Acetaminophen toxicity in rat and mouse hepatocytes in vitro

Context: Acetaminophen (APAP) hepatotoxicity is often studied in primary cultures of hepatocytes of various species, but there are only few works comparing interspecies differences in susceptibility of hepatocytes to APAP in vitro. Objectives: The aim of our work was to compare hepatotoxicity of APAP in rat and mouse hepatocytes in primary cultures. Materials and methods: Hepatocytes isolated from male Wistar rats and C57Bl/6J mice were exposed to APAP for up to 24?h. We determined lactate dehydrogenase (LDH) activity in culture medium, activity of cellular dehydrogenases (WST-1) and activity of caspases 3 in cell lysate as markers of cell damage/death. We assessed content of intracellular reduced glutathione, production of reactive oxygen species (ROS) and malondialdehyde (MDA). Respiration of digitonin-permeabilized hepatocytes was measured by high resolution respirometry and mitochondrial membrane potential (MMP) was visualized (JC-1). Results: APAP from concentrations of 2.5 and 0.75?mmol/L induced a decrease in viability of rat (p?p?Conclusion: APAP displayed dose-dependent toxicity in hepatocytes of both species. Mouse hepatocytes in primary culture however had approximately three-fold higher susceptibility to the toxic effect of APAP when compared to rat hepatocytes.     

Benoxaprofen induced toxicity in isolated rat hepatocytes

The toxicity of benoxaprofen, a non-steroidal anti-inflammatory compound was investigated using rat hepatic microsomal and isolated hepatocyte suspensions. In microsomes, benoxaprofen produced a Type I binding spectra and competitively inhibited (k_i 380 μM) the oxidative metabolism of aminopyrine. Marked toxicity was observed following incubation of benoxaprofen with isolated hepatocytes from either untreated, phenobarbitone (PB) or 3-methylcholanthrene (3-MC) pretreated male rats. In untreated hepatocytes increases in the intracellular lactate/pyruvate (L/P) ratio and alanine aminotransferase (ALT) release were related to the benoxaprofen concentration and duration of incubation. Alterations in L/P ratio preceded the release of cytosolic ALT and at 4 h a well defined dose-response relationship existed between the benoxaprofen concentration and the observed increases in the L/P ratio and ALT release. Pretreatment of animals with either PB or 3-MC did not affect the temporal nature nor the magnitude of the hepatocyte response to benoxaprofen. In addition, inhibitors of cytochrome P-450 isozymes (SKF-525A, metyrapone and -napthoflavone) were ineffective with regard to modifying the observed toxicity. The results of this study suggest that hepatic cytochrome P-450 mediated metabolism may not be implicated in the toxicity of benoxaprofen in isolated hepatocytes. However, alterations in the cellular redox state and evidence of plasma membrane bleb formation suggest that benoxaprofen may uncouple oxidative phosphorylation and disturb intracellular calcium ion homeostasis.     

异烟肼致肝脏毒性的蛋白质组学分析

目的:应用蛋白质组学技术研究异烟肼致大鼠肝脏损伤时肝脏蛋白质表达谱的变化,为从分子水平上寻找毒性损伤标志物以及阐明异烟肼毒性机制奠定基础。方法:将Wistar大鼠随机分为生理盐水正常对照组和异烟肼组(400 mg.kg-1),分别连续灌胃14 d后处死大鼠,提取肝脏总蛋白,双向凝胶电泳分离蛋白质组成分,采用考马斯亮蓝R-250染色,经ImageMaster2D Platinum5.0软件分析比较两组图谱,并对差异蛋白斑点进行基质辅助激光解吸/电离飞行时间质谱(MALD-TOF-MS)分析鉴定。结果:发现11个差异有统计学意义的蛋白质点,鉴定出8个蛋白,其中,异烟肼处理组比对照组表达升高的蛋白质有鸟氨酸转氨酶、葡萄糖调节蛋白、乙醛脱氢酶和3α-羟甾类脱氢酶,比对照组表达降低的蛋白质有抗氧化酶B166、谷胱苷肽转硫酶、醛铜还原酶和碳酸酐酶。结论:异烟肼可能造成肝脏抗氧化系统和氧化应激异常,这对阐明异烟肼的肝损伤机制具有十分重要的作用。     

