RESUMEN
Nanoparticles are extensively used in industrial products or as food additives. However, despite their contribution to improving our quality of life, concerns have been raised regarding their potential impact on occupational and public health. To speed up research assessing nanoparticle-related hazards, this study was undertaken to identify early markers of harmful effects on the lungs. Female Sprague Dawley rats were either exposed to crystalline silica DQ-12 with instillation, or to titanium dioxide P25, carbon black Printex-90, or multi-walled carbon nanotube Mitsui-7 with nose-only inhalation. Tissues were collected at three post-exposure time points to assess short- and long-term effects. All particles induced lung inflammation. Histopathological and biochemical analyses revealed phospholipid accumulation, lipoproteinosis, and interstitial thickening with collagen deposition after exposure to DQ-12. Exposure to the highest dose of Printex-90 and Mitsui-7, but not P25, induced some phospholipid accumulation. Comparable histopathological changes were observed following exposure to P25, Printex-90, and Mitsui-7. Comparison of overall gene expression profiles identified 15 potential early markers of adverse lung outcomes induced by spherical particles. With Mitsui-7, a distinct gene expression signature was observed, suggesting that carbon nanotubes trigger different toxicity mechanisms to spherical particles.
Asunto(s)
Nanotubos de Carbono , Ratas , Femenino , Animales , Nanotubos de Carbono/toxicidad , Calidad de Vida , Ratas Sprague-Dawley , Pulmón/patología , Dióxido de Silicio/farmacología , Exposición por Inhalación/efectos adversos , Líquido del Lavado Bronquioalveolar/químicaRESUMEN
Multi-walled carbon nanotubes (MWCNTs), which vary in length, diameter, functionalization and specific surface area, are used in diverse industrial processes. Since these nanomaterials have a high aspect ratio and are biopersistant in the lung, there is a need for a rapid identification of their potential health hazard. We assessed in Sprague-Dawley rats the pulmonary toxicity of two pristine MWCNTs (the "long and thick" NM-401 and the "short and thin" NM-403) following either intratracheal instillation or 4-week inhalation in order to gain insights into the predictability and intercomparability of the two methods. The deposited doses following inhalation were lower than the instilled doses. Both types of carbon nanotube induced pulmonary neutrophil influx using both exposure methods. This influx correlated with deposited surface area across MWCNT types and means of exposure at two different time points, 1-3â¯days and 28-30â¯days post-exposure. Increased levels of DNA damage were observed across doses and time points for both exposure methods, but no dose-response relationship was observed. Intratracheal instillation of NM-401 induced fibrosis at the highest dose while lower lung deposited doses obtained by inhalation did not induce such lung pathology. No fibrosis was observed following NM-403 exposure. When the deposited dose was taken into account, sub-acute inhalation and a single instillation of NM-401 and NM-403 produced very similar inflammation and DNA damage responses. Our data suggest that the dose-dependent inflammatory responses observed after intratracheal instillation and inhalation of MWCNTs are similar and were predicted by the deposited surface area.
Asunto(s)
Enfermedades Pulmonares/inducido químicamente , Nanotubos de Carbono/toxicidad , Animales , Líquido del Lavado Bronquioalveolar/citología , Ensayo Cometa , Daño del ADN/efectos de los fármacos , Vías de Administración de Medicamentos , Exposición por Inhalación , Ratas , Ratas Sprague-DawleyRESUMEN
The number of workers potentially exposed to nanoparticles (NPs) during industrial processes is increasing, although the toxicological properties of these compounds still need to be fully characterized. As NPs may be aerosolized during industrial processes, inhalation represents their main route of occupational exposure. Here, the short- and long-term pulmonary toxicological properties of titanium dioxide were studied, using conventional and molecular toxicological approaches. Fischer 344 rats were exposed to 10â¯mg/m3 of a TiO2 nanostructured aerosol (NSA) by nose-only inhalation for 6â¯h/day, 5â¯days/week for 4â¯weeks. Lung samples were collected up to 180 post-exposure days. Biochemical and cytological analyses of bronchoalveolar lavage (BAL) showed a strong inflammatory response up to 3 post-exposure days, which decreased overtime. In addition, gene expression profiling revealed overexpression of genes involved in inflammation that was maintained 6â¯months after the end of exposure (long-term response). Genes involved in oxidative stress and vascular changes were also up-regulated. Long-term response was characterized by persistent altered expression of a number of genes up to 180 post-exposure days, despite the absence of significant histopathological changes. The physiopathological consequences of these changes are not fully understood, but they should raise concerns about the long-term pulmonary effects of inhaled biopersistent NPs such as TiO2.
