A zebrafish phenotypic assay for assessing drug-induced hepatotoxicity

IntroductionNumerous studies have confirmed that zebrafish and mammalian toxicity profiles are strikingly similar and the transparency of larval zebrafish permits direct in vivo assessment of drug toxicity including hepatotoxicity in zebrafish.MethodsHepatotoxicity of 6 known mammalian hepatotoxic drugs (acetaminophen [APAP], aspirin, tetracycline HCl, sodium valproate, cyclophosphamide and erythromycin) and 2 non-hepatotoxic compounds (sucrose and biotin) were quantitatively assessed in larval zebrafish using three specific phenotypic endpoints of hepatotoxicity: liver degeneration, changes in liver size and yolk sac retention. Zebrafish liver degeneration was originally screened visually, quantified using an image-based morphometric analysis and confirmed by histopathology.ResultsAll the tested mammalian hepatotoxic drugs induced liver degeneration, reduced liver size and delayed yolk sac absorption in larval zebrafish, whereas the non-hepatotoxic compounds did not have observable adverse effect on zebrafish liver. The overall prediction success rate for hepatotoxic drugs and non-hepatotoxic compounds in zebrafish was 100% (8/8) as compared with mammalian results, suggesting that hepatotoxic drugs in mammals also caused similar hepatotoxicity in zebrafish.DiscussionLarval zebrafish phenotypic assay is a highly predictive animal model for rapidly in vivo assessment of compound hepatotoxicity. This convenient, reproducible animal model saves time and money for drug discovery and can serve as an intermediate step between cell-based evaluation and conventional animal testing of hepatotoxicity.     

基于斑马鱼模型的中药致肝脏毒性评价的研究进展

中药的广泛使用,提高了肝脏毒性发生风险。斑马鱼模型在肝脏毒性研究中显现出较大潜力的同时,也面临评价标准难以统一的问题。在使用斑马鱼进行药物肝损伤检测和评价之前应先明确其在不同生长阶段的肝功能状态,利用斑马鱼胚胎和幼鱼的通体透明的特点,以胚胎的孵化率、死亡率、畸形率作出初步的发育毒性判断,以幼鱼肝脏灰度值,肝脏面积大小,卵黄囊吸收程度作为药物是否具有肝脏毒性的判断标准,利用成鱼肝脏容易分离的特点研究肝脏毒性的作用机制。检测时可根据这些指标判断斑马鱼肝脏发育阶段,为其是否发生药物肝损伤提供依据,从而进一步提高临床用药安全性。综述了不同发育时期斑马鱼评估中药肝毒性的研究方法及研究进展,以期为后续中药的肝脏毒性评价提供参考。     

Zebrafish larva as a reliable model for in vivo assessment of membrane remodeling involvement in the hepatotoxicity of chemical agents

The easy‐to‐use in vivo model, zebrafish larva, is being increasingly used to screen chemical‐induced hepatotoxicity, with a good predictivity for various mechanisms of liver injury. However, nothing is known about its applicability in exploring the mechanism called membrane remodeling, depicted as changes in membrane fluidity or lipid raft properties. The aim of this study was, therefore, to substantiate the zebrafish larva as a suitable in vivo model in this context. Ethanol was chosen as a prototype toxicant because it is largely described, both in hepatocyte cultures and in rodents, as capable of inducing a membrane remodeling leading to hepatocyte death and liver injury. The zebrafish larva model was demonstrated to be fully relevant as membrane remodeling was maintained even after a 1‐week exposure without any adaptation as usually reported in rodents and hepatocyte cultures. It was also proven to exhibit a high sensitivity as it discriminated various levels of cytotoxicity depending on the extent of changes in membrane remodeling. In this context, its sensitivity appeared higher than that of WIF‐B9 hepatic cells, which is suited for analyzing this kind of hepatotoxicity. Finally, the protection afforded by a membrane stabilizer, ursodeoxycholic acid (UDCA), or by a lipid raft disrupter, pravastatin, definitely validated zebrafish larva as a reliable model to quickly assess membrane remodeling involvement in chemical‐induced hepatotoxicity. In conclusion, this model, compatible with a high throughput screening, might be adapted to seek hepatotoxicants via membrane remodeling, and also drugs targeting membrane features to propose new preventive or therapeutic strategies in chemical‐induced liver diseases. Copyright © 2016 John Wiley & Sons, Ltd.     

