Hepatic gene expression profiling and lipid homeostasis in mice exposed to steatogenic drug, tetracycline.

Tetracycline is one of a group of drugs known to induce microvesicular steatosis. In the present study, we investigated the effects of tetracycline on gene expression in mouse liver, using Applied Biosystems Mouse Genome Survey Microarrays. A single oral dose of 0.1 or 1 g/kg tetracycline was administered to male ICR mice, and liver samples were obtained after 6, 24, or 72 h. Histopathological evaluation showed microvesicular steatosis in the high-dose group at 24 h. In total, 96 genes were identified as tetracycline responsive. Their level of expression differed significantly from controls (two-way analysis of variance; p < 0.05), after adjustment by the Benjamini-Hochberg multiple testing correction, and displayed a twofold or greater induction or repression. The largest groups of gene products affected by tetracycline exposure were those involved in signal transduction, nucleic acid metabolism, developmental processes, and protein metabolism. The expression of genes known to be involved in lipid metabolism was examined, using two-sample Student's t-test for each treatment group versus a corresponding control group. The overall net effects on expression of lipid metabolism genes indicated an increase in cholesterol and triglyceride biosynthesis and a decrease in beta-oxidation of fatty acids. Our data support a proposed mechanism for tetracycline-induced steatogenic hepatotoxicity that involves these processes. Moreover, we demonstrated global changes in hepatic gene expression following tetracycline exposure; many of these genes have the potential to be used as biomarkers of exposure to steatogenic hepatotoxic agents.     

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.     

Gene expression profiles of murine fatty liver induced by the administration of valproic acid

Valproic acid (VPA) has been used as anticonvulsants, however, it induces hepatotoxicity such as microvesicular steatosis and necrosis in the liver. To explore the mechanisms of VPA-induced steatosis, we profiled the gene expression patterns of the mouse liver that were altered by treatment with VPA using microarray analysis. VPA was orally administered as a single dose of 100 mg/kg (low-dose) or 1000 mg/kg (high-dose) to ICR mice and the animals were killed at 6, 24, or 72 h after treatment. Serum alanine aminotransferase and aspartate aminotransferase levels were not significantly altered in the experimental animals. However, symptoms of steatosis were observed at 72 h with low-dose and at 24 h and 72 h with high-dose. After microarray data analysis, 1910 genes were selected by two-way ANOVA (P<0.05) as VPA-responsive genes. Hierarchical clustering revealed that gene expression changes depended on the time rather than the dose of VPA treatment. Gene profiling data showed striking changes in the expression of genes associated with lipid, fatty acid, and steroid metabolism, oncogenesis, signal transduction, and development. Functional categorization of 1156 characteristically up- and down-regulated genes (cutoff >1.5-fold) revealed that 60 genes were involved in lipid metabolism that was interconnected with biological pathways for biosynthesis of triglyceride and cholesterol, catabolism of fatty acid, and lipid transport. This gene expression profile may be associated with the known steatogenic hepatotoxicity of VPA and it may provide useful information for prediction of hepatotoxicity of unknown chemicals or new drug candidates through pattern recognition.     

In vitro - in vivo correlation of gene expression alterations induced by liver carcinogens

Although cultivated hepatocytes are widely used in the studies of drug metabolism, their application in toxicogenomics is considered as problematic, because previous studies have reported only little overlap between chemically induced gene expression alterations in liver in vivo and in cultivated hepatocytes. Here, we identified 22 genes that were altered in livers of rats after oral administration of the liver carcinogens aflatoxin B1 (AB1), 2-nitrofluorene (2-NF), methapyrilene (MP) or piperonyl-butoxide (PBO). The functions of the 22 genes have been classified into two groups. Genes related to stress response, DNA repair or metabolism and genes associated with cell proliferation, respectively. Next, rat hepatocyte sandwich cultures were exposed to AB1, 2-NF, MP or PBO for 24h and expression of the above mentioned genes was determined by RT-qPCR. Significant correlations between the degree of gene expression alterations in vivo and in vitro were obtained for the stress, DNA repair and metabolism associated genes at concentrations covering a range from cytotoxic concentrations to non-toxic/in vivo relevant concentrations. In contrast to the stress associated genes, no significant in vivo/in vitro correlation was obtained for the genes associated with cell proliferation. To understand the reason of this discrepancy, we compared replacement proliferation in vivo and in vitro. While hepatocytes in vivo, killed after administration of hepatotoxic compounds, are rapidly replaced by proliferating surviving cells, in vitro no replacement proliferation as evidenced by BrdU incorporation was observed after washing out hepatotoxic concentrations of MP. In conclusion, there is a good correlation between gene expression alterations induced by liver carcinogens in vivo and in cultivated hepatocytes. However, it should be considered that cultivated primary hepatocytes do not show replacement proliferation explaining the in vivo/in vitro discrepancy concerning proliferation associated genes.     

