), serum albumin and lactoferrin. In addition, several proteins binding to HHPA were found in NLFs from guinea pigs but these could not be identified from database searches. Hypotheses for development of airways diseases by adduction of this allergenic compound to the NLF proteins in humans were established.
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Resinas Epoxi/metabolismo , Resinas Epoxi/farmacología , Líquido del Lavado Nasal/química , Mucosa Nasal/efectos de los fármacos , Mucosa Nasal/metabolismo , Anhídridos Ftálicos/metabolismo , Anhídridos Ftálicos/farmacología , Proteínas/metabolismo , Animales , Cobayas , Humanos , Masculino , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiologíaRESUMEN
Cell marking is widely used to examine cell development and differentiation in developmental biology. We developed a new method for localizing cell markers in a semi-thin epoxy section with scanning electron microscopy. Cultured fibroblasts ingesting carbon particles were autologously transplanted into a rabbit transparent ear chamber, 6 mm in diameter and 100 microm in depth. Eight days after the transplantation, tissues in the chamber were fixed and embedded in epoxy resin. Semi-thin sections were cut and stained with toluidine blue. Fibroblasts in connective tissues which contained black spots were observed with a light microscope. These sections were subsequently ion-etched with an ion-coater and coated with platinum. The same fibroblasts were then visualized by secondary electron imaging using a scanning electron microscope. A nucleus with nuclear envelope, nuclear pores, a nucleolus and heterochromatin, mitochondria with cristae and rough endoplasmic reticulum were observed in the fibroblasts. The black spots in the fibroblasts were identified as bright bodies with the scanning electron microscope. The bright bodies were found to be a lump of tiny particles less than 100 nm in diameter. In order to analyse such particles with energy dispersive X-ray microanalysis, ion-etched sections were coated with carbon. X-ray energy spectrometry clearly demonstrated that these were carbon particles, which had been endocytosed by the fibroblast. This suggests that scanning electron microscopy combined with energy dispersive X-ray microanalysis is useful for detecting carbon particles in the cytoplasm at an ultrastructural level in semi-thin epoxy sections subsequent to ion etching and that this method may be applicable to other cell markers, such as gold particles to track cells in the field of cell development and cell differentiation.
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Biomarcadores/análisis , Microanálisis por Sonda Electrónica/métodos , Endocitosis , Resinas Epoxi/metabolismo , Fibroblastos/química , Fibroblastos/metabolismo , Microscopía Electrónica de Rastreo/métodos , Adhesión del Tejido/métodos , Animales , Fibroblastos/ultraestructura , ConejosRESUMEN
Hexahydrophthalic anhydride (HHPA; CAS No. 13149-00-3) is a highly allergenic compound commonly used in the chemical industry. Guinea pigs and rats were exposed to [3H2]HHPA by inhalation for 3-8 h and were killed at various intervals during 7 days. The tissue distribution of non-volatile and covalently bound radioactivity was studied by autoradiography. Tissue bound radioactivity was mainly found in the mucosa of the upper respiratory airways, whereas negligible levels were observed in the lungs. In addition, tissue bound radioactivity was present in the gastrointestinal tract and conjunctiva. Moreover, in the cortex of the kidneys in rats, but not in guinea pigs, a low level of tissue bound radioactivity was found. The radioactivity in the tissues persisted for at least 7 days after the end of exposure. Plasma proteins and soluble proteins from trachea, lung, and kidney from [3H2]HHPA-exposed animals were separated by gel filtration. The radioactivity in dialysed plasma was mainly found in the same fractions as albumin. The soluble proteins from trachea, lung, and kidney in both rats and guinea pigs showed a similar pattern as found in blood. The radioactivity in dialysed plasma from both guinea pigs and rats seemed to decay according to a two-compartment model. The non-extractable binding of [3H2]HHPA in the upper respiratory airways and conjunctiva may be of relevance for symptoms in workers with allergy, since they mainly develop symptoms and signs from the nose and eyes.
