Predictive models of hepatotoxicity using gene expression data from primary rat hepatocytes

With the aim of evaluating the usefulness of an in vitro system for assessing the potential hepatotoxicity of compounds, the paper describes several methods of obtaining mathematical models for the prediction of compound-induced toxicity in vivo. These models are based on data derived from treating rat primary hepatocytes with various compounds, and thereafter using microarrays to obtain gene expression 'profiles' for each compound. Predictive models were constructed so as to reduce the number of 'probesets' (genes) required, and subjected to rigorous cross-validation. Since there are a number of possible approaches to derive predictive models, several distinct modelling strategies were applied to the same data set, and the outcomes were compared and contrasted. While all the strategies tested showed significant predictive capability, it was interesting to note that the different approaches generated models based on widely disparate probesets. This implies that while these models may be useful in ascribing relative potential toxicity to compounds, they are unlikely to provide significant information on underlying toxicity mechanisms. Improved predictivity will be obtained through the generation of more comprehensive gene expression databases, covering more 'toxicity space', and by the development of models that maximize the observation, and combination, of individual differences between compounds.     

In vitro assay for drug‐induced hepatosteatosis using rat primary hepatocytes,a fluorescent lipid analog and gene expression analysis

To evaluate new drugs' potential for hepatosteatosis, we developed a cell‐based assay using a fluorescent fatty acid analog: BODIPY558/568 C₁₂. Rat primary hepatocytes were exposed to positive reference compounds [cyclosporine A (CsA), clofibrate (CFR), tetracycline (TC), valproic acid (VPA), carbon tetrachloride (CCl4), tamoxifen (TMX)] in the presence of BODIPY558/568 C₁₂. The formation of fluorsecent particles or lipid droplets in the cytoplasm was confirmed by confocal laser scanning microscopy and electron microscopy respectively. The accumulation of BODIPY558/568 C₁₂ was measured by fluorometry and high content imaging method. All positive reference compounds increased fluorescent particles in number and fluorescence intensity. High content imaging was more sensitive and selective method than fluorometry to detect fluorescent particles. Gene expression analysis of the hepatocytes showed two patterns: genes related to lipid metabolism/synthesis were down‐regulated by oxidative stress inducing compounds: CsA, TC and TMX, and up‐regulated by peroxisome proliferator‐activated receptor‐alpha agonists: CFR and VPA. From these findings, we concluded that the cell‐based assay developed in this study is an appropriate method to predict drugs' potential for hepatosteatosis, and gene expression analysis is useful to profile the mechanism of the hepatosteatosis. Copyright © 2009 John Wiley & Sons, Ltd.     

An improved model of predicting hepatocarcinogenic potential in rats by using gene expression data

Carcinogenicity studies using animals are expensive and time consuming. Therefore, the development of a highly accurate carcinogenicity prediction system to interpret short‐term test results would be beneficial. The Ames test is popular for mutagens; however, it cannot detect non‐genotoxic carcinogens. Previously, we reported a prediction system using gene expression data obtained from a short‐term (28‐day) study that screened candidate compounds for testing in long‐term carcinogenicity studies. In this study, our system was improved by adding more gene expression data. To establish our new system, we used the data of 93 test compounds (41 hepatocarcinogens and 52 non‐hepatocarcinogens). Analysis of liver gene expression data by dividing compounds into ‘for training’ and ‘for test’ categories (20 cases assigned randomly) using Support Vector Machine (SVM) identified a set of marker probe sets that could be used to predict hepatocarcinogenicity. The assigned 42 probe sets have included the cancer‐ or c‐Myc‐related genes such as Hsp90, Pink1, Hspc111, Fbx29, Hepsin, Syndecan2 and Synbindin. Compared with the older version, the improved system had a higher concordance rate with the training data and a good performance with the external test data. Copyright © 2015 John Wiley & Sons, Ltd.     