对乙酰氨基酚对大鼠原代肝细胞及BRL-3A细胞的毒性作用比较

目的 建立大鼠原代肝细胞提取鉴定体系,比较对乙酰氨基酚(APAP)对大鼠原代肝细胞和永生化细胞BRL-3A的毒性作用。方法 采用胶原酶原位两步灌流法提取大鼠原代肝细胞,通过过碘酸雪夫染色(PAS)和肝细胞双核结构进行鉴定;CCK 8法测定APAP对大鼠原代肝细胞及BRL-3A细胞毒性作用的IC₅₀;光学显微镜透射电镜观察APAP对2种细胞的损伤情况;全自动生化分析仪测定细胞上清AST、ALT、LDH、ALP、ALB、BUN、TP、GLU 8项生化指标的变化。结果 PAS糖原染色鉴定获取的大鼠原代肝细胞,双核结构,细胞存活率浮动在80%~95%;最佳接种密度为60000/cm²,在第3~5天为对数生长期;APAP作用于大鼠原代肝细胞24 h的IC₅₀为18.03 mmol/L,95%置信区间为(17.28~18.81)mmol/L,作用于BRL-3A的IC₅₀为20.05 mmol/L,95%置信区间为(18.99~21.17)mmol/L;透射电镜结果显示,在30 mmol/L APAP作用下,2种细胞细胞器肿胀,核膜破裂,细胞膜边界模糊不清;与对照组比较,大鼠原代肝细胞分泌的天冬氨酸氨基转移酶(AST)、尿素氮(BUN)、葡萄糖(GLU)、碱性磷酸酶(ALP)、乳酸脱氢酶(LDH)随着APAP浓度增加产生显著变化,而BRL-3A细胞几乎所有的酶学指标变化均差异不显著。结论 与永生化细胞BRL-3A比较,大鼠原代肝细胞更能体现药物的肝脏毒性作用,但其体外培养存活时间较短;BRL-3A细胞缺少肝脏重要酶类,增加细胞内肝脏酶类是提升其作为肝脏毒性筛选模型的更好手段之一。     

柑桔素抑制CYP450 3A4活性并减轻异烟肼和利福平合用的肝细胞毒性

目的研究柑桔素对异烟肼和利福平导致肝毒性增加的防治作用及CYP3A4在其中的作用机制。方法将培养的QSG-7701人肝细胞培养液中分别加入异烟肼和利福平,同时加入不同剂量(1、5、25mg.L-1)的柑桔素后继续培养48h。分别收集药物处理后的培养液和细胞,将细胞裂解后,用比色法分别测定培养液和细胞裂解液中的乳酸脱氢酶的活性,计算细胞外和细胞内乳酸脱氢酶的比值。药物处理48h后,将细胞与CYP3A4作用底物咪达唑仑共同孵育2h,采用高效液相色谱质谱联用法测定咪达唑仑浓度的变化,计算细胞内CYP3A4的活性。结果与对照组比较,异烟肼和利福平合用使肝细胞乳酸脱氢酶释放增加,CYP3A4活性增强;5、25mg.L-1的柑桔素均可减弱异烟肼和利福平升高乳酸脱氢酶的作用,但未使其恢复正常水平;5mg.L-1的柑桔素还减弱了异烟肼和利福平合用导致CYP3A4活性增强的作用,但也未使其恢复正常水平。结论异烟肼和利福平合用对人肝细胞具有细胞毒性作用,柑桔素可以通过抑制其升高CYP3A4活性的作用而减轻肝细胞的损伤。     

Apoptotic effect of cyanobacterial extract on rat hepatocytes and human lymphocytes

