Injection Of Doxorubicin Research Articles

Ceratonia siliqua pods (Carob) methanol extract alleviates doxorubicin-induced nephrotoxicity via antioxidant, anti-inflammatory and anti-apoptotic pathways in rats

Doxorubicin (DOX) is an anti-neoplastic therapy, but its use is limited by its deleterious toxic effects including nephrotoxicity and cardiotoxicity. This work aimed at assessing the potential protective effect of Ceratonia siliqua methanol extract (CME) on DOX-induced nephrotoxicity in 5 groups of Wistar rats. Nephrotoxicity was induced experimentally by intraperitoneal (IP) injection of DOX (15 mg/kg). DOX increased serum creatinine, urea, sodium, and potassium levels. It elevated MDA levels in the renal tissue but decreased the concentration of GSH and the activity of GST, CAT, and SOD. Meanwhile, it decreased the level of immunomodulatory anti-inflammatory mediators: IL-10 and TGF-β, as well as the activity of MPO but increased the level of IL-6, TNF-α, and caspase-3 in the renal tissue. DOX has upregulated COX-2, caspase-9, and Bax gene expression and downregulated the Bcl-2 gene expression. Immunolabeling of renal tubular epithelium in DOX-intoxicated rats was moderate to strong against Bax, COX-2, and NF-kβ and weak against Bcl-2. Treatment with CME significantly restored the levels of kidney function parameters and the levels of oxidative stress markers. It stimulated the production of IL-10 and TGF-β and decreased the level of IL-6 and TNF-α. CME reverted the gene expression of COX-2, caspase-9, and Bax. Microscopically, CME alleviated the DOX-induced renal damage. Phytochemical analysis revealed the presence of 26 compounds in the CME. No signs of acute toxicity were recorded by CME up to 4000 mg/kg b. wt. orally into mice. Finally, CME could effectively alleviate the deleterious effects of DOX on the kidney. The safety of carob extract encourages its use in the preparation of valuable therapeutic agents.

Lady's Bedstraw as a Powerful Antioxidant for Attenuation of Doxorubicin-Induced Cardiotoxicity.

This study aimed to examine the effects of a 14-day treatment with lady's bedstraw methanol extract on doxorubicin-induced cardiotoxicity through functional, biochemical and histological examinations. We used 24 male Wistar albino rats divided into the following groups: control (CTRL), doxorubicin (DOX), and DOX + GVE (Galium verum extract). GVE was administered orally at a dose of 50 mg/kg per day for 14 days, while a single dose of doxorubicin was injected into the DOX groups. After accomplishing treatment with GVE, cardiac function was assessed, which determined the redox state. During the autoregulation protocol on the Langendorff apparatus, ex vivo cardiodynamic parameters were measured. Our results demonstrated that the consumption of GVE effectively suppressed the disturbed response of the heart to changes in perfusion pressures caused by administration of DOX. Intake of GVE was associated with a reduction in most of the measured prooxidants in comparison to the DOX group. Moreover, this extract was capable of increasing the activity of the antioxidant defense system. Morphometric analyses showed that rat hearts treated with DOX showed more pronounced degenerative changes and necrosis compared to the CTRL group. However, GVE pretreatment seems to be able to prevent the pathological injuries caused by DOX injection via decrease in oxidative stress and apoptosis.

Development and validation of a UPLC-MS/MS method for simultaneous detection of doxorubicin and sorafenib in plasma: Application to pharmacokinetic studies in rats

An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the simultaneous quantitation of doxorubicin (DOX) and sorafenib (SOR) in rat plasma. Chromatographic separation was performed using a reversed-phase column C18 (1.7 μm, 1.0 × 100 mm Acquity UPLC BEH™). The gradient mobile phase system consisted of water containing 0.1% acetic acid (mobile phase A) and methanol (mobile phase B) with a flow rate of 0.40 mL/min over 8 min. Erlotinib (ERL) was used as an internal standard (IS). The quantitation of conversion of [M + H]+, which was the protonated precursor ion, to the corresponding product ions was performed using multiple reaction monitoring (MRM) with a mass-to-charge ratio (m/z) of 544 > 397.005 for DOX, 465.05 > 252.03 for SOR, and 394 > 278 for the IS. Different parameters were used to validate the method including accuracy, precision, linearity, and stability. The developed UPLC–MS/MS method was linear over the concentration ranges of 9–2000 ng/mL and 7–2000 ng/mL with LLOQ of 9 and 7 ng/mL for DOX and SOR, respectively. The intra-day and inter-day accuracy, expressed as % relative standard deviation (RSD%), was below 10% for both DOX and SOR in all QC samples that have drug concentrations above the LLOQ. The intra-day and inter-day precision, expressed as percent relative error (Er %), was within the limit of 15.0% for all concentrations above LLOQ. Four groups of Wistar rats (250–280 g) were used to conduct the pharmacokinetic study. Group I received a single intraperitoneal (IP) injection of DOX (5 mg/kg); Group II received a single oral dose of SOR (40 mg/kg), Group III received a combination of both drugs; and Group IV received sterile water for injection IP and 0.9% w/v sodium chloride solution orally to serve as a control. Non-compartmental analysis was used to calculate the different pharmacokinetic parameters. Data revealed that coadministration of DOX and SOR altered some of the pharmacokinetic parameters of both agents and resulted in an increase in the Cmax and AUC and reduction in the apparent clearance (CL/F). In conclusion, our newly developed method is sensitive, specific, and can reliably be used to simultaneously determine DOX and SOR concentrations in rat plasma. Moreover, the results of the pharmacokinetic study suggest that coadministration of DOX and SOR might cause an increase in exposure of both drugs.

