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.
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 B6C3F1 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 B6C3F1 mice.
► 24mg/kg was a cumulative cardiotoxic dose of doxorubicin in male B6C3F1 mice. ► Doxorubicin-induced hematological toxicity was in association with splenomegaly. ► Doxorubicin induced severe testicular toxicity in B6C3F1 male mice.
Age is a predisposing condition for susceptibility to chronic kidney disease and progression as well as acute kidney injury that may arise due to the adverse effects of some drugs. Age-related ...differences in kidney biology, therefore, are a key concern in understanding drug safety and disease progression. We hypothesize that the underlying suite of genes expressed in the kidney at various life cycle stages will impact susceptibility to adverse drug reactions. Therefore, establishing changes in baseline expression data between these life stages is the first and necessary step in evaluating this hypothesis. Untreated male F344 rats were sacrificed at 2, 5, 6, 8, 15, 21, 78, and 104 weeks of age. Kidneys were collected for histology and gene expression analysis. Agilent whole-genome rat microarrays were used to query global expression profiles. An ANOVA (p<0.01) coupled with a fold-change>1.5 in relative mRNA expression, was used to identify 3,724 unique differentially expressed genes (DEGs). Principal component analyses of these DEGs revealed three major divisions in life-cycle renal gene expression. K-means cluster analysis identified several groups of genes that shared age-specific patterns of expression. Pathway analysis of these gene groups revealed age-specific gene networks and functions related to renal function and aging, including extracellular matrix turnover, immune cell response, and renal tubular injury. Large age-related changes in expression were also demonstrated for the genes that code for qualified renal injury biomarkers KIM-1, Clu, and Tff3. These results suggest specific groups of genes that may underlie age-specific susceptibilities to adverse drug reactions and disease. This analysis of the basal gene expression patterns of renal genes throughout the life cycle of the rat will improve the use of current and future renal biomarkers and inform our assessments of kidney injury and disease.
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.
•Upregulation of miR-34a before doxorubicin-induced cardiac tissue injury•Apoptosis might be an early event in mouse heart during doxorubicin treatment.•Expression of miR-150 declined before doxorubicin-induced cardiac tissue injury.
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.
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.
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.
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.
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.
Traditional technologies or cardiac disease markers have low sensitivity in detecting early signs of subclinical changes in hearts of cancer patients treated with doxorubicin (DOX). The present study identified miRNA‐34a‐5p as a potential early circulating biomarker of DOX cardiotoxicity in mice 1 week after receiving 6, 9, 12, 18, or 24 mg/kg total cumulative doses. Higher expression of miR‐34a‐5p in plasma at 24 weeks after receiving 24 mg/kg total dose suggested its potential as an early biomarker of delayed‐onset cardiotoxicity.
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 8weeks (6, 9, 12, 18, or 24mg/kg cumulative DOX doses, respectively). Also, a group of mice was pretreated (intraperitoneally) with the cardio-protectant, dexrazoxane (DXZ; 60mg/kg) 30min 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 24mg/kg cumulative DOX doses, whereas myocardial alterations were observed only at the 24mg/kg dose. Of 1019 genes interrogated, 185, 109, 140, 184, and 451 genes were differentially expressed at 6, 9, 12, 18, and 24mg/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 24mg/kg cumulative DOX. This information on early transcriptional changes during DOX treatment may be useful in designing cardioprotective strategies targeting mitochondria.
•Altered mitochondria-related gene expression before heart injury by doxorubicin•Dexrazoxane mitigated doxorubicin-induced early expression changes in mitochondria.•Dexrazoxane completely ameliorated doxorubicin-induced pathology in mouse heart.
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.
•Significant increase in plasma NOTCH1 prior to and after myocardial injury by doxorubicin in mice.•Significant increase in plasma vWF level prior to and after myocardial injury by doxorubicin in mice.•Dexrazoxane completely prevented formation of cardiac lesions in doxorubicin-treated mice.•Dexrazoxane significantly attenuated increased plasma NOTCH1 level in doxorubicin-treated mice.•Dexrazoxane significantly attenuated increased plasma vWF level in doxorubicin-treated 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.
Subclinical cardiotoxicity elicited by low total cumulative doxorubicin (DOX) doses can occur years after treatment in cancer patients. In male B6C3F1 mice, a progressive deterioration in heart function measured by echocardiography was evidenced by a significant time‐dependent decline in systolic function during 24 weeks after treatment with 6‐ or 9‐mg/kg total cumulative DOX doses. A significant dose‐related downregulation of Ryr2 and Serca2 and increased number of apoptotic cardiomyocytes at 24 weeks after treatment may underlie delayed‐onset subclinical cardiac dysfunction.
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