RESUMEN
Alcoholic chronic pancreatitis (ACP) is a fibroinflammatory disease of the pancreas. However, metabolic basis of ACP is not clearly understood. In this study, we evaluated differential pancreatic injury in hepatic alcohol dehydrogenase-deficient (ADH-) deer mice fed chronic ethanol (EtOH), chronic plus binge EtOH, and chronic plus binge EtOH and fatty acid ethyl esters (FAEEs, nonoxidative metabolites of EtOH) to understand the metabolic basis of ACP. Hepatic ADH- and ADH normal (ADH+) deer mice were fed Lieber-DeCarli liquid diet containing 3% (wt/vol) EtOH for 3 mo. One week before the euthanization, chronic EtOH-fed mice were further administered with an oral gavage of binge EtOH with/without FAEEs. Blood alcohol concentration (BAC), pancreatic injury, and inflammatory markers were measured. Pancreatic morphology, ultrastructural changes, and endoplasmic reticulum (ER)/oxidative stress were examined using H&E staining, electron microscopy, immunostaining, and/or Western blot, respectively. Overall, BAC was substantially increased in chronic EtOH-fed groups of ADH- versus ADH+ deer mice. A significant change in pancreatic acinar cell morphology, with mild to moderate fibrosis and ultrastructural changes evident by dilatations and disruption of ER cisternae, ER/oxidative stress along with increased levels of inflammatory markers were observed in the pancreas of chronic EtOH-fed groups of ADH- versus ADH+ deer mice. Furthermore, chronic plus binge EtOH and FAEEs exposure elevated BAC, enhanced ER/oxidative stress, and exacerbated chronic EtOH-induced pancreatic injury in ADH- deer mice suggesting a role of increased body burden of EtOH and its metabolism under reduced hepatic ADH in initiation and progression of ACP.NEW & NOTEWORTHY We established a chronic EtOH feeding model of hepatic alcohol dehydrogenase-deficient (ADH-) deer mice, which mimics several fibroinflammatory features of human alcoholic chronic pancreatitis (ACP). The fibroinflammatory and morphological features exacerbated by chronic plus binge EtOH and FAEEs exposure provide a strong case for metabolic basis of ACP. Most importantly, several pathological and molecular targets identified in this study provide a much broader understanding of the mechanism and avenues to develop therapeutics for ACP.
Asunto(s)
Alcohol Deshidrogenasa , Pancreatitis Alcohólica , Alcohol Deshidrogenasa/metabolismo , Animales , Nivel de Alcohol en Sangre , Ésteres , Etanol , Ácidos Grasos/metabolismo , Peromyscus/metabolismoRESUMEN
The single most common cause of chronic pancreatitis (CP, a serious inflammatory disease) is chronic alcohol abuse, which impairs hepatic alcohol dehydrogenase (ADH, a major ethanol oxidizing enzyme). Previously, we found ~5 fold greater fatty acid ethyl esters (FAEEs), and injury in the pancreas of hepatic ADH deficient (ADH-) vs. hepatic normal ADH (ADH+) deer mice fed 3.5g% ethanol via liquid diet daily for two months. Therefore, progression of ethanol-induced pancreatic injury was determined in ADH- deer mice fed ethanol for four months to delineate the mechanism and metabolic basis of alcoholic chronic pancreatitis (ACP). In addition to a substantially increased blood alcohol concentration and plasma FAEEs, significant degenerative changes, including atrophy and loss of acinar cells in some areas, ultrastructural changes evident by such features as swelling and disintegration of endoplasmic reticulum (ER) cisternae and ER stress were observed in the pancreas of ethanol-fed ADH- deer mice vs. ADH+ deer mice. These changes are consistent with noted increases in pancreatic injury markers (plasma lipase, pancreatic trypsinogen activation peptide, FAEE synthase and cathepsin B) in ethanol-fed ADH- deer mice. Most importantly, an increased levels of pancreatic glucose regulated protein (GRP) 78 (a prominent ER stress marker) were found to be closely associated with increased phosphorylated eukaryotic initiation factor (eIF) 2α signaling molecule in PKR-like ER kinase branch of unfolded protein response (UPR) as compared to X box binding protein 1S and activating transcription factor (ATF)6 - 50kDa protein of inositol requiring enzyme 1α and ATF6 branches of UPR, respectively, in ethanol-fed ADH- vs. ADH+ deer mice. These results along with findings on plasma FAEEs, and pancreatic histology and injury markers suggest a metabolic basis of ethanol-induced pancreatic injury, and provide new avenues to understand metabolic basis and molecular mechanism of ACP.
Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Éteres de Etila/metabolismo , Páncreas/patología , Alcoholismo , Animales , Nivel de Alcohol en Sangre , Modelos Animales de Enfermedad , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Etanol/sangre , Ácidos Grasos/metabolismo , Hígado/metabolismo , Ratones , Páncreas/metabolismo , Peromyscus/fisiologíaRESUMEN
Tetrachloroethene (perchloroethylene, PCE), an ubiquitous environmental contaminant, has been implicated in inducing autoimmunity/autoimmune diseases (ADs), including systemic lupus erythematosus (SLE) and scleroderma in humans. However, experimental evidence suggesting the potential of PCE in mediating autoimmunity is lacking. This study was, therefore, undertaken to explore PCE's potential in inducing/exacerbating an autoimmune response. Six-week old female MRL+/+ mice, in groups of 6 each, were treated with PCE (0.5mg/ml) via drinking water for 12, 18 and 24weeks and markers of autoimmunity and oxidative stress were evaluated. PCE exposure led to significant increases in serum anti-nuclear antibodies (ANA), anti-dsDNA and anti-scleroderma-70 (anti-Scl-70) antibodies at 18weeks and, to a greater extent at 24weeks, suggesting that PCE exposure exacerbated autoimmunity in our animal model. The increases in autoantibodies were associated with time-dependent increases in malondialdehyde (MDA)-protein adducts and their antibodies, as well as significantly decreased levels of antioxidants GSH and SOD. The splenocytes isolated from mice treated with PCE for 18 and 24weeks showed greater Th17 cell proliferation and increased release of IL-17 in culture supernatants following stimulation with MDA-mouse serum albumin adducts, suggesting that MDA-modified proteins may act as an immunologic trigger by activating Th17 cells and contribute to PCE-mediated autoimmunity. Our studies thus provide an experimental evidence that PCE induces/exacerbates an autoimmune response and lipid-derived aldehydes (such as MDA) contribute to this response.
Asunto(s)
Autoinmunidad/efectos de los fármacos , Malondialdehído/metabolismo , Solventes/toxicidad , Tetracloroetileno/toxicidad , Animales , Anticuerpos/sangre , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Glutatión/sangre , Interleucina-17/metabolismo , Lípidos , Lupus Eritematoso Sistémico , Ratones , Unión Proteica , Esclerodermia Sistémica , Bazo/citología , Superóxido Dismutasa/sangre , Células Th17/efectos de los fármacosRESUMEN
The development of antibodies to low molecular weight haptens remains challenging due to both the low immunogenicity of many haptens and the cross-reactivity of the protein carriers used to generate the immune response. Recombinant antibodies and novel display technologies have greatly advanced antibody development; however, new techniques are still required to select rare hapten-specific antibodies from large recombinant libraries. In the present study, we used a combination of phage and yeast display to screen an immune antibody library (size, 4.4 × 10(6)) against hapten markers for petroleum contamination (phenanthrene and methylphenanthrenes). Selection via phage display was used first to enrich the library between 20- and 100-fold for clones that bound to phenanthrene-protein conjugates. The enriched libraries were subsequently transferred to a yeast display system and a newly developed competitive FACS procedure was employed to select rare hapten-specific clones. Competitive FACS increased the frequency of hapten-specific scFvs in our yeast-displayed scFvs from 0.025 to 0.005% in the original library to between 13 and 35% in selected pools. The presence of hapten-specific scFvs was confirmed by competitive ELISA using periplasmic protein. Three distinct antibody clones that recognize phenanthrene and methylphenanthrenes were selected, and their distinctive binding properties were characterized. To our knowledge, these are first antibodies that can distinguish between methylated (petrogenic) versus unmethylated (pyrogenic) phenanthrenes; such antibodies will be useful in detecting the sources of environmental contamination. This selection method could be generally adopted in the selection of other hapten-specific recombinant antibodies.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Haptenos/inmunología , Fenantrenos/inmunología , Anticuerpos de Cadena Única/inmunología , Animales , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Ratones , Biblioteca de Péptidos , Proteínas Recombinantes/inmunología , Técnicas del Sistema de Dos HíbridosRESUMEN
Exposure to trichloroethene (TCE), a ubiquitous environmental contaminant, is known to induce autoimmunity both in humans and animal models. However, mechanisms underlying TCE-mediated autoimmunity remain largely unknown. Previous studies from our laboratory in MRL+/+ mice suggest that oxidative stress may contribute to TCE-induced autoimmune response. The current study was undertaken to further assess the role of oxidative stress in TCE-induced autoimmunity by supplementing with an antioxidant N-acetylcysteine (NAC). Groups of female MRL+/+ mice were given TCE, NAC or TCE+NAC for 6 weeks (TCE, 10mmol/kg, i.p., every 4th day; NAC, 250mg/kg/day through drinking water). TCE exposure led to significant increases in serum levels of anti-nuclear, anti-dsDNA and anti-Sm antibodies. TCE exposure also led to significant induction of anti-malondiadelhyde (MDA)- and anti-hydroxynonenal (HNE)-protein adduct antibodies which were associated with increased ANA in the sera along with increased MDA-/HNE-protein adducts in the livers and kidneys, and increases in protein oxidation (carbonylation) in the sera, livers and kidneys, suggesting an overall increase in oxidative stress. Moreover, TCE exposure also resulted in increased release of IL-17 from splenocytes and increases in IL-17 mRNA expression. Remarkably, NAC supplementation attenuated not only the TCE-induced oxidative stress, IL-17 release and mRNA expression, but also the markers of autoimmunity, as evident from decreased levels of ANA, anti-dsDNA and anti-Sm antibodies in the sera. These results provide further support to a role of oxidative stress in TCE-induced autoimmune response. Attenuation of TCE-induced autoimmunity in mice by NAC provides an approach for preventive and/or therapeutic strategies.
