Effects of acetaminophen on hepatic gene expression in mice

Acetaminophen (APAP) is one of the most commonly used drugs for the safe and effective treatment of fever and pain. However, it is a well-established hepatotoxin. The objective of this study was to identify alternation in various genes in liver of mice after administration of low and high doses of APAP. Male C57BL/6J mice received APAP (30 or 300 mg/kg, i.p.). They were sacrificed after 6 hr and 24 hr for assessment of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), total RNA isolation, cDNA microarray analysis and histopathological analysis of liver injury. Low dose of APAP did not cause hepatotoxicity in mice. However, it was toxic at a high dose. Using microarray technology, we selected changed genes more than 1.5 fold. Gene expression changes were recorded even at a low dose treatment with APAP. Six (6) hr after APAP treatment at low dose, 6 genes were up-regulated and 25 genes were down-regulated. However, 24 hr after treatment at low dose 8 genes were up-regulated and 34 genes were down-regulated. 6 hr after of high dose treatment 29 genes were down-regulated and none was up-regulated. A 24 hr treatment with high dose up-regulated 6 genes and down-regulated 18 genes. These expression patterns provide information on high versus low dose mechanisms of APAP toxicity. Gene expression signatures recorded after a nontoxic dose of APAP strongly support the validity of gene expression changes as meaningful markers of hepatotoxicity.     

Systems toxicology: integrated genomic, proteomic and metabonomic analysis of methapyrilene induced hepatotoxicity in the rat

Administration of high doses of the histamine antagonist methapyrilene to rats causes periportal liver necrosis. The mechanism of toxicity is ill-defined and here we have utilized an integrated systems approach to understanding the toxic mechanisms by combining proteomics, metabonomics by 1H NMR spectroscopy and genomics by microarray gene expression profiling. Male rats were dosed with methapyrilene for 3 days at 150 mg/kg/day, which was sufficient to induce liver necrosis, or a subtoxic dose of 50 mg/kg/day. Urine was collected over 24 h each day, while blood and liver tissues were obtained at 2 h after the final dose. The resulting data further define the changes that occur in signal transduction and metabolic pathways during methapyrilene hepatotoxicity, revealing modification of expression levels of genes and proteins associated with oxidative stress and a change in energy usage that is reflected in both gene/protein expression patterns and metabolites. The difficulties of combining and interpreting multiomic data are considered.     

Gene expression profiling reveals multiple toxicity endpoints induced by hepatotoxicants

Microarray technology continues to gain increased acceptance in the drug development process, particularly at the stage of toxicology and safety assessment. In the current study, microarrays were used to investigate gene expression changes associated with hepatotoxicity, the most commonly reported clinical liability with pharmaceutical agents. Acetaminophen, methotrexate, methapyrilene, furan and phenytoin were used as benchmark compounds capable of inducing specific but different types of hepatotoxicity. The goal of the work was to define gene expression profiles capable of distinguishing the different subtypes of hepatotoxicity. Sprague-Dawley rats were orally dosed with acetaminophen (single dose, 4500 mg/kg for 6, 24 and 72 h), methotrexate (1mg/kg per day for 1, 7 and 14 days), methapyrilene (100mg/kg per day for 3 and 7 days), furan (40 mg/kg per day for 1, 3, 7 and 14 days) or phenytoin (300 mg/kg per day for 14 days). Hepatic gene expression was assessed using toxicology-specific gene arrays containing 684 target genes or expressed sequence tags (ESTs). Principal component analysis (PCA) of gene expression data was able to provide a clear distinction of each compound, suggesting that gene expression data can be used to discern different hepatotoxic agents and toxicity endpoints. Gene expression data were applied to the multiplicity-adjusted permutation test and significantly changed genes were categorized and correlated to hepatotoxic endpoints. Repression of enzymes involved in lipid oxidation (acyl-CoA dehydrogenase, medium chain, enoyl CoA hydratase, very long-chain acyl-CoA synthetase) were associated with microvesicular lipidosis. Likewise, subsets of genes associated with hepatotocellular necrosis, inflammation, hepatitis, bile duct hyperplasia and fibrosis have been identified. The current study illustrates that expression profiling can be used to: (1) distinguish different hepatotoxic endpoints; (2) predict the development of toxic endpoints; and (3) develop hypotheses regarding mechanisms of toxicity.     

