RESUMEN
Preplacement of compensatory tissue repair (CTR) by exposure to a nonlethal dose of a toxicant protects animals against a lethal dose of another toxicant. Although CTR is known to heteroprotect, the underlying molecular mechanisms are not completely known. Here, we investigated the mechanisms of heteroprotection using thioacetamide (TA): acetaminophen (APAP) heteroprotection model. Male Swiss Webster mice received a low dose of TA or distilled water (DW) vehicle 24 hours prior to a lethal dose of APAP. Liver injury, tissue repair, and promitogenic signaling were studied over a time course of 24 hours after APAP overdose to the TA- and DW-primed mice (TA + APAP and DW + APAP, respectively). Thioacetamide pretreatment afforded 100% protection against APAP overdose compared to 100% lethality in the DW + APAP-treated mice. Although hepatic Cyp2e1 was similar at the time of APAP administration, immediate activation of hepatic c-Jun N-terminal kinases (JNK) was observed in the TA + APAP-treated mice compared to its delayed activation in the DW + APAP group. In contrast to the DW + APAP group, the TA + APAP-treated mice exhibited extensive CTR, which was secondary to the timely activation of Wnt/ß-catenin pathway. Our data indicate that rapid activation and appropriate termination of Wnt/ß-catenin signaling and modulation of JNK activity underlie TA + APAP heteroprotection.
RESUMEN
Compensatory tissue repair (CTR) in thioacetamide (TA)-primed rats protects them against acetaminophen (APAP)-induced lethality. This study was aimed at investigating the mechanisms of CTR-mediated heteroprotection in mice. Male Swiss Webster mice received a priming dose of TA (40 mg/kg body weight [BW] in 10 mL distilled water [DW]/kg BW, intraperitoneally [IP]). Thioacetamide-induced liver injury, CTR, and expression of annexin A1 and A2 (ANX1 and ANX2), the endogenous inhibitors of the death protein secretory phospholipase A2 (sPLA2), were measured over a time course of 84 hours after TA priming. Both centrilobular necrosis and CTR peaked at 36 hours after TA priming as indicated by significantly increased plasma alanine transaminase (ALT) and aspartate transaminase (AST) activities, liver histology, and proliferating cell nuclear antigen immunostaining. Thioacetamide priming resulted in the overexpression of ANX1 and ANX2 at 36 to 84 hours and 12 to 60 hours, respectively. A lethal dose of APAP (600 mg/kg BW in 10 mL 0.45% NaCl/kg BW, IP) was given at 12, 24, or 36 hours after TA-priming. Thioacetamide priming did not affect the rise in plasma ALT, AST, sPLA2, and arachidonic acid levels seen at 2 hours after the APAP overdose. Neither these biochemical parameters nor histology suggested any escalation of hepatic injury at later time points (12 and 24 hours after APAP overdose), consistent with 100% survival of the TA + APAP-treated mice compared to DW + APAP-treated mice, which had 100% mortality. Inhibition of ANX1 and ANX2 biosynthesis using cycloheximide (40 mg/kg BW in 5 mL DW/kg BW, IP) abolished this heteroprotection. Our data indicate that hepatic overexpression of ANX1 and ANX2 inhibits APAP-induced expansion of liver injury.
Asunto(s)
Acetaminofén , Anexina A1/metabolismo , Anexina A2/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Fallo Hepático/metabolismo , Tioacetamida , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Membrana Celular/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Sistema Enzimático del Citocromo P-450/metabolismo , Glutatión/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Fallo Hepático/sangre , Fallo Hepático/inducido químicamente , Fallo Hepático/patología , Masculino , RatonesRESUMEN
Once initiated, how tissue injury expands after high toxicant doses, even after their complete elimination, is not understood. Free-radical generation was initially proposed to mediate progression of injury. However, mechanisms proposed thus far have remained unsubstantiated. Necrotic injury is characterized by loss of osmoregulation, cell swelling, blebbing, and cell rupture. This exposes cytosolic enzymes, including proteases, phospholipases, and lysosomal Ca(2+)-dependent enzymes, to high extracellular calcium (Ca(2+)). Activated hydrolytic enzymes, termed 'death proteins,' hydrolyze their substrates in the plasma membrane of neighboring cells, commencing self-perpetuated injury progression. Likewise, ischemia-reperfusion injury exposes the hydrolytic enzymes to high Ca(2+), fuelling the progression of tissue injury. This mechanism is independent of the offending toxicant that initiates the injury. I present here a case for therapeutic intervention with inhibitors directed against death proteins as a means to avert organ failure and death well after the poisoning event.