Asunto(s)
Perfilación de la Expresión Génica , Pulmón/patología , Nanoestructuras/toxicidad , Titanio/toxicidad , Aerosoles , Animales , Vasos Sanguíneos/efectos de los fármacos , Líquido del Lavado Bronquioalveolar , Regulación de la Expresión Génica/efectos de los fármacos , Exposición por Inhalación/efectos adversos , Ganglios Linfáticos/patología , Masculino , Análisis por Micromatrices , Estrés Oxidativo/genética , Ratas , Ratas Endogámicas F344 , Titanio/administración & dosificaciónRESUMEN
1. Multiple exposures are ubiquitous in industrial environments. In this article, we highlight the risks faced by workers and complete the data available on the metabolic impact of a common mixture: toluene (TOL) and methylethylketone (MEK). 2. Rats were exposed by inhalation under controlled conditions either to each solvent individually, or to mixtures of the two. How the interaction between the two solvents affected their fate in the blood and brain, their main relevant urinary metabolites (o-cresol, benzylmercapturic acid for TOL and 2,3-butanediols for MEK) and their hepatic metabolism were investigated. 3. Although the cytochrome P450 concentration was unchanged, and the activities of CYP1A2 and CYP2E1 isoforms were not additively or synergistically induced by co-exposure, TOL metabolism was inhibited by the presence of MEK (and vice versa). Depending on the relative proportions of each compound in the mixture, this sometimes resulted in a large increase in blood and brain concentrations. Apart from extreme cases (unbalanced mixtures), the amount of o-cresol and benzylmercapturic acid (and to a lesser extent 2,3-butanediols) excreted were proportional to the blood solvent concentrations. 4. In a co-exposure context, ortho-cresol and benzylmercapturic acid can be used as urinary biomarkers in biomonitoring for employees to relatively accurately assess TOL exposure.
Asunto(s)
Butanonas/metabolismo , Butanonas/toxicidad , Exposición por Inhalación , Tolueno/metabolismo , Tolueno/toxicidad , Animales , Bioensayo , Peso Corporal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Butanonas/sangre , Butanonas/orina , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Tamaño de los Órganos/efectos de los fármacos , Ratas Endogámicas BN , Tolueno/sangre , Tolueno/orinaRESUMEN
Methylethylketone (MEK) is widely used in industry, often in combination with other compounds. Although nontoxic, it can make other chemicals harmful. This study investigates the fate of MEK in rat blood, brain and urine as well as its hepatic metabolism following inhalation over 1 month (at 20, 200 or 1400 ppm). MEK did not significantly accumulate in the organism: blood concentrations were similar after six-hour or 1-month inhalation periods, and brain concentrations only increased slightly after 1 month's exposure. Urinary excretion, based on the major metabolites, 2,3-butanediols (± and meso forms), accounted for less than 2.4% of the amount inhaled. 2-Butanol, 3-hydroxy-2-butanone and MEK itself were only detectable in urine in the highest concentration conditions investigated, when metabolic saturation occurred. Although MEK exposure did not alter the total cytochrome P450 concentration, it induced activation of both CYP1A2 and CYP2E1 enzymes. In addition, the liver glutathione concentration (reduced and oxidized forms) decreased, as did glutathione S-transferase (GST) activity (at exposure levels over 200 ppm). These metabolic data could be useful for pharmacokinetic model development and/or verification and suggest the ability of MEK to influence the metabolism (and potentiate the toxicity) of other substances.
Asunto(s)
Butanonas/farmacocinética , Acetoína/orina , Administración por Inhalación , Animales , Biotransformación , Encéfalo/metabolismo , Butanoles/orina , Butanonas/administración & dosificación , Butanonas/sangre , Butanonas/orina , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Activación Enzimática , Glutatión/metabolismo , Glutatión Transferasa/metabolismo , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Ratas Endogámicas BN , Eliminación Renal , Distribución TisularRESUMEN
Calcium pyrophosphate dehydrate (CPPD) crystals are found in the synovial fluid of patients with articular chondrocalcinosis or sometimes with osteoarthritis. In inflammatory conditions, the synovial membrane (SM) is subjected to transient hypoxia, especially during movement. CPPD formation is supported by an increase in extracellular inorganic pyrophosphate (ePPi) levels, which are mainly controlled by the transporter Ank and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). We demonstrated previously that transforming growth factor (TGF)-ß1 increased ePPi production by inducing Ank and Enpp1 expression in chondrocytes. As the TGF-ß1 level raises in synovial fluid under hypoxic conditions, we investigated whether hypoxia may transform SM as a major source of ePPi production. Synovial fibroblasts and SM explants were exposed to 10 ng/mL of TGF-ß1 in normoxic or hypoxic (5% O2) culture conditions. Ank and Enpp1 expression were assessed by quantitative PCR, Western blot and immunohistochemistry. ePPi was quantified in culture supernatants. RNA silencing was used to define the respective roles of Ank and Enpp1 in TGF-ß1-induced ePPi generation. The molecular mechanisms involved in hypoxia were investigated using an Ank promoter reporter plasmid for transactivation studies, as well as gene overexpression and RNA silencing, the respective role of hypoxia-induced factor (HIF)-1 and HIF-2. Our results showed that TGF-ß1 increased Ank, Enpp1, and therefore ePPi production in synovial fibroblasts and SM explants. Ank was the major contributor in ePPi production compared to ENPP1. Hypoxia increased ePPi levels on its own and enhanced the stimulating effect of TGF-ß1. Hypoxic conditions enhanced Ank promoter transactivation in an HIF-1-dependent/HIF-2-independent fashion. We demonstrated that under hypoxia, SM is an important contributor to ePPi production in the joint through the induction of Enpp1 and Ank. These findings are of interest as a rationale for the beneficial effect of anti-inflammatory drugs on SM in crystal depositions.