Asunto(s)
Acetilcisteína/farmacología , Enfermedades Autoinmunes/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Tricloroetileno/toxicidad , Animales , Anticuerpos Antinucleares/sangre , Autoanticuerpos/sangre , Enfermedades Autoinmunes/inducido químicamente , Femenino , Interleucina-17/genética , Interleucina-17/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratones Endogámicos MRL lpr , Carbonilación Proteica/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Bazo/citología , Bazo/efectos de los fármacos , Bazo/metabolismoRESUMEN
Fatty liver is an early stage of alcoholic and nonalcoholic liver disease (ALD and NALD) that progresses to steatohepatitis and other irreversible conditions. In this study, we identified proteins that were differentially expressed in the livers of rats fed 5% ethanol in a Lieber-DeCarli diet daily for 1 and 3 months by discovery proteomics (two-dimensional gel electrophoresis and mass spectrometry) and non-parametric modeling (Multivariate Adaptive Regression Splines). Hepatic fatty infiltration was significantly higher in ethanol-fed animals as compared to controls, and more pronounced at 3 months of ethanol feeding. Discovery proteomics identified changes in the expression of proteins involved in alcohol, lipid, and amino acid metabolism after ethanol feeding. At 1 and 3 months, 12 and 15 different proteins were differentially expressed. Of the identified proteins, down regulation of alcohol dehydrogenase (-1.6) at 1 month and up regulation of aldehyde dehydrogenase (2.1) at 3 months could be a protective/adaptive mechanism against ethanol toxicity. In addition, betaine-homocysteine S-methyltransferase 2 a protein responsible for methionine metabolism and previously implicated in fatty liver development was significantly up regulated (1.4) at ethanol-induced fatty liver stage (1 month) while peroxiredoxin-1 was down regulated (-1.5) at late fatty liver stage (3 months). Nonparametric analysis of the protein spots yielded fewer proteins and narrowed the list of possible markers and identified d-dopachrome tautomerase (-1.7, at 3 months) as a possible marker for ethanol-induced early steatohepatitis. The observed differential regulation of proteins have potential to serve as biomarker signature for the detection of steatosis and its progression to steatohepatitis once validated in plasma/serum.
Asunto(s)
Hígado Graso Alcohólico/metabolismo , Proteómica/métodos , Animales , Modelos Animales de Enfermedad , Electroforesis en Gel Bidimensional , Etanol/administración & dosificación , Etanol/toxicidad , Hígado Graso Alcohólico/genética , Regulación de la Expresión Génica , Masculino , Ratas , Ratas Endogámicas F344 , Análisis de Regresión , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Chronic alcohol abuse is a 2nd major cause of liver disease resulting in significant morbidity and mortality. Alcoholic liver disease (ALD) is characterized by a wide spectrum of pathologies starting from fat accumulation (steatosis) in early reversible stage to inflammation with or without fibrosis and cirrhosis in later irreversible stages. Previously, we reported significant steatosis in the livers of hepatic alcohol dehydrogenase (ADH)-deficient (ADHâ») vs. hepatic ADH-normal (ADHâº) deer mice fed 4% ethanol daily for 2 months [Bhopale et al., 2006, Alcohol 39, 179-188]. However, ADHâ» deer mice fed 4% ethanol also showed a significant mortality. Therefore, a dose-dependent study was conducted to understand the mechanism and identify lipid(s) involved in the development of ethanol-induced fatty liver. ADHâ» and ADH⺠deer mice fed 1, 2 or 3.5% ethanol daily for 2 months and fatty infiltration in the livers were evaluated by histology and by measuring dry weights of extracted lipids. Lipid metabolomic changes in extracted lipids were determined by proton (¹H) and ³¹phosphorus (³¹P) nuclear magnetic resonance (NMR) spectroscopy. The NMR data was analyzed by hierarchical clustering (HC) and principle component analysis (PCA) for pattern recognition. Extensive vacuolization by histology and significantly increased dry weights of total lipids found only in the livers of ADHâ» deer mice fed 3.5% ethanol vs. pair-fed controls suggest a dose-dependent formation of fatty liver in ADHâ» deer mouse model. Analysis of NMR data of ADHâ» deer mice fed 3.5% ethanol vs. pair-fed controls shows increases for total cholesterol, esterified cholesterol, fatty acid methyl esters (FAMEs), triacylglycerides and unsaturation, and decreases for free cholesterol, phospholipids and allylic and diallylic protons. Certain classes of neutral lipids (cholesterol esters, fatty acyl chain (-COCH2-) and FAMEs) were also mildly increased in ADHâ» deer mice fed 1 or 2% ethanol. Only small increases were observed for allylic and diallylic protons, FAMEs and unsaturations in ADH⺠deer mice fed 3.5% ethanol vs. pair-fed controls. PCA of NMR data showed increased clustering by gradual separation of ethanol-fed ADHâ» deer mice groups from their respective pair-fed control groups and corresponding ethanol-fed ADH⺠deer mice groups. Our data indicate that dose of ethanol and hepatic ADH deficiency are two key factors involved in initiation and progression of alcoholic fatty liver disease. Further studies on characterization of individual lipid entities and associated metabolic pathways altered in our deer mouse model after different durations of ethanol feeding could be important to delineate mechanism(s) and identify potential biomarker candidate(s) of early stage ALD.
