Hyperthyroidism induces apoptosis in rat liver through activation of death receptor-mediated pathways

BACKGROUND/AIMS: The molecular basis of hepatic dysfunction in thyrotoxicosis is not fully understood. Here, we investigated the effect of altered thyroidal status on death receptor pathways including p75 neurotrophin receptor (p75NTR), a member of tumor necrosis factor (TNF) receptor superfamily, in rat liver. METHODS: Hyperthyroidism was induced in Sprague-Dawley rats by daily injections of triiodothyronine in a dose of 12.5 microg/100 g body weight for 10 days. RESULTS: Terminal deoxynucleotide-transferase-mediated dUTP nick end labeling assay and caspase-3 activation data confirmed apoptosis in hyperthyroid rat liver. We observed the elevated levels of death ligands, TNF-alpha, Fas ligand and their cognate receptors, TNF-receptor-1 and Fas, and 8-fold increase in caspase-8 activation in hyperthyroid rat liver (p<0.001). We demonstrated for the first time that hyperthyroidism elevates p75NTR levels and its ligands, pro-nerve growth factor and pro-brain-derived neurotrophic factor, in rat liver. Further we showed that most of the apoptotic cells in hyperthyroid liver express p75NTR. We also demonstrated that triiodothyronine administration to rats causes NF-kappaB activation, but persistent exposure (10 days) to triiodothyronine deactivates NF-kappaB leading to sustained c-Jun N-terminal kinase (JNK) activation. CONCLUSIONS: This study showed that hyperthyroidism-induced apoptosis in rat liver involves the activation of death receptor-mediated pathways, including p75NTR.     

甲亢重症肝损害报告1例

1病例资料 患者,女,49岁,因间断发热、皮肤巩膜黄染半个月入院。患者入院前半个月无明显诱因出现发热,未测体温,自服维C银翘片（1片/次,3次/d）1.5 d后退热。随后出现皮肤巩膜黄染,伴有乏力、厌食、恶心,无腹痛,无陶土样大便,     

Agmatine induces apoptosis in rat hepatocyte cultures.

BACKGROUND/AIMS: Agmatine, the compound formed by decarboxylation of arginine, is believed to be an endogenous neurotransmitter through interaction with the imidazoline receptors. However, it also appears to regulate rat hepatocyte polyamines by modifying both their synthesis and their catabolism. As the decrease in polyamine content has been correlated with apoptosis, we examined the possibility that agmatine has an effect on this phenomenon. METHODS: Apoptotic cells were detected by visualizing nuclear shrinkage/fragmentation in hepatocytes cultured at 21 and 5% oxygen tension. Caspase-3 activity, cleavage of PARP, release of cytochrome c and mitochondrial swelling were therefore measured in the two conditions and in the presence or not of agmatine. RESULTS: In rat hepatocytes agmatine promoted apoptosis, procaspase 3 processing and increase of caspase-3 like activity. This occurred through mitochondria swelling and release of cytochrome c. Cyclosporin A and catalase blocked the swelling. CONCLUSIONS: Our experiments show that agmatine, besides all the known biological effects, has also part, at least in hepatocytes, in the modulation of programmed cell death.     

Glucocorticoid induces apoptosis in rat leydig cells.

