RESUMEN
BACKGROUND AND AIMS: Pretreatment with clofibrate, a peroxisome proliferator-activated receptor alpha (PPARa) agonist, protects mice from acetaminophen (APAP) injury. Protection is not due to alterations in APAP metabolism and is dependent on PPARa expression. Gene array analysis revealed that mice receiving clofibrate have enhanced hepatic Vanin-1 (Vnn1) gene expression, a response that is also PPARa dependent. METHODS: We examined the role of Vnn1 by comparing the responses of Vnn1 knockout and wild-type mice following APAP hepatotoxicity. APAP metabolism, hepatotoxicity, and compensatory hepatocyte proliferation and immune responses were assessed. RESULTS: Vnn1 knockout mice are more susceptible to APAP hepatotoxicity despite no differences in hepatic glutathione content, gene expression of APAP metabolizing enzymes, or hepatic capacity to bioactivate or detoxify APAP ex vivo. Together, these data strongly suggest that the susceptibility of Vnn1 knockout mice is not due to differences in APAP metabolism. Immunochemistry revealed a lack of proliferating cell nuclear antigen-positive hepatocytes and F4/80-positive macrophages in and around areas of centrilobular necrosis in APAP-treated Vnn1 knockouts. Hepatic gene induction of pro-inflammatory cytokines was either significantly reduced or completely blunted in these mice. This was correlated with a reduction in early recruitment of cells positive for granulocyte differentiation antigen 1 or integrin alpha M. Heightened toxicity was also observed in CCl4 and ConA hepatitis models in the absence of Vnn1. CONCLUSIONS: These results indicate that mice lacking Vnn1 have deficiencies in compensatory repair and immune responses following toxic APAP exposure and that these mechanisms may contribute to the enhanced hepatotoxicity seen.
Asunto(s)
Acetaminofén/efectos adversos , Amidohidrolasas/deficiencia , Proliferación Celular/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/inmunología , Hígado/inmunología , Acetaminofén/farmacología , Amidohidrolasas/inmunología , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Clofibrato/farmacología , Proteínas Ligadas a GPI/deficiencia , Proteínas Ligadas a GPI/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Hepatocitos/inmunología , Hepatocitos/patología , Hígado/patología , Macrófagos/inmunología , Macrófagos/patología , Ratones , Ratones Noqueados , PPAR alfa/genética , PPAR alfa/inmunologíaRESUMEN
In liver, cysteamine in all probability represents a "low-capacity, high-affinity" scavenger of ROS. The available body of evidence suggests that reduced cysteamine and oxidized cystamine exist in equilibrium and that this ratio acts as an active redox sensor within the cell much like GSH. During normal liver homeostasis cysteamine's antioxidant properties are evident. Highly metabolic and/or pro-oxidative conditions, such as in mice treated with peroxisome proliferators, shift this equilibrium to favor the oxidized form. Under these conditions, cystamine is likely able to inactivate proteins involved in energy biogenesis through cysteaminylation of critical Cys residues as has been shown in vitro. This would allow cystamine to function as a "metabolic brake" to prevent the formation of additional ROS. In vivo, subcellular localization, pH, reducing capacity, FMO status and metabolic rate are all probable factors in determining the cysteamine:cystamine ratio. The availability of free cysteamine is also regulated by hydrolysis of pantetheine by pantetheinase. This cleavage results in the formation of pantothenic acid, a precursor to Coenzyme A which is prominently involved with lipid metabolism and energy production by the ß -oxidation pathway and TCA cycle, respectively. Expression of pantetheinase is controlled by the Vnn1 gene and is upregulated in response to free fatty acids, PPAR activation or oxidative stress. The use of Vnn1 knockout mice has provided clear evidence that Vnn1 modulates redox and immune pathways In vivo, both of which appear at least partially due to a loss of cysteamine/cystamine. Immunologically, Vnn1 expression may influence cell signaling indirectly through maintenance of disulfide bonds or directly by interaction of pantetheinase on the cell surface. Cysteamine treatment has been used clinically as an antidote to APAP poisoning and in animal models against hepatotoxicants including APAP, galactosamine and CCl4. Protection in animal models occurs even when administered up to 12 hours following intoxication, suggesting that protection is the result of effects that occur downstream of bioactivation and covalent binding of reactive metabolites to target cellular macromolecules. Currently, the downstream influences of Vnn1 expression and cysteamine at endogenous concentrations remain largely unknown. Vnn1 knockout mice represent a valuable tool available to researchers investigating these events. Future studies in the field are needed to elucidate the precise mechanisms by which pantetheinase and/or cysteamine impact immune cell recruitment, cell signaling and survival, though it is clear that these factors have far reaching implications in the fields of immunology and toxicology.