Microarray analysis of hepatic gene expression in pyrazole-mediated hepatotoxicity: identification of potential stimuli of Cyp2a5 induction

Cytochrome P450 2a5 (Cyp2a5) expression is induced during liver damage caused by hepatotoxins such as pyrazole, however, the mechanism underlying this overexpression is unclear. In order to identify pathophysiological and cellular responses to pyrazole that might alter Cyp2a5 expression, we examined the effect of pyrazole on mouse hepatic gene expression in C57BL/6 mice using Affymetrix 430 2.0 microarrays. Over 3000 differentially expressed genes were identified 24-h after pyrazole treatment that were associated with a variety of cellular pathways. Upregulated genes were primarily involved in the splicing and processing of RNA and the unfolded protein response pathway, while downregulated genes were associated with amino acid and lipid metabolism, and generation of precursor metabolites for energy production. We also examined the effects of pyrazole on cellular pathways linked to metabolic and histopathological changes observed with pyrazole toxicity. Increased mRNA levels were observed for genes involved in bilirubin production, whereas the major genes of the urea cycle were strongly decreased. Changes in genes involved in carbohydrate metabolism were also observed which could explain pyrazole-induced glycogen depletion and decreased serum glucose. In addition, over 100 genes involved in the cellular stress response were upregulated by pyrazole treatment, including genes involved in the unfolded protein response and redox status. Based on these results and previous evidence concerning the regulation of Cyp2a5, we have identified several pathophysiological changes including altered energy homeostasis, hyperbilirubinemia, ER stress, and altered redox status that are associated with CYP2A5 overexpression and may represent potential stimuli for the induction of Cyp2a5.     

Gene expression profiling of nephrotoxicity from the sevoflurane degradation product fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether ("compound A") in rats.

The major degradation product of the volatile anesthetic sevoflurane, the haloalkene fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (FDVE or "compound A"), is nephrotoxic in rats. FDVE undergoes complex metabolism and bioactivation, which mediates the nephrotoxicity. Nevertheless, the molecular and cellular mechanisms of FDVE toxification are unknown. This investigation evaluated the gene expression profile of kidneys in rats administered a nephrotoxic dose of FDVE. Male Fischer 344 rats (five per group) received 0.25 mmol/kg intraperitoneal FDVE or corn oil (controls) and were sacrificed after 24 or 72 h. Urine output and kidney histological changes were quantified. Kidney RNA was extracted for microarray analysis using Affymetrix GeneChip Rat Expression Array 230A arrays. Quantitative real-time PCR confirmed the modulation of several genes. FDVE caused significant diuresis and necrosis at 24 h, with normal urine output and evidence of tubular regeneration at 72 h. There were 517 informative genes that were differentially expressed >1.5-fold (p < 0.05) versus control at 24 h, of which 283 and 234 were upregulated and downregulated, respectively. Major classes of upregulated genes included those involved in apoptosis, oxidative stress, and inflammatory response (mostly at 24 h), and regeneration and repair; downregulated genes were generally associated with transporters and intermediary metabolism. Among the quantitatively most upregulated genes were kidney injury molecule, osteopontin, clusterin, tissue inhibitor of metalloproteinase 1, and TNF receptor 12, which have been associated with other forms of nephrotoxicity, and angiopoietin-like protein 4, glycoprotein nmb, ubiquitin hydrolase, and HSP70. Microarray results were confirmed by quantitative real-time PCR. FDVE causes rapid and brisk changes in gene expression, providing potential insights into the mechanism of FDVE toxification, and potential biomarkers for FDVE nephrotoxicity which are more sensitive than conventional measures of renal function.     