Toxic cyanobacterial blooms are an increasing problem in Poland. The production of cyanobacterial toxins and their presence in drinking and recreational waters represent a growing danger to human and animal health. This is connected with the increase of cyanobacterial biomass caused by excessive eutrophication of the water ecosystem. There is evidence that cyanobacterial hepatotoxins can act as a potent promoter of primary liver cancer. The apoptotic effect of microcystins in Polish cyanobacterial bloom samples on rat hepatocytes and human lymphocytes was observed using light and fluorescence microscopy, flow cytometry, and electrophoretic analysis. The incubation time needed to observe the first morphological apoptotic changes in hepatocytes was approximately 30 min; however, the characteristic biochemical changes in DNA were not observed even after 120 min. In lymphocyte cultures the morphological changes characteristic for apoptosis were observed after 24 h of incubation and a 48‐h incubation was found to be optimal for analysis of internucleosomal DNA fragmentation, which is one of the main biochemical hallmarks of programmed cell death. These cells are an easily isolated and inexpensive material for medical diagnostics. Therefore the apoptotic changes, together with the clastogenic effect seen in lymphocyte cultures, are proposed as a future analytical method for these toxins. © 2001 John Wiley & Sons, Inc. Environ Toxicol 16: 225–233, 2001     

Inhibition of cytochrome P450s enhances (+)‐usnic acid cytotoxicity in primary cultured rat hepatocytes

(+)‐Usnic acid (UA) is consumed as a dietary supplement to promote weight loss; however, dietary supplements containing UA have been associated with clinical cases of severe liver injury. UA has been shown to be hepatotoxic in rats and is extensively metabolized by hepatic cytochrome P450s (CYPs); therefore, we examined if UA metabolism results in the formation of cytotoxic metabolites or if metabolism is a detoxification process in primary rat hepatocytes. When CYP activity was suppressed by the non‐isoenzyme‐selective inhibitor SKF‐525A (20 μM), or the CYP1A inhibitor alpha‐naphthoflavone (10 μM), or the CYP3A inhibitor ketoconazole (25 μM), the cytotoxicity of UA at 3 ~ 6 μM after 3 ~ 20 h of exposure was significantly increased as measured by lactate dehydrogenase (LDH) leakage. At 2 h after UA exposure, an earlier time point prior to LDH release, these CYP inhibitors potentiated UA‐induced inhibition of cellular respiration as determined by the Clark type oxygen electrode. Cellular adenosine triphosphate (ATP) depletion by UA was also exacerbated by these CYP inhibitors. The CYP2B/2C inhibitor, ticlopidine at 20 μM, showed no effects in parallel experiments. These data demonstrate that UA is bio‐transformed to less toxic metabolites in rat primary hepatocytes, probably mainly by CYP1A and 3A, but not 2B/2C. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.     

Acrylamide toxicity in isolated rat hepatocytes

Acrylamide (ACR) is an important industrial chemical used primarily in the production of polymers and co-polymers. Acrylamide is mainly neurotoxic to experimental animals as well as humans and has also been shown to be mutagenic and carcinogenic. The present study was designed to investigate the toxicity of ACR on isolated rat hepatocytes. The hepatocytes were isolated by collagenase perfusion method and were incubated with different concentrations of ACR (0.1, 1, 10 m ) for 2 hours. Cell viability by trypan blue exclusion and leakage of the enzymes such as alanine transaminase (ALT) and aspartate transaminase (AST) were determined. Reduced glutathione (GSH), glutathione S-transferase (GST) activity were also measured. A significant decrease in the cell viability was observed after exposure to 10 m ACR for 30 min, while 1 m ACR caused a significant decrease in the viability after 60 min. ALT leakage was parallel to the cell viability. AST leakage was significantly increased at 30 min of incubation with 10 m ACR, whereas 2 hours of incubation was required for the leakage of AST from rats hepatocytes with 1 m ACR. 10 m ACR decreased significantly GSH as early as 30 min, while GSH level was decreased at 60 min after exposure to 1 m ACR. Also, the GST activity increased with increasing the dose of ACR. Cytochrome P450 concentration was decreased after exposure to 10 m ACR. The effect of ACR on cell viability, ALT and AST leakage, GSH and GST activity was time and dose dependent.     