In vitro CYP1A activity in the zebrafish: temporal but low metabolite levels during organogenesis and lack of gender differences in the adult stage

The zebrafish (Danio rerio) is increasingly used as a screening model for acute, chronic and developmental toxicity. More specifically, the embryo is currently investigated as a replacement of in vivo developmental toxicity studies, although its biotransformation capacity remains a point of debate. As the cytochrome P450 1 (CYP1) family plays an important role in the biotransformation of several pollutants and drugs, a quantitative in vitro protocol was refined to assess gender- and age-related CYP1A activity in the zebrafish using the ethoxyresorufin-o-deethylase (EROD) assay. Microsomal protein fractions were prepared from livers of adult males and females, ovaries and whole embryo homogenates of different developmental stages. A large biological variation but no gender-related difference in CYP1A activity was observed in adult zebrafish. Embryos showed distinct temporal but low CYP1A activity during organogenesis. These in vitro data raise questions on the bioactivation capacity of zebrafish embryos in developmental toxicity studies.     

Are zebrafish larvae suitable for assessing the hepatotoxicity potential of drug candidates?

Drug‐induced liver injury (DILI) is poorly predicted by single‐cell‐based assays, probably because of the lack of physiological interactions with other cells within the liver. An intact whole liver system such as one present in zebrafish larvae could provide added value in a screening strategy for DILI; however, the possible occurrence of other organ toxicities and the immature larval stage of the zebrafish might complicate accurate and fast analysis. We investigated whether expression analysis of liver‐specific fatty acid binding protein 10a (lfabp10a) was an appropriate endpoint for assessing hepatotoxic effects in zebrafish larvae. It was found that expression analysis of lfabp10a was a valid marker, as after treatment with hepatotoxicants, dose–response curves could be obtained and statistically significant abnormal lfabp10 expression levels correlated with hepatocellular histopathological changes in the liver. However, toxicity in other vital organs such as the heart could impact liver outgrowth and thus had to be assessed concurrently. Whether zebrafish larvae were suitable for assessing human relevant drug‐induced hepatotoxicity was assessed with hepatotoxicants and non‐hepatotoxicants that have been marketed for human use and classified according to their mechanism of toxicity. The zebrafish larva showed promising predictivity towards a number of mechanisms and was capable of distinguishing between hepatotoxic and non‐hepatotoxic chemical analogues, thus implying its applicability as a potential screening model for DILI. Copyright © 2015 John Wiley & Sons, Ltd.     

An investigation of the mechanism of hepatotoxicity of the antitumour agent N-methylformamide in mice

N-Methylformamide (NMF) has been reported to cause liver damage in animals and man. This hepatotoxicity was characterized in BALB/c mice by the release of liver enzymes into the plasma and by histopathological examination of livers after single and repeated administration of NMF. Whereas plasma levels of sorbitol dehydrogenase were elevated dramatically 24 hr after 400 mg/kg given as a single dose, the glutathione content of the livers was not different from controls even after repeated administration. Liver damage was apparent on gross inspection and was defined as periacinar necrosis on histopathology. A dose of 100 mg/kg did not cause damage even after repeated injections on five consecutive days. The hypothesis that NMF is metabolized to a chemically reactive species was tested. Incubation of mouse hepatocytes with 7 mM NMF for 80 min produced a decrease in intracellular glutathione. Exposure of hepatocytes to NMF for 240 min led to the production of breakdown products of lipid peroxides at levels significantly above controls. However, incubation of microsomes or mitochondria with NMF and NADPH did not lead to raised levels of lipid peroxides. The effects described were specific to NMF as incubation of N,N-dimethylformamide, N-hydroxymethylformamide or formamide with hepatocytes did not result in glutathione depletion or increased lipid peroxidation. NMF undergoes extensive metabolism in vivo and the results indicate that NMF forms a chemically reactive metabolite, even though incubation of the drug with liver fractions or hepatocytes did not lead to metabolites at levels which were analytically identifiable.     