RESUMEN
Carbon nanotubes (CNTs) are nanomaterials presenting an occupational inhalation risk during production or handling. The International Agency for Research on Cancer classified one CNT, Mitsui-7 (MWNT-7), as 'possibly carcinogenic to humans'. In recognition of their similarities, a proposal has been submitted to the risk assessment committee of ECHA to classify all fibers with 'Fibre Paradigm' (FP)-compatible dimensions as carcinogenic. However, there is a lack of clarity surrounding the toxicity of fibers that do not fit the FP criteria. In this study, we compared the effects of the FP-compatible Mitsui-7, to those of NM-403, a CNT that is too short and thin to fit the paradigm. Female Sprague Dawley rats deficient for p53 (GMO) and wild type (WT) rats were exposed to the two CNTs (0.25 mg/rat/week) by intratracheal instillation. Animals (GMO and WT) were exposed weekly for four consecutive weeks and were sacrificed 3 days or 8 months after the last instillation. Exposure to both CNTs induced acute lung inflammation. However, persistent inflammation at 8 months was only observed in the lungs of rats exposed to NM-403. In addition to the persistent inflammation, NM-403 stimulated hyperplasic changes in rat lungs, and no adenomas or carcinomas were detected. The degree and extent of hyperplasia was significantly more pronounced in GMO rats. These results suggest that CNT not meeting the FP criteria can cause persistent inflammation and hyperplasia. Consequently, their health effects should be carefully assessed.
Asunto(s)
Nanotubos de Carbono , Animales , Femenino , Ratas , Hiperplasia/patología , Inflamación , Exposición por Inhalación , Pulmón , Nanotubos de Carbono/toxicidad , Ratas Sprague-Dawley , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Transforming growth factor (TGF)-ß1 stimulates extracellular PP(i) (ePP(i)) generation and promotes chondrocalcinosis, which also occurs secondary to hyperparathyroidism-induced hypercalcemia. We previously demonstrated that ANK was up-regulated by TGF-ß1 activation of ERK1/2 and Ca(2+)-dependent protein kinase C (PKCα). Thus, we investigated mechanisms by which calcium could affect ePP(i) metabolism, especially its main regulating proteins ANK and PC-1 (plasma cell membrane glycoprotein-1). We stimulated articular chondrocytes with TGF-ß1 under extracellular (eCa(2+)) or cytosolic Ca(2+) (cCa(2+)) modulations. We studied ANK, PC-1 expression (quantitative RT-PCR, Western blotting), ePP(i) levels (radiometric assay), and cCa(2+) input (fluorescent probe). Voltage-operated Ca(2+)-channels (VOC) and signaling pathways involved were investigated with selective inhibitors. Finally, Ank promoter activity was evaluated (gene reporter). TGF-ß1 elevated cCa(2+) and ePP(i) levels (by up-regulating Ank and PC-1 mRNA/proteins) in an eCa(2+) dose-dependent manner. TGF-ß1 effects were suppressed by cCa(2+) chelation or L- and T-VOC blockade while being mostly reproduced by ionomycin. In the same experimental conditions, the activation of Ras, the phosphorylation of ERK1/2 and PKCα, and the stimulation of Ank promoter activity were affected similarly. Activation of SP1 (specific protein 1) and ELK-1 (Ets-like protein-1) transcription factors supported the regulatory role of Ca(2+). SP1 or ELK-1 overexpression or blockade experiments demonstrated a major contribution of ELK-1, which acted synergistically with SP1 to activate Ank promoter in response to TGF-ß1. TGF-ß1 promotes input of eCa(2+) through opening of L- and T-VOCs, to potentiate ERK1/2 and PKCα signaling cascades, resulting in an enhanced activation of Ank promoter and ePP(i) production in chondrocyte.
Asunto(s)
Calcio/metabolismo , Cartílago Articular/metabolismo , Condrocitos/metabolismo , Difosfatos/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Canales de Calcio/genética , Canales de Calcio/metabolismo , Cartílago Articular/patología , Células Cultivadas , Condrocalcinosis/genética , Condrocalcinosis/metabolismo , Condrocalcinosis/patología , Condrocitos/patología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Transporte Iónico/efectos de los fármacos , Transporte Iónico/genética , Ionomicina/farmacología , Ionóforos/farmacología , Masculino , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas de Transporte de Fosfato/genética , Proteínas de Transporte de Fosfato/metabolismo , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Regiones Promotoras Genéticas/genética , Proteína Quinasa C-alfa/genética , Proteína Quinasa C-alfa/metabolismo , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , Ratas , Ratas Wistar , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Proteína Elk-1 con Dominio ets/genética , Proteína Elk-1 con Dominio ets/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
The differentiated phenotype of chondrocyte is lost in pathological situations and after interleukin (IL)-1beta challenge. Wnt proteins and the inorganic pyrophosphate (PP(i)) transporter Ank regulate the differentiation process in many cell types. We investigated the possible contribution of Ank and/or PP(i) to the maintenance of the differentiated chondrocyte phenotype with special care to Wnt signaling. Primary articular chondrocytes lost their phenotype upon IL-1beta challenge, with cessation of type II collagen and Sox-9 expression. Ank expression and PP(i) transport were strongly reduced by IL-1beta, whereas Wnt-5a was the only Wnt protein increased. Transient overexpression of Ank counteracted most of IL-1beta effects on Type II collagen, Sox-9, and Wnt-5a expression. When resting chondrocytes were transfected with a siRNA against Ank, this reproduced the phenotype induced by IL-1beta. In both cases, no markers for hypertrophic chondrocytes were detected. The conditioned supernatant from chondrocytes knocked-down for Ank contained Wnt-5a, which activated Tcf/Lef reporter plasmids and promoted translocation of beta-catenin into the nucleus without activating the c-Jun N-terminal kinase (JNK) pathway. Supplementation with PP(i) compensated for most effects of Ank deficiency on Type II collagen, Sox-9, and Wnt-5 expression, both in IL-1beta and Ank knock-down conditions. Phenotype changes induced by IL-1beta were also supported by activation of the JNK pathway, but this latter was not sensitive to PP(i) supplementation. Altogether our data demonstrate that the transport of PP(i) by ANK contributed to the maintenance of the differentiated phenotype of chondrocyte by controlling the canonical Wnt pathway in a Wnt-5a-dependent manner.
