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.

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.

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.

Simultaneous Quantification of t(14;18) and HPRT Exon 2/3 Deletions in Human Lymphocytes.

Specific recurring chromosomal translocations and deletions are found in a variety of cancers. In hematopoietic malignancies, many of these chromosomal aberrations result from mistakes involving V(D)J recombination. V(D)J recombination is required for the formation of functional T-cell receptor genes in T-cells and antibody genes in B-cells. This is an inherently dangerous process, however, because double-strand breaks are introduced into the chromosomes. Molecular evidence indicates that failure of the fidelity of this process results in the activation of proto-oncogenes or the inactivation of tumor suppressor genes. Here we describe sensitive, quantitative PCR assays for the measurement of such events in human lymphocytes. One assay measures the frequency of t(14;18) translocations that result in the dysfunctional regulation of the anti-apoptotic gene BCL-2. The other assay measures the frequency of a deletion caused by illegitimate V(D)J recombination in the X-linked HPRT gene.

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.

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.

Sex and age differences in the expression of liver microRNAs during the life span of F344 rats.

BACKGROUND: Physiological factors such as age and sex have been shown to be risk factors for adverse effects in the liver, including liver diseases and drug-induced liver injury. Previously, we have reported age- and sex-related significant differences in hepatic basal gene expression in rats during the life span that may be related to susceptibility to such adverse effects. However, the underlying mechanisms of the gene expression changes were not fully understood. In recent years, increasing evidence for epigenetic mechanisms of gene regulation has fueled interest in the role of microRNAs (miRNAs) in toxicogenomics and biomarker discovery. We therefore proposed that significant age and sex differences exist in baseline liver miRNA expression, and that comprehensive profiling of miRNAs will provide insights into the epigenetic regulation of gene expression in rat liver. METHODS: To address this, liver tissues from male and female F344 rats were examined at 2, 5, 6, 8, 15, 21, 52, 78, and 104 weeks of age for the expression of 677 unique miRNAs. Following data processing, predictive pathway analysis was performed on selected miRNAs that exhibited prominent age and/or sex differences in expression. RESULTS: Of the 314 miRNAs found to be expressed, 214 were differentially expressed; 65 and 212 miRNAs showed significant (false discovery rate (FDR) <5% and ≥1.5-fold change) sex- and age-related differences in expression, respectively. Thirty-eight miRNAs showed 2-week-specific expression, of which 31 miRNAs were found to be encoded within the Dlk1-Dio3 cluster located on chromosome 6. This cluster has been associated with tissue proliferation and differentiation, and liver energy homeostasis in postnatal development. Predictive pathway analysis linked sex-biased miRNA expression with sexually dimorphic molecular functions and toxicological functions that may reflect sex differences in hepatic physiology and disease. The expression of miRNAs (miR-18a, miR-99a, and miR-203, miR-451) was also found to associate with specific sexually dimorphic hepatic histopathology. The expression of miRNAs involved in regulating cell death, cell proliferation, and cell cycle was found to change as the rats matured from adult to old age. CONCLUSIONS: Overall, significant age- and sex-related differences in liver miRNA expression were identified and linked to histopathological findings and predicted functional pathways that may underlie susceptibilities to liver toxicity and disease.

Transcriptomics analysis of early embryonic stem cell differentiation under osteoblast culture conditions: Applications for detection of developmental toxicity.

The mouse embryonic stem cell test (mEST) is a promising in vitro assay for predicting developmental toxicity. In the current study, early differentiation of D3 mouse embryonic stem cells (mESCs) under osteoblast culture conditions and embryotoxicity of cadmium sulfate were examined. D3 mESCs were exposed to cadmium sulfate for 24, 48 or 72h, and whole genome transcriptional profiles were determined. The results indicate a track of differentiation was identified as mESCs differentiate. Biological processes that were associated with differentiation related genes included embryonic development and, specifically, skeletal system development. Cadmium sulfate inhibited mESC differentiation at all three time points. Functional pathway analysis indicated biological pathways affected included those related to skeletal development, renal and reproductive function. In summary, our results suggest that transcriptional profiles are a sensitive indicator of early mESC differentiation. Transcriptomics may improve the predictivity of the mEST by suggesting possible modes of action for tested chemicals.

