Role of benoxaprofen and flunoxaprofen acyl glucuronides in covalent binding to rat plasma and liver proteins in vivo

Benoxaprofen (BNX) has been implicated in rare but serious hepatotoxicity which led to its withdrawal from the world market. Flunoxaprofen (FLX), a structural analog, appears to be less toxic. It has been postulated that the nonsteroidal antiinflammatory drugs associated toxicity may be related to covalent modification of proteins by their reactive acyl glucuronides, and the extent of covalent protein binding depends on both reactivity of the acyl glucuronide and the exposure to the reactive metabolite. The disposition of BNX and FLX in rats were compared upon intravenous administration of 20 mg/kg of BNX, FLX or their metabolites. Covalent binding of BNX and FLX to plasma and liver proteins were also determined, and an immunochemical approach was used to detect their hepatic targets. Similar concentrations of plasma protein adducts for BNX and FLX were detected even though the AUC of BNX-glucuronide (BNX-G) was almost twice that of FLX-glucuronide (FLX-G). Similar concentrations of liver protein adducts for BNX and FLX were also detected at 8 h, however, the hepatobiliary exposure of BNX-G was only 1/3rd that of FLX-G indicating that BNX-G was more reactive than FLX-G, which was in agreement with in vitro data. Proteins of 110 and 70 kDa were the major liver protein targets modified by covalent attachment of BNX and FLX. In conclusion, measuring covalent binding to tissue proteins in animals in addition to plasma adducts should be considered when evaluating and comparing carboxylic acid analogs that form reactive acyl glucuronides.     

The imidazoline-like drug S23515 affects lipid metabolism in hepatocyte by inhibiting the oxidosqualene: lanosterol cyclase activity

Imidazoline-like drugs are centrally-acting antihypertensive agents that inhibit the activity of the sympathetic nervous system by interacting with the alpha2-adrenoreceptor and also with a non-adrenergic imidazoline binding site called the imidazoline 1 receptor. Recently, these molecules were proposed to play an additional role in cardiovascular diseases by acting on glucose and lipid metabolism. We used S23515, a potent imidazoline-like molecule acting selectively on blood pressure through the imidazoline 1 receptor, to decipher the effects of these drugs on lipid metabolism. We found that S23515 inhibited specifically and dose-dependently cholesterol synthesis in cultured rodent and primate hepatocytes. This hypocholesterolemic effect was likely due to the inhibition of the oxido:lanosterol cyclase (OSC), a rate-limiting enzyme in the cholesterol biosynthetic pathway. Partial OSC inhibition induced by S23515 led to the generation of 24(S),25-epoxycholesterol, a potent ligand for the liver X receptor (LXR). Furthermore, S23515 increased in human macrophages the expression of both ABCA1 and G1, the 2 ATP binding cassette transporters, which play a pivotal role in the reverse cholesterol transport. Thus, these results suggest that S23515, and potentially other imidazoline-like drugs, could exert hypolipidemic effects in addition to their hypotensive activities.     

