Potential role of the apelin-APJ pathway in sex-related differential cardiotoxicity induced by doxorubicin in mice.

Preclinical and clinical findings suggest sexual dimorphism in cardiotoxicity induced by a chemotherapeutic drug, doxorubicin (DOX). However, molecular alterations leading to sex-related differential vulnerability of heart to DOX toxicity are not fully explored. In the present study, RNA sequencing in hearts of B6C3F1 mice indicated more differentially expressed genes in males than females (224 vs. 19; ≥1.5-fold, False Discovery Rate [FDR] < 0.05) at 1 week after receiving 24 mg/kg total cumulative DOX dose that induced cardiac lesions only in males. Pathway analysis further revealed probable inactivation of cardiac apelin fibroblast signaling pathway (p = 0.00004) only in DOX-treated male mice that showed ≥1.25-fold downregulation in the transcript and protein levels of the apelin receptor, APJ. In hearts of DOX-treated females, the transcript levels of apelin (1.24-fold) and APJ (1.47-fold) were significantly (p < 0.05) increased compared to saline-treated controls. Sex-related differential DOX effect was also observed on molecular targets downstream of the apelin-APJ pathway in cardiac fibroblasts and cardiomyocytes. In cardiac fibroblasts, upregulation of Tgf-ß2, Ctgf, Sphk1, Serpine1, and Timp1 (fibrosis; FDR < 0.05) in DOX-treated males and upregulation of only Tgf-ß2 and Timp1 (p < 0.05) in females suggested a greater DOX toxicity in hearts of males than females. Additionally, Ryr2 and Serca2 (calcium handling; FDR < 0.05) were downregulated in conjunction with 1.35-fold upregulation of Casp12 (sarcoplasmic reticulum-mediated apoptosis; FDR < 0.05) in DOX-treated male mice. Drug effect on the transcript level of these genes was less severe in female hearts. Collectively, these data suggest a likely role of the apelin-APJ axis in sex-related differential DOX-induced cardiotoxicity in our mouse model.

A comprehensive rat transcriptome built from large scale RNA-seq-based annotation.

The rat is an important model organism in biomedical research for studying human disease mechanisms and treatments, but its annotated transcriptome is far from complete. We constructed a Rat Transcriptome Re-annotation named RTR using RNA-seq data from 320 samples in 11 different organs generated by the SEQC consortium. Totally, there are 52 807 genes and 114 152 transcripts in RTR. Transcribed regions and exons in RTR account for ∼42% and ∼6.5% of the genome, respectively. Of all 73 074 newly annotated transcripts in RTR, 34 213 were annotated as high confident coding transcripts and 24 728 as high confident long noncoding transcripts. Different tissues rather than different stages have a significant influence on the expression patterns of transcripts. We also found that 11 715 genes and 15 852 transcripts were expressed in all 11 tissues and that 849 house-keeping genes expressed different isoforms among tissues. This comprehensive transcriptome is freely available at http://www.unimd.org/rtr/. Our new rat transcriptome provides essential reference for genetics and gene expression studies in rat disease and toxicity models.

MicroRNA-34a-5p as a promising early circulating preclinical biomarker of doxorubicin-induced chronic cardiotoxicity.

Cardiotoxicity is a serious adverse effect of an anticancer drug, doxorubicin (DOX), which can occur within a year or decades after completion of therapy. The present study was designed to address a knowledge gap concerning a lack of circulating biomarkers capable of predicting the risk of cardiotoxicity induced by DOX. Profiling of 2083 microRNAs (miRNAs) in mouse plasma revealed 81 differentially expressed miRNAs 1 week after 6, 9, 12, 18, or 24 mg/kg total cumulative DOX doses (early-onset model) or saline (SAL). Among these, the expression of seven miRNAs was altered prior to the onset of myocardial injury at 12 mg/kg and higher cumulative doses. The expression of only miR-34a-5p was significantly (false discovery rate [FDR] < 0.1) elevated at all total cumulative doses compared with concurrent SAL-treated controls and showed a statistically significant dose-related response. The trend in plasma miR-34a-5p expression levels during DOX exposures also correlated with a significant dose-related increase in cardiac expression of miR-34a-5p in these mice. Administration of a cardioprotective drug, dexrazoxane, to mice before DOX treatment, significantly mitigated miR-34a-5p expression in both plasma and heart in conjunction with attenuation of cardiac pathology. This association between plasma and heart may suggest miR-34a-5p as a potential early circulating marker of early-onset DOX cardiotoxicity. In addition, higher expression of miR-34a-5p (FDR < 0.1) in plasma and heart compared with SAL-treated controls 24 weeks after 24 mg/kg total cumulative DOX dose, when cardiac function was altered in our recently established delayed-onset cardiotoxicity model, indicated its potential as an early biomarker of delayed-onset cardiotoxicity.