Asunto(s)
Cartílago Articular/metabolismo , Condrocitos/metabolismo , Difosfatos/metabolismo , Proteínas de la Membrana/metabolismo , Fenotipo , Transducción de Señal , Animales , Cartílago Articular/citología , Diferenciación Celular , Células Cultivadas , Condrocitos/citología , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Medios de Cultivo Condicionados/farmacología , Interleucina-1beta/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de Transporte de Fosfato , ARN Interferente Pequeño/farmacología , Ratas , Ratas Wistar , Factor de Transcripción SOX9/genética , Factor de Transcripción SOX9/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Although aging is associated with a higher risk of developing respiratory pathologies, very few studies have assessed the impact of age on the adverse effects of inhaled nanoparticles. Using conventional and transcriptomic approaches, this study aimed to compare in young (12-13-week-old) and elderly (19-month-old) fisher F344 rats the pulmonary toxicity of an inhaled nanostructured aerosol of titanium dioxide (TiO2). Animals were nose-only exposed to this aerosol at a concentration of 10 mg/m3 for 6 h per day, 5 days per week for 4 weeks. Tissues were collected immediately (D0), and 28 days after exposure (D28). A pulmonary influx of neutrophilic granulocytes was observed in exposed rats at D0, but diminished with time while remaining significant until D28. Similarly, an increased expression of several genes involved in inflammation at the two post-exposure time-points was seen. Apart from an age-specific pulmonary influx of lymphocyte, only slight differences in physio-pathological responses following TiO2 exposure between young and elderly animals were noticed. Conversely, marked age-related differences in gene expression profiles were observed making possible to establish lists of genes specific to each age group and post-exposure times. These results highlight different signaling pathways that were disrupted in rats according to their age.
RESUMEN
Hexavalent chromium (Cr(VI)) compounds are classified as carcinogenic to humans. Whereas chromium measurements in urine and plasma attest to the last few hours of total chromium exposure (all oxidation states of chromium), chromium in red blood cells (RBC) is attributable specifically to Cr(VI) exposure over the last few days. Before recommending Cr in RBC (CrIE) as a biological indicator of Cr(VI) exposure, in vivo studies must be undertaken to assess its reliability. The present study examines the kinetics of Cr(VI) in rat after a single intravenous dose of ammonium dichromate. Chromium levels were measured in plasma, red blood cells and urine. The decay of the chromium concentration in plasma is one-phase-like (with half-life time of 0.55 day) but still measurable two days post injection. The excretion of urinary chromium peaks between five and six hours after injection and shows large variations. Intra-erythrocyte chromium (CrIE) was very constant up to a minimum of 2 days and half-life time was estimated to 13.3 days. Finally, Cr(III) does not interfere with Cr(VI) incorporation in RBC. On the basis of our results, we conclude that, unlike urinary chromium, chromium levels in RBC are indicative of the amount of dichromate (Cr(VI)) in blood.
Asunto(s)
Carcinógenos Ambientales/administración & dosificación , Carcinógenos Ambientales/metabolismo , Cromo/administración & dosificación , Cromo/sangre , Eritrocitos/metabolismo , Administración Intravenosa , Animales , Biomarcadores/sangre , Biomarcadores/orina , Carga Corporal (Radioterapia) , Carcinógenos Ambientales/farmacocinética , Carcinógenos Ambientales/toxicidad , Cromo/farmacocinética , Cromo/toxicidad , Masculino , Modelos Biológicos , Oxidación-Reducción , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Especificidad de la Especie , ToxicocinéticaRESUMEN
In response to inflammatory cytokines, chondrocytes and synovial fibroblasts produce high amounts of prostaglandins (PG) which self-perpetuate locally the inflammatory reaction. Prostaglandins act primarily through membrane receptors coupled to G proteins but also bind to nuclear Peroxisome Proliferator-Activated Receptors (PPARs). Amongst fatty acids, the cyclopentenone metabolite of PGD2, 15-deoxy-Delta12,14PGJ2 (15d-PGJ2), was shown to be a potent ligand of the PPARgamma isotype prone to inhibit the production of inflammatory mediators. As the stimulated synthesis of PGE2 originates from the preferential coupling of inducible enzymes, cyclooxygenase-2 (COX-2) and membrane PGE synthase-1 (mPGES-1), we investigated the potency of 15d-PGJ2 to regulate prostaglandins synthesis in rat chondrocytes stimulated with interleukin-1beta (IL-1beta). We demonstrated that 15d-PGJ2, but not the high-affinity PPARgamma ligand rosiglitazone, decreased almost completely PGE2 synthesis and mPGES-1 expression. The inhibitory potency of 15d-PGJ2 was unaffected by changes in PPARgamma expression and resulted from inhibition of NF-kappaB nuclear binding and IkappaBalpha sparing, secondary to reduced phosphorylation of IKKbeta. Consistently with 15d-PGJ2 being a putative endogenous regulator of the inflammatory reaction if synthesized in sufficient amounts, the present data confirm the variable PPARgamma-dependency of its effects in joint cells while underlining possible species and cell types specificities.