Asunto(s)
Etanol/toxicidad , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Animales , Colesterol/sangre , Ésteres del Colesterol/sangre , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Etanol/administración & dosificación , Ácidos Grasos/sangre , Hepatopatías Alcohólicas/metabolismo , Masculino , PeromyscusRESUMEN
Two diets simulating the recommendations of the American Heart Association to increase the intake of n-3 polyunsaturated fatty acids (n-3 PUFAs) were tested on Golden Syrian hamsters and compared to the diet simulating the current estimated consumption of fat in the United States. N-3 PUFAs were evaluated for their effects on serum and brain lipids and on the three cytochrome P450 enzymes (CYPs 7A1, 27A1, and 46A1) that play key roles in cholesterol elimination from different organs. Hamsters on the highest concentration of n-3 PUFAs had a statistically significant decrease in LDL and HDL cholesterol and no change in serum total cholesterol and triglycerides levels. CYP27A1 and CYP46A1 mRNA levels were increased in the liver and brain, respectively, whereas possible effects on CYP7A1 were obscured by a marked intergroup variability at mRNA, protein, and sterol product levels. Increased levels of CYP46A1 mRNA in the brain did not lead to significant changes in the levels of lathosterol, 24S-hydroxycholesterol or cholesterol in this organ. The data obtained are discussed in relation to inconsistent effects of n-3 PUFAs on serum lipids in human trials and reported positive effects of fish oil on cognitive function.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Ácidos Grasos Omega-3/farmacología , Isoenzimas/metabolismo , Hígado/enzimología , Microsomas Hepáticos/enzimología , Animales , Secuencia de Bases , Clonación Molecular , Cricetinae , Sistema Enzimático del Citocromo P-450/genética , Cartilla de ADN , Cromatografía de Gases y Espectrometría de Masas , Isoenzimas/genética , Masculino , MesocricetusRESUMEN
Aniline exposure is associated with toxicity to the spleen which is characterized by splenomegaly, hyperplasia, fibrosis, and a variety of sarcomas on chronic exposure in rats. However, mechanisms by which aniline elicits splenotoxic responses are not well understood. Earlier we have shown that aniline exposure leads to increased nitration of proteins in the spleen. However, nitrated proteins remain to be characterized. Therefore, in the current study using proteomic approaches, we focused on characterizing the nitrated proteins in the spleen of aniline-exposed rats. Aniline exposure led to increased tyrosine nitration of proteins, as determined by 2D Western blotting with anti-3-nitrotyrosine specific antibody, compared to the controls. The analyzed nitrated proteins were found in the molecular weight range of 27.7 to 123.6kDa. A total of 37 nitrated proteins were identified in aniline-treated and control spleens. Among them, 25 were found only in aniline-treated rats, 11 were present in both aniline-treated and control rats, while one was found in controls only. The nitrated proteins identified mainly represent skeletal proteins, chaperones, ferric iron transporter, enzymes, nucleic acids binding protein, and signaling and protein synthesis pathways. Furthermore, aniline exposure led to significantly increased iNOS mRNA and protein expression in the spleen, suggesting its role in increased reactive nitrogen species formation and contribution to increased nitrated proteins. The identified nitrated proteins provide a global map to further investigate alterations in their structural and functional properties, which will lead to a better understanding of the role of protein nitration in aniline-mediated splenic toxicity.
Asunto(s)
Compuestos de Anilina/toxicidad , Proteínas/metabolismo , Proteómica , Bazo/efectos de los fármacos , Animales , Western Blotting , Masculino , Óxido Nítrico Sintasa de Tipo II/genética , Ratas , Ratas Sprague-Dawley , Bazo/metabolismo , Estrés FisiológicoRESUMEN
Alcoholic liver disease (ALD) is a serious health problem with significant morbidity and mortality. In this study we examined the progression of ALD along with lipidomic changes in rats fed ethanol for 2 and 3 months to understand the mechanism, and identify possible biomarkers. Male Fischer 344 rats were fed 5% ethanol or caloric equivalent of maltose-dextrin in a Lieber-DeCarli diet. Animals were killed at the end of 2 and 3 months and plasma and livers were collected. Portions of the liver were fixed for histological and immunohistological studies. Plasma and the liver lipids were extracted and analyzed by nuclear magnetic resonance (NMR) spectroscopy. A time dependent fatty infiltration was observed in the livers of ethanol-fed rats. Mild inflammation and oxidative stress were observed in some ethanol-fed rats at 3 months. The multivariate and principal component analysis of proton and phosphorus NMR spectroscopy data of extracted lipids from the plasma and livers showed segregation of ethanol-fed groups from the pair-fed controls. Significant hepatic lipids that were increased by ethanol exposure included fatty acids and triglycerides, whereas phosphatidylcholine (PC) decreased. However, both free fatty acids and PC decreased in the plasma. In liver lipids unsaturation of fatty acyl chains increased, contrary to plasma, where it decreased. Our studies confirm that over-accumulation of lipids in ethanol-induced liver steatosis accompanied by mild inflammation on long duration of ethanol exposure. Identified metabolic profile using NMR lipidomics could be further explored to establish biomarker signatures representing the etiopathogenesis, progression and/or severity of ALD.