Doxorubicin-induced delayed-onset subclinical cardiotoxicity in mice.

Subclinical cardiotoxicity at low total cumulative doxorubicin (DOX) doses can manifest into cardiomyopathy in long-term cancer survivors. However, the underlying mechanisms are poorly understood. In male B6C3F1 mice, assessment of cardiac function by echocardiography was performed at 1, 4, 10, 17, and 24 weeks after exposure to 6, 9, 12, and 24 mg/kg total cumulative DOX doses or saline (SAL) to monitor development of delayed-onset cardiotoxicity. The 6- or 9-mg/kg total cumulative doses resulted in a significant time-dependent decline in systolic function (left ventricular ejection fraction (LVEF) and fractional shortening (FS)) during the 24-week recovery although there was not a significant alteration in % LVEF or % FS at any specific time point during the recovery. A significant decline in systolic function was elicited by the cardiotoxic cumulative DOX dose (24 mg/kg) during the 4- to 24-week period after treatment compared to SAL-treated counterparts. At 24 weeks after DOX treatment, a significant dose-related decrease in the expression of genes and proteins involved in sarcoplasmic reticulum (SR) calcium homeostasis (Ryr2 and Serca2) was associated with a dose-related increase in the transcript level of Casp12 (SR-specific apoptosis) in hearts. These mice also showed enhanced apoptotic activity in hearts indicated by a significant dose-related elevation in the number of apoptotic cardiomyocytes compared to SAL-treated counterparts. These findings collectively suggest that a steady decline in SR calcium handling and apoptosis might be involved in the development of subclinical cardiotoxicity that can evolve into irreversible cardiomyopathy later in life.

Hepatic Transcript Profiles of Cytochrome P450 Genes Predict Sex Differences in Drug Metabolism.

Safety assessments of new drug candidates are an important part of the drug development and approval process. Often, possible sex-associated susceptibilities are not adequately addressed, and better assessment tools are needed. We hypothesized that hepatic transcript profiles of cytochrome P450 (P450) enzymes can be used to predict sex-associated differences in drug metabolism and possible adverse events. Comprehensive hepatic transcript profiles were generated for F344 rats of both sexes at nine ages, from 2 weeks (preweaning) to 104 weeks (elderly). Large differences in the transcript profiles of 29 drug metabolizing enzymes and transporters were found between adult males and females (8-52 weeks). Using the PharmaPendium data base, 41 drugs were found to be metabolized by one or two P450 enzymes encoded by sexually dimorphic mRNAs and thus were candidates for evaluation of possible sexually dimorphic metabolism and/or toxicities. Suspension cultures of primary hepatocytes from three male and three female adult rats (10-13 weeks old) were used to evaluate the metabolism of 11 drugs predicted to have sexually dimorphic metabolism. The pharmacokinetics of the drug or its metabolite was analyzed by liquid chromatography/tandem mass spectrometry using multiple reaction monitoring. Of those drugs with adequate metabolism, the predicted significant sex-different metabolism was found for six of seven drugs, with half-lives 37%-400% longer in female hepatocytes than in male hepatocytes. Thus, in this rat model, transcript profiles may allow identification of potential sex-related differences in drug metabolism. SIGNIFICANCE STATEMENT: The present study showed that sex-different expression of genes coding for drug metabolizing enzymes, specifically cytochrome P450s, could be used to predict sex-different drug metabolism and, thus, provide a new tool for protecting susceptible subpopulations from possible adverse drug events.