Hydrogen inhalation enhances autophagy via the AMPK/mTOR pathway, thereby attenuating doxorubicin-induced cardiac injury.

Doxorubicin is a drug widely used in clinical cancer treatment, but severe cardiotoxicity limits its clinical application. Autophagy disorder is an important factor in the mechanism of doxorubicin-induced cardiac injury. As the smallest molecule in nature, hydrogen has various biological effects such as anti-oxidation, anti-apoptosis and regulation of autophagy. Hydrogen therapy is currently considered to be an emerging therapeutic method, but the effect and mechanism of hydrogen on doxorubicin-induced myocardial injury have not been determined. The purpose of this study was to investigate the protective effect of hydrogen inhalation on doxorubicin-induced chronic myocardial injury and its effect and mechanism on autophagy. In this study, we established a chronic heart injury model by intraperitoneal injection of doxorubicin in rats for 30days, accumulating 20mg/kg. The effect of hydrogen inhalation on the cardiac function in rats was explored by echocardiography, Elisa, and H&E staining. To clarify the influence of autophagy, we detected the expression of LC3 and related autophagy proteins in vivo and in vitro by immunofluorescence and western blot.In order to further explore the mechanism of autophagy, we added pathway inhibitors and used western blot to preliminarily investigate the protective effect of hydrogen inhalation on myocardial injury caused by doxorubicin. Hydrogen inhalation can improve doxorubicin-induced cardiac function decline and pathological structural abnormalities in rats. It was confirmed by immunofluorescence that hydrogen treatment could restore the expression of autophagy marker protein LC3 (microtubule-associated protein 1 light chain 3) in cardiomyocytes reduced by doxorubicin, while reducing cardiomyocyte apoptosis. Mechanistically, Western blot results consistently showed that hydrogen treatment up-regulated the ratio of p-AMPK (phosphorylated AMP-dependent protein kinase) to AMPK and down-regulated p-mTOR (phosphorylated mammalian target of rapamycin) and mTOR ratio. These results suggest that hydrogen inhalation can activate autophagy through the AMPK/mTOR pathway and protect against myocardial injury induced by doxorubicin. Hydrogen inhalation therapy may be a potential treatment for doxorubicin-induced myocardial injury.

Identification of Novel Targets for Treatment of Dilated Cardiomyopathy Based on the Ferroptosis and Immune Heterogeneity.

This study aimed to investigate the role of ferroptosis in dilated cardiomyopathy (DCM) and to identify new targets for treatment and diagnosis of DCM. GSE116250 and GSE145154 were downloaded from the Gene Expression Omnibus database. Unsupervised consensus clustering of DCM patients was used to confirm the impact of ferroptosis. Ferroptosis-related hub genes were identified by WGCNA and single cell sequencing analyses. Finally, we established a DCM mouse model via injection of Doxorubicin to verify the expression level of OTUD1 and colocalization between cell markers and OTUD1 in DCM mouse heart. A total of 13 ferroptosis-related differentially expressed genes (DEGs) were identified. The DCM patients were divided into two clusters according to the expression of 13 DEGs. The DCM patients in different clusters showed discrepancies in immune infiltration. Four hub genes were further identified by WGCNA analysis. Single cell data analysis revealed that OTUD1 may regulate B cells and DC cells and then participate in immune infiltration discrepancy. The upregulation of OTUD1 and the colocalization of OTUD1 with CD19 (B cell maker) and CD11c (DCs markers) markers were confirmed in DCM mouse hearts. Ferroptosis and the immune microenvironment are closely associated with DCM, and OTUD1 may play an important role through B cells and DCs.

Hepatoprotective Effects of Hyaluronic Acid-Preconditioned Bone Marrow Mesenchymal Stem Cells against Liver Toxicity via the Inhibition of Apoptosis and the Wnt/β-Catenin Signaling Pathway.

Doxorubicin (DOX) is widely used to treat a variety of malignancies in both adults and children, including those of the bladder, breast, stomach, and ovaries. Despite this, it has been reported to cause hepatotoxicity. The recent discovery of bone marrow-derived mesenchymal stem cells' (BMSCs) therapeutic effects in the context of liver diseases suggests that their administration plays a part in the mitigation and rehabilitation of drug-induced toxicities. This study investigated whether bone BMSCs could reduce DOX-induced liver damage by blocking the Wnt/β-catenin pathway that causes fibrotic liver. BMSCs were isolated and treated with hyaluronic acid (HA) for 14 days before injection. Thirty-five mature male SD rats were categorized into four groups; group one (control) rats were supplemented with saline 0.9% for 28 days, group two (DOX) rats were injected with DOX (20 mg/kg), group three (DOX + BMSCs) rats were injected with 2 × 106 BMSCs after 4 days of DOX injection, group four (DOX + BMSCs + HA) rats were injected with 0.1 mL BMSCs pretreated with HA after 4 days of DOX. After 28 days the rats were sacrificed, and blood and liver tissue samples were subjected to biochemical and molecular analysis. Morphological and immunohistochemical observations were also carried out. In terms of liver function and antioxidant findings, cells treated with HA showed considerable improvement compared to the DOX group (p < 0.05). Moreover, the expression of inflammatory markers (TGFβ1, iNos), apoptotic markers (Bax, Bcl2), cell tracking markers (SDF1α), fibrotic markers (β-catenin, Wnt7b, FN1, VEGF, and Col-1), and ROS markers (Nrf2, HO-1) was improved in BMSCs conditioned with HA in contrast to BMSCs alone (p < 0.05). Our findings proved that BMSCs treated with HA exert their paracrine therapeutic effects via their secretome, suggesting that cell-based regenerative therapies conditioned with HA may be a viable alternative to reduce hepatotoxicity.