Asunto(s)
Cartílago Articular/citología , Condrocitos/metabolismo , PPAR gamma/agonistas , Prostaglandinas/biosíntesis , Animales , Cartílago Articular/efectos de los fármacos , Cartílago Articular/metabolismo , Células Cultivadas , Condrocitos/efectos de los fármacos , Dinoprostona/biosíntesis , Interleucina-1/farmacología , Masculino , FN-kappa B/fisiología , PPAR gamma/fisiología , Prostaglandina D2/análogos & derivados , Prostaglandina D2/farmacología , Ratas , Ratas WistarRESUMEN
Synthetic amorphous silica nanomaterials (SAS) are extensively used in food and tire industries. In many industrial processes, SAS may become aerosolized and lead to occupational exposure of workers through inhalation in particular. However, little is known about the in vivo genotoxicity of these particulate materials. To gain insight into the toxicological properties of four SAS (NM-200, NM-201, NM-202, and NM-203), rats are treated with three consecutive intratracheal instillations of 3, 6, or 12 mg/kg of SAS at 48, 24, and 3 hrs prior to tissue collection (cumulative doses of 9, 18, and 36 mg/kg). Deoxyribonucleic acid (DNA) damage was assessed using erythrocyte micronucleus test and the standard and Fpg-modified comet assays on cells from bronchoalveolar lavage fluid (BALF), lung, blood, spleen, liver, bone marrow, and kidney. Although all of the SAS caused increased dose-dependent changes in lung inflammation as demonstrated by BALF neutrophilia, they did not induce any significant DNA damage. As the amount of SAS reaching the blood stream and subsequently the internal organs is probably to be low following intratracheal instillation, an additional experiment was performed with NM-203. Rats received three consecutive intravenous injections of 5, 10, or 20 mg/kg of SAS at 48, 24, and 3 hrs prior to tissue collection. Despite the hepatotoxicity, thrombocytopenia, and even animal death induced by this nanomaterial, no significant increase in DNA damage or micronucleus frequency was observed in SAS-exposed animals. It was concluded that under experimental conditions, SAS induced obvious toxic effects but did cause any genotoxicity following intratracheal instillation and intravenous injection.
Asunto(s)
Daño del ADN/efectos de los fármacos , Nanopartículas/efectos adversos , Estrés Oxidativo/efectos de los fármacos , Dióxido de Silicio/efectos adversos , Animales , Humanos , Inyecciones Intravenosas , Peroxidación de Lípido/efectos de los fármacos , Malondialdehído/sangre , Pruebas de Micronúcleos , Mutágenos/efectos adversos , Ratas , Dióxido de Silicio/síntesis química , Distribución Tisular/efectos de los fármacosRESUMEN
The cytogenetic alterations in leukocytes and the increased risk for leukemia, lymphoma, or all lymphohematopoietic cancer observed in workers occupationally exposed to styrene have been associated with its hepatic metabolisation into styrene-7,8-oxide, an epoxide which can induce DNA damages. However, it has been observed that styrene-7,8-oxide was also found in the atmosphere of reinforced plastic industries where large amounts of styrene are used. Since the main route of exposure to these compounds is inhalation, in order to gain new insights regarding their systemic genotoxicity, Fisher 344 male rats were exposed in full-body inhalation chambers, 6 h/day, 5 days/week for 4 weeks to styrene-7,8-oxide (25, 50, and 75 ppm) or styrene (75, 300, and 1000 ppm). Then, the induction of micronuclei in circulating reticulocytes and DNA strand breaks in leukocytes using the comet assay was studied at the end of the 3rd and 20th days of exposure. Our results showed that neither styrene nor styrene-7,8-oxide induced a significant increase of the micronucleus frequency in reticulocytes or DNA strand breaks in white blood cells. However, in the presence of the formamidopyridine DNA glycosylase, an enzyme able to recognize and excise DNA at the level of some oxidized DNA bases, a significant increase of DNA damages was observed at the end of the 3rd day of treatment in leukocytes from rats exposed to styrene but not to styrene-7,8-oxide. This experimental design helped to gather new information regarding the systemic genotoxicity of these two chemicals and may be valuable for the risk assessment associated with an occupational exposure to these molecules.