A temporal study on the histopathological, biochemical and molecular responses of CCl(4)-induced hepatotoxicity in Cyp2e1-null mice

Previous study using Cyp2e1-null mice showed that Cyp2e1 is required in CCl(4)-induced liver injury at 24h, what remains unclear are the temporal changes in liver damage and the spectrum of genes involved in this process. We investigated the time-dependent liver changes that occurred at morphological, histopathological, biochemical and molecular levels in both Cyp2e1(+/+) and Cyp2e1(-/-) mice after treating with either corn oil or CCl(4) (1 ml/kg) for 2, 6, 12, 24 and 48 h. A pale orange colored liver, indicative of fatty infiltration, was observed in Cyp2e1(+/+) mice treated with CCl(4) for 24 and 48 h, while the Cyp2e1(+/+) mice treated with corn oil and Cyp2e1(-/-) mice treated with either corn oil or CCl(4) showed normal reddish brown colored liver. Ballooned hepatocytes with multiple vacuoles in their cytoplasm were observed in the livers of Cyp2e1(+/+) mice 24 and 48 h after treating with CCl(4). The levels of serum alanine aminotransferase and aspartate aminotransferase, markers for liver injury, were significantly higher at 12h, peaked at 24h and gradually decreased at 48 h after CCl(4) intoxication. In contrast, this kind of damage was not apparent in the Cyp2e1(-/-) mice treated with CCl(4). Altered expressions of genes related to liver cirrhosis, apoptosis, oxidative stress, xenobiotic detoxification, lipid metabolism, chemsensory signaling or tumorigenesis, structural organization, regeneration and inflammatory response were identified, and the time-dependent changes in expression of these genes were varied. Overall, the present study provides insights into the mechanism of CCl(4)-induced hepatotoxicity in animal models.     

Cadmium-induced apoptosis and changes in expression of p53, c-jun and MT-I genes in testes and ventral prostate of rats

Apoptosis and a change in the expression of p53, c-jun and MT-I genes occurred in rats exposed to cadmium in a way known to cause carcinogenesis in testes and ventral prostate. In situ end labelling (ISEL), DNA electrophoresis, and RT-PCR methods were used in present study. Adult male Wistar rats were given a single (s.c.) injection of 0, 5, 10, or 20 micromol/kg CdCl2. Then 12, 48 or 96 h after administration of cadmium, animals were sacrificed. It was observed that cadmium markedly induced apoptosis in the testes at the dose of 5 micromol/kg while 10 and 20 micromol/kg cadmium caused more necrosis than apoptosis. Apoptosis in the ventral prostate was markedly induced by all the doses of cadmium and there was an obvious time- and dose-dependent relationship between apoptotic index (AI) and cadmium treatment. Far fewer apoptotic cells appeared in liver, compared to the testes and ventral prostate. p53 mRNA expression was clearly enhanced in the ventral prostate but clearly suppressed in the testes by cadmium exposure, and the time- and dose-effect was very clear. The expression level of p53 in the liver was not affected by cadmium treatment. Cadmium-induced overexpression of c-jun gene appeared at 12 h in the liver, but not until 96 h in the testes and ventral prostate. Although the MT-I gene was found to be expressed in all tissues, marked induction by cadmium of the expression of MT-I gene was only observed in the liver. These results indicate: (1) that apoptosis is an early mechanism of acute tissue damage by cadmium in the testes and ventral prostate; (2) that p53 and c-jun genes may be involved in cadmium-induced cytotoxicity (apoptosis) and related carcinogenicity in male reproductive tissues; and (3) that the enhanced expression of MT-I in the liver could protect this organ from cadmium-induced cytotoxicity (apoptosis) and carcinogenicity.     

The response of the rat liver in situ to bromobenzene--in vivo proton magnetic resonance imaging and 31P magnetic resonance spectroscopy studies