FGF10 mitigates doxorubicin-induced myocardial toxicity in mice via activation of FGFR2b/PHLDA1/AKT axis.

Doxorubicin is a common chemotherapeutic agent in clinic, but myocardial toxicity limits its use. Fibroblast growth factor (FGF) 10, a multifunctional paracrine growth factor, plays diverse roles in embryonic and postnatal heart development as well as in cardiac regeneration and repair. In this study we investigated the role of FGF10 as a potential modulator of doxorubicin-induced cardiac cytotoxicity and the underlying molecular mechanisms. Fgf10+/- mice and an inducible dominant negative FGFR2b transgenic mouse model (Rosa26rtTA; tet(O)sFgfr2b) were used to determine the effect of Fgf10 hypomorph or blocking of endogenous FGFR2b ligands activity on doxorubicin-induced myocardial injury. Acute myocardial injury was induced by a single injection of doxorubicin (25 mg/kg, i.p.). Then cardiac function was evaluated using echocardiography, and DNA damage, oxidative stress and apoptosis in cardiac tissue were assessed. We showed that doxorubicin treatment markedly decreased the expression of FGFR2b ligands including FGF10 in cardiac tissue of wild type mice, whereas Fgf10+/- mice exhibited a greater degree of oxidative stress, DNA damage and apoptosis as compared with the Fgf10+/+ control. Pre-treatment with recombinant FGF10 protein significantly attenuated doxorubicin-induced oxidative stress, DNA damage and apoptosis both in doxorubicin-treated mice and in doxorubicin-treated HL-1 cells and NRCMs. We demonstrated that FGF10 protected against doxorubicin-induced myocardial toxicity via activation of FGFR2/Pleckstrin homology-like domain family A member 1 (PHLDA1)/Akt axis. Overall, our results unveil a potent protective effect of FGF10 against doxorubicin-induced myocardial injury and identify FGFR2b/PHLDA1/Akt axis as a potential therapeutic target for patients receiving doxorubicin treatment.

Danggui Shaoyaosan attenuates doxorubicin induced Nephrotic Syndrome through regulating on PI3K/Akt Pathway.

The study aimed to explore the role and the underlying mechanism of Danggui Shaoyaosan (DSS) in nephrotic syndrome (NS). NS rat model was induced by doxorubicin injection twice. After DSS treatment, inflammation and oxidative stress index were detected via ELISA. Western blot was used for the protein detection. Go and KEGG analysis was applied to evaluate target gene and signaling of DSS. MCP-5 cell was applied for the cell rescue experiments and mechanism exploration. The 24h urine protein levels of NS rats increased significantly, which was reduced by DSS treatment in a concentration-dependent manner. After DSS treatment, levels of BUN, SCr, TG and TC were also decreased, and serum ALB and TP levels were increased in rats. GO and KEGG pathway enrichment identified PI3K-Akt to be the candidate signaling of DSS in the treatment of NS, which was activated in NS rats. The recuse experiments in MCP-5 demonstrated that IGF-1, the agonist of PI3K/AKT, abolished the beneficial role of DSS in podocyte cell viability, apoptosis, inflammation and oxidative stress. In conclusion,DSS exerts a protective role against the development of NS. The mechanism is related to the improvement of podocyte injury and the inhibition of PI3K/Akt pathway-related proteins.

The influence of sacubitril and sacubitril/valsartan on echocardiographic and electrocardiographic parameters in a rodent model of anthracycline-induced cardiotoxicity