氚标记小牛胸腺DNA在大鼠和犬体内的药动学研究

目的:研究氚标记小牛胸腺DNA(3H-小牛胸腺DNA)在大鼠和Beagle犬体内的药动学特征。方法:采用氚水交换法制备3H-小牛胸腺DNA。取大鼠尾静脉注射高、中、低剂量(15、5、1.67 mg/kg,n=5)的3H-小牛胸腺DNA,中、高剂量组大鼠连续给药7d(第1、7天给予3H-小牛胸腺DNA,其余时间给予未标记小牛胸腺DNA),低剂量大鼠仅单次给药;另取犬前肢静脉注射高、中、低剂量(1.5、0.5、0.167 mg/kg,n=3)的3H-小牛胸腺DNA,中剂量犬连续给药7 d,低、高剂量犬仅单次给药。分别于第1、7天给药后0.033、0.25、1、2、4、6、8、12、24 h取血,分离血浆后加入闪烁液并使用液闪计数仪分析,以WinNonlin软件计算药动学参数。结果:高、中、低剂量3H-小牛胸腺DNA在大鼠体内的药动学参数分别为:单次给药的AUC0-24 h为(11 742±2 245)、(3 571±851)、(727±202)ng-Eq·h/g,t1/2为(21.4±5.08)、(13±6.0)、(6.8±1.76)h;重复给药高、中剂量的AUC0-24 h为(5 706±1 009)、(7 601±1 861)ng-Eq·h/g,t1/2为(16.0±10.13)、(9±2.7)h。高、中、低剂量3H-小牛胸腺DNA在犬体内的药动学参数分别为:单次给药的AUC0-24 h为(4 444±999)、(2 719±139)、(501±101)ng-Eq·h/g,t1/2为(17.6±7.57)、(14.0±1.76)、(16.4±2.39)h;重复给药中剂量的AUC0-24 h为(3 073±200)ng-Eq·h/g,t1/2为(20.6±6.62)h。结论:3H-小牛胸腺DNA在大鼠与Beagle犬体内单次给药和重复给药均可被快速消除。     

The dietary antioxidants resveratrol and quercetin protect cells from exogenous pro-oxidative damage.

In the colorectal epithelium oxidative stress is observed endogenously in premalignant adenoma cells or induced by nutritional factors like fatty acid hydroperoxides (LOOH). Bioactive phenols like resveratrol and quercetin can quench reactive oxygen species and protect from pro-oxidative damage. Our study used colorectal adenoma and carcinoma cell lines to assess antioxidant protective effects of resveratrol and quercetin. It demonstrated that both compounds efficiently protect from oxidative stress induced by LOOH. Effective concentrations (10 microM resveratrol and 1 microM quercetin) can easily be reached in the intestinal lumen after consumption of plant foods or food supplements. Both compounds prevent LOOH-induced formation of intracellular H2O2, stimulation of cyclooxygenase-2 and vascular endothelial growth factor. For reduction of endogenous H2O2 levels in colorectal tumor cells higher antioxidant-concentrations are needed in all cell lines. Quercetin (10 microM) alone even increased H2O2 in LT97 adenoma cells and stimulated VEGF production. Resveratrol and quercetin also induced 10-30% and 40-60% cell loss respectively by apoptosis. In summary, this indicates that resveratrol and quercetin have little protective capacity in absence of exogenous stress. They are however highly efficient in protecting against nutrition induced oxidative stress damage suggesting that this constitutes the major part of their tumor protective activity.     

Arsenic trioxide in environmentally and clinically relevant concentrations interacts with calcium homeostasis and induces cell type specific cell death in tumor and non-tumor cells

While arsenic compounds are known as environmental toxicants (especially in drinking water) and as carcinogens, some arsenic compounds, like arsenic trioxide (As2O3), are clinically used in humans to treat some forms of cancer (e.g. leukemia). Although arsenic compounds have been studied intensively, their interactions with living cells are still not fully elucidated. We have previously proposed that modulation of intracellular calcium ([Ca2+]i) homeostasis induced by As2O3 could be an important mechanism to induce cytotoxicity. Here we demonstrate, using human cell models (neuroblastoma (SY-5Y) or embryonic kidney cells (HEK)) and confocal microscopy in combination with the calcium sensitive dye fluo 4-AM, that As2O3 interferes with calcium signaling at low (environmentally and clinically relevant concentrations of 100 pM to 1 microM). Within this concentration range, As2O3 had cell type specific cytotoxic effects, with neuroblastoma cells being more sensitive to As2O3 than HEK 293. In addition, by staining with Hoechst 33347 and counting micronucleated cells as well as apoptotic nuclei, As2O3 was found to increase the rate of apoptosis and DNA damage, which was also cell type specific. These results indicate that the As2O3-induced cell death could be triggered or mediated by [Ca2+]i signals and suggest that low concentrations of As2O3 are able to interfere with specific physiological processes in diverse cell models.     