Asunto(s)
Compuestos Epoxi/toxicidad , Estireno/toxicidad , Administración por Inhalación , Animales , Cámaras de Exposición Atmosférica , Recuento de Células Sanguíneas , Ensayo Cometa , Roturas del ADN/efectos de los fármacos , ADN Glicosilasas/metabolismo , Compuestos Epoxi/sangre , Eritrocitos/efectos de los fármacos , Eritrocitos/ultraestructura , Masculino , Pruebas de Micronúcleos , Mutágenos/toxicidad , Ratas , Ratas Endogámicas F344 , Reticulocitos/efectos de los fármacos , Reticulocitos/ultraestructura , Estireno/sangreRESUMEN
The balance between extracellular inorganic phosphate (ePi) and extracellular inorganic pyrophosphate (ePPi) is controlled by four membrane proteins: the transporters ANK (exporting PPi outside the cells) and PiT-1 (importing ePi into the cells), and the enzymes PC-1 (generating ePPi from nucleotides) and Tissue Non-specific Alkaline Phosphatase (TNAP, hydrolyzing ePPi into ePi). TGF-ß1 was shown to stimulate ANK and PC-1 expression in articular chondrocytes, and subsequent ePPi level, as well as to increase ePi uptake by inducing PiT-1 expression in a chondrogenic cell line. Thus, we investigated the ability of ePi to modulate the effect of TGF-ß1 on the regulatory proteins of the ePi/ePPi balance in chondrocytes. In the pathophysiological range of 0.01-1 mM, ePi was inactive by itself but potentiated the stimulatory effects of TGF-ß1 on ANK, PC-1 or PiT-1 mRNA (RT-qPCR) and protein (Western blot) levels. PC-1 activity was also increased by TGF-ß1 and further potentiated by ePi supplementation. TNAP mRNA and activity became undetectable in response to TGF-ß1. These data suggest that ePi could increase ePPi level by changing the control of ANK and PC-1 expression by TGF-ß1, further highlighting an adaptative regulation of the Pi/PPi balance to prevent basic calcium phosphate deposition into the joints.
Asunto(s)
Condrocitos/metabolismo , Difosfatos/metabolismo , Regulación de la Expresión Génica/fisiología , Fosfatos/metabolismo , Factor de Crecimiento Transformador beta1/farmacología , Animales , Células Cultivadas , Condrocitos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratas , Ratas WistarRESUMEN
BACKGROUND: Rosiglitazone and pioglitazone are high-affinity peroxisome proliferator-activated receptor (PPAR)-gamma agonists with potent anti-diabetic properties and potential anti-inflammatory effects. We compared the ability of a range of oral doses of these thiazolidinediones, including those sufficient to restore insulin sensitization, to inhibit the pathogenesis of adjuvant-induced arthritis (AIA). METHODS: AIA was induced in Lewis rats by a subcutaneous injection of 1 mg of complete Freund's adjuvant. Rats were treated orally for 21 days with pioglitazone 3, 10 or 30 mg/kg/day, rosiglitazone 3 or 10 mg/kg/day, or with vehicle only. The time course of AIA was evaluated by biotelemetry to monitor body temperature and locomotor activity, by clinical score and plethysmographic measurement of hindpaw oedema. At necropsy, RT-PCR analysis was performed on synovium, liver and subcutaneous fat. Changes in cartilage were evaluated by histological examination of ankle joints, radiolabelled sulphate incorporation (proteoglycan synthesis), glycosaminoglycan content (proteoglycan turnover) and aggrecan expression in patellar cartilage. Whole-body bone mineral content was measured by dual-energy X-ray absorptiometry. RESULTS: The highest doses of rosiglitazone (10 mg/kg/day) or pioglitazone (30 mg/kg/day) were required to reduce fever peaks associated with acute or chronic inflammation, respectively, and to decrease arthritis severity. At these doses, thiazolidinediones reduced synovitis and synovial expression of TNF-alpha, IL-1beta and basic fibroblast growth factor without affecting neovascularization or the expression of vascular endothelial growth factor. Thiazolidinediones failed to prevent cartilage lesions and arthritis-induced inhibition of proteoglycan synthesis, aggrecan mRNA level or glycosaminoglycan content in patellar cartilage, but reduced bone erosions and inflammatory bone loss. A trend towards lower urinary levels of deoxipyridinolin was also noted in arthritic rats treated with thiazolidinediones. Rosiglitazone 10 mg/kg/day or pioglitazone 30 mg/kg/day increased the expression of PPAR-gamma and adiponectin in adipose tissue, confirming that they were activating PPAR-gamma in inflammatory conditions, although an increase in fat mass percentage was observed for the most anti-arthritic dose. CONCLUSION: These data emphasize that higher dosages of thiazolidinediones are required for the treatment of arthritis than for restoring insulin sensitivity but that thiazolidinediones prevent inflammatory bone loss despite exposing animals to increased fatness possibly resulting from excessive activation of PPAR-gamma.