Anthracycline-induced cardiotoxicity (AIC) poses a clinical challenge in the management of cancer patients. Sacubitril/valsartan (Sac/Val), recently incorporated into the management of heart failure with reduced ejection fraction, was shown to provide an additive cardioprotective effect over Val monotherapy in preclinical models of AIC. However, Sac monotherapy was not assessed in this setting. Thus, we hypothesized that the administration of Sac will provide independent cardioprotective effects in a rodent model of AIC.We aimed to investigate the efficacy of Sac and Sac/Val in the primary prevention of AIC.Male 12-week-old Sprague-Dawley rats received doxorubicin (DOX) intraperitoneally (4 milligrams (mg)/kg weekly) over 4 weeks. The animals were randomly divided into 3 groups (control, Sac/Val, and Sac). Starting on the day of the first DOX injection, animals were gavaged daily with either saline, Sac/Val (20 mg/kg), or Sac (10 mg/kg) for 4 weeks. Echocardiography and electrocardiography (ECG) were performed at baseline and at 4 weeks.The echocardiographic parameters of the left ventricle (LV) systolic function in the control group depicted a DOX-induced deterioration, with a significant decrease of LV ejection fraction (EF) (76.91±2.58% vs. 66.33±5.91; p&lt;0.05), stroke volume/tibial length ratio (0.078±0.009 milliliters (ml)/centimetre (cm) vs 0.058±0.02; p&lt;0.05), and cardiac output/tibial length ratio (20.51±2.43 ml/min/cm vs 12.54±4.11; p&lt;0.05). Additionally, treatment with DOX was associated with prolongation of the QRS complex (25.50±2.81 milliseconds (ms) vs 27.92±4.08, p&lt;0.05) and QTc (64.19±5.42 vs 69.61±3.85; p&lt;0.05). In the treatment groups, a decrease in EF was prevented only in the Sac-treated group (EF: 77.56±2.91 vs 78.67± 4.16; p=0.24), which was not observed in the Sac/Val group (EF: 77.52±1.37 vs 69.57±5.09; p&lt;0.05). Both treatments were effective in preventing deterioration of the stroke volume/tibial length ratio (Sac: 0.075±0.008 vs 0.071±0.012; p=0.48; Sac/Val: 0.068±0.008 vs 0.068±0.008; p=0.36), while only Sac monotherapy prevented a decrease in the cardiac output/tibial length ratio (Sac: 22.81±2.74 vs 17.30±3.11; p=0.11; Sac/Val: 18.36±1.96 vs 15.34±1.15; p&lt;0.05). In both treatment groups, there were no changes in the duration of the QRS complex (Sac: 27.50±3.96 vs 28.00±1,00; p=0.15; Sac/Val: 25.29±3.15ms vs 26.25±0.43; p=0.09), and QTc (Sac: 65.15±3.43 vs 66.16±4.05; p=0.38; Sac/Val: 66.12±5.88 vs 70.18±6.87; p=0.09). Additionally, Sac monotherapy prevented DOX-associated weight loss (319±13.08g vs 309±21.62; p=0.31), which was not observed in the Sac/Val group (314±10,11 vs 275±20,44; p&lt;0.05).Both Sac and Sac/Val offered some protection against deterioration of echocardiographic parameters of LV systolic function and ECG changes in a rodent model of AIC. However, this effect was more pronounced in the group receiving Sac monotherapy, which prevented a decrease in all of the assessed parameters, including weight loss. This study was supported by the MD-PhD program No. 93/FS228/2020 based on cooperation between the Medical University of Warsaw and the National Centre for Research and Development (Poland). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Potential protective role of apelin-13 in the doxorubicin-induced cardiotoxicity in the rat

Due to its unselective action, chemotherapy affects not only cancer cells but also a variety of normal human cells causing several toxicities, thus leading to decreased anticancer efficacy. One of such toxicities is cardiotoxicity caused by anthracyclines, mainly doxorubicin (DOX). Doxorubicin-induced cardiotoxicity (DIC) generally occurs as a chronic process and resembles chronic heart failure with reduced ejection fraction. Based on the data about the role of the apelinergic system in the pathogenesis of cardiovascular diseases like heart failure, we hypothesize that it can be also involved in the DIC. Dysregulation of the apelinergic system has been observed in heart failure or myocardial infarction, which was confirmed in studies when apelinergic stimulation resulted in a cardioprotective effect.The aim of the present study was to investigate the role of apelin-13 on echocardiographic parameters in the DIC in the rat.The study was performed in a rat model (male Sprague Dawley, 12 weeks old) of cardiotoxicity. The animals were randomly divided on 3 groups (control, DOX, apelin 40 μg/kg). The cardiotoxicity was induced by 4 intraperitoneal injections of DOX in a dose of 3,5 mg/kg every 7 days with control group receiving saline. All animals were implanted with osmotic pumps to receive a continuous infusion of apelin-13 or saline (control) for 4 weeks, concomitantly with DOX. Immediately before the first dose echo examination was performed. The same procedure was repeated on day 28 days after the last dose of DOX. Later, animals were euthanized and heart samples were collected.Echocardiographic parameters in DOX group compered to control group showed left ventricle (LV) systolic dysfunction, with significant deterioration of LV ejection fraction (LVEF) (66±2.55% vs. 90±4.88), fractional shortening (%FS) (31,67±2.2% vs 55,33±4.45), stroke volume/tibial length ratio (0.041±0.008ml/cm vs 0.064±0.02), and cardiac output/tibial length ratio (10.35±2.12ml/min/cm vs 17.04±4.08). Apelin-13 at a dose of 40 μg/kg reduced the toxic effects of DOX. Furthermore, the heart weight in the group with DOX was significantly smaller than in the control group, while in the group receiving continuous infusion of apelin-13 at a dose 40 μg/kg the heart weight was very similar to control group.The results indicate that continuous exposure to apelin-13 in dose 40 μg/kg has a cardioprotective effect in echocardiographic and clinical parameters. Evaluating the role of the apelinergic system will help us to better understand the pathomechanisms of cardiotoxicity and in the future may result in the development of protective or curative therapies. Acknowledgments: This study was supported by the Grant No UMO-2020/37/B/NZ5/02529 from the National Science Center (Poland). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Changes in the gut microbiota structure and function in rats with doxorubicin-induced heart failure.