Identification of metabolites of evobrutinib in rat and human hepatocytes by using ultra‐high performance liquid chromatography coupled with diode array detector and Q Exactive Orbitrap tandem mass spectrometry

Evobrutinib is a highly selective inhibitor of Bruton's tyrosine kinase (BTK) which may be clinically effective in treating certain autoimmune diseases. The purpose of the present study was to investigate the metabolism of evobrutinib in rat and human hepatocytes. Evobrutinib was incubated with rat and human hepatocytes at 37°C for 2 hours after which the samples were analyzed by ultra‐high performance liquid chromatography with diode array detection and Q Exactive Orbitrap tandem mass spectrometry (UPLC–DAD–Q Exactive Orbitrap‐MS). The acquired data were processed by MetWorks? software using mass effect filter and background subtraction functions. Under these conditions, 23 metabolites were detected and their identities proposed. Among these metabolites, M13 and M15 were identified by comparison of their retention times, accurate masses, and fragment ions with those of authentic reference standards. The metabolic pathways of evobrutinib were proposed accordingly. Our results demonstrated that evobrutinib was metabolized via hydroxylation, hydrolysis, O‐dealkylation, glucuronidation, and GSH conjugation. Species‐related metabolic differences between rat and human hepatocytes were observed. M1–M4 were rat‐specific metabolites. M13 (hydroxyl‐evobrutinib) was the major metabolite whereas M15 (evobrutinib‐diol) was a minor metabolite in rat hepatocytes. On the other hand, M6, M11, M16, M17, and M19 were human‐specific metabolites. M15 was the most abundant metabolite whereas M13 was the minor metabolite in human hepatocytes. This study provides preliminary information regarding the metabolism of evobrutinib that may be helpful in understanding the pharmacology of evobrutinib.     

Primary rat hepatocytes as in vitro system for gene expression studies: comparison of sandwich, Matrigel and 2D cultures

Recent studies have presented evidence that in vivo obtained gene expression data can be used for carcinogen classification, for instance to differentiate between genotoxic and non-genotoxic carcinogens. However, although primary rat hepatocytes represent a well-established in vitro system for drug metabolism and enzyme induction, they have not yet been systematically optimized for toxicogenomic studies. The latter may be confounded by the fact that cultured hepatocytes show strong spontaneous alterations in gene expression patterns. Therefore, we addressed the following questions: (1) which culture system is optimal, comparing sandwich, Matrigel and 2D cultures, (2) how critical is the impact of culture period on substance-induced alterations in gene expression and (3) do these substance-induced alterations in cultured hepatocytes occur already at in vivo relevant concentrations? For this purpose we analyzed the expression of four genes, namely Abat, Gsk3β, Myd116 and Sult1a1 that recently have been reported to be influenced by the antihistamine and non-genotoxic carcinogen methapyrilene (MPy). The most reproducible effects of MPy were observed in sandwich cultures. Induction factors of Gsk3β and Myd116 at 100 μM MPy were 2 and 4 (medians), respectively, whereas expression of Abat and Sult1a1 were inhibited by factors of 7 and 5, respectively. Similar results were observed in hepatocytes maintained for 24 h or 3 weeks in sandwich culture with respect to the influence of MPy on the expression of Abat, Gsk3β, Myd116 and Sult1a1. To determine whether MPy influences gene expression at in vivo relevant concentrations, 3.5 mg/kg MPy were administered to male Wistar rats intraperitoneally, resulting in plasma concentrations ranging between 1.72 and 0.32 μM 5 and 80 min after injection. Inhibition of Abat and Sult1a1 expression in vitro already occurred at in vivo relevant concentrations of 0.39 μM MPy. Induction of Myd116 was observed at 6.25 μM which is higher but in the same order of magnitude as in vivo relevant concentrations. In conclusion, the presented data strongly suggest that sandwich cultures are most adequate for detection of MPy-induced gene expression alterations and the effect of MPy was detected at in vivo relevant concentrations.     