Toxicity of polycyclic aromatic hydrocarbons. IV. Effects of diphenaldehyde, a major product of ozonized phenanthrene, in rats

Diphenaldehyde is the major product of phenanthrene ozonized on silica gel. Male Sprague-Dawley rats were treated with a single ip injection of DMSO (3.0 ml/kg) or diphenaldehyde (90 mg/kg) in DMSO. Diphenaldehyde produced significant alterations in levels of serum aspartate aminotransferase, alanine aminotransferase, sorbitol dehydrogenase, gamma-glutamyl transpeptidase, and lactate dehydrogenase relative to DMSO-injected rats 24 hr after injection. These results, as well as gross observations on necropsy, suggest that diphenaldehyde exhibits significant hepatotoxicity.     

Short Term Training Attenuates Opening of the Mitochondrial Permeability Transition Pore Without Affecting Myocardial Function Following Ischemia-Reperfusion

Hepatotoxicity is one of the most serious adverse effects of antituberculosis drugs. The aim of this study was to produce a rat model of isoniazid-rifampicin (INH-RIF) induced hepatotoxicity. Materials and Methods: Wistar rats (100–150 g) were treated with different doses of INH i.e. 25, 50 and 75 mg/kg/day with a fixed dose of RIF i.e. 50 mg/kg/day intragastrically for a period of 28 days. Serum glutamate oxaloacetate aminotransferase (SGOT), glutamate pyruvate aminotransferase (SGPT), bilirubin (Bil) and alkaline phosphatase (ALP) were estimated at 0,14, 21 and 28 days in rats. Histological analysis was carried out to assess the liver. Results: Treatment of rats with INH–RIF (50 mg/kg/day each) induced hepatotoxicity as judged by elevated serum SGPT, SGOT, Bil and ALP as compared with their base line. Histological evaluation of INH–RIF induced hepatotoxicity also showed liver damage. Conclusion: The present study suggests that 50 mg/kg/day each of INH–RIF was selected as hepatotoxic dose (i.e. minimum dose with maximum hepatotoxicity) in wistar rats.     

Clustering of the SOM easily reveals distinct gene expression patterns: results of a reanalysis of lymphoma study

Background  

A method to evaluate and analyze the massive data generated by series of microarray experiments is of utmost importance to reveal the hidden patterns of gene expression. Because of the complexity and the high dimensionality of microarray gene expression profiles, the dimensional reduction of raw expression data and the feature selections necessary for, for example, classification of disease samples remains a challenge. To solve the problem we propose a two-level analysis. First self-organizing map (SOM) is used. SOM is a vector quantization method that simplifies and reduces the dimensionality of original measurements and visualizes individual tumor sample in a SOM component plane. Next, hierarchical clustering and K-means clustering is used to identify patterns of gene expression useful for classification of samples.     

Vascular endothelial growth factor and hepatocyte regeneration in acetaminophen toxicity

VEGF or VEGF-A is a major regulator of angiogenesis and has been recently shown to be important in organ repair. The potential role of VEGF in acetaminophen (APAP)-induced hepatotoxicity and recovery was investigated in B6C3F1 male mice. Mice were treated with APAP (300 mg/kg ip) and killed at various time points that reflect both the acute and recovery stages of toxicity. VEGF-A protein levels were increased 7-fold at 8 h and followed the development of hepatotoxicity. VEGF receptor 1, 2, and 3 (VEGFR1, VEGFR2, and VEGFR3, respectively) expression increased throughout the time course, with maximal expression at 48, 8, and 72 h, respectively. Treatment with the VEGF receptor inhibitor SU5416 (25 mg/kg ip at 3 h) had no effect on toxicity at 6 or 24 h. In further studies, the role of SU5416 on the late stages of toxicity was examined. Treatment of mice with APAP and SU5416 (25 mg/kg ip at 3 h) resulted in decreased expression of PCNA, a marker of cellular proliferation. Expression of platelet endothelial cell adhesion molecule, a measure of small vessel density, and endothelial nitric oxide synthase (NOS), a downstream target of VEGFR2, were increased at 48 and 72 h following toxic doses of APAP, and treatment with SU5416 decreased their expression. These data indicate that endogenous VEGF is critically important to the process of hepatocyte regeneration in APAP-induced hepatotoxicity in the mouse.     