The aim of the present study was to investigate whether glucocorticoid induces apoptosis in rat Leydig cells. To determine whether there are developmental differences in glucocorticoid sensitivity, Leydig cells were isolated at distinct stages of their differentiation [mesenchymal-like progenitors (PLC), immature Leydig cells (ILC), and adult Leydig cells (ALC)] from 21-, 35-, and 90-d-old Sprague Dawley rats, respectively. Glucocorticoid induction of apoptosis was evaluated after both in vitro and in vivo exposures. In the first set of experiments, PLC, ILC, and ALC were treated with 100 nM corticosterone (CORT) for either 4 or 24 h in vitro and then assessed for labeling with the apoptotic marker annexin V. PLC exposed to CORT had levels of annexin V-fluorescein isothiocyanate labeling that were unchanged relative to control values at both time points (P > 0.05). In contrast, CORT-treated ILC and ALC had increased frequencies of apoptosis: in ALC, a 22.1 +/- 1.7% incidence after 4 h and 30.5 +/- 2.3% after 24 h compared with 7.4 +/- 0.8% in untreated controls (P < 0.05). Similar trends were observed for ILC. Ultrastructural analysis confirmed that the increase in annexin V labeling was associated with characteristic signs of apoptosis, including nuclear fragmentation and formation of apoptotic bodies. A second line of experiments examined whether apoptosis was evident in purified Leydig cells after administration of CORT in vivo. Male rats were subjected to bilateral adrenalectomy and were treated with CORT by ip injection twice daily at doses ranging from 2.5-7.5 mg/100 g BW starting 3 d after surgery. The frequency of Leydig cell apoptosis was measured at 12, 24, 48, and 72 h after the first injection. Administration of the 2.5-mg dose raised circulating CORT 5-10 times above normal basal concentrations, and LH levels sampled at these times were not altered in the treated animals. Increased Leydig cell apoptosis was measurable after 24 h of treatment, with an incidence of 21.1 +/- 1.8% in ALC compared with 5.7 +/- 0.8% in untreated controls (P < 0.05). Sharp reductions in immunocytochemical staining intensity were observed in the treated animals for a Leydig cell marker, 11beta-hydroxysteroid dehydrogenase, which occurred concurrently with decreased serum T levels. This was consistent with the hypothesis that CORT-mediated induction of apoptosis leads to declines in Leydig cell numbers, thereby affecting T production. These results suggest that excessive exposure to CORT initiates apoptosis in rat Leydig cells, potentially contributing to suppression of circulating T levels during stress and other conditions in which glucocorticoid concentrations are elevated.     

NLRP3炎症小体在大鼠严重烫伤早期心肌内的表达及意义

目的 探讨严重烫伤大鼠早期心肌组织内NLRP3炎症小体信号通路的表达及意义。方法 采用大鼠95℃热水浴18 s,制成40%总体表面积Ⅲ度烫伤模型,将24只健康雄性SD大鼠按随机数表法分为对照组、烫伤组、干预组。烫伤组大鼠立即腹腔注射10 ml生理盐水补液;干预组大鼠注射10 ml生理盐水+(BAY11-7082) 10 μl(0.2 mg/ml,10 mg/kg);对照组动物背部置于25℃温水浴18 s模拟烫伤过程。伤后8 h分别取各组大鼠心肌组织及血清,采用蛋白免疫印迹法和实时荧光定量RT-PCR法检测心肌组织中NLRP3、半胱氨酸天冬氨酸蛋白酶（caspase）-1蛋白表达水平和NLRP3、caspase-1、IL-1β、TNF-α mRNA表达水平,同时观察HE染色组织大体形态,酶联免疫吸附试验法检测血清肌酸激酶同工酶（CK-MB）、乳酸脱氢酶（LDH）含量。结果 ①烫伤组大鼠心肌组织内NLRP3、caspase-1蛋白表达量显著高于对照组（P<0.01）;与烫伤组比较,干预组大鼠心肌组织内NLRP3、caspase-1明显降低（P<0.01）;②烫伤组大鼠心肌组织中NLRP3、caspase-1、IL-1β和TNF-α的mRNA表达量分别为3.15±0.46,2.47±0.24,3.26±0.22和2.17±0.32,显著高于对照组的1.00±0.09,1.00±0.06,1.00±0.08和1.00±0.07（均P<0.01）;干预组大鼠心肌组织中NLRP3、caspase-1、IL-1β和TNF-α的mRNA表达量分别为1.68±0.37,1.55±0.17,1.21±0.15和1.38±0.23,显著低于烫伤组（均P<0.01）;③对照组大鼠心肌组织呈不规则圆柱形、排列整齐、横纹清晰,结构完整。烫伤组大鼠部分心肌纤维排列紊乱,细胞和间质可见水肿,见点片状溶解灶及炎性细胞浸润。BAY11-7082干预组部分心肌纤维排列紊乱,心肌细胞和间质水肿、心肌纤维溶解及炎细胞浸润程度均较烫伤组减轻;④烫伤组大鼠血清CK-MB、LDH含量分别为(2753±825)U/L,(2618±238)U/L,显著高于对照组的(247±76)U/L,(721±59)U/L（均P<0.01）;干预组大鼠血清CK-MB、LDH含量分别为(1267±248)U/L,(1982±183)U/L,显著低于单纯烫伤组（均P<0.01）。结论 NLRP3炎症小体在严重烫伤大鼠早期心肌组织内活性增高,使用BAY11-7082干预后,可抑制NLRP3炎症小体的活化,减轻心肌组织损伤,降低心肌炎症反应。     