Stably Expressed Genes Involved in Basic Cellular Functions.

Stably Expressed Genes (SEGs) whose expression varies within a narrow range may be involved in core cellular processes necessary for basic functions. To identify such genes, we re-analyzed existing RNA-Seq gene expression profiles across 11 organs at 4 developmental stages (from immature to old age) in both sexes of F344 rats (n = 4/group; 320 samples). Expression changes (calculated as the maximum expression / minimum expression for each gene) of >19000 genes across organs, ages, and sexes ranged from 2.35 to >109-fold, with a median of 165-fold. The expression of 278 SEGs was found to vary ≤4-fold and these genes were significantly involved in protein catabolism (proteasome and ubiquitination), RNA transport, protein processing, and the spliceosome. Such stability of expression was further validated in human samples where the expression variability of the homologous human SEGs was significantly lower than that of other genes in the human genome. It was also found that the homologous human SEGs were generally less subject to non-synonymous mutation than other genes, as would be expected of stably expressed genes. We also found that knockout of SEG homologs in mouse models was more likely to cause complete preweaning lethality than non-SEG homologs, corroborating the fundamental roles played by SEGs in biological development. Such stably expressed genes and pathways across life-stages suggest that tight control of these processes is important in basic cellular functions and that perturbation by endogenous (e.g., genetics) or exogenous agents (e.g., drugs, environmental factors) may cause serious adverse effects.

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.

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 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 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.

Effects of oral exposure to bisphenol A on gene expression and global genomic DNA methylation in the prostate, female mammary gland, and uterus of NCTR Sprague-Dawley rats.

Bisphenol A (BPA), an industrial chemical used in the manufacture of polycarbonate and epoxy resins, binds to the nuclear estrogen receptor with an affinity 4-5 orders of magnitude lower than that of estradiol. We reported previously that "high BPA" [100,000 and 300,000 µg/kg body weight (bw)/day], but not "low BPA" (2.5-2700 µg/kg bw/day), induced clear adverse effects in NCTR Sprague-Dawley rats gavaged daily from gestation day 6 through postnatal day (PND) 90. The "high BPA" effects partially overlapped those of ethinyl estradiol (EE2, 0.5 and 5.0 µg/kg bw/day). To evaluate further the potential of "low BPA" to induce biological effects, here we assessed the global genomic DNA methylation and gene expression in the prostate and female mammary glands, tissues identified previously as potential targets of BPA, and uterus, a sensitive estrogen-responsive tissue. Both doses of EE2 modulated gene expression, including of known estrogen-responsive genes, and PND 4 global gene expression data showed a partial overlap of the "high BPA" effects with those of EE2. The "low BPA" doses modulated the expression of several genes; however, the absence of a dose response reduces the likelihood that these changes were causally linked to the treatment. These results are consistent with the toxicity outcomes.

Age and sex differences in kidney microRNA expression during the life span of F344 rats.