异鼠李素对人肝微粒体CYPs活性及大鼠原代肝细胞毒性作用研究

目的 研究异鼠李素对肝脏6种CYPs的体外抑制作用,以及对大鼠原代肝细胞的毒性作用。方法 采用人肝微粒体（HLMs）体外温孵法研究异鼠李素对6种细胞色素P450酶（CYPs）——CYP2C19、CYP2D6、CYP3A4、CYP2E1、CYP1A2和CYP2C9的体外抑制作用;使用HPLC-MS/MS法检测异鼠李素和HLMs共同孵育后的代谢产物;利用体外培养的低CYPs活性的大鼠原代肝细胞,考察不同剂量异鼠李素对细胞培养液中乳酸脱氢酶（LDH）、丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）的影响。结果 50 μmol/L的异鼠李素对CYP2E1和CYP1A2有一定的抑制作用,抑制率分别为59.48%和39.91%;异鼠李素和HLMs共同孵育后,产生去甲基化代谢产物3,3'',4'',5,7-五羟基黄酮,转化为极性和水溶性较高的代谢物;30、100、300 μmol/L的异鼠李素会使大鼠原代肝细胞培养液中的ALT和LDH显著上升（P＜0.01）,100、300 μmol/L异鼠李素使AST显著上升（P＜0.05、0.01）,呈浓度相关性。结论 异鼠李素在体外主要经HLMs代谢,同时对CYP2E1和CYP1A2有一定的抑制作用,可能会使CYP2E1和CYP1A2的底物药物在体内的浓度产生变化,导致一系列药物的相互作用;大量使用异鼠李素可能会造成一定程度的肝细胞损伤,且呈现浓度相关性。临床应用应合理设置剂量,并注意潜在的药物之间的相互作用。     

Comparison of receptor-mediated endocytosis kinetics between wild-type t-PA and recombinant pamiteplase in isolated rat hepatocytes and liver cell plasma membranes

1. Differences in receptor-mediated endocytosis kinetics between pamiteplase, an engineered t-PA, and an unmodified rt-PA were examined using liver cell plasma membranes and isolated rat hepatocytes. 2. Whereas the binding site of pamiteplase on hepatocytes was the same as that of rtPA, the K_d of pamiteplase was 5.1-7.7 times larger than that of rt-PA, indicating a lower affinity of pamiteplase for the t-PA receptor. 3. k_e for pamiteplase measured using parenchymal cells or non-parenchymal cells was slightly smaller than that for rt-PA, whereas k on for pamiteplase were much lower than that of rt-PA, suggesting that the interaction between pamiteplase and the receptor is slower than that of rt-PA because of its structural modification. 4. Therefore, the difference in drug disposition between pamiteplase and rt-PA is mainly due to the difference in the hepatic clearance caused by a change in the interaction rate between the ligand and its cell-surface receptor.     

Diphenylhydantoin potentiates the protective effect of diazepam against pentylenetetrazol but not against bicuculline and isoniazid-induced seizures in mice

The anticonvulsant activity of diazepam alone, or in combination with diphenylhydantoin was studied in pentylenetetrazol-, bicuculline- and isoniazid-induced Scizures. Alone, diphenylhydantoin did not influence the chemically-induced convulsions but enhanced the antipentylenetetrazol action of diazepam whilst failing to affect the protective effect of the benzodiazepine against bicuculline- and isoniazid-induced convulsions. It is suggested that a diphenylhydantoin-induced increase in the total number of specific benzodiazepine binding sites might be responsible for the enhancement of the antipentylenetetrazol activity of diazepam. The anticonvulsant action of diazepam against bicuculline and isoniazid-induced Scizures does not seem to involve an interaction with benzodiazepine receptors.     