Proton magnetic resonance imaging (MRI) and 31P magnetic resonance spectroscopy (MRS) have been used to study the response of the rat liver in situ to bromobenzene, a classic hepatotoxicant. A localized region of high proton signal intensity was seen in the perihilar region of the liver 24 hr after injection of a sublethal dose of bromobenzene. The signal intensity of the entire liver was increased at 48 hr with a gradual return approaching control values by 120 hr. These results are consistent with acute hepatic edema followed by repair of the damaged tissue. In vivo 31P MRS studies of the same rat livers were performed under conditions whereby localized, quantitative spectra could be obtained without surgical intervention. Initial concentrations of the major endogenous phosphorus-containing metabolites within the livers of control rats were 2.97 +/- 0.43 mM for the phosphomonoesters (PME), 2.92 +/- 0.56 mM for inorganic phosphate, 11.3 +/- 1.0 mM for phosphodiesters (PDE), 4.09 +/- 0.54 mM for ATP, and 0.56 +/- 0.50 mM for ADP and the intracellular pH was 7.39 +/- 0.14 (mean +/- SD, n = 10). Bromobenzene was found to cause statistically significant (p less than 0.05) changes in several of these metabolites: a decrease in hepatic ATP levels (20% at 24 hr; 27% at 48 hr), a decrease in PDE levels (15% at 24 hr; 18% at 48 hr), and an increase in the PME (63% at 24 hr; 84% at 48 hr). Both the proton MRI and the 31P MRS changes have an onset of 15-20 hr and maximum effect at 25-60 hr, but the MRS changes returned to normal well before the MRI changes. The decreased ATP levels indicate deleterious effects of bromobenzene on the bioenergetic status of the liver in situ, while the increase in PME, due to a selective increase in phosphocholine, suggests the activation of a phosphatidylcholine-specific phospholipase C in response to tissue damage. Trolox C, a potent inhibitor of lipid peroxidation, prevented the bromobenzene-induced hepatic edema (i.e., the increase in proton MRI signal intensity) and the bioenergetic deterioration (i.e., the decrease in ATP levels). However, the bromobenzene-induced increase in PME levels was not prevented by Trolox C. These results indicate that the process of lipid peroxidation plays a significant role in the hepatotoxicity of bromobenzene within the intact animal.     

施普睿达对荷H22肝癌小鼠基因表达谱的影响

目的研究苦豆子生物碱衍生物施普睿达对荷H22肝癌小鼠基因表达谱的影响,探讨施普睿达抗肝癌的分子机制。方法应用包含4096个小鼠基因的cDNA芯片检测施普睿达对荷H22肝癌小鼠基因表达谱的影响,并对差异基因进行GO聚类和统计分析。结果施普睿达治疗前后共有358个基因出现差异表达,83个基因的表达差异达到3倍以上,表达差异在2倍以上的基因共有275个。经GO分析,差异表达基因包括许多与细胞凋亡、细胞周期、代谢、免疫应答和DNA修复等相关的基因。结论施普睿达主要是通过调控肿瘤细胞周期进程、影响代谢和免疫应答、调节肿瘤细胞凋亡相关基因的表达等多种途径抑制肿瘤的增殖和转移。     

New insights into generalized hepatoprotective effects of oleanolic acid: key roles of metallothionein and Nrf2 induction

Oleanolic acid (OA) is a natural triperpenoid that protects against a variety of hepatotoxicants such as carbon tetrachloride, cadmium, acetaminophen, and bromobenzene. To gain insight into the molecular mechanisms of this generalized hepatoprotection, genomic analysis was performed on mouse and rat livers after OA treatment. Mice and rats were given OA at a hepatoprotective dose (50 micromol/kg, s.c., daily for 4 days) and hepatic RNA was isolated, purified, and subjected to gene expression analysis. OA treatment produced changes in 5% of the genes on custom-designed mouse liver array and rat toxicology-II array. Changes in key gene expressions were further analyzed by real-time RT-PCR. OA treatment dramatically increased expression of hepatic metallothionein (Mt), and increased the expression of the nuclear factor E2-related factor 2 (Nrf2), NAD(P)H:quinone oxidoreductase 1 (Nqo1), heme oxygenase-1 (Hmox1), and glutamate-cysteine ligases (Gclc and Gclm). OA treatment also increased the expression of genes related to cell proliferation and suppressed the expression of several cytochrome P450 genes possibly to switch cellular metabolic energy to an acute-phase response. Hepatic MT protein was increased 60- and 15-fold in mice and rats, respectively, together with a 30% increase in mouse liver zinc. These gene expression changes, particularly the dramatic induction of MT and the Nrf2 signaling, occur with hepatoprotection doses of OA, and likely are important in the generalized protective effects of OA against hepatotoxicants.     