Toxicity of polycyclic aromatic hydrocarbons. V. Protective effect of beta-naphthoflavone against hepatotoxicity induced by diphenaldehyde in rats

Male Sprague-Dawley rats were treated ip with beta-naphthoflavone (40 mg/kg/day) in corn oil or in DMSO for three days. Diphenaldehyde (90 mg/kg in DMSO) was injected ip 24 hr after pretreatment. The increase in the levels of aspartate aminotransferase, alanine aminotransferase, sorbitol dehydrogenase, gamma glutamyl transpeptidase, and lactate dehydrogenase was significantly lower in rats pretreated with BNF. This suggests that the BNF-induced P-450 isozyme systems have a protective effect against the acute hepatotoxicity of diphenaldehyde.     

Temporal expression of fumonisin B(1)-induced tumor necrosis factor-alpha and interferon gamma in mice

Fumonisin B(1) (FB(1)), a toxic metabolite of Fusarium verticillioides, is a carcinogen and causative agent of various animal diseases. Our previous studies indicated the involvement of tumor necrosis factor-alpha (TNF alpha) in FB(1)-induced toxic responses. To further investigate the time-course of TNF alpha production and signaling, mice (four/group) were treated subcutaneously (s.c.) or per os (p.o.) with either vehicle or 25 mg/kg of FB(1) as a single dose and sacrificed at 0, 2, 4, 8, 12 and 24 h after treatment. The TNF alpha expression was increased in liver and kidney after both routes of FB(1) exposure without any alterations in spleen. The p.o.-route FB(1) treatment caused greater hepatotoxicity compared to the s.c. route, as depicted by increased alanine aminotransferase and aspartate aminotransferase level in plasma, observed only after p.o. FB(1) treatment. The increase in enzymes at 8 h after p.o. treatment correlated with the highest TNF alpha expression, also noted at 8 h after p.o. treatment, thus further confirming the involvement of TNF alpha in FB(1) toxicity. The interferon (IFN)-gamma expression was increased in liver at 4 h after p.o. FB(1) treatment, suggesting a possible combined role of TNF alpha and IFN gamma in their induction and hepatotoxicity.     

Microarray analysis of gene expression during early adipocyte differentiation

The molecular mechanisms that regulate cellular differentiation during development and throughout life are complex. It is now recognized that precise patterns of differentially expressed genes ultimately direct a particular cell toward a given lineage and many of these are regulated during the earliest stages of differentiation. Using a microarray-based expression analysis, we have examined gene expression profiles during the first 24 h of 3T3-L1 adipocyte differentiation. RNA was isolated at times 0, 2, 8, 16, and 24 h following stimulation of differentiation and hybridized in duplicate to high density Affymetrix microarray gene chips containing a series of 13,179 cDNA/expressed sequence tag (EST) probe sets. Two hundred and eighty-five cDNA/ESTs were shown to have at least a fivefold change in expression levels during this time course and both hierarchical and self-organizing map clustering analysis was performed to categorize them by expression profiles. Several genes known to be regulated during this time period were confirmed and Western blot analysis of the proteins encoded by some of the identified genes revealed expression profiles similar to their mRNA counterparts. As expected, many of the genes identified have not been examined in such a critical time period during adipogenesis and may well represent novel adipogenic mediators.     

Hepatoprotective effect of allicin against acetaminophen‐induced liver injury: Role of inflammasome pathway,apoptosis, and liver regeneration

