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.

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.

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.

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.

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.

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.

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.

Age and sex dependent changes in liver gene expression during the life cycle of the rat.

BACKGROUND: Age- and sex-related susceptibility to adverse drug reactions and disease is a key concern in understanding drug safety and disease progression. We hypothesize that the underlying suite of hepatic genes expressed at various life cycle stages will impact susceptibility to adverse drug reactions. Understanding the basal liver gene expression patterns is a necessary first step in addressing this hypothesis and will inform our assessments of adverse drug reactions as the liver plays a central role in drug metabolism and biotransformation. Untreated male and female F344 rats were sacrificed at 2, 5, 6, 8, 15, 21, 52, 78, and 104 weeks of age. Liver tissues were collected for histology and gene expression analysis. Whole-genome rat microarrays were used to query global expression profiles. RESULTS: An initial list of differentially expressed genes was selected using criteria based upon p-value (p < 0.05) and fold-change (+/- 1.5). Three dimensional principal component analyses revealed differences between males and females beginning at 2 weeks with more divergent profiles beginning at 5 weeks. The greatest sex-differences were observed between 8 and 52 weeks before converging again at 104 weeks. K-means clustering identified groups of genes that displayed age-related patterns of expression. Various adult aging-related clusters represented gene pathways related to xenobiotic metabolism, DNA damage repair, and oxidative stress. CONCLUSIONS: These results suggest an underlying role for genes in specific clusters in potentiating age- and sex-related differences in susceptibility to adverse health effects. Furthermore, such a comprehensive picture of life cycle changes in gene expression deepens our understanding and informs the utility of liver gene expression biomarkers.

Underlying mitochondrial dysfunction triggers flutamide-induced oxidative liver injury in a mouse model of idiosyncratic drug toxicity.

Flutamide, a widely used nonsteroidal anti-androgen, but not its bioisostere bicalutamide, has been associated with idiosyncratic drug-induced liver injury. Although the susceptibility factors are unknown, mitochondrial injury has emerged as a putative hazard of flutamide. To explore the role of mitochondrial sensitization in flutamide hepatotoxicity, we determined the effects of superimposed drug stress in a murine model of underlying mitochondrial abnormalities. Male wild-type or heterozygous Sod2(+/-) mice were injected intraperitoneously with flutamide (0, 30 or 100 mg/kg/day) for 28 days. A kinetic pilot study revealed that flutamide (100 mg/kg/day) caused approximately 10-fold greater exposure than the reported therapeutic mean plasma levels. Mutant (5/10), but not wild-type, mice in the high-dose group exhibited small foci of hepatocellular necrosis and an increased number of apoptotic hepatocytes. Hepatic GSSG/GSH, protein carbonyl levels, and serum lactate levels were significantly increased, suggesting oxidant stress and mitochondrial dysfunction. Measurement of mitochondrial superoxide in cultured hepatocytes demonstrated that mitochondria were a significant source of flutamide-enhanced oxidant stress. Indeed, mitochondria isolated from flutamide-treated Sod2(+/-) mice exhibited decreased aconitase activity as compared to vehicle controls. A transcriptomics analysis using MitoChips revealed that flutamide-treated Sod2(+/-) mice exhibited a selective decrease in the expression of all complexes I and III subunits encoded by mitochondrial DNA. In contrast, Sod2(+/-) mice receiving bicalutamide (50 mg/kg/day) did not reveal any hepatic changes. These results are compatible with our concept that flutamide targets hepatic mitochondria and exerts oxidant stress that can lead to overt hepatic injury in the presence of an underlying mitochondrial abnormality.

Effect of short-term exposure to zidovudine (AZT) on the expression of mitochondria-related genes in skeletal muscle of neonatal mice.

Zidovudine (3'-azido-3'-deoxythymidine; AZT) is the main anti-retroviral drug given to HIV-1-infected pregnant women during pregnancy and to their infants after birth to reduce mother-to-child transmission of the virus. In animal studies, however, a significant mitochondrial morphological damage has been reported in skeletal muscle as a consequence of transplacental or perinatal exposure to AZT. Because proper muscle function is highly dependent on efficient mitochondrial function and information on AZT-induced mitochondrial toxicity during neonatal exposure is limited, we investigated the effect of AZT on the expression of 542 mitochondria-related genes encoded by both nuclear and mitochondrial DNA in the skeletal muscle of infant male and female mice using microarray technology. Animals were treated orally by gavage with AZT at 0, 10, 50, 100, and 200mg/kg body weight/day from postnatal day (PND) 1 through 8 and were sacrificed at 1- and 2-h following the last dose on PND 8. These doses in mice correspond to 0, 1.1, 5.5, 11.0, and 22.0mg/kg AZT in human infants [Center for Drug Evaluation and Research (CDER) 2005. Pharmacology and Toxicology, Guidance for industry. Estimating the maximum safe dose in initial clinical trials for therapeutics in adult healthy volunteers, p. 7. http://www.fda.gov/cder/guidance/index.htm.]. Microarray data were analyzed for effects of time, sex, treatment, and their interactions using a fixed effect linear model. The results showed modest, but significant, dose-related responses in the expression level of genes associated with apoptosis, fatty acid metabolism, mitochondrial DNA maintenance, and various mitochondrial membrane transporters. The transcription levels were not significantly different at both time points and were not sex dependent. The results suggest that changes in expression of mitochondria-related genes in skeletal muscle may be an initial response to short-term AZT exposure in infant mice.