高CYPs酶活性大鼠原代肝细胞模型的建立及其在药物肝毒性评价中的应用

目的 建立一种灵敏、快速的高细胞色素P450(CYPs)酶活性的大鼠原代肝细胞模型,用于评价经CYPs酶代谢后产生肝毒性的药物。方法 分别采用苯巴比妥和β-萘黄酮两种CYPs酶广谱诱导剂构建大鼠原代肝细胞模型;应用“cocktail”探针底物法考察两种诱导剂对CYPs酶的影响;以基于CYPs酶代谢导致肝毒性的药物他克林(TAC)、双氯芬酸钠(DIC)和对乙酰氨基酚(PAR)为模型药物,评价所构建的高CYPs酶活性大鼠原代肝细胞模型与普通大鼠原代肝细胞间灵敏性的差异;应用所构建的高CYPs酶活性大鼠原代肝细胞模型评价维拉帕米(VER)的肝毒性。结果 与普通大鼠原代肝细胞相比,3种模型药物在高CYPs酶活性大鼠原代肝细胞中,在较低剂量时可使细胞上清液中乳酸脱氢酶(LDH)、细胞中活性氧(ROS)水平升高,线粒体膜电位(MMP)水平下降,或相同剂量下得到更严重的损伤结果,CYPs酶抑制剂1-aminobenzotriazole(ABT)和metyrapone(MET)能抑制这3种损伤的发生;100 μmol/L维拉帕米在普通大鼠原代肝细胞中并未引起LDH、ROS和MMP的改变,但在高CYPs活性大鼠原代肝细胞模型中,会引起细胞损伤,ABT和MET能抑制这种损伤。结论 经诱导建立的两种高CYPs酶活性大鼠原代肝细胞模型能更灵敏的评价基于CYPs酶代谢致毒药物的肝毒性;两种诱导剂诱导得到的高CYPs酶活性大鼠原代肝细胞模型对经不同CYPs酶亚型代谢的药物评价结果有各自优势。应用高CYPs酶活性大鼠原代肝细胞模型证实维拉帕米的致毒机制是经CYPs酶代谢产生肝毒性。     

A model describing the disposition of phenytoin in isolated rat hepatocytes

The use of isolated rat hepatocytes in studies of drug metabolism has become well documented in the past few years. However, in part because of modelling difficulties due to the simultaneously occurring substrate transferring processes, its predictability of in vivo situations has not been emphasized. Much controversy surrounds the metabolism of phenytoin (5,5-diphenylhydantoin), a widely used anticonvulsant, and an appropriate pharmacokinetic model to describe the disposition of this drug still lacks general acceptance. In the present study, metabolism of phenytoin in the isolated rat hepatocyte system was followed by assaying either the unchanged drug or the pooled metabolites in both the suspending medium and the cells. A model was developed which can describe the time course of the different species sampled. Inhibition of biotransformation by the major metabolic product [5-(p-hydroxyphenyl)-5-phenylhydantoin or p-HPPH] and the uptake and release of the latter were also studied, in order to elucidate the role of product inhibition in determining the dose-dependent pharmacokinetic behaviour of the drug. The results obtained strongly suggest that only concentrations of p-HPPH higher than the ones attained by phenytoin biotransformation alone can significantly inhibit the main enzymatic reaction.     

丹参对GSH-Px/MDA比值的影响及其在人肝细胞胶原合成中的作用

利用四氯化碳（CCl4）造成肝细胞脂质过氧化损伤，测定原代培养人胚肝细胞匀浆中羟脯氨酸（Hyp）含量及培养液中Ⅲ型前胶原（PCⅢ）、脂质过氧化物（MDA）、谷胱甘肽过氧化物酶（GSH－Px）活性水平．观察丹参对GSH－Px／MDA比值的影响及其与PCⅢ、Hyp水平的关系。结果：与损伤模型组比较，丹参（1mg／ml）预作用4h，可明显减少受损肝细胞产生PCⅢ、Hyp及MDA，提高GSH－Px活性，GSH－Px／MDA比值增加非常显著（P＜0．01）。提示丹参可抑制CCl4所致人胚肝细胞胶原合成。     

Effects of model inducers on thyroxine UDP-glucuronosyl-transferase activity in vitro in rat and mouse hepatocyte cultures.

Thyroxine (T₄)-UDP-glucuronosyltransferase (UGT) activity was measured directly in cultured male Sprague–Dawley rat and OF-1 mouse hepatocyte monolayers. The activity of T₄-UGT (pmol/min/g liver) in vitro in hepatocyte cultures was, after 24 hr in culture, equivalent to that previously measured in vivo in rat and mouse liver microsomes (Viollon-Abadie et al., 1999). A progressive decline in T₄-UGT activity occurred over time in both rat and mouse hepatocyte cultures. Treatment of cultures with various model inducers such as phenobarbital (PB), β-naphthoflavone (NF) and clofibric acid (CLO) induced a strong increase in T₄-UGT activity in rat hepatocyte monolayers. In addition, and as expected from available in vivo data, treatment of rat hepatocyte cultures with NF also increased p-nitrophenol (PNP)-UGT activity and treatment with PB or CLO increased bilirubin (Bili)-UGT activity. In contrast, T₄-UGT activity in mouse hepatocyte monolayers was not affected by the treatments, neither were PNP- and Bili- UGT activities. These in vitro data confirm our previous in vivo observations that these inducers increase rat but not mouse liver T₄-UGT activities (Viollon-Abadie et al., 1999). The present study thus demonstrates that hepatocyte monolayers are appropriated for the evaluation and inter-species comparison of the effects of xenobiotics on T₄-UGT activities.     