Asunto(s)
Antiinflamatorios/farmacología , Artritis Experimental/complicaciones , Resorción Ósea/etiología , Resorción Ósea/prevención & control , Hipoglucemiantes/farmacología , Tiazolidinedionas/farmacología , Adiponectina/genética , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Animales , Antiinflamatorios/administración & dosificación , Artritis Experimental/epidemiología , Artritis Experimental/patología , Artritis Experimental/fisiopatología , Biomarcadores/metabolismo , Densidad Ósea/efectos de los fármacos , Remodelación Ósea/efectos de los fármacos , Huesos/efectos de los fármacos , Huesos/metabolismo , Cartílago Articular/efectos de los fármacos , Cartílago Articular/patología , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Fiebre/fisiopatología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Miembro Posterior , Hipoglucemiantes/administración & dosificación , Incidencia , Masculino , PPAR gamma/genética , Pioglitazona , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas Lew , Rosiglitazona , Membrana Sinovial/metabolismo , Sinovitis/patología , Tiazolidinedionas/administración & dosificación , Aumento de Peso/efectos de los fármacosRESUMEN
Peroxisome proliferators activated receptors (PPAR) are ligand-inducible nuclear transacting factors comprising three subtypes, PPARalpha, PPARbeta/delta and PPARgamma, which play a key role in lipids and glucose homeostasis. All PPAR subtypes have been identified in joint or inflammatory cells and their activation resulted in a transcriptional repression of pro-inflammatory cytokines (IL-1, TNFalpha), early inflammatory genes (NOS(2), COX-2, mPGES-1) or matrix metalloproteases (MMP-1, MMP-13), at least for the gamma subtype. PPAR full agonists were also shown to stimulate IL-1 receptor antagonist (IL-1Ra) production by cytokine-stimulated articular cells in a subtype-dependent manner. These anti-inflammatory and anti-catabolic properties were confirmed in animal models of joint diseases where PPAR agonists reduced synovial inflammation while preventing cartilage destruction or inflammatory bone loss, although many effects required much higher doses than needed to restore insulin sensitivity or to lower circulating lipid levels. However, these promising effects of PPAR full agonists were hampered by their ability to reduce the growth factor-dependent synthesis of extracellular matrix components or to induce chondrocyte apoptosis, by the possible contribution of immunosuppressive properties to their anti-arthritic effects, by the increased adipocyte differentiation secondary to prolonged stimulation of PPARgamma, and by a variable contribution of PPAR subtypes depending on the system. Clinical data are scarce in rheumatoid arthritis (RA) patients whereas thousands of patients worldwilde, treated with PPAR agonists for type 2 diabetes or dyslipidemia, are paradoxically prone to suffer from osteoarthritis (OA). Whereas high dosage of full agonists may expose RA patients to cardiovascular adverse effects, the proof of concept that PPAR agonists have therapeutical relevance to OA may benefit from an epidemiological follow-up of joint lesions in diabetic or hyperlipidemic patients treated for long periods of time with glitazones or fibrates. Additionally, cellular and animal studies are required to assess whether partial agonists of PPAR (SPPARMs) may preserve therapeutical properties with potentially less safety concern.
Asunto(s)
Artropatías/fisiopatología , Receptores Activados del Proliferador del Peroxisoma/agonistas , Receptores Activados del Proliferador del Peroxisoma/fisiología , Animales , Artritis Reumatoide/tratamiento farmacológico , Cromanos/farmacología , Cromanos/uso terapéutico , Dimerización , Matriz Extracelular/fisiología , Humanos , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Osteoartritis/tratamiento farmacológico , PPAR alfa/fisiología , PPAR-beta/efectos de los fármacos , PPAR-beta/fisiología , Receptores Activados del Proliferador del Peroxisoma/genética , Rosiglitazona , Tiazolidinedionas/farmacología , Tiazolidinedionas/uso terapéutico , TroglitazonaRESUMEN
INTRODUCTION: Interleukin-6 (IL-6) is thought to play a pathogenic role in rheumatoid arthritis and synovium is a major source of IL-6 release. We investigated the ability of retinoids to suppress IL-6 expression in IL-1-stimulated synovial fibroblasts, with special care to the contribution of retinoic acid receptor (RAR) and retinoid X receptor (RXR) subtypes, and the implication of the mitogen-activated protein kinase (MAPK) pathway. METHODS: RAR-alpha, -beta, and -gamma and RXR-alpha, -beta, and -gamma levels were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) or Western blot in rat synovial fibroblasts stimulated with 10 ng/mL of IL-1beta. Stimulated levels of IL-6 were assessed by RT-qPCR or immunoassays in the presence or absence of 1 microM all-trans retinoic acid (ATRA) (RAR agonist) or 0.3 microM BMS-649 (RXR agonist). The contribution of RAR subtypes was checked with selective agonists or small interfering RNAs. The effect of ATRA on upstream MAPK (p38 MAPK, c-Jun N-terminal kinase [JNK], and extracellularly regulated kinase 1/2 [ERK1/2]) was assessed by Western blot, and the contribution of the ERK1/2 pathway to the activation of pro-inflammatory transcription factors was studied by TransAm assays. RESULTS: Synovial fibroblasts expressed all RAR and RXR subtypes except RXR-gamma. In IL-1-stimulated cells, ATRA, but not BMS-649, reduced IL-6 expression whereas selective RAR agonists were inactive. The inhibitory effect of ATRA on IL-6 was not affected by the silencing of RAR subtypes. ATRA also reduced the phosphorylation of ERK1/2, but not of p38 MAPK or of JNK. The suppressive effect of ATRA on the activation of activator protein-1 (AP-1) and nuclear factor-IL-6 (NF-IL-6) was reproduced by the MEK1 (mitogen-activated protein extracellularly regulated kinase kinase 1) inhibitor PD-98059, whereas ATRA and PD-98059 had no effect on NF-kappaB activation. CONCLUSIONS: Among RAR and RXR agonists, only ATRA inhibited IL-1-induced IL-6 expression in rat synovial fibroblasts by inhibiting ERK1/2 pathway and subsequent activation of AP-1 and NF-IL-6 independently of RAR.