Asunto(s)
Etanol/toxicidad , Metabolismo de los Lípidos/efectos de los fármacos , Hepatopatías Alcohólicas/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Peso Corporal/efectos de los fármacos , Etanol/administración & dosificación , Inmunohistoquímica , Hepatopatías Alcohólicas/enzimología , Espectroscopía de Resonancia Magnética , Masculino , Metabolómica/métodos , Tamaño de los Órganos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Análisis de Componente Principal , Ratas , Ratas Endogámicas F344RESUMEN
OBJECTIVE: Free radical-mediated reactions have been implicated as contributors in a number of autoimmune diseases, including systemic lupus erythematosus (SLE). However, the potential for oxidative/nitrosative stress to elicit an autoimmune response or to contribute to disease pathogenesis, and thus be useful when determining a prognosis, remains largely unexplored in humans. This study was undertaken to investigate the status and contribution of oxidative/nitrosative stress in patients with SLE. METHODS: Sera from 72 SLE patients with varying levels of disease activity according to the SLE Disease Activity Index (SLEDAI) and 36 age- and sex-matched healthy controls were evaluated for serum levels of oxidative/nitrosative stress markers, including antibodies to malondialdehyde (anti-MDA) protein adducts and to 4-hydroxynonenal (anti-HNE) protein adducts, MDA/HNE protein adducts, superoxide dismutase (SOD), nitrotyrosine (NT), and inducible nitric oxide synthase (iNOS). RESULTS: Serum analysis showed significantly higher levels of both anti-MDA/anti-HNE protein adduct antibodies and MDA/HNE protein adducts in SLE patients compared with healthy controls. Interestingly, not only was there an increased number of subjects positive for anti-MDA or anti-HNE antibodies, but also the levels of both of these antibodies were statistically significantly higher among SLE patients whose SLEDAI scores were > or = 6 as compared with SLE patients with lower SLEDAI scores (SLEDAI score <6). In addition, a significant correlation was observed between the levels of anti-MDA or anti-HNE antibodies and the SLEDAI score (r = 0.734 and r = 0.647, respectively), suggesting a possible causal relationship between these antibodies and SLE. Furthermore, sera from SLE patients had lower levels of SOD and higher levels of iNOS and NT compared with healthy control sera. CONCLUSION: These findings support an association between oxidative/nitrosative stress and SLE. The stronger response observed in serum samples from patients with higher SLEDAI scores suggests that markers of oxidative/nitrosative stress may be useful in evaluating the progression of SLE and in elucidating the mechanisms of disease pathogenesis.
Asunto(s)
Lupus Eritematoso Sistémico/sangre , Estrés Oxidativo , Adulto , Anciano , Biomarcadores/sangre , Progresión de la Enfermedad , Femenino , Estado de Salud , Humanos , Lupus Eritematoso Sistémico/patología , Lupus Eritematoso Sistémico/fisiopatología , Masculino , Malondialdehído/sangre , Persona de Mediana Edad , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/sangre , Índice de Severidad de la Enfermedad , Superóxido Dismutasa/sangre , Tirosina/análogos & derivados , Tirosina/sangre , Adulto JovenRESUMEN
Pancreatitis caused by activation of digestive zymogens in the exocrine pancreas is a serious chronic health problem in alcoholic patients. However, mechanism of alcoholic pancreatitis remains obscure due to lack of a suitable animal model. Earlier, we reported pancreatic injury and substantial increases in endogenous formation of fatty acid ethyl esters (FAEEs) in the pancreas of hepatic alcohol dehydrogenase (ADH)-deficient (ADH(-)) deer mice fed 4% ethanol. To understand the mechanism of alcoholic pancreatitis, we evaluated dose-dependent metabolism of ethanol and related pancreatic injury in ADH(-) and hepatic ADH-normal (ADH(+)) deer mice fed 1%, 2% or 3.5% ethanol via Lieber-DeCarli liquid diet daily for 2months. Blood alcohol concentration (BAC) was remarkably increased and the concentration was â¼1.5-fold greater in ADH(-) vs. ADH(+) deer mice fed 3.5% ethanol. At the end of the experiment, remarkable increases in pancreatic FAEEs and significant pancreatic injury indicated by the presence of prominent perinuclear space, pyknotic nuclei, apoptotic bodies and dilation of glandular ER were found only in ADH(-) deer mice fed 3.5% ethanol. This pancreatic injury was further supported by increased plasma lipase and pancreatic cathepsin B (a lysosomal hydrolase capable of activating trypsinogen), trypsinogen activation peptide (by-product of trypsinogen activation process) and glucose-regulated protein 78 (endoplasmic reticulum stress marker). These findings suggest that ADH-deficiency and high alcohol levels in the body are the key factors in ethanol-induced pancreatic injury. Therefore, determining how this early stage of pancreatic injury advances to inflammation stage could be important for understanding the mechanism(s) of alcoholic pancreatitis.
Asunto(s)
Alcohol Deshidrogenasa/deficiencia , Etanol/toxicidad , Hígado/enzimología , Páncreas/efectos de los fármacos , Páncreas/metabolismo , Pancreatitis Alcohólica/enzimología , Acetaldehído/sangre , Acetaldehído/metabolismo , Alcohol Deshidrogenasa/metabolismo , Animales , Catepsina B/sangre , Catepsina B/metabolismo , Modelos Animales de Enfermedad , Retículo Endoplásmico/metabolismo , Etanol/sangre , Éteres de Etila/metabolismo , Ácidos Grasos/metabolismo , Proteínas HSP70 de Choque Térmico/sangre , Proteínas HSP70 de Choque Térmico/metabolismo , Lipasa/sangre , Lipasa/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Proteínas de la Membrana/sangre , Proteínas de la Membrana/metabolismo , Ratones , Oligopéptidos/sangre , Oligopéptidos/metabolismo , Páncreas/enzimología , Páncreas/patología , Pancreatitis Alcohólica/sangre , Pancreatitis Alcohólica/metabolismo , Pancreatitis Alcohólica/patologíaRESUMEN
BACKGROUND: Hepatic steatosis (fatty liver), an early and reversible stage of alcoholic liver disease, is characterized by triglyceride deposition in hepatocytes, which can advance to steatohepatitis, fibrosis, cirrhosis, and ultimately to hepatocellular carcinoma. In the present work, we studied altered plasma and hepatic lipid metabolome (lipidome) to understand the mechanisms and lipid pattern of early-stage alcohol-induced-fatty liver. METHODS: Male Fischer 344 rats were fed 5% alcohol in a Lieber-DeCarli diet. Control rats were pair-fed an equivalent amount of maltose-dextrin. After 1 month, animals were killed and plasma collected. Livers were excised for morphological, immunohistochemical, and biochemical studies. The lipids from plasma and livers were extracted with methyl-tert-butyl ether and analyzed by 750/800 MHz proton nuclear magnetic resonance (¹H NMR) and phosphorus (³¹P) NMR spectroscopy on a 600 MHz spectrometer. The NMR data were then subjected to multivariate statistical analysis. RESULTS: Hematoxylin and Eosin and Oil Red O stained liver sections showed significant fatty infiltration. Immunohistochemical analysis of liver sections from ethanol-fed rats showed no inflammation (absence of CD3 positive cells) or oxidative stress (absence of malondialdehyde reactivity or 4-hydroxynonenal positive staining). Cluster analysis and principal component analysis of ¹H NMR data of lipid extracts of both plasma and livers showed a significant difference in the lipid metabolome of ethanol-fed versus control rats. ³¹P NMR data of liver lipid extracts showed significant changes in phospholipids similar to ¹H NMR data. ¹H NMR data of plasma and liver reflected several changes, while comparison of ¹H NMR and ³¹P NMR data offered a correlation among the phospholipids. CONCLUSIONS: Our results show that alcohol consumption alters metabolism of cholesterol, triglycerides, and phospholipids that could contribute to the development of fatty liver. These studies also indicate that fatty liver precedes oxidative stress and inflammation. The similarities observed in plasma and liver lipid profiles offer a potential methodology for detecting early-stage alcohol-induced fatty liver disease by analyzing the plasma lipid profile.