Testing for treatment effects on gene ontology.

In studies that use DNA arrays to assess changes in gene expression, it is preferable to measure the significance of treatment effects on a group of genes from a pathway or functional category such as gene ontology terms (GO terms, http://www.geneontology.org) because this facilitates the interpretation of effects and may markedly increase significance. A modified meta-analysis method to combine p-values was developed to measure the significance of an overall treatment effect on such functionally-defined groups of genes, taking into account the correlation structure among genes. For hypothesis testing that allows gene expression to change in both directions, p-values are calculated under the null distribution generated by a Monte Carlo method. As a test of this procedure, we attempted to distinguish altered pathways in microarray studies performed with Mitochips, oligonucleotide microarrays specific to mitochondrial DNA-encoded transcripts. We found that our analytic method improves the specificity of selection for altered pathways, due to incorporation of the inter-gene correlation structure in each pathway. It is thus a practical method to measure treatment effects on GO groups. In many actual applications, microarray experiments measure treatment effects under complicated design structures and with small sample sizes. For such applications to real data of limited statistical power, and also in computer simulations, we demonstrate that our method gives reasonable test results.

Nucleoside reverse transcriptase inhibitors (NRTIs)-induced expression profile of mitochondria-related genes in the mouse liver.

Mitochondrial dysfunction has been implicated in the adverse effects of nucleoside reverse transcriptase inhibitors (NRTIs) used to treat HIV-1 infections. To gain insight into the mechanism by which NRTIs alter mitochondrial function, the expression level of 542 genes associated with mitochondrial structure and functions was determined in the livers of p53 haplodeficient (+/-) C3B6F1 female mouse pups using mouse mitochondria-specific oligonucleotide microarray. The pups were transplacentally exposed to zidovudine (AZT) at 240 mg/kg bw/day or a combination of AZT and lamivudine (3TC) at 160 and 100mg/kg bw/day, respectively, from gestation day 12 through 18, followed by continuous treatment by oral administration from postnatal day 1-28. In addition, AZT/3TC effect was investigated in wild-type (+/+) C3B6F1 female mice. The genotype did not significantly affect the gene expression profile induced by AZT/3TC treatment. However, the transcriptional level of several genes associated with oxidative phosphorylation, mitochondrial tRNAs, fatty acid oxidation, steroid biosynthesis, and a few transport proteins were significantly altered in pups treated with AZT and AZT/3TC compared to their vehicle counterparts. Interestingly, AZT/3TC altered the expression level of 153 genes with false discovery rate of less than 0.05, in contrast to only 20 genes by AZT alone. These results suggest that NRTI-related effect on expression level of genes associated with mitochondrial functions was much greater in response to AZT/3TC combination treatment than AZT alone.

Effect of p53 genotype on gene expression profiles in murine liver.

The tumor suppressor protein p53 is a key regulatory element in the cell and is regarded as the "guardian of the genome". Much of the present knowledge of p53 function has come from studies of transgenic mice in which the p53 gene has undergone a targeted deletion. In order to provide additional insight into the impact on the cellular regulatory networks associated with the loss of this gene, microarray technology was utilized to assess gene expression in tissues from both the p53(-/-) and p53(+/-) mice. Six male mice from each genotype (p53(+/+), p53(+/-), and p53(-/-)) were humanely killed and the tissues processed for microarray analysis. The initial studies have been performed in the liver for which the Dunnett test revealed 1406 genes to be differentially expressed between p53(+/+) and p53(+/-) or between p53(+/+) and p53(-/-) at the level of p < or = 0.05. Both genes with increased expression and decreased expression were identified in p53(+/-) and in p53(-/-) mice. Most notable in the gene list derived from the p53(+/-) mice was the significant reduction in p53 mRNA. In the p53(-/-) mice, not only was there reduced expression of the p53 genes on the array, but genes associated with DNA repair, apoptosis, and cell proliferation were differentially expressed, as expected. However, altered expression was noted for many genes in the Cdc42-GTPase pathways that influence cell proliferation. This may indicate that alternate pathways are brought into play in the unperturbed liver when loss or reduction in p53 levels occurs.

In Vitro Modulation of Redox and Metabolism Interplay at the Brain Vascular Endothelium: Genomic and Proteomic Profiles of Sulforaphane Activity.