Asunto(s)
Interleucina-1/farmacología , Interleucina-6/biosíntesis , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Receptores de Ácido Retinoico/metabolismo , Membrana Sinovial/metabolismo , Tretinoina/farmacología , Animales , Células Cultivadas , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Interleucina-6/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Ratas , Ratas Wistar , Receptores de Ácido Retinoico/agonistas , Membrana Sinovial/citología , Membrana Sinovial/efectos de los fármacosRESUMEN
ANK is a multipass transmembrane protein transporter thought to play a role in the export of intracellular inorganic pyrophosphate and so to contribute to the pathophysiology of chondrocalcinosis. As transforming growth factor-beta-1 (TGF-beta1) was shown to favor calcium pyrophosphate dihydrate deposition, we investigated the contribution of ANK to the production of extracellular inorganic pyrophosphate (ePPi) by chondrocytes and the signaling pathways involved in the regulation of Ank expression by TGF-beta1. Chondrocytes were exposed to 10 ng/mL of TGF-beta1, and Ank expression was measured by quantitative polymerase chain reaction and Western blot. ePPi was quantified in cell supernatants. RNA silencing was used to define the respective roles of Ank and PC-1 in TGF-beta1-induced ePPi generation. Finally, selective kinase inhibitors and dominant-negative/overexpression plasmid strategies were used to explore the contribution of several signaling pathways to Ank induction by TGF-beta1. TGF-beta1 strongly increased Ank expression at the mRNA and protein levels, as well as ePPi production. Using small interfering RNA technology, we showed that Ank contributed approximately 60% and PC-1 nearly 20% to TGF-beta1-induced ePPi generation. Induction of Ank by TGF-beta1 required activation of the extracellular signal-regulated kinase (ERK) pathway but not of p38-mitogen-activated protein kinase or of protein kinase A. In line with the general protein kinase C (PKC) inhibitor calphostin C, Gö6976 (a Ca2+-dependent PKC inhibitor) diminished TGF-beta1-induced Ank expression by 60%, whereas a 10% inhibition was observed with rottlerin (a PKCdelta inhibitor). These data suggest a regulatory role for calcium in TGF-beta1-induced Ank expression. Finally, we demonstrated that the stimulatory effect of TGF-beta1 on Ank expression was inhibited by the suppression of the Ras/Raf-1 pathway, while being enhanced by their constitutive activation. Transient overexpression of Smad 7, an inhibitory Smad, failed to affect the inducing effect of TGF-beta1 on Ank mRNA level. These data show that TGF-beta1 increases ePPi levels, mainly by the induction of the Ank gene, which requires activation of Ras, Raf-1, ERK, and Ca2+-dependent PKC pathways in chondrocytes.
Asunto(s)
Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Difosfatos/metabolismo , Proteínas de la Membrana/biosíntesis , Factor de Crecimiento Transformador beta1/farmacología , Animales , Secuencia de Bases , Células Cultivadas , Condrocalcinosis/etiología , Condrocalcinosis/genética , Condrocalcinosis/metabolismo , Cartilla de ADN/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Cinética , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de Transporte de Fosfato , Hidrolasas Diéster Fosfóricas/genética , Hidrolasas Diéster Fosfóricas/metabolismo , Proteínas Proto-Oncogénicas c-raf/metabolismo , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Ratas , Ratas Wistar , Proteínas ras/metabolismoRESUMEN
OBJECTIVE: To study the potency of 2 peroxisome proliferator-activated receptor gamma (PPARgamma) agonists, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15-deoxy-PGJ(2)) and rosiglitazone, to modulate the expression of interleukin-1 receptor antagonist (IL-1Ra) in rat synovial fibroblasts. METHODS: Levels of messenger RNA for IL-1Ra and PPAR isotypes (alpha, beta/delta, gamma) were assessed by real-time polymerase chain reaction in rat synovial fibroblasts exposed to 10 ng/ml of IL-1beta. PPAR levels were assessed by Western blotting and secreted IL-1Ra levels by immunoassay. The potency of PPARgamma agonists and the PPARbeta/delta agonist GW-501516 on IL-1Ra levels was tested in the range of 1-10 microM and at 100 pM, respectively. The contribution of PPARgamma to the effects of rosiglitazone on IL-1Ra secretion was examined either by its overexpression or by inhibition using wild-type or dominant-negative constructs and the antagonist GW-9662 (10 microM), respectively. The dominant-negative strategy was also performed to investigate the possible contribution of PPARbeta/delta and NF-kappaB activation. RESULTS: IL-1beta-induced IL-1Ra production was increased by 10 microM rosiglitazone but was reduced dose-dependently by 15-deoxy-PGJ(2). Both agonists lowered IL-1beta secretion, but rosiglitazone alone reduced the imbalance of IL-1beta/IL-1Ra toward basal levels. Enhancement of IL-1beta-induced IL-1Ra production by rosiglitazone was not affected by PPARgamma overexpression or by its inhibition with dominant-negative PPARgamma or GW-9662. Inhibition of NF-kappaB was also ineffective against rosiglitazone but abolished the stimulating effect of IL-1beta on IL-1Ra. All PPAR isotypes were expressed constitutively in rat synoviocytes, but PPARgamma decreased dramatically upon IL-1beta exposure, whereas PPARbeta/delta remained stable. Dominant-negative PPARbeta/delta abolished the enhancement of IL-1Ra by rosiglitazone, whereas GW-501516 reproduced the effect of rosiglitazone on IL-1Ra secretion. CONCLUSION: Rosiglitazone stimulates IL-1Ra production by a PPARbeta/delta mechanism in activated rat synovial fibroblasts, further contributing to its potential antiarthritic properties and opening new perspectives for the modulation of inflammatory genes by specific PPAR agonists in articular cells.