抗结核病药物利福平致大鼠肝脏损伤的基因表达谱分析

目的:利用基因芯片技术研究抗结核病药物利福平对大鼠肝脏毒性效应的分子机制。方法:20只Wistar大鼠随机分为对照组和利福平组,每组10只,分别灌胃给予利福平(100 mg.kg-1)和等体积生理盐水,连续14 d。以cDNA微阵列技术研究利福平对大鼠肝脏基因表达谱的影响,根据差异表达基因的生物学功能探讨利福平对大鼠肝脏的毒性机制。结果:利福平组与对照组杂交的表达差异基因共19个,其中上调基因11个,下调基因8个,功能已知基因18条,主要包括一些与肝药酶活性、氧化应激和特异性免疫等相关的基因。结论:利福平可导致大鼠肝脏基因表达谱明显变化。该研究对阐明利福平的肝损伤机制具有十分重要的作用。     

Effects of clofibric acid on mRNA expression profiles in primary cultures of rat,mouse and human hepatocytes

The mRNA expression profile in control and clofibric acid (CLO)-treated mouse, rat, and human hepatocytes was analyzed using species-specific oligonucleotide DNA microarrays (Affymetrix). A statistical empirical Bayes procedure was applied in order to select the significantly differentially expressed genes. Treatment with the peroxisome proliferator CLO induced up-regulation of genes involved in peroxisome proliferation and in cell proliferation as well as down-regulation of genes involved in apoptosis in hepatocytes of rodent but not of human origin. CLO treatment induced up-regulation of microsomal cytochrome P450 4a genes in rodent hepatocytes and in two of six human hepatocyte cultures. In addition, genes encoding phenobarbital-inducible cytochrome P450s were also up-regulated by CLO in rodent and human hepatocyte cultures. Up-regulation of phenobarbital-inducible UDP-glucuronosyl-transferase genes by CLO was observed in both rat and human but not in mouse hepatocytes. CLO treatment induced up-regulation of L-fatty acid binding protein (L-FABP) gene in hepatocytes of both rodent and human origin. However, while genes of the cytosolic, microsomal, and mitochondrial pathways involved in fatty acid transport and metabolism were up-regulated by CLO in both rodent and human hepatocyte cultures, genes of the peroxisomal pathway of lipid metabolism were up-regulated in rodents only. An up-regulation of hepatocyte nuclear factor 1alpha (HNF1alpha) by CLO was observed only in human hepatocyte cultures, suggesting that this trans-activating factor may play a key role in the regulation of fatty acid metabolism in human liver as well as in the nonresponsiveness of human liver to CLO-induced regulation of cell proliferation and apoptosis.     

l-carnitine protects rat hepatocytes from oxidative stress induced by T-2 toxin

Context: Oxidative stress and mitochondrial dysfunction are thought to be the main mechanism of T-2 toxin toxicity. T-2 toxin is the most potent trichothecene mycotoxin which is present in agricultural products. L-carnitine, besides its anti-oxidative properties, facilitates the transportation of long-chain fatty acids in to mitochondrial matrix. Objective: In this study we tested whether L-carnitine, an antioxidant and a facilitator for long-chain fatty acid transportation across mitochondrial membranes, could protect rat hepatocytes against toxicity induced by T-2 toxin. Materials and methods: L-carnitine in low and high doses (50 and 500?mg/kg) was administered for five consecutive days to male Wistar rats. Hepatocytes were isolated and freshly exposed to appropriate concentration of T-2 toxin for 2?h followed by oxidative stress and cell death evaluations. Results: Glutathione depletion, ROS overproduction and mitochondrial membrane potential collapse were determined under T-2 toxin exposure. Pretreatment with L-carnitine particularly at high-dose reduced toxicity and prevented the hepatocytes from abnormal caspase-3 activity and apoptosis. Conclusion: Low toxicity of L-carnitine and its mitochondrial protective effects promises an effective way to reduce or prevent the toxicity induced by certain environmental pollutants, including T-2 toxin.     