Nevirapine induces apoptosis in liver(HepG2) cells

Objective: To generate insights into the mechanism of NVP induced hepatotoxicity. Methods: Liver(HepG2) cells were cultured with various concentrations of NVP. This cell line was chosen because it has low expression of cytochrome P450, allowing evaluation of the effects of NVP rather than specific metabolites. Cytotoxicity was determined using a proliferation assay and cell numbers were monitored using trypan blue exclusion assay for long term culture experiments and apoptosis induction was determined by morphological and biochemical investigation. Results: HepG2 cells treated with the highest concentration of NVP tested(819 μM) initially showed a rounded morphology and all cells had died by week three of exposure. Nuclear condensation and fragmentation, increased Annexin V/propidium iodide staining and caspase 9 activation all supported the induction of apoptosis in HepG2 cells in response to NVP treatment. Conclusions: There is a clear induction of apoptosis in response to NVP which suggests that NVP has significant cytotoxicity, over and above any cytotoxicity of metabolites and may contribute directly to patient hepatotoxicity.     

Hepatocyte proliferation and apoptosis in rat liver after liver injury

BACKGROUND/AIMS: In this paper the early phase of proliferate response and apoptosis of hepatocytes after partial liver resection, during reperfusion after ischemia and during sepsis is demonstrated. METHODOLOGY: Experiments were conducted in a rat model with regeneration times of 0.5-24 hours after injury. Proliferation was analyzed by Ki-67 immunohistochemistry and confirmed by double staining with CK18 in FACS. Apoptosis was analyzed by TUNEL technique. RESULTS: Periportal hepatocytes enter the cell cycle already 0.5-2 hours after injury in all three models. This early proliferative response is predominant periportally localized. During reperfusion and during sepsis there was a strict pericentral apoptosis of hepatocytes found. CONCLUSIONS: An early periportal proliferation of hepatocytes is a common reaction of the liver to injury. This proliferation takes place much earlier then the main proliferative response 24-72 h after partial resection. This predominant periportal proliferation together with the pericentral apoptosis fit to the concept of the "streaming liver" in liver regeneration.     

Effect of hypothyroidism and hyperthyroidism on triiodothyronine production in perfused rat liver

This study was undertaken to evaluate the effect of altered thyroid states on hepatic T3 production in a functioning intact organ system, the isolated perfused liver. Thyroidectomized rats were treated for 3-4 weeks with vehicle, T4, 1.5 micrograms/100 g-1 day-1, or T4, 20 micrograms/100 g-1 day-1, to produce hypothyroidism, euthyroidism, or hyperthyroidism. Livers were perfused for 1 h with medium containing T4, 10 micrograms/dl, and T3 production was estimated by RIA. T3 production in the hypothyroid, euthyroid, and hyperthyroid groups, respectively, was 1.61 +/- (SE) 0.50, 5.18 +/- 0.55, and 15.62 +/- 1.61 ng/g-1 liver h-1. These differences in T3 production resulted entirely from changes in percent conversion of T4 to T3 which were 0.87 +/- 0.25%, 3.21 +/- 0.38%, and 12.02 +/- 1.82% in the hypothyroid, euthyroid, and hyperthyroid groups, respectively. The measured hepatic uptake of T4 decreased slightly with T4 administration from 188 +/- 13 to 162 +/- 7 and 144 +/- 10 ng/g liver in these same groups. The changes in T3 production were not accounted for by differences in biliary excretion or deiodination of T3. These studies demonstrate a stimulatory effect of T4 on the conversion of T4 to T3 which is important to altering net hepatic T3 production.     