Candidate early predictive plasma protein markers of doxorubicin-induced chronic cardiotoxicity in B6C3F1 mice.

Cardiotoxicity is a serious adverse effect of doxorubicin (DOX) treatment in cancer patients. Currently, there is a lack of sensitive biomarkers to predict the risk of DOX-induced cardiotoxicity. Using SOMAmer-based proteomic technology, 1129 proteins were profiled to identify potential early biomarkers of cardiotoxicity in plasma from male B6C3F1 mice given a weekly intravenous dose of 3 mg/kg DOX or saline (SAL) for 2, 3, 4, 6, or 8 weeks (6, 9, 12, 18, or 24 mg/kg cumulative DOX doses, respectively). Also, a group of mice received the cardio-protectant, dexrazoxane (DXZ; 60 mg/kg; intraperitoneal) 30 min before a weekly DOX or SAL dose. Proteomic analysis in plasma collected a week after the last dose showed a significant ≥1.2-fold change in level of 18 proteins in DOX-treated mice compared to SAL-treated counterparts during 8-week exposure. Of these, neurogenic locus notch homolog protein 1 (NOTCH1), von Willebrand factor (vWF), mitochondrial glutamate carrier 2, Wnt inhibitory factor 1, legumain, and mannan-binding lectin serine protease 1 were increased in plasma at 6 mg/kg cumulative DOX dose, prior to the release of myocardial injury marker, cardiac troponin I at 12 mg/kg and higher cumulative doses. These six proteins also remained significantly elevated following myocardial injury or pathology at 24 mg/kg. Pretreatment of mice with DXZ significantly attenuated DOX-induced elevated levels of only NOTCH1 and vWF with mitigation of cardiotoxicity. This suggests NOTCH1 and vWF as candidate early biomarkers of DOX cardiotoxicity, which may help in addressing a clinically important question of identifying cancer patients at risk for cardiotoxicity.

Status of hepatic DNA methylome predetermines and modulates the severity of non-alcoholic fatty liver injury in mice.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a major health problem and a leading cause of chronic liver disease in the United States and Western countries. In humans, genetic factors greatly influence individual susceptibility to NAFLD; nonetheless, the effect of inter-individual differences in the normal liver epigenome with regard to the susceptibility to NAFLD has not been determined. RESULTS: In the present study, we investigated the association between the DNA methylation status in the livers of A/J and WSB/EiJ mice and the severity of NAFLD-associated liver injury. We demonstrate that A/J and WSB/EiJ mice, which are characterized by significant differences in the severity of liver injury induced by a choline- and folate-deficient (CFD) diet exhibit substantial differences in cytosine DNA methylation in their normal livers. Furthermore, feeding A/J and WSB/EiJ mice a CFD diet for 12 weeks resulted in different trends and changes in hepatic cytosine DNA methylation. CONCLUSION: Our findings indicate a primary role of hepatic DNA methylation in the pathogenesis of NAFLD and suggest that individual variations in DNA methylation across the genome may be a factor determining and influencing the vulnerability to NAFLD.

Early transcriptional changes in cardiac mitochondria during chronic doxorubicin exposure and mitigation by dexrazoxane in mice.