Protective effect of 16,16-dimethyl prostaglandin E2 on the hepatotoxicity of bromobenzene in mice

It has been suggested that 16,16-dimethyl prostaglandin E2 may have a cytoprotective effect in the liver. To assess this hypothesis, we determined the effects of this prostaglandin on the metabolism and toxicity of bromobenzene in mice. Administration of 16,16-dimethyl prostaglandin E2 (50 micrograms/kg s.c., 30 min before, and every 6 hr after, the administration of bromobenzene) did not modify the disappearance curves of unchanged bromobenzene from plasma and liver, and did not modify the amount of bromobenzene metabolites covalently bound to hepatic proteins 1-24 hr after the administration of a toxic dose of bromobenzene (0.36 ml/kg i.p.). The prostaglandin, however, markedly reduced serum alanine aminotransferase activity, the extent of liver cell necrosis, the depletion of glutathione, and the disappearance of cytochrome P-450 after administration of this toxic dose of bromobenzene (0.36 ml/kg i.p.). It also markedly reduced mortality after administration of a lethal dose of bromobenzene (0.43 ml/kg i.p.). We conclude that 16,16-dimethyl prostaglandin E2 can prevent hepatic necrosis without decreasing the covalent binding of bromobenzene metabolites to hepatic proteins. The mechanism for this dissociation between covalent binding and toxicity remains unknown.     

Dose-dependent metabolic excretion of bromobenzene and its possible relationship to hepatotoxicity in rats

Male Sprague-Dawley rats received an intraperitoneal injection of 0.25-, 0.5-, 1.0-, 2.5-, and 5.0-mmol/kg dose of bromobenzene in corn oil. The metabolic fate of bromobenzene was studied by measuring its various urinary metabolites 24 h following bromobenzene administration. The hepatotoxicity of bromobenzene was estimated by determination of the serum glutamic-oxaloacetic and glutamic-pyruvic transaminase activities (SGOT and SGPT) 24 h after dosing. Treatment of rats with bromobenzene at up to 0.5 mmol/kg did not influence the transaminase activities, but significant increases in such activities began to manifest at a dose of 1 mmol/kg. However, no further increase in hepatotoxic response was induced on exposure to higher doses (2.5 and 5.0 mmol/kg) of bromobenzene. The urinary excretion of toxic doses of bromobenzene was nonlinear, based on the quantitative composition of various urinary metabolites. Furthermore, the fraction of the dose converted to thioethers, p-bromophenol, m-bromophenol, and total phenolic metabolites decreased with increasing toxic dose, suggesting their formation to be capacity-limited. The ratios of thioethers to total phenolic metabolites, of thioethers to p-bromophenol, and of thioethers to o-bromophenol decreased with increasing dose of bromobenzene. The correlation of the dose-dependent fate of metabolic excretion of bromobenzene with the results of the dose-hepatotoxic response curves supports the conclusion that there exists an apparent threshold dose (approximately 1-2.5 mmol/kg) for the toxic effects of bromobenzene that coincides with saturation of the metabolic pathways involving both glutathione/glutathione S-transferase(s) and formation of certain phenolic derivatives for its detoxification. All these results further suggest a role of a saturable, metabolic activation process involving 3,4-epoxide rather than 2,3-epoxide of bromobenzene in the development of its hepatotoxicity.     

Expression of clusterin (testosterone-repressed prostate message-2) mRNA during growth and regeneration of rat liver

 Clusterin has been used as a marker for apoptosis (often denoted “active”“or programmed” cell death) in the prostate, mammary gland and other solid organs. The protein is thought to be involved in membrane remodelling during separation of apoptotic cells from their vital neighbours and fragmentation into apoptotic bodies. In the present study, we have looked at the expression of clusterin during the growth and regression of rat liver induced by short term administration of the hepatomitogen, cyproterone acetate. The steady state level of clusterin mRNA, as measured by Northern and slot blot analysis, is low in control hepatocytes. Following administration of cyproterone acetate, the clusterin mRNA level is increased during both liver growth and regression. In situ hybridization reveals that clusterin is expressed in all hepatocytes, indicating that it is not confined to cell death by apoptosis. These results suggest that the gene product may be involved in maintaining membrane integrity, which is necessary during both mitosis and apoptosis. To determine whether clusterin mRNA is induced by membrane remodelling independent of either mitosis or apoptosis, we examined the expression of clusterin mRNA in the liver after a necrogenic dose of carbon tetrachloride. During the first 24–48 h of this time period, necrosis is the predominant form of cell death and liver regeneration starts after approximately 24 h. Elevated levels of clusterin mRNA are found as early as 12 h after carbon tetrachloride administration and persist for at least 72 h. Clusterin expression in tissues such as the prostate and mammary gland appears to be confined to the apoptotic pathway; however, our results suggest that in hepatocytes, the expression of the gene is induced in processes other than apoptosis, including mitosis and necrosis. These processes involve substantial membrane remodelling, suggesting that clusterin expression may be induced by perturbations in the normal membrane structure. Received: 9 May 1993 / Accepted: 5 October 1994