Acetaminophen (APAP) overdose leads to liver injury. NLRP3 inflammasome is a key player in APAP‐induced inflammation. Also, apoptosis and liver regeneration play an important role in liver injury. Therefore, we assessed allicin's protective effect on APAP‐induced hepatotoxicity and studied its effect on NLRP3 inflammasome and apoptosis. Mice in the APAP group were injected by APAP (250 mg/kg, intraperitoneal). The allicin‐treated group received allicin orally (10 mg/kg/d) during 7 days before APAP injection. Serum and hepatic tissues were separated 24 hours after APAP injection. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, alkaline phosphatase (ALP), and hepatic malondialdehyde (MDA) were assessed using the colorimetric method. Hepatic NLRP3 inflammasome, caspase‐1, and interleukin‐1β (IL‐1β) were estimated using enzyme‐linked immunosorbent assay. Hepatic Bcl‐2 and Ki‐67 were investigated by immunohistochemistry. APAP significantly increased AST, ALT, and ALP, whereas allicin significantly decreased their levels. Also, APAP significantly decreased albumin and allicin significantly improved it. APAP produced changes in liver morphology, including inflammation and massive coagulative necrosis. Allicin protected the liver from APAP‐induced necrosis, apoptosis, and hepatocellular degeneration via increasing Bcl‐2 and Ki‐67 levels. APAP significantly increased the hepatic MDA, whereas allicin significantly prevented this increase. APAP markedly activated the NLRP3 inflammasome pathway and consequently increased the production of caspase‐1 and IL‐1β. Interestingly, we found that allicin significantly inhibited NLRP3 inflammasome activation, which resulted in decreased caspase‐1 and IL‐1β levels. Allicin has a hepatoprotective effect against APAP‐induced liver injury via the decline of oxidative stress and inhibition of the inflammasome pathway and apoptosis. Therefore, allicin might be a novel tool to halt the progression of APAP‐stimulated hepatotoxicity.     

FLAME,a novel fuzzy clustering method for the analysis of DNA microarray data

Background  

Data clustering analysis has been extensively applied to extract information from gene expression profiles obtained with DNA microarrays. To this aim, existing clustering approaches, mainly developed in computer science, have been adapted to microarray data analysis. However, previous studies revealed that microarray datasets have very diverse structures, some of which may not be correctly captured by current clustering methods. We therefore approached the problem from a new starting point, and developed a clustering algorithm designed to capture dataset-specific structures at the beginning of the process.     

Methods for assessing reproducibility of clustering patterns observed in analyses of microarray data

MOTIVATION: Recent technological advances such as cDNA microarray technology have made it possible to simultaneously interrogate thousands of genes in a biological specimen. A cDNA microarray experiment produces a gene expression 'profile'. Often interest lies in discovering novel subgroupings, or 'clusters', of specimens based on their profiles, for example identification of new tumor taxonomies. Cluster analysis techniques such as hierarchical clustering and self-organizing maps have frequently been used for investigating structure in microarray data. However, clustering algorithms always detect clusters, even on random data, and it is easy to misinterpret the results without some objective measure of the reproducibility of the clusters. RESULTS: We present statistical methods for testing for overall clustering of gene expression profiles, and we define easily interpretable measures of cluster-specific reproducibility that facilitate understanding of the clustering structure. We apply these methods to elucidate structure in cDNA microarray gene expression profiles obtained on melanoma tumors and on prostate specimens.     

An integrated study of acute effects of valproic acid in the liver using metabonomics, proteomics, and transcriptomics platforms

An integrated omics approach was undertaken in order to elucidate a systems biology level understanding of the acute hepatotoxcity of valproic acid (VPA). Metabonomics, proteomics and gene expression microarray platforms were employed in this systems biology study. CD-1 female pregnant mice were injected subcutaneously with 600 mg/kg VPA or vehicle control. Urine, serum, and liver tissue were collected at 6, 12, and 24 h after dosing. Principal component analysis (PCA) of the metabonomics data showed clustering of the dosed groups away from the controls for the urine samples. Looser clustering was seen in the other sample sets investigated. However, VPA administration resulted in altered glucose concentrations in urine samples at 12 and 24 h and in aqueous liver tissue extracts at 12 h after VPA administration. Proteomics studies identified two proteins, glycogen phosphorylase and amylo-1,6-glucosidase, which were increased in dosed animals relative to control. Both of these proteins are involved in converting glycogen to glucose. Examination of the expression of 20,000 liver genes did not reveal significantly altered expression at 6, 12, or 24 h after VPA exposure. The combined studies indicated a perturbation in the glycogenolysis pathway following administration of VPA.     

Antioxidant activity of medicinal herb Rhodococcum vitis-idaea on galactosamine-induced liver injury in rats.