Denbinobin induces apoptosis in human lung adenocarcinoma cells via Akt inactivation, Bad activation, and mitochondrial dysfunction

Increasing evidence demonstrated that denbinobin, isolated from Ephemerantha lonchophylla, exert cytotoxic effects in cancer cells. The purpose of this study was to investigate whether denbinobin induces apoptosis and the apoptotic mechanism of denbinobin in human lung adenocarcinoma cells (A549). Denbinobin (1-20microM) caused cell death in a concentration-dependent manner. Flow cytometric analysis and annexin V labeling demonstrated that denbinobin increased the percentage of apoptotic cells. A549 cells treated with denbinobin showed typical characteristics of apoptosis including morphological changes and DNA fragmentation. Denbinobin induced caspase 3 activation, and N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a broad-spectrum caspase inhibitor, prevented denbinobin-induced cell death. Denbinobin induced the loss of the mitochondrial membrane potential and the release of mitochondrial apoptotic proteins including cytochrome c, second mitochondria derived activator of caspase (Smac), and apoptosis-inducing factor (AIF). In addition, denbinobin-induced Bad activation was accompanied by the dissociation of Bad with 14-3-3 and the association of Bad with Bcl-xL. Furthermore, denbinobin induced Akt inactivation in a time-dependent manner. Transfection of A549 cells with both wild-type and constitutively active Akt significantly suppressed denbinobin-induced Bad activation and cell apoptosis. These results suggest that Akt inactivation, followed by Bad activation, mitochondrial dysfunction, caspase 3 activation, and AIF release, contributes to denbinobin-induced cell apoptosis.     

小牛血清去蛋白注射液对局灶性脑缺血大鼠脑血管储备能力的影响研究

目的本文主要研究小牛血清去蛋白注射液对局灶性脑缺血大鼠脑血管储备能力的影响。方法随机将88只大鼠分为治疗组40只、缺血组40只、假手术组8只,治疗组在手术前的三天给予腹腔注射小牛血清去蛋白注射液,剂量为2毫升,一天一次,最后一次进药时间是手术前的五小时。在完成模后的三天内分别处死缺血组和治疗组,最后采取血管内皮生长因子（VEGF）的免疫组化检测。结果在手术后缺血的三小时,治疗组逐渐出现阳性反应,各时间点脑组织表达明显多于缺血组和假手术组,具有统计意义（P〈0．05）。结论小牛血清去蛋白注射液可以提高局灶性脑缺血大鼠脑血管储备能力,对缺血性脑损伤具有保护功能。     

Study of cytotoxic and apoptogenic properties of saffron extract in human cancer cell lines

Saffron (dried stigmas of Crocus sativus L.) has been used as a spice, food colorant and medicinal plant for millennia. In this study cytotoxic effect of saffron extract was evaluated in HepG2 and HeLa cell lines. Meanwhile role of apoptosis and ROS were explored. Malignant and non-malignant cells (L929) were cultured in DMEM medium and incubated with different concentrations of ethanolic saffron extract. Cell viability was quantitated by MTT assay. Apoptotic cells were determined using PI staining of DNA fragmentation by flow cytometry (sub-G1 peak). ROS was measured using DCF-DA by flow cytometry analysis. Saffron could decrease cell viability in malignant cells as a concentration and time-dependent manner. The IC50 values against HeLa and HepG2 were determined 800 and 950 μg/ml after 48 h, respectively. Saffron induced a sub-G1 peak in flow cytometry histogram of treated cells compared to control indicating apoptotic cell death is involved in saffron toxicity. This toxicity was also independent of ROS production. It might be concluded that saffron could cause cell death in HeLa and HepG2 cells, in which apoptosis or programmed cell death plays an important role. Saffron could be also considered as a promising chemotherapeutic agent in cancer treatment in future.