Asunto(s)
Hígado Graso Alcohólico/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Peso Corporal/efectos de los fármacos , Depresores del Sistema Nervioso Central/farmacología , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Análisis por Conglomerados , Interpretación Estadística de Datos , Etanol/farmacología , Hígado Graso Alcohólico/patología , Inmunohistoquímica , Lípidos/sangre , Hígado/efectos de los fármacos , Hígado/patología , Espectroscopía de Resonancia Magnética , Masculino , Tamaño de los Órganos/efectos de los fármacos , Estrés Oxidativo/fisiología , Isótopos de Fósforo , Análisis de Componente Principal , Protones , Ratas , Ratas Endogámicas F344RESUMEN
AIM: To compare the clinical and radiographic response of primary teeth to vital pulpotomy using mineral trioxide aggregate (MTA) or formocresol (FC). METHODOLOGY: A group of 17 children aged 4-9 were selected from those referred to the Paedodontic Department at Shahid Beheshti University, Dental School. Cases with at least two matching teeth were selected (40 teeth), showing signs of pulp involvement. A pulpotomy procedure was carried out in all cases with FC in control teeth whilst MTA was placed in experimental teeth. Clinical and radiographic evaluations were performed at 1-, 6-, 12- and 24-month recall. Statistical analysis using a Fischer exact test was performed on the data to determine significant differences between the groups. RESULTS: Overall, 22 second and 18 first primary molars were included. The gender ratio was one male to three female. No significant difference was found between the clinical and radiographic outcomes of the two groups at 6-, 12- and 24-month follow-up (P > 0.05). Internal resorption was seen significantly more often in FC cases after 12 months than MTA cases. Overall radiographic appearance of normal structures at 24th month was seen in more than 95% of the cases in MTA and 90% in the FC-treated group (P > 0.05). CONCLUSION: Mineral trioxide aggregate for pulp treatment of primary teeth can be considered a replacement for FC.
Asunto(s)
Compuestos de Aluminio/uso terapéutico , Compuestos de Calcio/uso terapéutico , Formocresoles/uso terapéutico , Óxidos/uso terapéutico , Pulpotomía/métodos , Silicatos/uso terapéutico , Diente Primario/patología , Estudios de Casos y Controles , Niño , Preescolar , Coronas , Amalgama Dental , Caries Dental/terapia , Pulpa Dental/efectos de los fármacos , Restauración Dental Permanente , Restauración Dental Provisional , Combinación de Medicamentos , Femenino , Estudios de Seguimiento , Humanos , Masculino , Diente Molar/diagnóstico por imagen , Diente Molar/patología , Ligamento Periodontal/diagnóstico por imagen , Radiografía de Mordida Lateral , Resorción Dentaria/diagnóstico por imagen , Resorción Dentaria/etiología , Diente Primario/diagnóstico por imagen , Resultado del Tratamiento , Cemento de Óxido de Zinc-EugenolRESUMEN
The cycling and fate of polycyclic aromatic hydrocarbons (PAHs) is not well understood in estuarine systems. It is critical now more than ever given the increased ecosystem pressures on these critical coastal habitats. A budget of PAHs and cycling has been created for Galveston Bay (Texas) in the northwestern Gulf of Mexico, an estuary surrounded by 30-50% of the US capacity of oil refineries and chemical industry. We estimate that approximately 3 to 4 mt per year of pyrogenic PAHs are introduced to Galveston Bay via gaseous exchange from the atmosphere (ca. 2 mt/year) in addition to numerous spills of petrogenic PAHs from oil and gas operations (ca. 1.0 to 1.9 mt/year). PAHs are cycled through and stored in the biota, and ca. 20 to 30% of the total (0.8 to 1.5 mt per year) are estimated to be buried in the sediments. Oysters concentrate PAHs to levels above their surroundings (water and sediments) and contain substantially greater concentrations than other fish catch (shrimp, blue crabs and fin fish). Smaller organisms (infaunal invertebrates, phytoplankton and zooplankton) might also retain a significant fraction of the total, but direct evidence for this is lacking. The amount of PAHs delivered to humans in seafood, based on reported landings, is trivially small compared to the total inputs, sediment accumulation and other possible fates (metabolic remineralization, export in tides, etc.), which remain poorly known. The generally higher concentrations in biota from Galveston Bay compared to other coastal habitats can be attributed to both intermittent spills of gas and oil and the bay's close proximity to high production of pyrogenic PAHs within the urban industrial complex of the city of Houston as well as periodic flood events that transport PAHs from land surfaces to the Bay.