Asunto(s)
Traumatismos de los Tejidos Blandos/inducido químicamente , Traumatismos de los Tejidos Blandos/metabolismo , Acetaminofén/farmacocinética , Acetaminofén/toxicidad , Animales , Calpaína/metabolismo , Muerte Celular/fisiología , Progresión de la Enfermedad , Radicales Libres/metabolismo , Humanos , Traumatismos de los Tejidos Blandos/patologíaRESUMEN
We have previously reported that among the other death proteins, hepatic secretory phospholipase A2 (sPLA2) is a leading mediator of progression of liver injury initiated by CCl4 in rats. The aim of our present study was to test the hypothesis that increased hepatic sPLA2 released after acetaminophen (APAP) challenge mediates progression of liver injury in wild type (WT) and COX-2 knockout (KO) mice. COX-2 WT and KO mice were administered a normally non lethal dose (400 mg/kg) of acetaminophen. The COX-2 KO mice suffered 60% mortality compared to 100% survival of the WT mice, suggesting higher susceptibility of COX-2 KO mice to sPLA2-mediated progression of acetaminophen hepatotoxicity. Liver injury was significantly higher at later time points in the KO mice compared to the WT mice indicating that the abatement of progression of injury requires the presence of COX-2. This difference in hepatotoxicity was not due to increased bioactivation of acetaminophen as indicated by unchanged cyp2E1 protein and covalently bound ¹4C-APAP in the livers of KO mice. Hepatic sPLA2 activity and plasma TNF-α were significantly higher after APAP administration in the KO mice. This was accompanied by a corresponding fall in hepatic PGE2 and lower compensatory liver regeneration and repair (³H-thymidine incorporation) in the KO mice. These results suggest that hindered compensatory tissue repair and poor resolution of inflammation for want of beneficial prostaglandins render the liver very vulnerable to sPLA2-mediated progression of liver injury. These findings are consistent with the destructive role of sPLA2 in the progression and expansion of tissue injury as a result of continued hydrolytic breakdown of plasma membrane phospholipids of perinecrotic hepatocytes unless mitigated by sufficient co-induction of COX-2.
Asunto(s)
Acetaminofén/toxicidad , Analgésicos no Narcóticos/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Ciclooxigenasa 2/genética , Fosfolipasas A2 Secretoras/metabolismo , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/enzimología , Enfermedad Hepática Inducida por Sustancias y Drogas/fisiopatología , Dinoprostona/metabolismo , Progresión de la Enfermedad , Regeneración Hepática/fisiología , Masculino , Ratones , Ratones Noqueados , Sobrevida , Factores de Tiempo , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Often, chemotherapy by doxorubicin (Adriamycin) is limited due to life threatening cardiotoxicity in patients during and posttherapy. Recently, we have shown that moderate diet restriction remarkably protects against doxorubicin-induced cardiotoxicity. This cardioprotection is accompanied by decreased cardiac oxidative stress and triglycerides and increased cardiac fatty-acid oxidation, ATP synthesis, and upregulated JAK/STAT3 pathway. In the current study, we investigated whether a physiological intervention by feeding 40% high fat diet (HFD), which induces obesity in male Sprague-Dawley rats (250-275 g), sensitizes to doxorubicin-induced cardiotoxicity. A LD(10) dose (8 mg doxorubicin/kg, ip) administered on day 43 of the HFD feeding regimen led to higher cardiotoxicity, cardiac dysfunction, lipid peroxidation, and 80% mortality in the obese (OB) rats in the absence of any significant renal or hepatic toxicity. Doxorubicin toxicokinetics studies revealed no change in accumulation of doxorubicin and doxorubicinol (toxic metabolite) in the normal diet-fed (ND) and OB hearts. Mechanistic studies revealed that OB rats are sensitized due to: (1) higher oxyradical stress leading to upregulation of uncoupling proteins 2 and 3, (2) downregulation of cardiac peroxisome proliferators activated receptor-alpha, (3) decreased plasma adiponectin levels, (4) decreased cardiac fatty-acid oxidation (666.9+/-14.0 nmol/min/g heart in ND versus 400.2+/-11.8 nmol/min/g heart in OB), (5) decreased mitochondrial AMP-alpha2 protein kinase, and (6) 86% drop in cardiac ATP levels accompanied by decreased ATP/ADP ratio after doxorubicin administration. Decreased cardiac erythropoietin and increased SOCS3 further downregulated the cardioprotective JAK/STAT3 pathway. In conclusion, HFD-induced obese rats are highly sensitized to doxorubicin-induced cardiotoxicity by substantially downregulating cardiac mitochondrial ATP generation, increasing oxidative stress and downregulating the JAK/STAT3 pathway.
Asunto(s)
Grasas de la Dieta/efectos adversos , Doxorrubicina/toxicidad , Cardiopatías/sangre , Cardiopatías/inducido químicamente , Obesidad/sangre , Animales , Biomarcadores/sangre , Cardiopatías/complicaciones , Masculino , Obesidad/complicaciones , Ratas , Ratas Sprague-DawleyRESUMEN
The objective was to investigate if the hepatotoxic sensitivity in nonalcoholic steatohepatitic mice to acetaminophen (APAP) is due to downregulation of nuclear receptor PPARalpha via lower cell division and tissue repair. Male Swiss Webster mice fed methionine and choline deficient diet for 31 days exhibited NASH. On the 32nd day, a marginally toxic dose of APAP (360 mg/kg, ip) yielded 70% mortality in steatohepatitic mice, while all non steatohepatitic mice receiving the same dose survived. (14)C-APAP covalent binding, CYP2E1 protein, and enzyme activity did not differ from the controls, obviating increased APAP bioactivation as the cause of amplified APAP hepatotoxicity. Liver injury progressed only in steatohepatitic livers between 6 and 24 h. Cell division and tissue repair assessed by (3)H-thymidine incorporation and PCNA were inhibited only in the steatohepatitic mice given APAP suggesting that higher sensitivity of NASH liver to APAP-induced hepatotoxicity was due to lower tissue repair. The hypothesis that impeded liver tissue repair in steatohepatitic mice was due to downregulation of PPARalpha was tested. PPARalpha was downregulated in NASH. To investigate whether downregulation of PPARalpha in NASH is the critical mechanism of compromised liver tissue repair, PPARalpha was induced in steatohepatitic mice with clofibrate (250 mg/kg for 3 days, ip) before injecting APAP. All clofibrate pretreated steatohepatitic mice receiving APAP exhibited lower liver injury, which did not progress and the mice survived. The protection was not due to lower bioactivation of APAP but due to higher liver tissue repair. These findings suggest that inadequate PPARalpha expression in steatohepatitic mice sensitizes them to APAP hepatotoxicity.