Inhaled ethylene oxide induces preneoplastic foci in rat liver

Summary The metabolite of E, EO, has been shown to be an extrahepatic carcinogen in rats in long-term studies. By means of a rat liver foci bioassay with 3 to 4 days old Sprague-Dawley rats, EO showed an initiating capacity in the livers of female, but not of male rats, measured as incidence of foci deficient in ATPase. After inhalation of 55 and 100 ppm EO, 8 h daily, 5 days weekly, and over 3 weeks, 1 week of pause, and another 8 weeks of promotion with polychlorinated biphenyls, foci incidence was generally low. But it was concentration dependently higher than in controls 12 weeks after starting the experiment. A linear concentration-effect relationship existed with cocorrelation coefficient of r=0.991. With 33 ppm EO the number of foci was not enhanced significantly. The administration 10000 ppm E did not result in an enhanced foci incidence. In general the carcinogenic potential of EO, which has not been shown so far to cause hepatic tumors in rats. could be demonstrated in rat liver using a sensitive rat liver foci bioassay.Abbreviations EO ethylene oxide - E ethylene - ATPase adenosine-5-triphosphatase (EC 3.6.1.3) - GGTase gamma-glutamyltranspeptidase (EC 2.3.2.2.) Dedicated to Professor Werner Kunz on the occasion of his 65th birthdaySupported by grants from Umweltbundesamt, Berlin (UBA 10606043)     

Prolactin induces its own receptors in rat liver.

Attempts to restore PRL receptors (PRL-R) in liver membranes of hypophysectomized rats with injections of PRL have so far been only partly successful. In the present study, bovine PRL (bPRL) was mixed with polyvinylpyrrolidone (PVP) in order to sustain PRL blood levels with a single injection a day. PRL-R were measured by displacement of the binding of [125I]ovine PRL (lactoperoxidase oxidation) to a 5000 x g particulate fraction of liver by unlabeled ovine PRL. The number of binding sites was calculated by Scatchard analysis and expressed as femtomoles per mg protein. PRL-R were 85 +/- 12 fmol/mg in normal intact female rats. Seven days posthypophysectomy, PRL-R were undetectable. Daily injections of bPRL with PVP for 10 days fully restored PRL-R (117 +/- 32 fmol/mg). No significant change in PRL-R was noted when bPRL was injected with bovine GH (bGH; 120 +/- 23 fmol/mg), bovine LH (bLH; 84 +/- 14 fmol/mg), bGH plus bLH (90 +/- 12 fmol/mg), or estrogens (79 +/- 12 fmol/mg). Daily injections of bGH or bLH, alone or in combination, or estrogens with PVP failed to restore PRL-R. These results demonstrate a direct role of PRL in stimulating the production of its own receptors when a sustained blood level of the hormone is achieved.     

Nitric oxide from rat liver sinusoidal endothelial cells induces apoptosis in IFN gamma-sensitized CC531s colon carcinoma cells

BACKGROUND/AIMS: Investigation of apoptosis is pivotal in searching for mechanisms that eliminate colon cancer cells getting trapped in liver sinusoids at the time of surgical removal of the primary tumor. This study focuses on nitric oxide (NO), Fas/FasL and the involvement of interferon-gamma (IFNgamma) in liver sinusoidal endothelial cells (LSECs) and in the colon carcinoma cell line CC531s. METHODS: Apoptosis was quantified and visualized in vitro by specific DNA fragmentation, specific staining and electron microscopy. In vivo experiments were also conducted. RESULTS: In co-cultures of LSECs with CC531s, apoptosis of CC531s was observed only when they were pre-treated with IFNgamma, and was unaffected by blocking the Fas/FasL pathway. However, LSECs continuously produced NO, and apoptosis was inhibited by NO-inhibitors (NMMA and dexamethasone). When IFNgamma-sensitized CC531s were injected into rats, liver weight was lower, in contrast to control conditions where liver weight was higher. CONCLUSIONS: (i) LSECs induce apoptosis in IFNgamma-sensitized CC531s in vitro; (ii) LSECs express FasL; (iii) Fas on CC531s becomes active after IFNgamma-treatment; however, (iv) blocking the Fas/FasL pathway had no effect; (v) apoptosis was inhibited by NO-inhibitors; (vi) the immune system uses this IFNgamma-activated pathway to support LSECs in killing tumor cells.     