Asunto(s)
Bahías/química , Monitoreo del Ambiente/estadística & datos numéricos , Hidrocarburos Policíclicos Aromáticos/metabolismo , Contaminantes Químicos del Agua/metabolismo , Animales , Organismos Acuáticos/química , Organismos Acuáticos/metabolismo , Atmósfera/química , Braquiuros/química , Braquiuros/metabolismo , Peces/metabolismo , Sedimentos Geológicos/química , Golfo de México , Ostreidae/química , Ostreidae/metabolismo , Contaminación por Petróleo/estadística & datos numéricos , Texas , Contaminantes Químicos del Agua/análisisRESUMEN
Reactive trichloroethene (TCE) metabolites and oxidative stress are involved in TCE-mediated autoimmunity, as evident from our earlier studies in MRL+/+ mice. However, molecular mechanisms underlying the autoimmunity remain largely unknown. Cytochrome P450 2E1 (CYP2E1), the major enzyme responsible for TCE metabolism, could contribute to TCE-induced toxic response through free radical generation. The current study was, therefore, aimed to further evaluate the significance of TCE metabolism leading to oxidative stress and autoimmune response by using MRL+/+ mice that lack CYP2E1. The Cyp2e1-null MRL+/+ mice were generated by backcrossing Cyp2e1-null mice (B6N; 129S4-Cyp2e1) to MRL +/+ mice. Female MRL+/+ and Cyp2e1-null MRL+/+ mice were given TCE (10 mmol/kg, i.p., every 4th day) for 6 weeks; their respective controls received corn oil only. TCE treatment in MRL+/+ mice induced oxidative stress, evident from significantly increased serum malondiadelhyde (MDA)-protein adducts, their antibodies and reduced liver GSH levels. TCE treatment also modulated Nrf2 pathway with decreased Nrf2 and HO-1, and elevated NF-κB (p65) expression in the liver. TCE exposure also led to increases in serum antinuclear antibodies (ANA) and anti-double stranded DNA antibodies (anti-dsDNA). Although TCE treatment in Cyp2e1-null MRL+/+ mice also led to increases in serum MDA-protein adducts and their antibodies, changes in liver GSH, Nrf2, HO-1 and NF-κB along with increases in serum ANA, anti-dsDNA, the alterations in the oxidative stress and autoimmunity markers in these mice were less pronounced compared to those in MRL+/+ mice. These findings support the contribution of CYP2E1-mediated TCE metabolism in autoimmune response and an important role of Nrf2 pathway in TCE-mediated autoimmunity.
Asunto(s)
Autoanticuerpos/inmunología , Enfermedades Autoinmunes/epidemiología , Autoinmunidad/inmunología , Citocromo P-450 CYP2E1/fisiología , Estrés Oxidativo , Tricloroetileno/toxicidad , Anestésicos por Inhalación/toxicidad , Animales , Enfermedades Autoinmunes/inducido químicamente , Enfermedades Autoinmunes/patología , Femenino , Glutatión/metabolismo , Incidencia , Peroxidación de Lípido , Ratones , Ratones Noqueados , Transducción de SeñalRESUMEN
A community-based participatory research was utilized to address the coastal community's concern regarding Deepwater Horizon oil contamination of seafood. Therefore, we analyzed polycyclic aromatic hydrocarbons (PAHs), major toxic constituents of crude oil, in the seafood collected from gulf coast (Louisiana, Alabama and Mississippi) during December 2011-February 2014. PAHs were extracted from edible part of shrimp, oysters, and crabs by the QuEChERS/dsPE procedure and analyzed by gas chromatography-mass spectrometry. The total PAHs data were further analyzed using the General Linear Mixed Model procedure of the SAS (Version 9.3, SAS Institute, Inc., Cary, NC) statistical software. Brown shrimp showed statistically significant differences in PAHs levels with respect to time and locations while white shrimp showed differences at various time points. PAHs levels in oyster and crab samples were not statistically different at the Type I error of 0.05. Overall, the PAHs levels are far below FDA levels of concern for human consumption.
Asunto(s)
Contaminación de Alimentos/análisis , Hidrocarburos Policíclicos Aromáticos/análisis , Alimentos Marinos/análisis , Contaminantes Químicos del Agua/análisis , Alabama , Animales , Braquiuros/química , Monitoreo del Ambiente/métodos , Cromatografía de Gases y Espectrometría de Masas , Louisiana , Mississippi , Ostreidae/química , Penaeidae/química , Contaminación por Petróleo/análisisRESUMEN
Lipid peroxidation is implicated in the pathogenesis of various autoimmune diseases. Lipid peroxidation-derived aldehydes such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are highly reactive and bind to proteins, but their role in eliciting an autoimmune response and their contribution to disease pathogenesis remain unclear. To investigate the role of lipid peroxidation in the induction and/or exacerbation of autoimmune response, 6-week-old autoimmune-prone female MRL+/+ mice were treated for 4 weeks with trichloroethene (TCE; 10 mmol/kg, ip, once a week), an environmental contaminant known to induce lipid peroxidation. Sera from TCE-treated mice showed significant levels of antibodies against MDA-and HNE-adducted proteins along with antinuclear antibodies. This suggested that TCE exposure not only caused increased lipid peroxidation, but also accelerated autoimmune responses. Furthermore, stimulation of cultured splenic lymphocytes from both control and TCE-treated mice with MDA-adducted mouse serum albumin (MDA-MSA) or HNE-MSA for 72 h showed significant proliferation of CD4+ T cells in TCE-treated mice as analyzed by flow cytometry. Also, splenic lymphocytes from TCE-treated mice released more IL-2 and IFN-gamma into cultures when stimulated with MDA-MSA or HNE-MSA, suggesting a Th1 cell activation. Thus, our data suggest a role for lipid peroxidation-derived aldehydes in TCE-mediated autoimmune responses and involvement of Th1 cell activation.