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Alérgenos/metabolismo , Resinas Epoxi/metabolismo , Anhídridos Ftálicos/metabolismo , Sistema Respiratorio/metabolismo , Administración por Inhalación , Animales , Autorradiografía , Sistema Digestivo/metabolismo , Resinas Epoxi/administración & dosificación , Cobayas , Masculino , Membrana Mucosa/metabolismo , Anhídridos Ftálicos/administración & dosificación , Unión Proteica , Ratas , Ratas Endogámicas BNRESUMEN
Hexahydrophthalic anhydride (HHPA) and methylhexahydrophthalic anhydride (MHHPA) are highly allergenic compounds used in the chemical industry. The aim of this study was to characterize the protein adducts in erythrocytes following exposure to HHPA and MHHPA. Blood and urine samples were obtained from 51 HHPA- and MHHPA-exposed workers. Erythrocytic proteins from HHPA- and MHHPA-exposed workers were fractionated by gel filtration and ion exchange chromatography. In vitro synthesized conjugates between tritium-labeled and unlabeled HHPA and hemoglobin (Hb) were hydrolyzed by acid or digested by Pronase E. Levels of in vivo formed anhydride-Hb adducts and urinary/plasma levels of the corresponding acids were analyzed by gas chromatography-mass spectrometry (GC-MS) and correlated. The decay of adducts was studied in workers leaving employment or during vacation. More than 85% of the adduct forming protein in vivo coeluted with Hb in gel filtration and ion exchange chromatography. At least 70% of the HHPA in the in vitro formed adducts was found on lysine by GC-MS. Similar findings were obtained using Pronase E-digested tritium-labeled Hb-HHPA. The adduct levels in workers ranged 0-26 pmol/g Hb (mean 2. 7 pmol/g Hb) for HHPA, and the range for MHHPA was 0-55 pmol/g Hb (mean 4.1 pmol/g Hb). The Spearman's rank correlation coefficient between urine data and adducts was for HHPA rs = 0.80 and for MHHPA, rs = 0.78. For the plasma, the correlation using HHPA data was rs = 0.80 and for MHHPA, rs = 0.69. The adducts seemed to be stable in vivo. The adduct levels may be used as biomarkers of exposure to HHPA and MHHPA.
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Proteínas Sanguíneas/metabolismo , Resinas Epoxi/farmacología , Eritrocitos/metabolismo , Hemoglobinas/metabolismo , Lisina/metabolismo , Anhídridos Ftálicos/metabolismo , Anhídridos Ftálicos/farmacología , Alérgenos , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Resinas Epoxi/metabolismo , Eritrocitos/efectos de los fármacos , Humanos , Hidrólisis , Lisina/análisis , Exposición Profesional , Anhídridos Ftálicos/sangre , Anhídridos Ftálicos/orina , Factores de TiempoRESUMEN
A method for the simultaneous determination of hexahydrophthalic acid (HHP acid) and methylhexahydrophthalic acid (MHHP acid) in human plasma was developed. The procedure was a rapid, single step extractive derivatisation with pentafluorobenzyl bromide as the derivatisation agent. The formed pentafluorobenzyl esters were analysed by gas chromatography-mass spectrometry in negative ion chemical ionisation mode with ammonia as the moderating gas. Deuterium-labeled HHP acid and MHHP acid were used as internal standards. The detection limit was 0.4 ng/ml for HHP acid (m/z 153) and 0.3 ng/ml for MHHP acid (m/z 365). The within-day precision of the method was between 2 and 3% and the between-day precision was between 3 and 12%. The overall recovery was between 65 and 83%. A comparison between HHP acid determinations with a previous and this method showed that the methods gave similar results. The method was applicable for analysis of plasma from occupationally exposed workers.
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Ácidos Ciclohexanocarboxílicos/sangre , Fluorobencenos , Ácidos Ftálicos/sangre , Resinas Epoxi/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Humanos , Anhídridos Ftálicos/metabolismo , Compuestos de Amonio CuaternarioRESUMEN
Two cytochemical methods (malachite green fixation and PLA2-gold complex technique) were combined in order to detect the presence and ultrastructural distribution of phospholipids in epiphyseal cartilage and metaphyseal bone of 25-day-old rats. Chondrocytes and osteoblasts showed a more intense PLA2-gold complex labeling of rough endoplasmic reticulum than mitochondria and plasma membranes. Roundish osmiophilic, electron-dense, lightly labeled structures were visible at the periphery of the cells. In areas of early mineralization of cartilage, not all the matrix vesicles were labeled by PLA2-gold complex. Calcification nodules showed intense labeling in comparison with the surrounding, lightly labeled uncalcified matrix; gold particles were chiefly found at their periphery. Calcification nodules of metaphyseal bone were deeply electron-dense after glutaraldehyde-malachite green fixation; PLA2-gold labeling was mainly found in connection with crystals, whereas the uncalcified matrix was weakly labeled. Calcified bone matrix showed a heavy labeling with randomly scattered gold particles. When pre-embedding decalcification was carried out in the presence of malachite green, a good preservation of organic component was obtained. The PLA2-gold positivity of decalcified areas of cartilage and bone confirmed the presence of phospholipids in mineralized matrix.