Inhibition of carrier-mediated uptake of epirubicin reduces cytotoxicity in primary culture of rat hepatocytes

Epirubicin, an antineoplastic drug, is considered to be taken up by tumor cells via a common carrier by facilitated diffusion and is then pumped out in an energy-dependent manner because epirubicin is a substrate for P-glycoprotein (P-gp). However, this study investigated the details of the influx mechanism of epirubicin and demonstrated that epirubicin uptake was mediated by active carrier systems in addition to facilitated diffusion in the primary culture of rat hepatocytes. The uptake of epirubicin gradually increased in a saturated manner when the concentrations were between 1 x 10(-7) M and 1 x 10(-6) M. In contrast, the uptake increased progressively in a linear manner when the concentration was high (greater than 1 x 10(-6) M). The uptake of epirubicin at a clinical concentration (7.5 x 10(-7) M) was significantly reduced at 4 degrees C and significantly inhibited when pretreated with metabolic inhibitors (carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), rotenone and sodium azide) by nearly 25%. Furthermore, an organic anion transporter inhibitor, namely, 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS); organic anion transport substrates, namely, para-aminohippurate (PAH), taurocholic acid and estradiol 17-beta-D-glucuronide; and organic cation transporter inhibitors, namely, verapamil and tetraethylammonium significantly reduced the uptake of epirubicin. Furthermore, pretreatment with verapamil and PAH significantly prevented epirubicin-induced reduction of proliferative activity in rat hepatocytes. These results indicated that the uptake of epirubicin was induced, at least in part, by the active transport protein in rat hepatocytes; the inhibition of the probable transport protein protected the intact normal cells from the injury induced by the cytotoxicity of epirubicin.     

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。     

葡萄糖和胰岛素调节三明治肝细胞中胆酸相关转运体表达及功能

目的:测定葡萄糖、胰岛素等糖尿病血糖中的异常因素对体外培养肝细胞中胆汁酸相关转运体表达及功能的影响.方法:分离并用三明治法培养大鼠原代肝细胞,用不同浓度葡萄糖或胰岛素培养72 h,使用胆盐类似物Cholyl-lysyl-fluorescein(CLF)进行摄取转运体钠/胆汁酸共转运体(Na+/bile acid cotransporter,ntcp)及外排转运体胆盐外排泵(Bile salt export pump,bsep)转运体功能学实验,并用荧光定量PCR法测定胆汁酸相关转运体ntcp、bsep及调节胆汁酸代谢转运的核受体(Farnesoid X receptor,fxr)的mRNA表达变化.结果:高浓度的葡萄糖和胰岛素均使CLF的胆泡排泄减少.高糖和高胰岛素均能够降低ntcp的表达)3～4倍),增加bsep的表达(约2倍),高糖能够降低fxr的水平(约0.5倍),高胰岛素增加fxr的水平(约1.5倍).结论:葡萄糖和胰岛素水平异常均会对胆汁酸相关转运体表达和功能造成影响;三明治原代肝细胞可用于胆酸和药物肝胆分布研究.     

Protective effect of JBP485 on concanavalin A-induced hepatocyte toxicity in primary cultured rat hepatocytes

Cyclo-trans-4-L-hydroxyprolyl-L-serine (JBP485) is a dipeptide isolated from Laennec, and Laennec is a hydrolyzate of human placenta. Evidence has indicated that JBP485 exhibits potent anti-hepatitis activity. In this study, we investigated the protective effect and possible mechanisms of action of JBP485 in Concanavalin A (Con A)-induced hepatotoxicity in vitro. Two in vitro models were established. Model I: primary cultured female rat hepatocytes were only incubated with Con A (50 microg/ml); model II: co-culture system of hepatocytes and autologous splenic lymphocytes, both were stimulated with Con A (20 microg/ml). JBP485 (25 microM) was pre-incubated with the two models. Our results showed that JBP485 reduced cellular aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and tumor necrosis factor alpha (TNF-alpha) leakage following the application of Con A in both of the models. Potential protective mechanisms were elucidated by measuring DNA fragmentations, immunocytochemistry and RT-PCR. We showed that DNA fragmentations in hepatocytes were attenuated in the JBP485 pre-incubated groups, and at the same time, immunocytochemistry and RT-PCR indicated that expression levels of caspase-3 protein and mRNA in the JBP485 treated groups were decreased compared with those in the untreated groups. Moreover, intercellular adhesion molecule-1 (ICAM-1) was also down-regulated by this dipeptide. The results indicate that JBP485 exhibits hepatoprotective effect through inhibition of hepatocyte apoptosis and ICAM-1 expression.     