Identification of early biomarkers of cardiotoxicity could help initiate means to ameliorate the cardiotoxic actions of clinically useful drugs such as doxorubicin (DOX). Since DOX has been shown to target mitochondria, transcriptional levels of mitochondria-related genes were evaluated to identify early candidate biomarkers in hearts of male B6C3F1 mice given a weekly intravenous dose of 3mg/kg DOX or saline (SAL) for 2, 3, 4, 6, or 8 weeks (6, 9, 12, 18, or 24 mg/kg cumulative DOX doses, respectively). Also, a group of mice was pretreated (intraperitoneally) with the cardio-protectant, dexrazoxane (DXZ; 60 mg/kg) 30 min before each weekly dose of DOX or SAL. At necropsy a week after the last dose, increased plasma concentrations of cardiac troponin T (cTnT) were detected at 18 and 24 mg/kg cumulative DOX doses, whereas myocardial alterations were observed only at the 24 mg/kg dose. Of 1019 genes interrogated, 185, 109, 140, 184, and 451 genes were differentially expressed at 6, 9, 12, 18, and 24 mg/kg cumulative DOX doses, respectively, compared to concurrent SAL-treated controls. Of these, expression of 61 genes associated with energy metabolism and apoptosis was significantly altered before and after occurrence of myocardial injury, suggesting these as early genomics markers of cardiotoxicity. Much of these DOX-induced transcriptional changes were attenuated by pretreatment of mice with DXZ. Also, DXZ treatment significantly reduced plasma cTnT concentration and completely ameliorated cardiac alterations induced by 24 mg/kg cumulative DOX. This information on early transcriptional changes during DOX treatment may be useful in designing cardioprotective strategies targeting mitochondria.

Sex-related differential susceptibility to doxorubicin-induced cardiotoxicity in B6C3F1 mice.

Sex is a risk factor for development of cardiotoxicity, induced by the anti-cancer drug, doxorubicin (DOX), in humans. To explore potential mechanisms underlying differential susceptibility to DOX between sexes, 8-week old male and female B6C3F1 mice were dosed with 3mg/kg body weight DOX or an equivalent volume of saline via tail vein once a week for 6, 7, 8, and 9 consecutive weeks, resulting in 18, 21, 24, and 27mg/kg cumulative DOX doses, respectively. At necropsy, one week after each consecutive final dose, the extent of myocardial injury was greater in male mice compared to females as indicated by higher plasma concentrations of cardiac troponin T at all cumulative DOX doses with statistically significant differences between sexes at the 21 and 24mg/kg cumulative doses. A greater susceptibility to DOX in male mice was further confirmed by the presence of cytoplasmic vacuolization in cardiomyocytes, with left atrium being more vulnerable to DOX cardiotoxicity. The number of TUNEL-positive cardiomyocytes was mostly higher in DOX-treated male mice compared to female counterparts, showing a statistically significant sex-related difference only in left atrium at 21mg/kg cumulative dose. DOX-treated male mice also had an increased number of γ-H2A.X-positive (measure of DNA double-strand breaks) cardiomyocytes compared to female counterparts with a significant sex effect in the ventricle at 27mg/kg cumulative dose and right atrium at 21 and 27mg/kg cumulative doses. This newly established mouse model provides a means to identify biomarkers and access potential mechanisms underlying sex-related differences in DOX-induced cardiotoxicity.

Early metabolomics changes in heart and plasma during chronic doxorubicin treatment in B6C3F1 mice.

The present study aimed to identify molecular markers of early stages of cardiotoxicity induced by a potent chemotherapeutic agent, doxorubicin (DOX). Male B6C3F1 mice were dosed with 3 mg kg(-1) DOX or saline via tail vein weekly for 2, 3, 4, 6 or 8 weeks (cumulative DOX doses of 6, 9, 12, 18 or 24 mg kg(-1) , respectively) and euthanized a week after the last dose. Mass spectrometry-based and nuclear magnetic resonance spectrometry-based metabolic profiling were employed to identify initial biomarkers of cardiotoxicity before myocardial injury and cardiac pathology, which were not noted until after the 18 and 24 mg kg(-1) cumulative doses, respectively. After a cumulative dose of 6 mg kg(-1) , 18 amino acids and four biogenic amines (acetylornithine, kynurenine, putrescine and serotonin) were significantly increased in cardiac tissue; 16 amino acids and two biogenic amines (acetylornithine and hydroxyproline) were significantly altered in plasma. In addition, 16 acylcarnitines were significantly increased in plasma and five were significantly decreased in cardiac tissue compared to saline-treated controls. Plasma lactate and succinate, involved in the Krebs cycle, were significantly altered after a cumulative dose of 6 mg kg(-1) . A few metabolites remained altered at higher cumulative DOX doses, which could partly indicate a transition from injury processes at 2 weeks to repair processes with additional injury happening concurrently before myocardial injury at 8 weeks. These altered metabolic profiles in mouse heart and plasma during the initial stages of injury progression due to DOX treatment may suggest these metabolites as candidate early biomarkers of cardiotoxicity. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Transcriptomic responses provide a new mechanistic basis for the chemopreventive effects of folic acid and tributyrin in rat liver carcinogenesis.