Asunto(s)
Amidohidrolasas/metabolismo , Biocatálisis , Hígado/enzimología , Hígado/metabolismo , Estrés Oxidativo , Animales , Proteínas Ligadas a GPI/metabolismo , HumanosRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, representing a spectrum of liver pathologies that include simple hepatic steatosis and the more advanced nonalcoholic steatohepatitis (NASH). The current study was conducted to determine whether pediatric NASH also results in altered disposition of acetaminophen (APAP) and its two primary metabolites, APAP-sulfate and APAP-glucuronide. Pediatric patients with hepatic steatosis (n = 9) or NASH (n = 3) and healthy patients (n = 12) were recruited in a small pilot study design. All patients received a single 1000-mg dose of APAP. Blood and urine samples were collected at 1, 2, and 4 hours postdose, and APAP and APAP metabolites were determined by high-performance liquid chromatography. Moreover, human liver tissues from patients diagnosed with various stages of NAFLD were acquired from the Liver Tissue Cell Distribution System to investigate the regulation of the membrane transporters, multidrug resistance-associated protein 2 and 3 (MRP2 and MRP3, respectively). Patients with the more severe disease (i.e., NASH) had increased serum and urinary levels of APAP-glucuronide along with decreased serum levels of APAP-sulfate. Moreover, an induction of hepatic MRP3 and altered canalicular localization of the biliary efflux transporter, MRP2, describes the likely mechanism for the observed increase in plasma retention of APAP-glucuronide, whereas altered regulation of sulfur activation genes may explain decreased sulfonation activity in NASH. APAP-glucuronide and APAP-sulfate disposition is altered in NASH and is likely due to hepatic membrane transporter dysregulation as well as altered intracellular sulfur activation.
Asunto(s)
Acetaminofén/farmacocinética , Analgésicos no Narcóticos/farmacocinética , Hígado/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Regulación hacia Arriba , Acetaminofén/análogos & derivados , Acetaminofén/sangre , Acetaminofén/orina , Adolescente , Analgésicos no Narcóticos/sangre , Analgésicos no Narcóticos/orina , Canalículos Biliares/metabolismo , Canalículos Biliares/patología , Biotransformación , Niño , Estudios de Cohortes , Hígado Graso/sangre , Hígado Graso/metabolismo , Hígado Graso/patología , Hígado Graso/orina , Femenino , Humanos , Hígado/patología , Masculino , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Enfermedad del Hígado Graso no Alcohólico/sangre , Enfermedad del Hígado Graso no Alcohólico/patología , Enfermedad del Hígado Graso no Alcohólico/orina , Proyectos Piloto , Transporte de ProteínasRESUMEN
Germ cells are unique in their ability to transfer genetic information and traits from generation to generation. As such, the proper development of germ cells and the integrity of their genome are paramount to the health of organisms and the survival of species. Germ cells are also exquisitely sensitive to environmental influences although the testing of germ cell toxicity, especially in females, has proven particularly challenging. In this review, we first describe the remarkable odyssey of germ cells in mammals, with an emphasis on the female germline, from their initial specification early during embryogenesis to the generation of mature gametes in adults. We also describe the current methods used in germ cell toxicity testing and their limitations in examining the complex features of mammalian germ cell development. To bypass these challenges, we propose the use of alternative model systems such as Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, and in vitro germ cell methods that have distinct advantages over traditional toxicity models. We discuss the benefits and limitations of each approach, their application to germ cell toxicity studies, and the need for computational approaches to maximize the usefulness of these models. Together, the inclusion of these alternative germ cell toxicity models will be invaluable for the examination of stages not easily accessible in mammals as well as the large scale, high-throughput investigation of germ cell toxicity.
Asunto(s)
Células Germinativas/efectos de los fármacos , Células Germinativas/fisiología , Pruebas de Toxicidad/métodos , Animales , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/genética , Células Cultivadas , Drosophila melanogaster/genética , Femenino , Masculino , Mamíferos , Meiosis , Pruebas de Mutagenicidad/métodos , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Toxicología/métodosRESUMEN
The UDP-glucuronosyltransferases (UGTs) and sulfotransferases (SULTs) represent major phase II drug-metabolizing enzymes that are also responsible for maintaining cellular homeostasis by metabolism of several endogenous molecules. Perturbations in the expression or function of these enzymes can lead to metabolic disorders and improper management of xenobiotics and endobiotics. Nonalcoholic fatty liver disease (NAFLD) represents a spectrum of liver damage ranging from steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis. Because the liver plays a central role in the metabolism of xenobiotics, the purpose of the current study was to determine the effect of human NAFLD progression on the expression and function of UGTs and SULTs in normal, steatosis, NASH (fatty), and NASH (not fatty/cirrhosis) samples. We identified upregulation of UGT1A9, 2B10, and 3A1 and SULT1C4 mRNA in both stages of NASH, whereas UGT2A3, 2B15, and 2B28 and SULT1A1, 2B1, and 4A1 as well as 3'-phosphoadenosine-5'-phosphosulfate synthase 1 were increased in NASH (not fatty/cirrhosis) only. UGT1A9 and 1A6 and SULT1A1 and 2A1 protein levels were decreased in NASH; however, SULT1C4 was increased. Measurement of the glucuronidation and sulfonation of acetaminophen (APAP) revealed no alterations in glucuronidation; however, SULT activity was increased in steatosis compared with normal samples, but then decreased in NASH compared with steatosis. In conclusion, the expression of specific UGT and SULT isoforms appears to be differentially regulated, whereas sulfonation of APAP is disrupted during progression of NAFLD.