幽门螺杆菌体外诱导大鼠胃黏膜上皮细胞凋亡

目的:研究幽门螺杆菌(Hpylori)在大鼠胃黏膜上皮诱导细胞凋亡中的作用,并初步探讨其中凋亡相关基因表达的情况,为胃癌发病机制提供依据.方法:Hpylori超声提取液来自SydneySS-1Hpylori菌株.大鼠胃黏膜细胞OUMS-37为永生化细胞,当细胞生长至60%融合时,加入不同浓度的Hpylori超声提取液,同时设置空白,于培养的24-48h收集细胞进行形态观察.Westhernblotting检测P53蛋白表达,Northernblotting检测bax、bcl-2mRNA的表达.结果:细胞经Hpylori作用48h后在高倍镜下观察到细胞核碎裂成大小不等的块状,表现出细胞凋亡如细胞皱缩,胞浆嗜碱性,核染色质固缩致密,核染色质断裂,形成大小不等的胞内核小体,部分细胞核膜消失,核染色质聚集中细胞中央,呈现分裂期的形态学等形态学特征,对照组未出现以上特征性改变.培养细胞经过Hpylori作用后提取DNA,经15g/L琼脂糖凝胶电泳,在紫外线灯下观察呈现不连续的梯状结构电泳条带.培养细胞经过Hpylori作用后,Westhernblotting显示P53蛋白表达随Hpylori超声提取液浓度而升高,Northernblotting显示baxmRNA表达随Hpylori浓度而增加,bcl-2mRNA表达随Hpylori浓度而降低.结论:Hpylori超声提取液可在体外诱导鼠胃黏膜上皮细胞凋亡.其机制可能通过上调野生型P53蛋白和凋亡促进基因baxmRNA表达,并下调凋亡抑制基因bcl-2mRNA表达.提示Hpylori感染可通过干扰胃上皮细胞增殖与凋亡之间的平衡在胃癌病因学中发挥作用.     

Nitric oxide induces caspase-dependent apoptosis and necrosis in neonatal rat cardiomyocytes

Excessive nitric oxide (NO) production has been implicated in the pathophysiology of cardiomyocyte (CMC) apoptosis and necrosis induced by ischemia/reperfusion, inflammation and NO-donating chemicals. Although caspases are known to be involved in apoptosis, the present study examined whether caspases also play a role in NO-induced CMC necrosis. Neonatal rat CMCs were labeled with Annexin-V and propidium iodide, and apoptosis and necrosis were analyzed by confocal images and fluorescence activated cell sorter analysis. CMC apoptosis and necrosis were also evaluated by determining DNA fragmentation in the cell and the supernatant fractions. Treatment of CMCs with the NO donor, diethylenetriamine NO (DETA/NO) or S-nitroso-N-acetyl-penicillamine (SNAP) at concentrations of 10 and 100 microM for 24h induced predominantly apoptosis over necrosis, but a higher concentration (1mM) of DETA/NO or SNAP provoked both apoptosis and necrosis. The lower doses of DETA/NO-induced apoptosis was associated with a gradual increase in caspase-3 activity over 24h without appreciable activation of poly ADP-ribose polymerase (PARP), while the higher dose of DETA/NO induced a marked increase in caspase-3 activity and CMC apoptosis until 2h after the treatment, and increased necrotic CMCs thereafter associated with robust activation of PARP. The caspase inhibitor Z-DEVD-FMK but not the poly ADP-ribose polymerase (PARP) inhibitor 3-aminobenzamide (3-AB) abolished caspase-3 activation and CMC apoptosis induced by 100 microM DETA/NO. However, both Z-DEVD-FMK and 3-AB abolished PARP activation and CMC necrosis induced by 1mM DETA/NO. The amount of nicotinamide adenine dinucleotide (NAD) and adenine nucleotides in CMCs was not significantly affected by treatment with 10 and 100 microM DETA/NO, but was significantly reduced by treatment with 1mM DETA/NO without a decline of adenylate energy charge. The depletion of NAD and adenine nucleotides was abrogated by Z-DEVD-FMK and 3-AB. These results suggest that caspase activation play a crucial role in CMC apoptosis induced by lower concentrations of NO as well as in CMC necrosis induced by a higher concentration of and a longer exposure to NO. NO-induced CMC necrosis is likely mediated by PARP activation which occurs as a consequence of caspase activation.     