Asunto(s)
Acetaminofén/toxicidad , Clofibrato/farmacología , Hígado Graso/metabolismo , Hígado/efectos de los fármacos , PPAR alfa/biosíntesis , Adenosina Trifosfato/análisis , Alanina Transaminasa/sangre , Animales , Citocromo P-450 CYP2E1/metabolismo , Citoprotección , Replicación del ADN , Masculino , Ratones , Estrés Oxidativo , Antígeno Nuclear de Célula en Proliferación/análisisRESUMEN
Administration of a low priming dose of 2-butoxyethanol (BE, 500 mg/kg, p.o.) 7 days prior to a larger LD(90) dose (1500 mg BE/kg, p.o.) offers protection against the lethal dose-induced hemolysis and death in female Sprague Dawley rats because of prompt and efficient replacement of red blood cells (RBCs) with new resilient RBCs. The objective of the present work was to analyze the altered proteome of RBCs upon priming with BE in order to identify the potential anti-hemolytic survival proteins induced in the primed rat RBCs (P-RBCs) as opposed to vehicle-treated RBCs (V-RBCs). The RBCs from the two groups were fractionated into membrane and cytosolic fractions. The cytosolic fractions were further fractionated for proteomic analysis into 3 fractions. The fractions were labeled with Cy3 and Cy5 fluorescent dyes and subjected to 2-dimensional differential gel electrophoresis (DIGE) to analyze the protein profiles. Seven membrane and 8 cytosolic proteins were found to be significantly increased (> or =2.5 fold) in P-RBCs as compared to V-RBCs. The identified proteins can be classified into antioxidant, membrane skeleton, protein turnover, lipid raft, and energy metabolism components. Increased levels of the proteins from antioxidant and membrane skeleton groups were confirmed by Western blot analysis. The study provides the first report on protein profiling of rat RBCs as well as on alteration of the proteome upon exposure to a priming dose of hemotoxicant. Further studies are needed to prove the protective role of the identified proteins and will initiate the field of survival/protective/anti-hemolytic proteins in RBCs.
Asunto(s)
Proteínas Sanguíneas/análisis , Eritrocitos/efectos de los fármacos , Glicoles de Etileno/toxicidad , Proteoma , Animales , Catalasa/sangre , Citosol/química , Metabolismo Energético , Eritrocitos/química , Femenino , Glutatión/metabolismo , Hematócrito , Microdominios de Membrana/química , Proteínas de la Membrana/análisis , Ratas , Ratas Sprague-DawleyRESUMEN
Our previous study [Bhave, V. S., Donthamsetty, S., Latendresse, J. R., Muskhelishvili, L., and Mehendale, H. M. 2008-this issue. Secretory phospholipase A(2) mediates progression of acute liver injury in the absence of sufficient COX-2. Toxicol Appl Pharmacol] showed that in the absence of sufficient induction and co-presence of cyclooxygenase-2 (COX-2), secretory phospholipase A(2) (sPLA(2)) appearing in the intercellular spaces for cleanup of post-necrotic debris seems to contribute to the progression of toxicant-initiated liver injury, possibly by hydrolysis of membrane phospholipids of hepatocytes in the perinecrotic areas. To further test our hypothesis on the protective role of COX-2, male Fisher-344 rats were administered a selective COX-2 inhibitor, NS-398, and then challenged with a moderately toxic dose of CCl(4). This led to a 5-fold increase in the susceptibility of the COX-2 inhibited rats to CCl(4) hepatotoxicity and mortality. The CCl(4) bioactivating enzyme CYP2E1 protein, CYP2E1 enzyme activity, and the (14)CCl(4)-derived radiolabel covalently bound to the liver proteins were unaffected by the COX-2 inhibitor suggesting that the increased hepatotoxic sensitivity of the COX-2 inhibited rats was not due to higher bioactivation of CCl(4). Further investigation showed that this increased mortality was due to higher plasma and hepatic sPLA(2) activities, inhibited PGE(2) production, and progression of liver injury as compared to the non-intervened rats(.) In conclusion, inhibition of COX-2 mitigates the tissue protective mechanisms associated with COX-2 induction, which promotes sPLA(2)-mediated progression of liver injury in an acute liver toxicity model. Because increased sPLA(2) activity in the intercellular space is associated with increased progression of injury, and induced COX-2 is associated with hepatoprotection, ratios of hepatic COX-2 and sPLA(2) activities may turn out to be a useful tool in predicting the extent of hepatotoxicities.