红霉素和阿奇霉素诱导斑马鱼肝毒性及作用机制研究

目的 利用模式生物斑马鱼模型探索红霉素和阿奇霉素导致肝毒性及作用机制,并比较红霉素和阿奇霉素诱导肝毒性的差异。方法 选用肝脏转基因斑马鱼Tg(fapb10: dsRed)作为实验动物,将发育正常的3dpf斑马鱼幼鱼暴露于不同浓度的红霉素和阿奇霉素溶液中72h后,统计各组斑马鱼死亡率,计算红霉素和阿奇霉素对斑马鱼的LD50;荧光显微镜下活体观察斑马鱼幼鱼给药后肝脏形态的变化;采用整体油红O染色观察肝脏脂肪含量变化;利用qRT-PCR检测肝脏病理标志基因和凋亡相关基因在转录水平的变化。结果 红霉素和阿奇霉素对斑马鱼的LD50分别为3.82mmol/L和3.10mmol/L;活体观察显示红霉素和阿奇霉素均能导致幼鱼肝脏形态和荧光强度变化;整体油红O染色显示红霉素和阿奇霉素均能导致肝细胞脂肪堆积,并呈剂量依赖性;qRT-PCR结果显示红霉素和阿奇霉素均能影响脂肪肝、肝纤维化和细胞凋亡相关基因的表达。结论 红霉素和阿奇霉素均能诱导斑马鱼肝毒性,作用机制可能与两者诱导肝细胞变性或凋亡有关,但两者影响的相关基因通路可能有所不同。     

Cyclosporine A treated in vitro models induce cholestasis response through comparison of phenotype-directed gene expression analysis of in vivo Cyclosporine A-induced cholestasis

In vitro models for hepatotoxicity testing are a necessity for advancement of toxicological research. Assessing the in vitro response requires in vivo validated gene sets reflective of the hepatotoxic phenotype. Cholestasis, the impairment of bile flow, is induced in C57BL/6J mice treated with cyclosporine A (CsA) to identify phenotype reflective gene sets. CsA treatment through oral gavage for 25 days induced cholestasis, as confirmed by histopathology and serum chemistry. Over 1, 4, and 11 days of CsA exposure gradual increases in serum markers were correlated to gene expression. This phenotype-directed analysis identified gene sets specific to the onset and progression of cholestasis, such as PPAR related processes and drug metabolism, by circumventing other effects of CsA, such as immunosuppression, found in dose*time group analysis. In vivo gene sets are enriched in publicly available data sets of CsA-treated HepaRG and primary mouse hepatocytes. However, genes identified within these gene sets did not overlap between in vivo and in vitro. In vitro regulated genes represent the initial response to cholestasis, whereas in vivo genes represent the later adaptive response. We conclude that the applicability of in vitro models for hepatotoxicity testing fully depends on a solid in vivo phenotype anchored analysis.     

Zebrafish embryos as models for embryotoxic and teratological effects of chemicals

The experimental virtues of the zebrafish embryo such as small size, development outside of the mother, cheap maintenance of the adult made the zebrafish an excellent model for phenotypic genetic and more recently also chemical screens. The availability of a genome sequence and several thousand mutants and transgenic lines together with gene arrays and a broad spectrum of techniques to manipulate gene functions add further to the experimental strength of this model. Pioneering studies suggest that chemicals can have in many cases very similar toxicological and teratological effects in zebrafish embryos and humans. In certain areas such as cardiotoxicity, the zebrafish appears to outplay the traditional rodent models of toxicity testing. Several pilot projects used zebrafish embryos to identify new chemical entities with specific biological functions. In combination with the establishment of transgenic sensor lines and the further development of existing and new automated imaging systems, the zebrafish embryos could therefore be used as cost-effective and ethically acceptable animal models for drug screening as well as toxicity testing.     

Application of cDNA microarray to the study of arsenic-induced liver diseases in the population of Guizhou, China.