The rat model of heart failure (HF) induced by doxorubicin (DOX), a broad spectrum and highly effective chemotherapeutic anthracycline with high-affinity to myocardial tissue that causes severe dose-dependent irreversible cardiotoxicity has been widely recognized and applied in HF pathogenesis and drug therapy studies. The gut microbiota (GM) has attracted significant attention due to its potential role in HF, and research in this area may provide beneficial therapeutic strategies for HF. Considering the differences in the route, mode, and total cumulative dose of DOX administration used to establish HF models, the optimal scheme for studying the correlation between GM and HF pathogenesis remains to be determined. Therefore, focusing on establishing the optimal scheme, we evaluated the correlation between GM composition/function and DOX-induced cardiotoxicity (DIC). Three schemes were investigated: DOX (at total cumulative doses of 12, 15 or 18 mg/kg using a fixed or alternating dose via a tail vein or intraperitoneal injection) was administered to Sprague Dawley (SD) for six consecutive weeks. The M-mode echocardiograms performed cardiac function evaluation. Pathological changes in the intestine were observed by H&E staining and in the heart by Masson staining. The serum levels of N-terminal pre-B-type natriuretic peptide (NT-proBNP) and cardiac troponin I (cTnI) were measured by ELISA. The GM was analysed by 16S rRNA gene sequencing. Strikingly, based on the severity of cardiac dysfunction, there were marked differences in the abundance and grouping of GM under different schemes. The HF model established by tail vein injection of DOX (18 mg/kg, alternating doses) was more stable; moreover, the degree of myocardial injury and microbial composition were more consistent with the clinical manifestations of HF. The model of HF established by tail vein injection of doxorubicin, administered at 4mg/kg body weight (2mL/kg) at weeks 1, 3 and 5, and at 2mg/kg body weight (1mL/kg) at weeks 2, 4 and 6, with a cumulative total dose of 18mg/kg, is a better protocol to study the correlation between HF and GM.

The chemotherapeutic drug doxorubicin does not exacerbate p16Ink4a-positive senescent cell accumulation and cardiometabolic disease development in young adult female LDLR-deficient mice

Cancer survivors who received chemotherapy, such as the anthracycline doxorubicin (DOX), have an increased risk of developing complications later in life, including the development of chronic metabolic diseases. Although the etiology of this increased risk for late metabolic complications in cancer survivors is poorly understood, a causal role of therapy-induced senescent cells has been suggested.To study the role of cellular senescence in chemotherapy-induced metabolic complications, young adult female low-density lipoprotein receptor-deficient (Ldlr−/−)-p16-3MR mice, in which p16Ink4a-positive (p16Ink4a+) senescent cells can be genetically eliminated, were treated with four weekly injections of DOX (2.5 mg/kg) followed by a high-fat high-cholesterol diet for 12 weeks. While DOX treatment induced known short-term effects, such as reduction in body weight, gonadal fat mass, and adipose tissue inflammation, it was not associated with significant long-term effects on glucose homeostasis, hepatic steatosis, or atherosclerosis. We further found no evidence of DOX-induced accumulation of p16Ink4a+-senescent cells at 1 or 12 weeks after DOX treatment. Neither did we observe an effect of elimination of p16Ink4a+-senescent cells on the development of diet-induced cardiometabolic complications in DOX-treated mice. Other markers for senescence were generally also not affected except for an increase in p21 and Cxcl10 in gonadal white adipose tissue long-term after DOX treatment.Together, our study does not support a significant role for p16Ink4a+-senescent cells in the development of diet-induced cardiometabolic disease in young adult DOX-treated female Ldlr−/− mice. These findings illustrate the need of further studies to understand the link between cancer therapy and cardiometabolic disease development in cancer survivors.

Integration of transcriptomics, metabolomics, and lipidomics reveals the mechanisms of doxorubicin-induced inflammatory responses and myocardial dysfunction in mice

Doxorubicin (DOX) is an anthracycline antineoplastic agent that has limited clinical utility due to its dose-dependent cardiotoxicity. Although the exact mechanism remains unknown, inflammatory responses have been implicated in DOX-induced cardiotoxicity (DIC). In this study, we analyzed the transcriptomic, metabolomic as well as lipidomic changes in the DOX-treated mice to explore the underlying mechanisms of DIC. We found that continuous intraperitoneal DOX injections (3 mg/kg/d) for a period of five days significantly induced cardiac dysfunction and cardiac injury in male C57BL/6 J mice (8 weeks old). This corresponded to a significant increase in the myocardial levels of IL-4, IL-6, IL-10, IL-17 and IL-12p70. Furthermore, inflammation-related genes such as Ptgs2, Il1b, Cxcl5, Cxcl1, Cxcl2, Mmp3, Ccl2, Ccl12, Nfkbia, Fos, Mapk11 and Tnf were differentially expressed in the DOX-treated group, and enriched in the IL-17 and TNF signaling pathways. Besides, amino acids, peptides, imidazoles, toluenes, hybrid peptides, fatty acids and lipids such as Hex1Cer, Cer, SM, PG and ACCa were significantly associated with the expression pattern of inflammation-related genes. In conclusion, the integration of transcriptomic, metabolomic and lipidomic data identified potential new targets and biomarkers of DIC.