Asunto(s)
Inhibidores de la Ciclooxigenasa 2/toxicidad , Ciclooxigenasa 2/metabolismo , Hígado/efectos de los fármacos , Fosfolipasas A2 Secretoras/metabolismo , Enfermedad Aguda , Alanina Transaminasa/sangre , Animales , Western Blotting , Radioisótopos de Carbono , Tetracloruro de Carbono/administración & dosificación , Tetracloruro de Carbono/toxicidad , Intoxicación por Tetracloruro de Carbono/mortalidad , Aceite de Maíz/administración & dosificación , Inhibidores de la Ciclooxigenasa 2/administración & dosificación , Citocromo P-450 CYP2E1/metabolismo , Dinoprostona/metabolismo , Progresión de la Enfermedad , Sinergismo Farmacológico , Hígado/lesiones , Hígado/metabolismo , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Nitrobencenos/administración & dosificación , Nitrobencenos/toxicidad , Nitrofenoles/metabolismo , Ratas , Ratas Endogámicas F344 , Sulfonamidas/administración & dosificación , Sulfonamidas/toxicidad , Tasa de SupervivenciaRESUMEN
Previous studies have shown that injury initiated by toxicants progresses even after most of the toxicant is eliminated from the body. One mechanism of progression of injury is the extracellular appearance of hydrolytic enzymes following leakage or upon cell lyses. Under normal conditions, after exposure to low to moderate doses of toxicants, secretory phospholipase A(2) (sPLA(2)) and other hydrolytic enzymes are known to appear in the extracellular spaces in order to cleanup the post-necrotic debris in tissues. We tested the hypothesis that sPLA(2) contributes to progression of toxicant-initiated liver injury because of hydrolysis of membrane phospholipids of hepatocytes in the perinecrotic areas in the absence of sufficient cyclooxygenase-2 (COX-2). Male Sprague-Dawley rats were administered either a moderately hepatotoxic dose (MD, 2 ml CCl(4)/kg, ip) or a highly hepatotoxic dose (HD, 3 ml CCl(4)/kg, ip) of CCl(4). After MD, liver sPLA(2) and COX-2 were co-localized in the necrotic and perinecrotic areas and their activities in plasma and liver increased before decreasing in tandem with liver injury (ALT and histopathology) leading to 100% survival. In contrast, after the HD, high extracellular and hepatic sPLA(2) activities were accompanied by minimal COX-2 activity and localization in the liver throughout the time course. This led to progression of liver injury and 70% mortality. These data suggested a destructive role of sPLA(2) in the absence of sufficient COX-2. Time- and dose-dependent destruction of hepatocytes by sPLA(2) in isolated hepatocyte incubations confirmed the destructive ability of sPLA(2) when present extracellularly, suggesting its ability to spread injury in vivo. These findings suggest that sPLA(2), secreted for cleanup of necrotic debris upon initiation of hepatic necrosis, requires the co-presence of sufficiently induced COX-2 activity to prevent the run-away destructive action of sPLA(2) in the absence of the tissue protective mechanisms afforded by COX-2 induction.
Asunto(s)
Ciclooxigenasa 2/metabolismo , Hepatocitos/efectos de los fármacos , Hígado/efectos de los fármacos , Fosfolipasas A2 Secretoras/metabolismo , Enfermedad Aguda , Alanina Transaminasa/metabolismo , Animales , Tetracloruro de Carbono/administración & dosificación , Tetracloruro de Carbono/toxicidad , Intoxicación por Tetracloruro de Carbono/mortalidad , Supervivencia Celular/efectos de los fármacos , Ciclooxigenasa 1/metabolismo , Dinoprostona/metabolismo , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Hepatocitos/metabolismo , Hepatocitos/patología , Inmunohistoquímica , Inyecciones Intraperitoneales , L-Lactato Deshidrogenasa/metabolismo , Hígado/lesiones , Hígado/metabolismo , Masculino , Modelos Biológicos , Ratas , Ratas Sprague-Dawley , Tasa de Supervivencia , Factores de TiempoRESUMEN
Membrane transporters are critical for the uptake as well as elimination of chemicals and by-products of metabolism from the liver and kidneys. Since these proteins are important determinants of chemical disposition, changes in their expression in different disease states can modulate drug pharmacokinetics. The present study investigated alterations in the renal and hepatic expression of organic anion and cation transporters (Oats/Octs), multidrug resistance-associated proteins (Mrps), breast cancer resistance protein (Bcrp), P-glycoprotein (Pgp), and hepatic Na(+)-taurocholate cotransporting polypeptide (Ntcp) in type 2 diabetic rats. For this purpose, type 2 diabetes was induced by feeding male Sprague-Dawley rats a high fat diet followed by a single dose of streptozotocin (45 mg/kg, i.p., in 0.01 M citrate buffer pH 4.3) on day 14. Controls received normal diet and vehicle. Kidney and liver samples were collected on day 24 for generation of crude plasma membrane fractions and Western blot analysis of Oat, Oct, Mrp, Bcrp, Pgp, and Ntcp proteins. With regards to renal uptake transporters, type 2 diabetes increased levels of Oat2 (2.3-fold) and decreased levels of Oct2 to 50% of control kidneys. Conversely, efflux transporters Mrp2, Mrp4, and Bcrp were increased 5.4-fold, 2-fold, and 1.6-fold, respectively in type 2 diabetic kidneys with no change in levels of Mrp1, Mrp5, or Pgp. Studies of hepatic transporters in type 2 diabetic rats reveal that the protein level of Mrp5 was reduced to 4% of control livers with no change in levels of Bcrp, Mrp1, Mrp2, Mrp4, Ntcp, or Pgp. The changes reported in this study may have implications in type 2 diabetic patients.