The steady increase in the incidence and mortality of hepatocellular carcinoma (HCC) signifies a crucial need to understand better its pathogenesis to improve clinical management and prevention of the disease. The aim of this study was to investigate molecular mechanisms for the chemopreventive effects of folic acid and tributyrin alone or in combination on rat hepatocarcinogenesis. Male Wistar rats were subjected to a classic "resistant hepatocyte" model of liver carcinogenesis and treated with folic acid and tributyrin alone or in combination for 5 weeks during promotion stage. Treatment with folic acid and tributyrin alone or in combination strongly inhibited the development of glutathione-S-transferase placental form (GSTP)-positive foci. Microarray analysis showed significant changes in gene expression. A total of 498, 655 and 940 of differentially expressed genes, involved in cell cycle, p53-signaling, angiogenesis and Wnt pathways, was identified in the livers of rats treated with folic acid, tributyrin or folic acid and tributyrin. A detailed analysis of these differentially expressed genes revealed that treatments inhibited angiogenesis in the preneoplastic livers. This was evidenced by the fact that 30 out of 77 differentially expressed genes common to all three treatments are involved in the regulation of the angiogenesis pathway. The inhibition of angiogenesis was confirmed by reduced levels of CD34 protein. In conclusion, the tumor-suppressing activity of folic acid and tributyrin is associated with inhibition of angiogenesis at early stages of rat liver carcinogenesis. Importantly, the combination of folic acid and tributyrin has stronger chemopreventive effect than each of the compounds alone.

Early biomarkers of doxorubicin-induced heart injury in a mouse model.

Cardiac troponins, which are used as myocardial injury markers, are released in plasma only after tissue damage has occurred. Therefore, there is a need for identification of biomarkers of earlier events in cardiac injury to limit the extent of damage. To accomplish this, expression profiling of 1179 unique microRNAs (miRNAs) was performed in a chronic cardiotoxicity mouse model developed in our laboratory. Male B6C3F1 mice were injected intravenously with 3mg/kg doxorubicin (DOX; an anti-cancer drug), or saline once a week for 2, 3, 4, 6, and 8weeks, resulting in cumulative DOX doses of 6, 9, 12, 18, and 24mg/kg, respectively. Mice were euthanized a week after the last dose. Cardiac injury was evidenced in mice exposed to 18mg/kg and higher cumulative DOX dose whereas examination of hearts by light microscopy revealed cardiac lesions at 24mg/kg DOX. Also, 24 miRNAs were differentially expressed in mouse hearts, with the expression of 1, 1, 2, 8, and 21 miRNAs altered at 6, 9, 12, 18, and 24mg/kg DOX, respectively. A pro-apoptotic miR-34a was the only miRNA that was up-regulated at all cumulative DOX doses and showed a significant dose-related response. Up-regulation of miR-34a at 6mg/kg DOX may suggest apoptosis as an early molecular change in the hearts of DOX-treated mice. At 12mg/kg DOX, up-regulation of miR-34a was associated with down-regulation of hypertrophy-related miR-150; changes observed before cardiac injury. These findings may lead to the development of biomarkers of earlier events in DOX-induced cardiotoxicity that occur before the release of cardiac troponins.

Toxicogenomics and cancer susceptibility: advances with next-generation sequencing.