Left ventricular gene expression profile of healthy and cardiovascular compromised rat models used in air pollution studies

Abstract

The link between pollutant exposure and cardiovascular disease (CVD) has prompted mechanistic research with animal models of CVD. We hypothesized that the cardiac gene expression patterns of healthy and genetically compromised, CVD-prone rat models, with or without metabolic impairment, will reveal underlying disease processes that facilitate understanding of the mechanisms of air pollution susceptibility differences. Left ventricular gene expression was examined using Affymetrix rat 230A-gene arrays in male, age-matched (12–14 weeks old) healthy Wistar Kyoto (WKY) and CV-compromised spontaneously hypertensive (SH), stroke-prone SH (SHSP), obese SH heart failure (SHHF) and obese insulin-resistant (JCR) rats. Principle component analysis separated strains in three clusters: (1) WKY, (2) JCR and (3) SH, SHSP and SHHF. Gene expression pattern in JCR differed from all other CVD strains. Both SHHF and JCR strains presented the most differentially expressed genes from WKY, but generally with opposing directional pattern suggesting that the CVD in these strains arise through different mechanisms. Hierarchical clustering of nuclear factor-kappaB target genes indicated varying degrees of, but similar directional changes, in SH, SHSP and SHHF relative to WKY rats, which may relate to the severity of their CVD. The JCR strain had less pronounced expressions of these genes suggesting milder cardiac disease. No unique expression pattern could be identified for genes implicated in stroke and heart failure in SHSP and SHHF rats, respectively. The data show that the CVD pathophysiological mechanisms differ in models with different genetic backgrounds, and therefore, the mechanisms by which air pollutants affect the cardiopulmonary system are likely to vary.     

肝细胞生长因子在LO2细胞表达的实验研究

目的 将肝细胞生长因子(hepatocyte growth factor,HGF)真核表达栽体,转染培养的LO2肝细胞,观察其表达。方法 将含肝细胞生长因子cDNA真核表达栽体PEGFP-HCF,脂质体(Lipofectauline)转染培养的LO2肝细胞;PCR鉴定外源基因的导入,绿荧光蛋白检测肝细胞生长因子的表达。结果 (1)重组质粒PEGFP-C1-HGF转染峨肝细胞,5d后,细胞内可检测到绿荧光蛋白。(2)PCR结果显示：转染PEGFP-C1-HGF质粒组可见306bp特征条带与阳性对照大小一样,假转染组与空质粒组无特征条带。结论 人肝细胞生长因子基因真核表达栽体,PEGFP-C1-HGF,能成功转染LO2细胞并获表达。     

Relationship between hepatic gene expression profiles and hepatotoxicity in five typical hepatotoxicant-administered rats.