BACKGROUND: Growing evidence suggests that epigenetic mechanisms of gene regulation may play a role in susceptibilities to specific toxicities and adverse drug reactions. MiRNAs in particular have been shown to be important regulators in cancer and other diseases and show promise as predictive biomarkers for diagnosis and prognosis. In this study, we characterized the global kidney miRNA expression profile in untreated male and female F344 rats throughout the life span. These findings were correlated with sex-specific susceptibilities to adverse renal events, such as male-biased renal fibrosis and inflammation in old age. METHODS: Kidney miRNA expression was examined in F344 rats at 2, 5, 6, 8, 15, 21, 78, and 104 weeks of age in both sexes using Agilent miRNA microarrays. Differential expression was determined using filtering criteria of ≥1.5 fold change and ANOVA or pairwise t-test (FDR <5%) to determine significant age and sex effects, respectively. Pathway analysis software was used to investigate the possible roles of these target genes in age- and sex-specific differences. RESULTS: Three hundred eleven miRNAs were found to be expressed in at least one age and sex. Filtering criteria revealed 174 differentially expressed miRNAs in the kidney; 173 and 34 miRNAs exhibiting age and sex effects, respectively. Principal component analysis revealed age effects predominated over sex effects, with 2-week miRNA expression being much different from other ages. No significant sexually dimorphic miRNA expression was observed from 5 to 8 weeks, while the most differential expression (13 miRNAs) was observed at 21 weeks. Potential target genes of these differentially expressed miRNAs were identified. CONCLUSIONS: The expression of 56% of detected renal miRNAs was found to vary significantly with age and/or sex during the life span of F344 rats. Pathway analysis suggested that 2-week-expressed miRNAs may be related to organ and cellular development and proliferation pathways. Male-biased miRNA expression at older ages correlated with male-biased renal fibrosis and mononuclear cell infiltration. These miRNAs showed high representation in renal inflammation and nephritis pathways, and included miR-214, miR-130b, miR-150, miR-223, miR-142-5p, miR-185, and miR-296*. Analysis of kidney miRNA expression throughout the rat life span will improve the use of current and future renal biomarkers and inform our assessments of kidney injury and disease.

Sexual dimorphism in the expression of mitochondria-related genes in rat heart at different ages.

Cardiovascular disease (CVD) is the leading cause of mortality worldwide. Moreover, sex and age are considered major risk factors in the development of CVDs. Mitochondria are vital for normal cardiac function, and regulation of mitochondrial structure and function may impact susceptibility to CVD. To identify potential role of mitochondria in sex-related differences in susceptibility to CVD, we analyzed the basal expression levels of mitochondria-related genes in the hearts of male and female rats. Whole genome expression profiling was performed in the hearts of young (8-week), adult (21-week), and old (78-week) male and female Fischer 344 rats and the expression of 670 unique genes related to various mitochondrial functions was analyzed. A significant (p<0.05) sexual dimorphism in expression levels of 46, 114, and 41 genes was observed in young, adult and old rats, respectively. Gene Ontology analysis revealed the influence of sex on various biological pathways related to cardiac energy metabolism at different ages. The expression of genes involved in fatty acid metabolism was significantly different between the sexes in young and adult rat hearts. Adult male rats also showed higher expression of genes associated with the pyruvate dehydrogenase complex compared to females. In young and adult hearts, sexual dimorphism was not noted in genes encoding oxidative phosphorylation. In old rats, however, a majority of genes involved in oxidative phosphorylation had higher expression in females compared to males. Such basal differences between the sexes in cardiac expression of genes associated with energy metabolism may indicate a likely involvement of mitochondria in susceptibility to CVDs. In addition, female rats showed lower expression levels of apoptotic genes in hearts compared to males at all ages, which may have implications for better preservation of cardiac mass in females than in males.

Persistence of furan-induced epigenetic aberrations in the livers of F344 rats.

Furan is a heterocyclic organic compound produced in the chemical manufacturing industry and also found in a broad range of food products, including infant formulas and baby foods. Previous reports have indicated that the adverse biological effects of furan, including its liver tumorigenicity, may be associated with epigenetic abnormalities. In the present study, we investigated the persistence of epigenetic alterations in rat liver. Male F344 rats were treated by gavage 5 days per week with 8 mg furan/kg body weight (bw)/day for 90 days. After the last treatment, rats were divided randomly into 4 groups; 1 group of rats was sacrificed 24 h after the last treatment, whereas other groups were maintained without further furan treatment for an additional 90, 180, or 360 days. Treatment with furan for 90 days resulted in alterations in histone lysine methylation and acetylation, induction of base-excision DNA repair genes, suggesting oxidative damage to DNA, and changes in the gene expression in the livers. A majority of these furan-induced molecular changes was transient and disappeared after the cessation of furan treatment. In contrast, histone H3 lysine 9 and H3 lysine 56 showed a sustained and time-depended decrease in acetylation, which was associated with formation of heterochromatin and altered gene expression. These results indicate that furan-induced adverse effects may be mechanistically related to sustained changes in histone lysine acetylation that compromise the ability of cells to maintain and control properly the expression of genetic information.