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Regulación de la Expresión Génica , Proteínas de Transporte de Membrana/genética , Animales , Western Blotting , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Tipo 2/fisiopatología , Riñón/metabolismo , Riñón/fisiopatología , Hígado/metabolismo , Hígado/fisiopatología , Masculino , Proteínas de Transporte de Membrana/metabolismo , Preparaciones Farmacéuticas/metabolismo , Ratas , Ratas Sprague-Dawley , EstreptozocinaRESUMEN
Clinical use of doxorubicin (Adriamycin), an antitumor agent, is limited by its oxyradical-mediated cardiotoxicity. We tested the hypothesis that moderate diet restriction protects against doxorubicin-induced cardiotoxicity by decreasing oxidative stress and inducing cardioprotective mechanisms. Male Sprague-Dawley rats (250-275 g) were maintained on diet restriction [35% less food than ad libitum]. Cardiotoxicity was estimated by measuring biomarkers of cardiotoxicity, cardiac function, lipid peroxidation, and histopathology. A LD(100) dose of doxorubicin (12 mg/kg, ip) administered on day 43 led to 100% mortality in ad libitum rats between 7 and 13 days due to higher cardiotoxicity and cardiac dysfunction, whereas all the diet restricted rats exhibited normal cardiac function and survived. Toxicokinetic analysis revealed equal accumulation of doxorubicin and doxorubicinol (toxic metabolite) in the ad libitum and diet restricted hearts. Mechanistic studies revealed that diet restricted rats were protected due to (1) lower oxyradical stress from increased cardiac antioxidants leading to downregulation of uncoupling proteins 2 and 3, (2) induction of cardiac peroxisome proliferators activated receptor-alpha and plasma adiponectin increased cardiac fatty acid oxidation (666.9+/-14.0 nmol/min/g heart in ad libitum versus 1035.6+/-32.3 nmol/min/g heart in diet restriction) and mitochondrial AMPalpha2 protein kinase. The changes led to 51% higher cardiac ATP levels (17.7+/-2.1 micromol/g heart in ad libitum versus 26.7+/-1.9 micromol/g heart in diet restriction), higher ATP/ADP ratio, and (3) increased cardiac erythropoietin and decreased suppressor of cytokine signaling 3, which upregulates cardioprotective JAK/STAT3 pathway. These findings collectively show that moderate diet restriction renders resiliency against doxorubicin cardiotoxicity by lowering oxidative stress, enhancing ATP synthesis, and inducing the JAK/STAT3 pathway.
Asunto(s)
Antibióticos Antineoplásicos/toxicidad , Dieta , Doxorrubicina/toxicidad , Cardiopatías/inducido químicamente , Proteínas Quinasas Activadas por AMP , Adenosina Trifosfato/metabolismo , Adiponectina/sangre , Animales , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacocinética , Ácidos Grasos/metabolismo , Regulación de la Expresión Génica , Cardiopatías/prevención & control , Canales Iónicos/genética , Quinasas Janus/metabolismo , Masculino , Proteínas Mitocondriales/genética , Complejos Multienzimáticos/metabolismo , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , PPAR alfa/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Proteína Desacopladora 2 , Proteína Desacopladora 3RESUMEN
Protection against a high dose of a toxicant by prior exposure to another toxicant is called heteroprotection. Our objective was to establish a heteroprotection model in RBCs. Female Sprague Dawley rats treated with an LD90 dose of 2-butoxyethanol (BE, 1500 mg/kg in water, 5 ml/kg po) 14 days after priming with 0.9% NaCl suffered 90% mortality by 15 days, whereas all rats receiving the LD90 dose of BE 14 days after priming with phenylhydrazine (PHZ, 125 mg/kg in 0.9% NaCl, 3 ml/kg po) survived. Hematocrit decreased from normal 45% to 24% by day 3 after PHZ priming and improved thereafter. Increasing the time interval between the priming and LD90 dose to 21 days abolished the heteroprotection. RBCs obtained on days 7 and 14 after PHZ priming unlike those on day 21 were resilient to the hemotoxic metabolite of BE, butoxyacetic acid (BAA). Unaltered hepatic alcohol and aldehyde dehydrogenase activities upon PHZ priming suggested that bioactivation of BE to BAA was unaffected. Lower renal (6 and 12 h) and hepatic (12 h) BAA levels and 3 fold higher excretion of BAA in PHZ-primed rat urine suggested a protective role of toxicokinetics. Higher erythropoietin, reticulocytes, and resiliency of PHZ-primed rat RBCs indicated that newly formed RBCs are resilient to hemolytic BAA. The antioxidant levels in the PHZ-primed rat RBCs did not indicate a protective role in heteroprotection. In conclusion, the resistance of PHZ-primed rats against BE-induced hemotoxicity and lethality is mediated by a combination of altered toxicokinetics, robust erythropoiesis, and resiliency of new RBCs.