The aim of this review is to comprehensively summarize the recent achievements in the field of toxicogenomics and cancer research regarding genetic-environmental interactions in carcinogenesis and detection of genetic aberrations in cancer genomes by next-generation sequencing technology. Cancer is primarily a genetic disease in which genetic factors and environmental stimuli interact to cause genetic and epigenetic aberrations in human cells. Mutations in the germline act as either high-penetrance alleles that strongly increase the risk of cancer development, or as low-penetrance alleles that mildly change an individual's susceptibility to cancer. Somatic mutations, resulting from either DNA damage induced by exposure to environmental mutagens or from spontaneous errors in DNA replication or repair are involved in the development or progression of the cancer. Induced or spontaneous changes in the epigenome may also drive carcinogenesis. Advances in next-generation sequencing technology provide us opportunities to accurately, economically, and rapidly identify genetic variants, somatic mutations, gene expression profiles, and epigenetic alterations with single-base resolution. Whole genome sequencing, whole exome sequencing, and RNA sequencing of paired cancer and adjacent normal tissue present a comprehensive picture of the cancer genome. These new findings should benefit public health by providing insights in understanding cancer biology, and in improving cancer diagnosis and therapy.

Metzincins and related genes in experimental renal ageing: towards a unifying fibrosis classifier across species.

BACKGROUND: We have previously described a transcriptomic classifier consisting of metzincins and related genes (MARGS) discriminating kidneys and other organs with or without fibrosis from human biopsies. We now apply our MARGS-based algorithm to a rat model of age-associated interstitial renal fibrosis. METHODS: Untreated Fisher 344 rats (n = 76) were sacrificed between 2 to 104 weeks of age. For gene expression studies, we used single colour (Cy3) Agilent Whole Rat Genome 4 × 44k microarrays; 4-5 animals of each sex were profiled at each of the following ages: 2, 5, 6, 8, 15, 21, 78 and 104 weeks. Intensity data were subjected to variance stabilization (www.Partek.com). Data were analysed with ANOVA and other statistical methods. RESULTS: Sixty MARGS were differentially expressed across age groups. More MARGS were differentially expressed in older males than in older females. Principal component analysis showed gene expression induced segregation of age groups by sex from 6 to 104 weeks of age. The expression level of MMP7 correlated best with fibrosis grade. Severity of fibrosis was determined in 20 animals at 78 and 104 weeks of age. Expression values of 15 of 19 genes of the original classifier present on the Agilent array, in conjunction with linear discriminant analysis, was sufficient to correctly classify these 20 samples into non-fibrosis and fibrosis. Overrepresentation of MMP2 protein and CD44 protein in fibrosis was confirmed by immunofluorescence. CONCLUSIONS: Based on these results and our previous work, the MARGS classifier represents a cross-organ and cross-species classifier of fibrosis irrespective of aetiology. This finding provides evidence for a common pathway leading to fibrosis and will help to design a PCR-based clinical test.

Differential gene expression in human hepatocyte cell lines exposed to the antiretroviral agent zidovudine.

Zidovudine (3'-azido-3'-deoxythymidine; AZT) is the most widely used nucleoside reverse transcriptase inhibitor for the treatment of AIDS patients and prevention of mother-to-child transmission of HIV-1. Previously, we demonstrated that AZT had significantly greater growth inhibitory effects upon the human liver carcinoma cell line HepG2 as compared to the immortalized human liver cell line THLE2. We have now used gene expression profiling to determine the molecular pathways associated with toxicity in both cell lines. HepG2 cells were incubated with 0, 2, 20, or 100 µM AZT for 2 weeks; THLE2 cells were treated with 0, 50, 500, or 2,500 µM AZT, concentrations that were equi-toxic to those used in the HepG2 cells. After the treatment, total RNA was isolated and subjected to microarray analysis. Global analysis of gene expression, with a false discovery rate ≤0.01 and a fold change ≥1.5, indicated that 6- to 70-fold more genes were differentially expressed in a significant concentration-dependent manner in HepG2 cells when compared to THLE2 cells. Comparative analysis indicated that 7 % of these genes were common to both cell lines. Among the common differentially expressed genes, 70 % changed in the same direction, most of which were associated with cell death and survival, cell cycle, cell growth and proliferation, and DNA replication, recombination, and repair. As determined by the uptake of [methyl-(3)H]AZT, the intracellular levels of total AZT were approximately twofold higher in THLE2 cells than in HepG2 cells. The expression of thymidine kinase 1 (TK1) and UDP-glucuronosyltransferase 2B7 (UGT2B7) genes that regulate the metabolic activation and deactivation of AZT, respectively, was increased in HepG2 cells but decreased in THLE2 cells after treatment with AZT. This differential response in AZT metabolism was confirmed by real-time PCR, western blotting, and/or enzymatic assays. These data indicate that molecular pathways involved with cell death and survival, cell cycle, cell growth and proliferation, and DNA replication, recombination, and repair are involved in the toxicities associated with AZT in both human cell lines, and that the difference in expression of TK1 and UGT2B7 in response to AZT treatment in HepG2 cells and THLE2 cells might explain why HepG2 cells are more sensitive than THLE2 cells to the toxicity of AZT.