Sulforaphane (SFN) has been shown to protect the brain vascular system and effectively reduce ischemic injuries and cognitive deficits. Given the robust cerebrovascular protection afforded by SFN, the objective of this study was to profile these effects in vitro using primary mouse brain microvascular endothelial cells and focusing on cellular redox, metabolism and detoxification functions. We used a mouse MitoChip array developed and validated at the FDA National Center for Toxicological Research (NCTR) to profile a host of genes encoded by nuclear and mt-DNA following SFN treatment (0-5 µM). Corresponding protein expression levels were assessed (ad hoc) by qRT-PCR, immunoblots and immunocytochemistry (ICC). Gene ontology clustering revealed that SFN treatment (24 h) significantly up-regulated ~50 key genes (>1.5 fold, adjusted p < 0.0001) and repressed 20 genes (<0.7 fold, adjusted p < 0.0001) belonging to oxidative stress, phase 1 & 2 drug metabolism enzymes (glutathione system), iron transporters, glycolysis, oxidative phosphorylation (OXPHOS), amino acid metabolism, lipid metabolism and mitochondrial biogenesis. Our results show that SFN stimulated the production of ATP by promoting the expression and activity of glucose transporter-1, and glycolysis. In addition, SFN upregulated anti-oxidative stress responses, redox signaling and phase 2 drug metabolism/detoxification functions, thus elucidating further the previously observed neurovascular protective effects of this compound.

Transcript profiling in the testes and prostates of postnatal day 30 Sprague-Dawley rats exposed prenatally and lactationally to 2-hydroxy-4-methoxybenzophenone.

2-hydroxy-4-methoxybenzophenone (HMB) is an ultraviolet light-absorbing compound that is used in sunscreens, cosmetics and plastics. HMB has been reported to have weak estrogenic activity by in vivo and in vitro studies, making it a chemical with potential reproductive concern. To explore if prenatal and lactational HMB exposure alters gene expression profiles of the developing reproductive organs, we performed microarray analysis using the prostate and testis of postnatal day (PND) 30 male Sprague-Dawley rats offspring exposed to 0, 3000, or 30,000 ppm of HMB from gestational day 6 through PND 21. Gene expression profiles of the prostate and testis were differentially affected by HMB dose with significant alterations observed at the 30,000 ppm HMB group. Tissue-specific gene expression was also identified. These genes, whose expression was altered by HMB exposure, may be considered as candidate biomarker(s) for testicular or prostatic toxicity; however, further studies are necessary to explore this potential.

Elimination of laboratory ozone leads to a dramatic improvement in the reproducibility of microarray gene expression measurements.

BACKGROUND: Environmental ozone can rapidly degrade cyanine 5 (Cy5), a fluorescent dye commonly used in microarray gene expression studies. Cyanine 3 (Cy3) is much less affected by atmospheric ozone. Degradation of the Cy5 signal relative to the Cy3 signal in 2-color microarrays will adversely reduce the Cy5/Cy3 ratio resulting in unreliable microarray data. RESULTS: Ozone in central Arkansas typically ranges between approximately 22 ppb to approximately 46 ppb and can be as high as 60-100 ppb depending upon season, meteorological conditions, and time of day. These levels of ozone are common in many areas of the country during the summer. A carbon filter was installed in the laboratory air handling system to reduce ozone levels in the microarray laboratory. In addition, the airflow was balanced to prevent non-filtered air from entering the laboratory. These modifications reduced the ozone within the microarray laboratory to approximately 2-4 ppb. Data presented here document reductions in Cy5 signal on both in-house produced microarrays and commercial microarrays as a result of exposure to unfiltered air. Comparisons of identically hybridized microarrays exposed to either carbon-filtered or unfiltered air demonstrated the protective effect of carbon-filtration on microarray data as indicated by Cy5 and Cy3 intensities. LOWESS normalization of the data was not able to completely overcome the effect of ozone-induced reduction of Cy5 signal. Experiments were also conducted to examine the effects of high humidity on microarray quality. Modest, but significant, increases in Cy5 and Cy3 signal intensities were observed after 2 or 4 hours at 98-99% humidity compared to 42% humidity. CONCLUSION: Simple installation of carbon filters in the laboratory air handling system resulted in low and consistent ozone levels. This allowed the accurate determination of gene expression by microarray using Cy5 and Cy3 fluorescent dyes.

Development of mitochondria-specific mouse oligonucleotide microarray and validation of data by real-time PCR.

This study describes the development of a mitochondria-specific microarray, MitoChip, to measure transcripts of mitochondria-associated genes in various diseases and drug-induced toxicities in the mouse. The array consists of 542 oligonucleotides that represent genes from the mitochondrial and nuclear genomes associated with mitochondrial structure and functions. The expression of mitochondrial genes was measured in the liver of both p53 haplodeficient (+/-) and wild-type (+/+) C3B6F(1) female mice exposed to antiretroviral agents, Zidovudine (AZT) and Lamivudine (3TC). Among genes whose expression was significantly altered, a set was selected for real-time PCR analysis to verify their differential gene expression. The real-time PCR data confirmed the observations by microarray analysis suggesting that the MitoChip may be an important tool for examining mitochondrial involvement in diseases and drug-induced toxicities.