Arsenic is an environmental toxicant and a human carcinogen. Epidemiology studies link human arsenic exposure to various diseases and cancers, including liver diseases and hepatocellular carcinoma. However, the molecular mechanisms for arsenic toxicity and carcinogenicity are poorly understood. To better understand these mechanisms, we used the human cancer cDNA expression array to profile aberrant gene expression in arsenic-exposed populations in Guizhou, China. The selected patients had a history of exposure to environmental arsenic for at least 6-10 years, and had arsenic-induced skin lesions and hepatomegaly. Samples were obtained by liver needle biopsy. Histology showed degenerative liver lesions, such as chronic inflammation, vacuolation, and focal necrosis. The University of North Carolina Hospitals provided normal human liver tissues from surgical resection or rejected transplants. Microarray was performed with total RNA from liver samples, and signal intensities were analyzed with AtlasImage software and normalized with 9 housekeeping genes. Means and SEM were calculated for statistical analysis. Approximately 60 genes (10%) were differentially expressed in arsenic-exposed human livers compared to controls. The differentially expressed genes included those involved in cell-cycle regulation, apoptosis, DNA damage response, and intermediate filaments. The observed gene alterations appear to be reflective of hepatic degenerative lesions seen in the arsenic-exposed patients. This array analysis revealed important patterns of aberrant gene expression occurring with arsenic exposure in human livers. Aberrant expressions of several genes were consistent with the results of array analysis of chronic arsenic-exposed mouse livers and chronic arsenic-transformed rat liver cells. Clearly, a variety of gene expression changes may play an integral role in arsenic hepatotoxicity and possibly carcinogenesis.     

Histological analysis of acute toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in zebrafish

Previous studies have demonstrated that acute exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by injection leads to inhibition of caudal fin regeneration in zebrafish. Since the TCDD exposure in these studies is systemic, it is possible that pathology in organs other than the fin could result in inhibition of fin regeneration. Therefore, histopathology of adult zebrafish (Danio rerio) organs was characterized following abdominal cavity injection of a TCDD dose (70ng/g). The most pronounced histopathologic changes 5 days post-injection included lipidosis and hypertrophy of liver hepatocytes and hypertrophy of gill lamellae. Effects of TCDD exposure on immunolocalization of the zebrafish aryl hydrocarbon receptor nuclear translocator (ARNT2), the heterodimer partner of the aryl hydrocarbon receptor (AHR2), and an AHR regulated gene cytochrome P450 1A (CYP1A) was also determined. ARNT2 was immunolocalized to the gastrointestinal tract, gill lamellae, kidney, ventricle of the heart, caudal fin, brain and liver of zebrafish. TCDD exposure had no measurable effect on ARNT2 abundance or localization. CYP1A was immunolocalized in TCDD exposed fish as a biomarker for cells with an activated AHR pathway. CYP1A was not detected in any tissue from vehicle exposed fish. Significant TCDD-dependent induction of CYP1A was detected in the proximal tubules of the kidney, in liver hepatocytes and in the gastrointestinal tract of TCDD exposed fish. Significant but lower TCDD-dependent CYP1A expression was evident in the gill, caudal fin and ventricle of the heart. Overall, TCDD exposure in adult zebrafish leads to histopathology similar to that reported in other fish species, and it appears unlikely that the histopathology in these organs completely explains the inhibition of fin regeneration.     

采用斑马鱼模型评价对乙酰氨基酚的肝脏毒性

目的研究对乙酰氨基酚（APAP）对斑马鱼幼鱼的肝脏毒性,并验证斑马鱼模型用于药物肝毒性快速评价的实用性。方法以发育72 hpf的肝脏荧光转基因斑马鱼幼鱼（L-FABP：EGFP）为实验对象,不同浓度的APAP分别处理斑马鱼幼鱼,于处理后24、48、72 h,体视显微镜下观察幼鱼的死亡率、肝脏形态学变化和卵黄囊吸收情况,荧光显微镜下观察APAP对幼鱼肝脏荧光的影响。结果 APAP对斑马鱼幼鱼存活率的影响呈剂量和时间相关性。APAP处理后的幼鱼肝脏形态出现异常、肝脏颜色变暗,卵黄囊肿。与空白对照组相比,APAP处理后的幼鱼肝组织L-FABP荧光表达明显下降,肝脏明显萎缩退化。结论 APAP对斑马鱼幼鱼具有肝脏毒性。肝脏荧光转基因斑马鱼模型快速评价药物的肝脏毒性具有较好的应用前景。     