Influence of doxorubicin on the extracellular matrix of the liver of rats under conditions of chronic alcoholic hepatitis

The liver has a high regenerative potential that is dependent on many factors, in particular adenosine monophosphate kinase signaling, however, long-term alteration, such as daily alcohol consumption, turns regeneration into a chronic disease such as fibrosis, the end stage of which is cirrhosis. Hepatic extracellular matrix proteins are important triggers of enhanced stellate cell function during the progression of liver fibrosis. The experiments were performed on Wistar rats divided into four groups: control group; doxorubicin injection group (1.25 mg/kg); chronic alcohol hepatitis group; injection of doxorubicin during alcoholic hepatitis group, where we studied the total concentration of glycosaminoglycans, concentration of heparin-heparan, keratan-dermatan and chondroitin fractions of glycosaminoglycans, free oxyproline and sialic acids in the liver tissue homogenate. Cells with Mallory-Denk bodies were present in the liver of rats from the alcohol hepatitis group, which consisted of tangled balls of intermediate filaments and showed eosinophilia in the cytoplasm of degenerative hepatocytes. There were also necrotic changes in cells. Sinusoidal capillaries were locally dilated. In the central sections of the liver lobules of rats injected with doxorubicin against the background of chronic alcoholic hepatitis, the central veins were expanded, their endothelium was thinned. Sinusoidal capillaries were locally expanded, there were no blood cells in their lumens. The nuclei of the vast majority of hepatocytes were in a state of karyopyknosis, karyorrhexis, and karyolysis. Mallory-Denk bodies were present in the cytoplasm of cells. Administration of doxorubicin to animals with chronic alcoholic hepatitis leads to an increase in the total concentration of glycosaminoglycans, the concentration of chondroitin sulfates, a decrease in the heparin-heparan fraction of glycosaminoglycans and no changes in concentration of the keratan-dermatan fraction in the liver of rats compared to the control group. In the group of rats injected with doxorubicin against the background of chronic alcoholic hepatitis, the concentrations of total glycosaminoglycans, heparin-heparan fraction, and chondroitin fraction significantly increased, and the content of the keratan-dermatan fraction of glycosaminoglycans significantly decreased compared to animals with chronic alcoholic hepatitis. In rats injected with doxorubicin against the background of chronic alcoholic hepatitis, the content of free oxyproline decreased by 1.25 times, sialic acids – by 1.36 times, compared to the group of animals with chronic alcoholic hepatitis. Administration of ethanol or doxorubicin in combination with ethanol to rats causes morphological changes in the liver that are characteristic of chronic alcoholic hepatitis. Administration of doxorubicin to rats leads to degenerative morphological changes in the liver lobules. The administration of doxorubicin prevents alcohol-induced collagenolysis and the breakdown of glycoproteins, but increases the breakdown of proteoglycans due to an increase in the content of chondroitin and heparin-heparan fractions.

Chlorogenic Acid Attenuates Doxorubicin-Induced Oxidative Stress and Markers of Apoptosis in Cardiomyocytes via Nrf2/HO-1 and Dityrosine Signaling.

(1) Background: Doxorubicin (DOX) is extensively used for cancer treatments; however, its clinical application is limited because of its cardiotoxic adverse effects. A combination of DOX and agents with cardioprotective properties is an effective strategy to ameliorate DOX-related cardiotoxicity. Polyphenolic compounds are ideal for the investigation of novel cardioprotective agents. Chlorogenic acid (CGA), an essential dietary polyphenol found in plants, has been previously reported to exert antioxidant, cardioprotective, and antiapoptotic properties. The current research evaluated CGA's in vivo cardioprotective properties in DOX-induced cardiotoxicity and the probable mechanisms underlying this protection. (2) Methods: CGA's cardioprotective properties were investigated in rats that were treated with CGA (100 mg/kg, p.o.) for fourteen days. The experimental model of cardiotoxicity was induced with a single intraperitoneal (15 mg/kg i.p.) injection of DOX on the 10th day. (3) Results: Treatment with CGA significantly improved the DOX-caused altered cardiac damage markers (LDH, CK-MB, and cTn-T), and a marked improvement in cardiac histopathological features accompanied this. DOX downregulated the expression of Nrf2/HO-1 signaling pathways, and the CGA reversed this effect. Consistently, caspase-3, an apoptotic-related marker, and dityrosine expression were suppressed, while Nrf2 and HO-1 expressions were elevated in the cardiac tissues of DOX-treated rats after treatment with the CGA. Furthermore, the recovery was confirmed by the downregulation of 8-OHdG and dityrosine (DT) expressions in immunohistochemical findings. (4) Conclusions: CGA demonstrated a considerable cardioprotective effect against DOX-induced cardiotoxicity. One of the possible mechanisms for these protective properties was the upregulation of the Nrf2/HO-1-dependent pathway and the downregulation of DT, which may ameliorate oxidative stress and cardiomyocyte apoptosis. These findings suggest that CGA may be cardioprotective, particularly in patients receiving DOX-based chemotherapy.