那格列奈在大鼠游离肝细胞中的代谢特点

目的:研究新型非磺酰脲类降血糖药那格列奈(nateglinide)在大鼠游离肝细胞的代谢特点.方法:为考察该药的体外代谢和参与代谢的CYP450同工酶的种类,167μmol·L-1 的CYP450同工酶抑制剂或底物(奎尼丁、磺胺嘧啶、奥美拉唑、普奈洛尔、西米替丁、左氧氟沙星、红霉素)和那格列奈在8×106个肝细胞*mL-1的混悬液中37℃时孵育60min,并于10,20,30,40,60min取样,HPLC法测定其中母体药物--那格列奈的浓度,并同空白对照组比较.结果:那格列奈的体外代谢迅速,10min内细胞悬液中原形药物仅占初始浓度的10%;奎尼丁、奥美拉唑、西米替丁能明显抑制那格列奈的肝脏代谢,使混悬液中母体药物浓度明显提高(P<0.01),磺胺嘧啶和普奈洛尔的加入,也使母体药物浓度显著提高(P<0.05).结论:CYP450同工酶CYP2D6和CYP2C9在那格列奈羟化过程中起重要作用.CYP1A2,CYP2C19和CYP3A4同工酶是否参与代谢尚需进一步实验研究.     

Screening of amide analogues of Trichostatin A in cultures of primary rat hepatocytes: search for potent and safe HDAC inhibitors

Summary  The vast majority of preclinical studies of HDAC inhibitors (HDAC-I) focus on the drug–target (cancer) cell interaction, whereas little attention is paid to the effects on non-target healthy cells, which could provide decisive information to eliminate potential cytotoxic compounds at a very early stage during drug development. In the current study we used cultures of primary rat hepatocytes as a read out system to select for the most potent HDAC-I in the group of structural analogues of an archetypal HDAC-I, namely Trichostatin A. This kind of approach allowed selecting compounds with high biological activity and with no apparent toxicity towards cultured hepatocytes. Joanna Fraczek and Sarah Deleu contributed equally to this article. T. Vanhaecke is a postdoctoral research fellow of the Fund for Scientific Research Flanders (FWO-Vlaanderen, Belgium)     

Effects of model inducers on thyroxine UDP-glucuronosyl-transferase activity in vitro in rat and mouse hepatocyte cultures

Thyroxine (T₄)-UDP-glucuronosyltransferase (UGT) activity was measured directly in cultured male Sprague–Dawley rat and OF-1 mouse hepatocyte monolayers. The activity of T₄-UGT (pmol/min/g liver) in vitro in hepatocyte cultures was, after 24 hr in culture, equivalent to that previously measured in vivo in rat and mouse liver microsomes (Viollon-Abadie et al., 1999). A progressive decline in T₄-UGT activity occurred over time in both rat and mouse hepatocyte cultures. Treatment of cultures with various model inducers such as phenobarbital (PB), β-naphthoflavone (NF) and clofibric acid (CLO) induced a strong increase in T₄-UGT activity in rat hepatocyte monolayers. In addition, and as expected from available in vivo data, treatment of rat hepatocyte cultures with NF also increased p-nitrophenol (PNP)-UGT activity and treatment with PB or CLO increased bilirubin (Bili)-UGT activity. In contrast, T₄-UGT activity in mouse hepatocyte monolayers was not affected by the treatments, neither were PNP- and Bili- UGT activities. These in vitro data confirm our previous in vivo observations that these inducers increase rat but not mouse liver T₄-UGT activities (Viollon-Abadie et al., 1999). The present study thus demonstrates that hepatocyte monolayers are appropriated for the evaluation and inter-species comparison of the effects of xenobiotics on T₄-UGT activities.