Asunto(s)
Eritrocitos/efectos de los fármacos , Glicoles de Etileno/toxicidad , Hemólisis/efectos de los fármacos , Oxidantes/farmacología , Fenilhidrazinas/farmacología , Aldehído Deshidrogenasa/efectos de los fármacos , Aldehído Deshidrogenasa/metabolismo , Animales , Antioxidantes/metabolismo , Eritrocitos/metabolismo , Eritropoyesis/efectos de los fármacos , Glicoles de Etileno/farmacocinética , Femenino , Glicolatos/farmacocinética , Glicolatos/toxicidad , Hematócrito , Ratas , Ratas Sprague-Dawley , Reticulocitos/efectos de los fármacos , Reticulocitos/metabolismo , Factores de TiempoAsunto(s)
Desarrollo Embrionario/efectos de los fármacos , Hidroxiurea/toxicidad , National Institute of Environmental Health Sciences (U.S.) , Reproducción/efectos de los fármacos , Toxicología , Animales , Vías de Administración de Medicamentos , Femenino , Humanos , Hidroxiurea/administración & dosificación , Hidroxiurea/efectos adversos , Hidroxiurea/química , Masculino , Embarazo , Estados UnidosRESUMEN
Previous studies (Vaidya VS, Shankar K, Lock EA, Bucci TJ, Mehendale HM. Toxicol Sci 74: 215-227, 2003; Korrapati MC, Lock EA, Mehendale HM. Am J Physiol Renal Physiol 289: F175-F185, 2005; Korrapati MC, Chilakapati J, Lock EA, Latendresse JR, Warbritton A, Mehendale HM. Am J Physiol Renal Physiol 291: F439-F455, 2006) demonstrated that renal repair stimulated by a low dose of S-(1,2-dichlorovinyl)l-cysteine (DCVC; 15 mg/kg i.p.) 72 h before administration of a normally lethal dose (75 mg/kg i.p.) protects mice from acute renal failure (ARF) and death (autoprotection). The present study identified the proteins indicative of DCVC-induced ARF and autoprotection in male Swiss Webster mice. Renal dysfunction and injury were assessed by plasma creatinine and histopathology, respectively. Whole-kidney homogenates were run on two-dimensional gel electrophoresis gels, and the expression of 18 common proteins was maximally changed (> or =10-fold) in all the treatment groups and they were conclusively identified by liquid chromatography tandem mass spectrometry. These proteins were mildly downregulated after low dose alone and in autoprotected mice in contrast to severe downregulation with high dose alone. Glucose-regulated protein 75 and proteasome alpha-subunit type 1 were further investigated by immunohistochemistry for their localization in the kidneys of all the groups. These proteins were substantially higher in the proximal convoluted tubular epithelial cells in the low-dose and autoprotected groups compared with high-dose alone group. Proteins involved in energetics were downregulated in all the three groups of mice, leading to a compromise in cellular energy. However, energy is recovered completely in low-dose and autoprotected mice. This study provides the first report on proteomics of DCVC-induced ARF and autoprotection in mice and reflects the application of proteomics in mechanistic studies as well as biomarker development in a variety of toxicological paradigms.
Asunto(s)
Lesión Renal Aguda/inducido químicamente , Lesión Renal Aguda/metabolismo , Cisteína/análogos & derivados , Riñón/metabolismo , Proteómica , Lesión Renal Aguda/mortalidad , Animales , Proteínas Reguladoras de la Apoptosis , Proteínas Portadoras/metabolismo , Coenzima A Ligasas/metabolismo , Creatinina/sangre , Cisteína/efectos adversos , Cisteína/farmacología , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/fisiología , Proteínas de Transporte de Ácidos Grasos/metabolismo , Depuradores de Radicales Libres/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Hemopexina/metabolismo , Riñón/efectos de los fármacos , Riñón/patología , Masculino , Proteínas de la Membrana/metabolismo , RatonesRESUMEN
Many biological subdisciplines that regularly assess dose-response relationships have identified an evolutionarily conserved process in which a low dose of a stressful stimulus activates an adaptive response that increases the resistance of the cell or organism to a moderate to severe level of stress. Due to a lack of frequent interaction among scientists in these many areas, there has emerged a broad range of terms that describe such dose-response relationships. This situation has become problematic because the different terms describe a family of similar biological responses (e.g., adaptive response, preconditioning, hormesis), adversely affecting interdisciplinary communication, and possibly even obscuring generalizable features and central biological concepts. With support from scientists in a broad range of disciplines, this article offers a set of recommendations we believe can achieve greater conceptual harmony in dose-response terminology, as well as better understanding and communication across the broad spectrum of biological disciplines.