Effect of the Anticancer Drug Doxorubicin (Adriamycin) on Antioxidant Studies and Ultrastructural Investigation in the Liver, Kidney, and Heart Tissues of Male Rats

Doxorubicin is a well-known antineoplastic agent that has proved to be successful in the treatment of various types of cancer. I used rats as the model to evaluate the effect of doxorubicin on antioxidant studies and ultrastructural investigations in the liver, kidney, and heart tissues. Male albino rats were given 1.0 mg/kg body weight of the anticancer drug doxorubicin intraperitoneally three times a week for 52 days. This was for a total of 18 doses. Control animals received 52 doses of 0.5 ml of saline over 52 days. The body weights of rats injected with doxorubicin experienced a significant decrease after the last dose compared to the control group of rats. In this study, the weights of the heart, kidneys, and liver were measured. Except for cardiac tissues, the protein content in the aforementioned tissues in treated rats was significantly different from the control. Glutathione (GSH) levels in the kidneys of experimental rats were not significantly lower (7.946 ± 0.781) compared to controls (8.06 ± 0.74) but there was a non-significant increase in GSH levels in the liver (17.095 ± 1.066) compared to controls (13.8 ± 1.3). In addition, the mean GSH levels in doxorubicin-treated hearts were significantly lower (7.9462 ± 0.781) compared to controls (8.06 ± 0.74). Lipid peroxidation (Lpx) and malondialdehyde content (MDA), a marker of lipid peroxidation, were found in much lower concentrations in the liver organ of the doxorubicin-treated group (0.0162 ± 0.00086) as compared to (0.20 ± 0.02) controls, and MDA content in the kidney was decreased (0.0239 ± 0.0003) compared to control rats (0.31 ± 0.03), as well as heat production (0.0398 ± 0.00097) compared to (47.451 ± 1.708) controls. Glutathione reductase (GR) levels were significantly elevated in the same tissue treatment group. Glutathione-S-Transferase (G-S-T) activity was assessed and significantly increased in all tissues in the doxorubicin model. Glutathione peroxidase (GPx) activity showed a significant decrease in all the above tissues after doxorubicin injection. The catalase (CAT) activity of doxorubicin was greatly increased in one treated rat. In the doxorubicin-treated group, levels of cytochrome p450 (CYTp450) were significantly decreased in liver and kidney tissue and significantly elevated in heart tissue. After doxorubicin treatment, cytochrome b5 (CYTb5) levels in liver tissues increased significantly (837.177± 61.197) compared to controls (615 ± 37.0), and the contents of cytochrome b5 in rats' kidneys increased significantly (447.685 ± 35.215) compared to controls (2605.5± 259.2). and cytochrome b5 in heart tissues was lower (165.352± 8.7) when compared to controls (88± 0.4). The results showed that there were few obvious changes in histological, ultrastructural, and biochemical changes in liver tissue in the doxorubicin model. Long-term doxorubicin treatment in kidney tissue results in no significant changes at the light microscopic level, but the electron microscopic level reveals no change from a histological point of view.

A low-salt diet with candesartan administration is associated with acute kidney injury in nephritis by increasing nitric oxide

A low-salt diet may activate the renin-angiotensin-aldosterone system (RAAS) and is often applied simultaneously with RAAS inhibitors, especially for treatment of proteinuric nephritis. To explore the effect of a low-salt diet combined with angiotensin receptor blockers (ARB) on kidney function, the proteinuric nephritis model was induced by single intravenous injection of doxorubicin, and then the SD rats were administrated with candesartan intraperitoneal injection and fed with different salt diets. Rats with low-salt plus candesartan, not either alone, experienced acute kidney injury (AKI) at day 7 and could not self-restore when extending the experiment time from 7 days to 21 days, unless switching low-salt to normal-salt. Among three nitric oxide synthetases (NOS), endothelial NOS (eNOS) was obviously elevated and PI3K-Akt-eNOS signal pathway was activated. NG-Nitro-L-Arginine Methyl Ester (L-NAME), an eNOS inhibitor, reversed the decreased blood pressure and recovered the kidney dysfunction induced by low-salt with candesartan. The increased TUNEL-positive cells, Bax/Bcl-2 and cleaved-caspase3 protein abundance was ameliorated by L-NAME in vivo. In vitro, sodium nitroprusside, a nitric oxide donor, can also increase Bax/Bcl-2 and cleaved-caspase3 protein level in HK-2 cell. Thus, low-salt diet combined with candesartan in nephritis rats led to AKI, and the mechanism involved the increase of eNOS/NO, which linked to the decrease of blood pressure and the increase of apoptosis. This study provides practical guidance for salt intake in cases of RAS inhibitor usage clinically.

Exposure to Doxorubicin Modulates the Cardiac Response to Isoproterenol in Male and Female Mice.

Sex is a salient risk factor in the development of doxorubicin-induced cardiotoxicity. Sex differences in the heart's ability to respond to hypertrophic stimuli in doxorubicin-exposed animals have not been reported. We identified the sexual dimorphic effects of isoproterenol in mice pre-exposed to doxorubicin. Male and female intact or gonadectomized C57BL/6N mice underwent five weekly intraperitoneal injections of 4 mg/kg doxorubicin followed by a five-week recovery period. Fourteen days of subcutaneous isoproterenol injections (10 mg/kg/day) were administered after the recovery period. Echocardiography was used to assess heart function one and five weeks after the last doxorubicin injection and on the fourteenth day of isoproterenol treatment. Thereafter, mice were euthanized, and the hearts were weighed and processed for histopathology and gene expression analysis. Doxorubicin did not produce overt cardiac dysfunction in male or female mice before starting isoproterenol treatment. The chronotropic response to a single isoproterenol injection was blunted by doxorubicin, but the inotropic response was maintained in both males and females. Pre-exposure to doxorubicin caused cardiac atrophy in both control and isoproterenol-treated male mice but not in female mice. Counterintuitively, pre-exposure to doxorubicin abrogated isoproterenol-induced cardiac fibrosis. However, there were no sex differences in the expression of markers of pathological hypertrophy, fibrosis, or inflammation. Gonadectomy did not reverse the sexually dimorphic effects of doxorubicin. Additionally, pre-exposure to doxorubicin abrogated the hypertrophic response to isoproterenol in castrated male mice but not in ovariectomized female mice. Therefore, pre-exposure to doxorubicin caused male-specific cardiac atrophy that persisted after isoproterenol treatment, which could not be prevented by gonadectomy.