Development of doxorubicin-induced chronic cardiotoxicity in the B6C3F1 mouse model.

Serum levels of cardiac troponins serve as biomarkers of myocardial injury. However, troponins are released into the serum only after damage to cardiac tissue has occurred. Here, we report development of a mouse model of doxorubicin (DOX)-induced chronic cardiotoxicity to aid in the identification of predictive biomarkers of early events of cardiac tissue injury. Male B6C3F(1) mice were administered intravenous DOX at 3mg/kg body weight, or an equivalent volume of saline, once a week for 4, 6, 8, 10, 12, and 14weeks, resulting in cumulative DOX doses of 12, 18, 24, 30, 36, and 42mg/kg, respectively. Mice were sacrificed a week following the last dose. A significant reduction in body weight gain was observed in mice following exposure to a weekly DOX dose for 1week and longer compared to saline-treated controls. DOX treatment also resulted in declines in red blood cell count, hemoglobin level, and hematocrit compared to saline-treated controls after the 2nd weekly dose until the 8th and 9th doses, followed by a modest recovery. All DOX-treated mice had significant elevations in cardiac troponin T concentrations in plasma compared to saline-treated controls, indicating cardiac tissue injury. Also, a dose-related increase in the severity of cardiac lesions was seen in mice exposed to 24mg/kg DOX and higher cumulative doses. Mice treated with cumulative DOX doses of 30mg/kg and higher showed a significant decline in heart rate, suggesting drug-induced cardiac dysfunction. Altogether, these findings demonstrate the development of DOX-induced chronic cardiotoxicity in B6C3F(1) mice.

Interstrain differences in the severity of liver injury induced by a choline- and folate-deficient diet in mice are associated with dysregulation of genes involved in lipid metabolism.

Nonalcoholic fatty liver disease (NAFLD) is a major health problem and a leading cause of chronic liver disease in the United States and developed countries. In humans, genetic factors greatly influence individual susceptibility to NAFLD. The goals of this study were to compare the magnitude of interindividual differences in the severity of liver injury induced by methyl-donor deficiency among individual inbred strains of mice and to investigate the underlying mechanisms associated with the variability. Feeding mice a choline- and folate-deficient diet for 12 wk caused liver injury similar to NAFLD. The magnitude of liver injury varied among the strains, with the order of sensitivity being A/J ≈ C57BL/6J ≈ C3H/HeJ < 129S1/SvImJ ≈ CAST/EiJ < PWK/PhJ < WSB/EiJ. The interstrain variability in severity of NAFLD liver damage was associated with dysregulation of genes involved in lipid metabolism, primarily with a down-regulation of the peroxisome proliferator receptor α (PPARα)-regulated lipid catabolic pathway genes. Markers of oxidative stress and oxidative stress-induced DNA damage were also elevated in the livers but were not correlated with severity of liver damage. These findings suggest that the PPARα-regulated metabolism network is one of the key mechanisms determining interstrain susceptibility and severity of NAFLD in mice.

Characterization of whole genome amplified (WGA) DNA for use in genotyping assay development.