Asunto(s)
Adaptación Fisiológica , Biología , Relación Dosis-Respuesta a Droga , Estrés Fisiológico , Terminología como Asunto , Animales , HumanosRESUMEN
The aim was to study the subchronic toxicity of perchloroethylene (Perc) by measuring injury and repair in liver and kidney in relation to disposition of Perc and its major metabolites. Male SW mice (25-29g) were given three dose levels of Perc (150, 500, and 1000 mg/kg day) via aqueous gavage for 30 days. Tissue injury was measured during the dosing regimen (0, 1, 7, 14, and 30 days) and over a time course of 24-96h after the last dose (30 days). Perc produced significant liver injury (ALT) after single day exposure to all three doses. Liver injury was mild to moderate and regressed following repeated exposure for 30 days. Subchronic Perc exposure induced neither kidney injury nor dysfunction during the entire time course as evidenced by normal renal histology and BUN. TCA was the major metabolite detected in blood, liver, and kidney. Traces of DCA were also detected in blood at initial time points after single day exposure. With single day exposure, metabolism of Perc to TCA was saturated with all three doses. AUC/dose ratio for TCA was significantly decreased with a concomitant increase in AUC/dose of Perc levels in liver and kidney after 30 days as compared to 1 day exposures, indicating inhibition of metabolism upon repeated exposure to Perc. Hepatic CYP2E1 expression and activity were unchanged indicating that CYP2E1 is not the critical enzyme inhibited. Hepatic CYP4A expression, measured as a marker of peroxisome proliferation was increased transiently only on day 7 with the high dose, but was unchanged at later time points. Liver tissue repair peaked at 7 days, with all three doses and was sustained after medium and high dose exposure for 14 days. These data indicate that subchronic Perc exposure via aqueous gavage does not induce nephrotoxicity and sustained hepatotoxicity suggesting adaptive hepatic repair mechanisms. Enzymes other than CYP2E1, involved in the metabolism of Perc may play a critical role in the metabolism of Perc upon subchronic exposure in SW mice. Liver injury decreased during repeated exposure due to inhibition of metabolism and possibly due to adaptive tissue repair mechanisms.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas , Contaminantes Ambientales/toxicidad , Enfermedades Renales/inducido químicamente , Tetracloroetileno/toxicidad , Alanina Transaminasa/sangre , Animales , Nitrógeno de la Urea Sanguínea , Citocromo P-450 CYP2E1/metabolismo , Citocromo P-450 CYP4A/metabolismo , Replicación del ADN/fisiología , Contaminantes Ambientales/administración & dosificación , Contaminantes Ambientales/farmacocinética , Glutatión/metabolismo , Histocitoquímica , Enfermedades Renales/enzimología , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Hepatopatías/enzimología , Hepatopatías/metabolismo , Hepatopatías/patología , Masculino , Ratones , Microsomas Hepáticos/enzimología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Tetracloroetileno/administración & dosificación , Tetracloroetileno/farmacocinética , Timidina/metabolismo , Ácido Tricloroacético/metabolismoRESUMEN
Liver injury initiated by non-lethal doses of CCl(4) and thioacetamide (TA) progresses to hepatic failure and death of type 2 diabetic (DB) rats due to failed advance of liver cells from G(0)/G(1) to S-phase and inhibited tissue repair. Objective of the present study was to investigate cellular signaling mechanisms of failed cell division in DB rats upon hepatotoxicant challenge. In CCl(4)-treated non-diabetic (non-DB) rats, increased IL-6 levels, sustained activation of extracellular regulated kinases 1/2 (ERK1/2) MAPK, and sustained phosphorylation of retinoblastoma protein (p-pRB) via cyclin D1/cyclin-dependent kinase (cdk) 4 and cyclin D1/cdk6 complexes stimulated G(0)/G(1) to S-phase transition of liver cells. In contrast to the non-DB rats, CCl(4) administration led to lower plasma IL-6, decreased ERK1/2 activation, lower cyclin D1, and cdk 4/6 expression resulting in decreased p-pRB and inhibition of liver cell division in the DB rats. Furthermore, higher TGFbeta1 expression and p21 activation may also contribute to decreased p-pRB in DB rats compared to non-DB rats. Similarly, after TA administration to DB rats, down-regulation of cyclin D1 and p-pRB leads to markedly decreased advance of liver cells from G(0)/G(1) to S-phase and tissue repair compared to the non-DB rats. Hepatic ATP levels did not differ between the DB and non-DB rats obviating its role in failed tissue repair in the DB rats. In conclusion, decreased p-pRB may contribute to blocked advance of cells from G(0)/G(1) to S-phase and failed cell division in DB rats exposed to CCl(4) or TA, leading to progression of liver injury and hepatic failure.