Triptolide‐induced hepatotoxicity via apoptosis and autophagy in zebrafish

Previous research about the development of triptolide (TP) as a natural active compound has often focused on hepatotoxicity. Among its various mechanisms, autophagy and apoptosis are two important signaling pathways. In this study, we used zebrafish to establish a TP‐induced hepatotoxicity model, and investigated the roles of autophagy and apoptosis in the progress of liver injury. Zebrafish exposed to TP showed increased mortality and malformation because of the increased drug dose and duration of exposure. Meanwhile, we found that TP induced liver injury in a time‐ and dose‐dependent manner, which was observed as a reduction in liver area, slow yolk absorption, upregulation of transaminase and local neurosis. With the application of the high‐content imaging system (HCIS) technique in liver 3D imaging in vivo, clear imaging of the zebrafish liver was achieved. The results showed a decrease in volume and location of necrosis in the liver after TP exposure. Increased expression of inflammatory cytokines genes tumor necrosis factor (Tnf)α, Il1β and Il6 were shown, particularly Tnfα. The Fas‐Caspase8 signaling pathway was activated. The apoptosis‐related gene Bcl‐2 was increased, and Bax, Caspase9 and Caspase3 were increased. However, autophagy related genes Beclin1, Atg5, Atg3 and Lc3 were increased more significantly, and the changes of Beclin1 and Atg5 were the most severe. This study successfully established a TP‐induced zebrafish hepatotoxicity model and applied the HCIS technique in a zebrafish hepatotoxicity study. The result indicated Fas might be the main target of TP‐induced hepatotoxicity. Autophagy played a more important role than apoptosis and was characterized by the overexpression of Beclin1 and Atg5.     

Oxmetidine (H2 receptor antagonist) induced cytotoxicity in isolated rat hepatocytes

Oxmetidine, a new and more potent analogue of the H2 receptor antagonist, cimetidine, was recently withdrawn from clinical trials because of associated hepatotoxicity. We investigated the potential hepatotoxicity of the drug in vitro and in vivo in the rat. In addition, we investigated, in in vitro experiments, the potential hepatoxicity of other gastric acid inhibitory drugs (cimetidine, ranitidine, omeprazole and nolinium bromide). In in vitro experiments, oxmetidine, at various concentrations, was added to isolated hepatocyte incubations and cytotoxicity was assayed by trypan blue exclusion. In in vivo experiments, oxmetidine was administered both i.p. and orally, and hepatotoxicity was assessed by serum biochemical measures (transaminases, alkaline phosphatase, 5' nucleotidase, gamma glutamyl transpeptidase) and liver histopathology. In the in vitro studies, the addition of oxmetidine to the hepatocyte incubations was associated with significant (P less than 0.001) dose and time dependent cytotoxicity. However, the in vivo experiments revealed no significant changes in serum biochemistry and no significant alterations in liver histopathology up to 72 h following the administration three different dosages of oxmetidine. Of the other gastric acid inhibitory drugs, only nolinium bromide was associated with significant (P less than 0.001) in vitro cytotoxicity. Our in vitro observations establish that oxmetidine is cytotoxic to isolated rat hepatocytes and suggest that nolinium bromide be further evaluated for potential hepatotoxicity.     

Attenuation of cadmium-induced liver injury in senescent male fischer 344 rats: role of metallothionein and glutathione

The influence of aging on the sensitivity of the liver to the acute toxicity of cadmium has not been studied previously in adult rats. In this study hepatotoxicity caused by a single sc injection of CdCl(2) was compared in 5-, 18-, and 28-month-old male Fischer 344 rats. Doses of Cd were adjusted on the basis of the mean lean body mass for each age group of rats, and liver injury was evaluated 24 h after treatment. Cd treatment produced substantial increases in serum alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) activities in 5- and 18-month-old rats, whereas no significant increases were observed in 28-month-old rats. Histologic examination of representative livers from each age group confirmed the findings for serum enzyme activity; hepatocellular necrosis was observed only in livers from 5- and 18-month-old rats. The attenuation of Cd hepatotoxicity in senescent rats did not appear to be related to pretreatment levels of metallothionein or glutathione. Likewise, resistance to Cd could not be explained on the basis of metallothionein induction, which decreased as a function of aging. Thus, the mechanisms that account for the postmaturational decline in sensitivity to Cd do not appear to be associated with alterations in levels of the major factors that protect against Cd-induced hepatotoxicity.     