Asunto(s)
Aldehídos/química , Autoinmunidad , Linfocitos T CD4-Positivos/citología , Peroxidación de Lípido , Tricloroetileno/química , Animales , Femenino , Radicales Libres , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Ratones , Modelos Biológicos , Solventes/farmacología , Bazo/citología , Células TH1/metabolismoRESUMEN
AIMS: To understand the mechanism(s) of alcoholic pancreatitis and role of fatty acid ethyl esters (FAEEs, non-oxidative metabolites of ethanol) in ethanol-induced pancreatic injury. METHODS: A time- and concentration-dependent synthesis of FAEEs and the cytotoxicity of ethanol and its predominant fatty acid esters were studied in rat pancreatic tumour (AR42J) cells in cultures. Role of FAEEs in ethanol-induced cytotoxicity was investigated by measuring the synthesis of FAEEs, injury markers and apoptosis in cells incubated simultaneously with ethanol and FAEE synthase inhibitor, 3-benzyl-6-chloro-2-pyrone. The cells were pre-incubated with caspase-3 inhibitor (N-acetyl-DEVD-CHO) to measure the effect of caspase-3 inhibition on ethanol-induced apoptosis. RESULTS: The levels of FAEEs synthesized in cell cultures incubated with 800 mg% ethanol for 6 h were approximately 10-fold higher (60 nmol/25 x 10(6) cells) than those in cells incubated with 100 mg% ethanol (5.4 nmol/25 x 10(6) cells). Ethanol exposure resulted in a concentration-dependent apoptosis (10, 12 and 13% at 200, 400 and 800 mg% ethanol, respectively, vs 5% in controls). A similar concentration-dependent apoptosis was also found in the cells incubated with ethyl oleate (one of the predominant FAEEs reported in alcoholic patients). Inhibition of FAEE synthesis and resultant apoptosis was found in the cells incubated simultaneously with pancreatic FAEE synthase inhibitor and ethanol. Ethanol-induced apoptosis was significantly inhibited in cells pre-incubated with caspase-3 inhibitor. CONCLUSIONS: These results support our hypothesis that ethanol-induced cytotoxicity in AR42J cells is mediated by the non-oxidative metabolite(s) of ethanol, and caspase-3 mediated apoptosis could be one of the mechanisms involved in ethanol-induced pancreatic injury.
Asunto(s)
Citotoxinas/toxicidad , Etanol/toxicidad , Ácidos Grasos/toxicidad , Páncreas Exocrino/efectos de los fármacos , Páncreas Exocrino/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Línea Celular Tumoral , Citotoxinas/biosíntesis , Citotoxinas/fisiología , Relación Dosis-Respuesta a Droga , Ésteres , Ácidos Grasos/biosíntesis , Ácidos Grasos/fisiología , Humanos , Páncreas Exocrino/citología , RatasRESUMEN
Exposure to trichloroethene (TCE), an occupational and ubiquitous environmental contaminant, is associated with the development of several autoimmune diseases, including autoimmune hepatitis (AIH). However, mechanisms contributing to TCE-mediated AIH are not known. Earlier, we have shown that dichloroacetyl chloride (DCAC), one of the reactive metabolites of TCE with strong acylating capability, can elicit an autoimmune response at much lower dose than TCE in female MRL+/+ mice. Furthermore, Kupffer cells (KCs), the liver resident macrophages, are crucial for hepatic homeostasis, but can also participate in the immunopathogenesis of AIH. However, contribution of KCs in TCE-mediated AIH and the underlying mechanisms are not understood. We hypothesized that increased apoptosis and delayed clearance of apoptotic bodies, due to compromised KC function, will result in the breakdown of self-tolerance, autoimmunity, and ultimately AIH. Therefore, using an in vitro model of immortalized mouse KCs, we investigated the contribution of DCAC in TCE-mediated AIH. KCs were treated with different concentrations of DCAC and apoptosis was measured by Annexin V and PI staining. Also, the impact of DCAC on phagocytic potential of KCs was evaluated. Furthermore, markers of inflammasome (NLRP3 and caspase1) were analyzed by real-time PCR and Western blot analysis. DCAC treatment resulted in significantly increased early and late-stage apoptosis, accompanied with inflammasome activation (NLRP3 increases). DCAC treatment resulted in decreased phagocytic function of KCs in a dose-dependent manner, with reduced MFG-E8 levels (phagocytotic function). Furthermore, DCAC exposure led to induction of phos-ERK and phos-AKT signaling. These findings suggest that DCAC induces apoptosis and inflammasome activation, while compromising the phagocytic function of KCs. Our data support that increased apoptosis and impaired KC function by DCAC could be contributory to TCE-mediated AIH.