Aim of the study was to evaluate in vivo antioxidant action of medicinal herb Rhodococcum vitis-idaea (Rh.v) on galactosamine (GalN)-induced rat liver toxicity. The results showed that the hepatotoxicity and oxidative stress induced by GalN (700 mg/kg, s.c.) after 24 h evidenced by an increase in serum alanine aminotransferase and glutathione (GSH) S-transferase activities, and lipid peroxidation in liver homogenate were significantly inhibited, when 10 times diluted Rh.v. extract (5 ml/kg, i.p.) was given to rats 12 and 1 h before GalN treatment demonstrating that the extract of Rh.v is a potent antioxidant and protective against GalN-induced hepatotoxicity. The main antioxidant compound of the herb water extract used in the experiment was determined as arbutin, which possess 8% of dry weight of the herb. The electron spin resonance (ESR) spectrometer analysis revealed that the arbutin isolated from Rh.v exhibited strong superoxide and hydroxyl radical scavenging ability.     

Information criterion-based clustering with order-restricted candidate profiles in short time-course microarray experiments

Background  

Time-course microarray experiments produce vector gene expression profiles across a series of time points. Clustering genes based on these profiles is important in discovering functional related and co-regulated genes. Early developed clustering algorithms do not take advantage of the ordering in a time-course study, explicit use of which should allow more sensitive detection of genes that display a consistent pattern over time. Peddada et al. [1] proposed a clustering algorithm that can incorporate the temporal ordering using order-restricted statistical inference. This algorithm is, however, very time-consuming and hence inapplicable to most microarray experiments that contain a large number of genes. Its computational burden also imposes difficulty to assess the clustering reliability, which is a very important measure when clustering noisy microarray data.     

Hepatic Mrp4 induction following acetaminophen exposure is dependent on Kupffer cell function

During acetaminophen (APAP) hepatotoxicity, increased expression of multidrug resistance-associated proteins 2, 3, and 4 (Mrp2-4) occurs. Mrp4 is the most significantly upregulated transporter in mouse liver following APAP treatment. Although the expression profiles of liver transporters following APAP hepatotoxicity are well characterized, the regulatory mechanisms contributing to these changes remain unknown. We hypothesized that Kupffer cell-derived mediators participate in the regulation of hepatic transporters during APAP toxicity. To investigate this, C57BL/6J mice were pretreated with clodronate liposomes (0.1 ml iv) to deplete Kupffer cells and then challenged with APAP (500 mg/kg ip). Liver injury was assessed by plasma alanine aminotransferase and hepatic transporter protein expression was determined by Western blot and immunohistochemistry. Depletion of Kupffer cells by liposomal clodronate increased susceptibility to APAP hepatotoxicity. Although increased expression of several efflux transporters was observed after APAP exposure, only Mrp4 was found to be differentially regulated following Kupffer cell depletion. At 48 and 72 h after APAP dosing, Mrp4 levels were increased by 10- and 33-fold, respectively, in mice receiving empty liposomes. Immunohistochemistry revealed Mrp4 staining confined to centrilobular hepatocytes. Remarkably, Kupffer cell depletion completely prevented Mrp4 induction by APAP. Elevated plasma levels of TNF-alpha and IL-1beta were also prevented by Kupffer cell depletion. These findings show that Kupffer cells protect the liver from APAP toxicity and that Kupffer cell mediators released in response to APAP are likely responsible for the induction of Mrp4.     

Activity of a nitroalkene derivative, 1-(5-bromofur-2-il)-2-bromo-2-nitroethene, in the Salmonella/microsome assay and the mouse bone marrow micronucleus test

Mutagenicity of a substituted nitroalkene, 1-(5-bromofur-2-il)-2-bromo-2-nitroethene (BNF) was tested in the Salmonella/microsome assay using the strains TA 98, TA 100 and TA 100NR (nitroreductase deficient). BNF was a direct mutagen in TA 98 and TA 100; the response was lowered when exogenous metabolic activation (S9) was used. A further decrease in mutagenicity was observed in strain TA 100NR, as compared to the parental TA 100, which showed the involvement of nitroreduction in the overall response elicited by BNF. The micronucleus assay was carried out in Swiss male mice which were given a single i.p. dose of 10–20 mg/kg of BNF dissolved in peanut oil, bone marrow being sampled 24 and 48 h later. The micronucleated polychromatic erythrocyte counts (MNPCE) showed a weak response in the dose range of 10–17.5 mg/kg at the second sampling (48 h) and a significant rise for 20 mg/kg at 24 and 48 h.