BACKGROUND: Genotyping assays often require substantial amounts of DNA. To overcome the problem of limiting amounts of available DNA, Whole Genome Amplification (WGA) methods have been developed. The multiple displacement amplification (MDA) method using Φ29 polymerase has become the preferred choice due to its high processivity and low error rate. However, the uniformity and fidelity of the amplification process across the genome has not been extensively characterized. RESULTS: To assess amplification uniformity, we used array-based comparative genomic hybridization (aCGH) to evaluate DNA copy number variations (CNVs) in DNAs amplified by two MDA kits: GenomiPhi and REPLI-g. The Agilent Human CGH array containing nearly one million probes was used in this study together with DNAs from a normal subject and 2 cystic fibrosis (CF) patients. Each DNA sample was amplified 4 independent times and compared to its native unamplified DNA. Komogorov distances and Phi correlations showed a high consistency within each sample group. Less than 2% of the probes showed more than 2-fold CNV introduced by the amplification process. The two amplification kits, REPLI-g and GenomiPhi, generate very similar amplified DNA samples despite the differences between the unamplified and amplified DNA samples. The results from aCGH analysis indicated that there were no obvious CNVs in the CFTR gene region due to WGA when compared to unamplified DNA. This was confirmed by quantitative real-time PCR copy number assays at 10 locations within the CFTR gene. DNA sequencing analysis of a 2-kb region within the CFTR gene showed no mutations introduced by WGA. CONCLUSION: The relatively high uniformity and consistency of the WGA process, coupled with the low replication error rate, suggests that WGA DNA may be suitable for accurate genotyping. Regions of the genome that were consistently under-amplified were found to contain higher than average GC content. Because of the consistent differences between the WGA DNA and the native unamplified DNA, characterization of the genomic region of interest, as described here, will be necessary to ensure the reliability of genotyping results from WGA DNA.

The liver toxicity biomarker study phase I: markers for the effects of tolcapone or entacapone.

The Liver Toxicity Biomarker Study is a systems toxicology approach to discover biomarkers that are indicative of a drug's potential to cause human idiosyncratic drug-induced liver injury. In phase I, the molecular effects in rat liver and blood plasma induced by tolcapone (a "toxic" drug) were compared with the molecular effects in the same tissues by dosing with entacapone (a "clean" drug, similar to tolcapone in chemical structure and primary pharmacological mechanism). Two durations of drug exposure, 3 and 28 days, were employed. Comprehensive molecular analysis of rat liver and plasma samples yielded marker analytes for various drug-vehicle or drug-drug comparisons. An important finding was that the marker analytes associated with tolcapone only partially overlapped with marker analytes associated with entacapone, despite the fact that both drugs have similar chemical structures and the same primary pharmacological mechanism of action. This result indicates that the molecular analyses employed in the study are detecting substantial "off-target" markers for the two drugs. An additional interesting finding was the modest overlap of the marker data sets for 3-day exposure and 28-day exposure, indicating that the molecular changes in liver and plasma caused by short- and long-term drug treatments do not share common characteristics.

Expression analysis of hepatic mitochondria-related genes in mice exposed to acrylamide and glycidamide.

Acrylamide (AA) is an industrial chemical that has been extensively investigated for central nervous system (CNS), reproductive, and genetic toxicity. However, AA effects on the liver, a major organ of drug metabolism, have not been adequately explored. In addition, the role of mitochondria in AA-mediated toxicity is still unclear. Changes in expression levels of genes associated with hepatic mitochondrial function of male transgenic Big Blue (BB) mice administered 500 mg/L AA or an equimolar concentration (600 mg/L) of its reactive metabolite glycidamide (GA) in drinking water for 3 and 4 wk, respectively, were examined. Transcriptional profiling of 542 mitochondria-related genes indicated a significant downregulation of genes associated with the 3-beta-hydroxysteroid dehydrogenase family in AA- and GA-treated mice, suggesting a possible role of both chemicals in altering hepatic steroid metabolism in BB mice. In addition, genes associated with lipid metabolism were altered by both treatments. Interestingly, only the parental compound (AA) significantly induced expression levels of genes associated with oxidative phosphorylation, in particular ATP synthase, which correlated with elevated ATP levels, indicating an increased energy demand in liver during AA exposure. Acrylamide-treated mice also showed significantly higher activity of glutathione S-transferase in association with decreased levels of reduced glutathione (GSH), which may imply an enhanced rate of conjugation of AA with GSH in liver. These results suggest different hepatic mechanisms of action of AA and GA and provide important insights into the involvement of mitochondria during their exposures.