In the field of gene expression analysis, DNA microarray technology has a major impact on many different areas including toxicogenomics, such as in predicting the adverse effects of new drug candidates and improving the process of risk assessment and safety evaluation. In this study, we investigated whether there is relationship between the hepatotoxic phenotypes and gene expression profiles of hepatotoxic chemicals measured by DNA microarray analyses. Sprague-Dawley rats (6 weeks old) were administered five hepatotoxicants: acetaminophen (APAP), bromobenzene, carbon tetrachloride, dimethylnitrosamine, and thioacetamide. Serum biochemical markers for liver toxicity were measured to estimate the maximal toxic time of each chemical. Hepatic mRNA was isolated, and the gene expression profiles were analyzed by DNA microarray containing 1,097 drug response genes, such as cytochrome P450s, other phase I and phase II enzymes, nuclear receptors, signal transducers, and transporters. All the chemicals tested generated specific gene expression patterns. APAP was sorted to a different cluster from the other four chemicals. From the gene expression profiles and maximal toxic time estimated by serum biochemical markers, we identified 10 up-regulated genes and 10 down-regulated genes as potential markers of hepatotoxicity. By Quality-Threshold (QT) clustering analysis, we identified major up- and down-regulated expression patterns in each group. Interestingly, the average gene expression patterns from the QT clustering were correlated with the mean value profiles from the biochemical markers. Furthermore, this correlation was observed at any extent of hepatotoxicity. In this study, we identified 17 potential toxicity markers, and those expression profiles could estimate the maximal toxic time independently of the hepatotoxicity levels. This expression profile analysis could be one of the useful tools for evaluating a potential hepatotoxicant in the drug development process.     

乙醇抑制原代大鼠星形细胞神经营养因子的基因表达

为了阐明乙醇中毒对神经系统损害的机理 ,用0 .5- 5.0 g· L-1浓度的乙醇处理原代培养的 CD新生大鼠大脑皮层星形细胞 ,用反转录 PCR和Northern印迹杂交方法同时测定神经营养因子( NGF)的基因表达水平 .结果显示 0 .5g·L-1乙醇在染毒 2 4 h后 ,NGF m RNA水平开始降低 ,5.0 g· L-1在染毒 48或 72 h后 ,NGF m RNA水平与对照组比较降低了 50 % - 58% .结果表明 ,乙醇中毒在体外可降低新生大鼠星形细胞 NGF m RNA的生成 ,这种作用可干扰神经元与胶质细胞间的正常交互作用并可能与胚胎发育期乙醇接触导致的神经病理改变有关 .     

Comparative gene expression of intestinal metabolizing enzymes

The purpose of this study was to compare the expression profiles of drug‐metabolizing enzymes in the intestine of mouse, rat and human. Total RNA was isolated from the duodenum and the mRNA expression was measured using Affymetrix GeneChip oligonucleotide arrays. Detected genes from the intestine of mouse, rat and human were ca. 60% of 22690 sequences, 40% of 8739 and 47% of 12559, respectively. Total genes of metabolizing enzymes subjected in this study were 95, 33 and 68 genes in mouse, rat and human, respectively. Of phase I enzymes, the mouse exhibited abundant gene expressions for Cyp3a25, Cyp4v3, Cyp2d26, followed by Cyp2b20, Cyp2c65 and Cyp4f14, whereas, the rat showed higher expression profiles of Cyp3a9, Cyp2b19, Cyp4f1, Cyp17a1, Cyp2d18, Cyp27a1 and Cyp4f6. However, the highly expressed P450 enzymes were CYP3A4, CYP3A5, CYP4F3, CYP2C18, CYP2C9, CYP2D6, CYP3A7, CYP11B1 and CYP2B6 in the human. For phase II enzymes, glucuronosyltransferase Ugt1a6, glutathione S‐transferases Gstp1, Gstm3 and Gsta2, sulfotransferase Sult1b1 and acyltransferase Dgat1 were highly expressed in the mouse. The rat revealed predominant expression of glucuronosyltransferases Ugt1a1 and Ugt1a7, sulfotransferase Sult1b1, acetyltransferase Dlat and acyltransferase Dgat1. On the other hand, in human, glucuronosyltransferases UGT2B15 and UGT2B17, glutathione S‐transferases MGST3, GSTP1, GSTA2 and GSTM4, sulfotransferases ST1A3 and SULT1A2, acetyltransferases SAT1 and CRAT, and acyltransferase AGPAT2 were dominantly detected. Therefore, current data indicated substantial interspecies differences in the pattern of intestinal gene expression both for P450 enzymes and phase II drug‐metabolizing enzymes. This genomic database is expected to improve our understanding of interspecies variations in estimating intestinal prehepatic clearance of oral drugs. Copyright © 2009 John Wiley & Sons, Ltd.