Gene expression profiling in the liver of CD-1 mice to characterize the hepatotoxicity of triazole fungicides

Four triazole fungicides used in agricultural or pharmaceutical applications were examined for hepatotoxic effects in mouse liver. Besides organ weight, histopathology, and cytochrome P450 (CYP) enzyme induction, DNA microarrays were used to generate gene expression profiles and hypotheses on potential mechanisms of action for this class of chemicals. Adult male CD-1 mice were exposed daily for 14 days to fluconazole, myclobutanil, propiconazole, or triadimefon at three dose levels by oral gavage. Doses were based on previous studies that resulted in liver hypertrophy or hepatotoxicity. All four triazoles caused hepatocyte hypertrophy, and all except triadimefon increased relative liver/body weight ratios at the middle and high dose levels. CYP enzyme activities were also induced by all four triazoles at the middle and high doses as measured by the dealkylations of four alkoxyresorufins, although some differences in substrate specificity were observed. Consistent with this common histopathology and biochemistry, several CYP and xenobiotic metabolizing enzyme (XME) genes were differentially expressed in response to all four (Cyp2d26 and Cyp3a11), or three of the four (Cyp2c40, Cyp2c55, Ces2, Slco1a4) triazoles. Differential expression of numerous other CYP and XME genes discriminated between the various triazoles, consistent with differences in CYP enzyme activities, and indicative of possible differences in mechanisms of hepatotoxicity or dose response. Multiple isoforms of Cyp1a, 2b, 2c, 3a, and other CYP and XME genes regulated by the nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) were differentially expressed following triazole exposure. Based on these results, we expanded on our original hypothesis that triazole hepatotoxicity was mediated by CYP induction, to include additional XME genes, many of which are modulated by CAR and PXR.     

The role of the liver in mediating the acute toxicity of the pesticide methyl parathion in the mouse

Mouse livers perfused in situ with the pesticide methyl parathion (O,O-dimethyl O-P-nitrophenyl phosphorothioate) resulted in the appearance of the toxic metabolite, methyl paraoxon (O,O-dimethyl-O-P-nitrophenyl phosphate), in the effluent perfusate. Mouse whole blood rapidly detoxified methyl paraoxon in vitro, but not at a rate sufficient to prevent transport of at least some of this toxic metabolite from liver to other tissues in vivo. The hepatic disposition and biotransformation of methyl parathion in perfused livers were altered markedly by changes in protein binding of methyl parathion to perfusate, but only slightly by changes in perfusate flow rates that maintained viable livers. Pretreatment of mice with phenobarbital daily for 4 days (80 mg/kg, ip) induced hepatic microsomal activation of methyl parathion to methyl paraoxon in vitro and increased the clearance of methyl parathion by perfused mouse livers. However, in contrast to perfusion of methyl parathion into livers from saline-pretreated mice, perfusion of methyl parathion into livers from phenobarbital-pretreated mice did not lead to the appearance of methyl paraoxon in effluent perfusate. Nevertheless, methyl paraoxon was produced intrahepatically during these perfusions since hepatic cholinesterase activities were depressed compared to livers from phenobarbital-pretreated mice perfused without methyl parathion. Furthermore, phenobarbital pretreatment antagonized the acute toxicity of methyl parathion in vivo in the mouse. These data demonstrate that the net result of the biotransformation of methyl parathion by livers in untreated mice is metabolic activation, whereas the net result by livers of phenobarbital-pretreated mice is detoxification.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gene expression profiles of murine fatty liver induced by the administration of methotrexate

Methotrexate (MTX) is used to treat a variety of chronic inflammatory and neoplastic diseases. However, it can induce hepatotoxicity such as microvesicular steatosis and necrosis. To explore the mechanisms of MTX-induced hepatic steatosis, we used microarray analysis to profile the gene expression patterns of mouse liver after MTX treatment. MTX was administered orally as a single dose of 10mg/kg (low dose) or 100 mg/kg (high dose) to ICR mice, and the livers were obtained 6 h, 24 h, and 72 h after treatment. Serum alanine aminotransferase, aspartate aminotransferase and triacylglycerol levels were not significantly altered in the experimental animals. Signs of steatosis were observed at 24 h after administration of high dose of MTX. From microarray data analysis, 908 genes were selected as MTX-responsive genes (P<0.05, two-way ANOVA; cutoff > or =1.5-fold). Database for Annotation, Visualization and Integrated Discovery (DAVID) analysis revealed that the predominant biological processes associated with these genes are response to unfolded proteins, phosphate metabolism, and cellular lipid metabolism. Functional categorization of these genes identified 28 genes involved in lipid metabolism that was interconnected with the biological pathways of biosynthesis, catabolism, and transport of lipids and fatty acids. Taken together, these data provide a better understanding of the molecular mechanisms of MTX-induced steatogenic hepatotoxicity, and useful information for predicting hepatotoxicity through pattern recognition.