Astragaloside IV Inhibits NLRP3 Inflammasome-Mediated Pyroptosis via Activation of Nrf-2/HO-1 Signaling Pathway and Protects against Doxorubicin-Induced Cardiac Dysfunction.

Doxorubicin (DOX) is an effective broad-spectrum antitumor drug, but its clinical application is limited due to the side effects of cardiac damage. Astragaloside IV (AS-IV) is a significant active component of Astragalus membranaceus that exerts cardioprotective effects through various pathways. However, whether AS-IV exerts protective effects against DOX-induced myocardial injury by regulating the pyroptosis is still unknown and is investigated in this study. The myocardial injury model was constructed by intraperitoneal injection of DOX, and AS-IV was administered via oral gavage to explore its specific protective mechanism. Cardiac function and cardiac injury indicators, including lactate dehydrogenase (LDH), cardiac troponin I (cTnI), creatine kinase isoenzyme (CK-MB), and brain natriuretic peptide (BNP), and histopathology of the cardiomyocytes were assessed 4 weeks post DOX challenge. Serum levels of IL-1β, IL-18, superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione (GSH) and the expression of pyroptosis and signaling proteins were also determined. Cardiac dysfunction was observed after the DOX challenge, as evidenced by reduced ejection fraction, increased myocardial fibrosis, and increased BNP, LDH, cTnI, and CK-MB levels (p < 0.05, N = 3-10). AS-IV attenuated DOX-induced myocardial injury. The mitochondrial morphology and structure were also significantly damaged after DOX treatment, and these changes were restored after AS-IV treatment. DOX induced an increase in the serum levels of IL-1β, IL-18, SOD, MDA and GSH as well as an increase in the expression of pyroptosis-related proteins (p < 0.05, N = 3-6). Besides, AS-IV depressed myocardial inflammatory-related pyroptosis via activation of the expressions of nuclear factor E2-related factor 2 (Nrf-2) and heme oxygenase 1 (HO-1) (p < 0.05, N = 3). Our results showed that AS-IV had a significant protective effect against DOX-induced myocardial injury, which may be associated with the activation of Nrf-2/HO-1 to inhibit pyroptosis.

Abstract P3-08-08: Establishment of a Breast Cancer Rat Model of Chemotherapy-Induced Cardiotoxicity

Abstract Establishment of a Breast Cancer Rat Model of Chemotherapy-Induced Cardiotoxicity Katherine Wallis, Marjan Boerma, Antiño Allen, Joseph Su, Sam Makhoul, Ping-Ching Hsu Background. Doxorubicin (DOX) is a highly effective chemotherapy agent that is commonly used in combination with precision medicine to treat a wide range of cancers, including 32% of breast cancer (BC) cases. Although the treatment has greatly increased the number of long-term cancer survivors, it has also increased the number of patients experiencing DOX-induced cardiotoxicity (DIC). Animal models have played a vital role in the development of basic and translational breast cancer research in humans, and rat is a preferred animal to study breast cancer for the similarity to human neoplasms and metabolism. However, most studies were established using mouse models, male rats and commonly non-cancer bearing animals. Currently no single universal cancer-bearing female rat model is utilized for DOX-induced chronic cardiotoxicity. The number of cancer cells to be injected to mimic human experience is not known. Large numbers of injected cancer cells grow quickly and kill the animal before the investigators have the time to assess for cardiac toxicity. Here we aimed to identify the minimal number of cancer cell injections to mimic tumor growth of BC patients and to achieve chemotherapy-induced toxicities. Methods. Various numbers (5 × 104 – 1 × 106) of MAT B III adenocarcinoma cancer cells derived from Fischer 344 (F344) rats were injected in the mammary fat pad of female F344 rats, followed by six intraperitoneal (IP) injections of either DOX (1.25mg/kg per dose) or saline post tumor cell injection. The treatments were administered on day 4 from cell injection, and every third day after for a total of six injections. Echocardiography (Vevo 3100, VisualSonics) was conducted before and after the end of chemotherapy. Results. Tumor burden reduced significantly among rats treated with DOX compared with the saline group. In the Kaplan-Meier survival curve, an injection of 5 × 104 cancer cells was associated with tumor growth but the tumor burden was low enough to allow long-term follow-up of both DOX and saline treated rats and assess signs of cardiotoxicity. At 4 days after the final DOX administration, increased left ventricular ejection fraction and fractional shortening in the absence of increased stroke volume were an indication of adverse cardiac effects in the DOX animals but not the saline-treated rats. Plasma was collected from the animals to be subjected to metabolomics testing so that a preliminary assessment of metabolites associated with DIC could be gathered. Conclusion. We have established a breast cancer rat model in which DIC can be studied. Research in this model can aid in identifying mechanisms of DIC and testing novel interventions. Citation Format: Katherine Wallis, Marjan Boerma, Antino Allen, Joseph Su, Sam Makhoul, Ping-Ching Hsu. Establishment of a Breast Cancer Rat Model of Chemotherapy-Induced Cardiotoxicity [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-08.