Alcohol induces liver neoplasia in a novel alcohol-preferring rat model

Background: Alcohol is a significant risk factor for the development of hepatocellular carcinoma (HCC). To date, no rodent model has demonstrated the formation of hepatic neoplasia in the setting of chronic alcohol consumption alone. Methods: We investigated whether rats selectively bred for high alcohol preference (P rats), allowed free access to water, or water and 10% (v/v) alcohol, for 6, 12, or 18 months, develop hepatic neoplasia. Results: At necropsy, liver tumor incidence and multiplicity were significantly increased in 18‐month alcohol‐consuming versus water‐consuming P rats. These data were confirmed histologically by glutathione‐S‐transferase pi‐class (GSTp) staining. Phosphorylated mitogen‐activated protein kinase/extracellular signal‐regulated kinase 1/2 (MAPK/ERK) staining was also increased in the sinusoidal lining cells within livers of alcohol‐consuming versus water only P rats. In addition, cytochrome p450IIE1 (CYP2E1) mRNA, protein expression/activity, and intrahepatic oxidative stress were significantly increased in alcohol‐consuming P rat livers versus water only. In contrast, acetaldehyde dehydrogenase expression decreased in alcohol‐consuming versus water only P rats. No significant difference in alcohol dehydrogenase expression was detected. Conclusions: These data demonstrate that chronic alcohol consumption is associated with hepatic neoplasia, MAPK/ERK activation, increased CYP2E1 activity, and intrahepatic oxidative stress in P rats. As these rats are well characterized as a model of alcoholism, these findings identify a novel rodent model of alcohol or “alcoholism”‐induced liver neoplasia.     

Bilirubin induces apoptosis via the mitochondrial pathway in developing rat brain neurons

Increased levels of unconjugated bilirubin, the end-product of heme catabolism, are detrimental to the central nervous system. To examine the role of apoptosis in bilirubin-induced toxicity and to characterize the biochemical pathway of cell death, we exposed developing rat brain neurons to purified unconjugated bilirubin at concentrations below and above saturation of human serum albumin. Isolated neurons treated with bilirubin showed increased levels of apoptosis. Mitochondrial cytochrome c was extensively released and accumulated in cytosol. Consistent with this observation, caspase-3 was activated and the full-length substrate poly(ADP)ribose polymerase (PARP) degraded, even in the presence of very modestly elevated concentrations of bilirubin. In parallel, all events were prevented in cells preincubated with ursodeoxycholate. Further experiments showed that bilirubin diminished mitochondrial transmembrane potential (DeltaPsi(m)) and increased mitochondrial-associated Bax protein levels, while directly disrupting membrane lipid and protein structure. In conclusion, bilirubin induces mitochondrial depolarization and Bax translocation via physical interaction with membranes, mediating the mitochondrial pathway of apoptosis in neurons exposed to bilirubin. These results provide a novel insight into the mechanism of bilirubin-induced toxicity.     

Subcellular changes and apoptosis induced by ethanol in rat liver

The livers of rats given ethanol for 5 weeks showed marked structural alterations of hepatocytes of acinar zone 3 including mitochondrial pleomorphism, increased smooth endoplasmic reticulum and deposition of small (less than 0.5 micron) lipid droplets. In addition, apoptotic bodies involving altered parenchymal cells were frequently observed, together with prominent mononuclear infiltrates adjacent to the terminal hepatic veins. It is suggested that 'age' of liver cells may play a role in the preferential perivenular localization of early ethanol-induced liver damage.     

Severe cholestatic jaundice in hyperthyroidism after treatment with 131-iodine

A 39-year-old white man was referred to our hospital for evaluation of his jaundice and pruritus. The patient was treated with I for diffuse toxic goiter prior to his referral to our hospital. Clinical examination and laboratory investigations excluded viral hepatitis, autoimmune hepatitis, granulomatous disease, primary biliary disease, extrahepatic biliary obstruction, and heart failure. Liver biopsy showed severe intrahepatic and canalicular cholestasis with minimal inflammatory changes. The patient's jaundice promptly resolved with therapy for hyperthyroidism and thyroid storm as bilirubin levels decreased from 35 mg/dL (normal: 0.5-1.2 mg/dL) to 0.4 mg/dL. Thyrotoxicosis can be an uncommon cause of profound cholestasis. Our case differs from all other reports in the literature because of the severity of the cholestasis and its prompt resolution with treatment for thyrotoxicosis.