Asunto(s)
Intoxicación por Tetracloruro de Carbono/patología , Enfermedad Hepática Inducida por Sustancias y Drogas , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 2/patología , Hepatopatías/patología , Tioacetamida/toxicidad , Adenosina Trifosfato/metabolismo , Animales , Intoxicación por Tetracloruro de Carbono/enzimología , Intoxicación por Tetracloruro de Carbono/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Ciclina D1/biosíntesis , Quinasas Ciclina-Dependientes/biosíntesis , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/enzimología , Diabetes Mellitus Tipo 2/metabolismo , Activación Enzimática/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Immunoblotting , Interleucina-6/sangre , Hepatopatías/enzimología , Hepatopatías/metabolismo , Masculino , Fosforilación/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Proteína de Retinoblastoma/metabolismo , Factor de Crecimiento Transformador beta1/biosíntesis , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Thioacetamide (TA) undergoes saturation toxicokinetics in ad libitum (AL) fed rats. Diet restriction (DR) protects rats from lethal dose of TA despite increased bioactivation-mediated liver injury via CYP2E1 induction. While a low dose (50 mg TA/kg) produces 6-fold higher initial injury, a 12-fold higher dose produces delayed and mere 2.5-fold higher injury. The primary objective was to determine if this less-than-expected increase in injury is due to saturation toxicokinetics. Rats on AL and DR for 21 days received either 50 or 600 mg TA/kg i.p. T(1/2) and AUCs for TA and TA-S-oxide were consistent with saturable kinetics. Covalent binding of (14)C-TA-derived-radiolabel to liver macromolecules after low dose was 2-fold higher in DR than AL rats. However, following lethal dose, no differences were found between AL and DR. This lack of dose-dependent response appears to be due to saturation of bioactivation at the higher dose. The second objective was to investigate the effect of phenobarbital pretreatment (PB) on TA-initiated injury following a sub-lethal dose (500 mg/kg). PB induced CYP2B1/2 approximately 350-fold, but did not increase covalent binding of (14)C-TA, TA-induced liver injury and mortality, suggesting that CYP2B1/2 has no major role in TA bioactivation. The third objective was to investigate the role of CYP2E1 using cyp2e1 knockout mice (KO). Injury was assessed over time (0-48 h) in wild type (WT) and KO mice after LD(100) dose (500 mg/kg) in WT. While WT mice exhibited robust injury which progressed to death, KO mice exhibited neither initiation nor progression of injury. These findings confirm that CYP2E1 is responsible for TA bioactivation.
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Restricción Calórica , Carcinógenos/farmacocinética , Citocromo P-450 CYP2E1/metabolismo , Fenobarbital/farmacología , Tioacetamida/farmacocinética , Alanina Transaminasa/sangre , Animales , Hidrocarburo de Aril Hidroxilasas/metabolismo , Aspartato Aminotransferasas/sangre , Biotransformación/efectos de los fármacos , Carcinógenos/toxicidad , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Citocromo P-450 CYP2B1/metabolismo , Citocromo P-450 CYP2E1/genética , ADN Complementario/biosíntesis , ADN Complementario/genética , Semivida , Cinética , Masculino , Ratones , Ratones Noqueados , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Ratas , Ratas Sprague-Dawley , Esteroide Hidroxilasas/metabolismo , Sulfóxidos/metabolismo , Tioacetamida/toxicidadRESUMEN
UNLABELLED: This study tested whether hepatic steatosis sensitizes liver to toxicant-induced injury and investigated the potential mechanisms of hepatotoxic sensitivity. Male Sprague-Dawley rats were fed a methionine- and choline-deficient diet for 31 days to induce steatosis. On the 32nd day, administration of a nonlethal dose of CCl4 (2 mL/kg, intraperitoneally) yielded 70% mortality in steatotic rats 12-72 hours after CCl4 administration, whereas all nonsteatotic rats survived. Neither CYP2E1 levels nor covalent binding of [14C] CCl4-derived radio-label differed between the groups, suggesting that increased bioactivation is not the mechanism for this amplified toxicity. Cell division and tissue repair, assessed by [3H]thymidine incorporation and proliferative cell nuclear antigen assay, were inhibited in the steatotic livers after CCl4 administration and led to progressive expansion of liver injury culminating in mortality. The hypothesis that fatty hepatocytes undergo cell cycle arrest due to (1) an inability to replenish ATP due to overexpressed uncoupling protein-2 (UCP-2) or (2) induction of growth inhibitor p21 leading to G1/S phase arrest was tested. Steatotic livers showed 10-fold lower ATP levels due to upregulated UCP-2 throughout the time course after CCl4 administration, leading to sustained inhibition of cell division. Western blot analysis revealed an up-regulation of p21 due to overexpression of TGF beta1 and p53 and down-regulation of transcription factor Foxm 1b in steatotic livers leading to lower phosphorylated retinoblastoma protein. Thus, fatty hepatocytes fail to undergo compensatory cell division, rendering the liver susceptible to progression of liver injury. CONCLUSION: Impaired tissue repair sensitizes the steatotic livers to hepatotoxicity.
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Tetracloruro de Carbono/toxicidad , Tolerancia a Medicamentos/fisiología , Hígado Graso/fisiopatología , Hígado/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , División Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Citocromo P-450 CYP2E1/metabolismo , ADN/metabolismo , Hígado Graso/complicaciones , Hígado Graso/metabolismo , Glucógeno/metabolismo , Hepatocitos/fisiología , Canales Iónicos/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Masculino , Proteínas Mitocondriales/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas , Ratas Sprague-Dawley , Proteína Desacopladora 2RESUMEN
Many hepatotoxicants like acetaminophen, chloroform, carbon tetrachloride, halothane, and thioacetamide cause hepatotoxicity through covalent binding of their reactive metabolites to proteins. The covalent binding to proteins may lead to dysfunction of critical proteins such as enzymes, transporters, receptors, and regulatory molecules. Because most reactive metabolites covalently bind to tissue macromolecules and tend to be unstable, they can not be isolated, and direct quantitation of the formation of reactive metabolites is not possible. Measuring their covalent binding to proteins offers a convenient way to estimate the amount of reactive metabolite formation. Such estimates have been used to quantify the bioactivation-based injury due to such hepatotoxicants. There are various methods by which covalent binding may be measured. This unit describes a protocol in which a radiolabeled compound can be utilized to measure covalent binding. Alternate protocols involve immunoblotting and immunohistochemistry. The time and method of measuring covalent binding play an important role in the evaluation.