Obese mammary tumour-bearing mice are highly sensitive to doxorubicin-induced hepatotoxicity

Obese mammary tumour-bearing mice are highly sensitive to doxorubicin-induced hepatotoxicity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Obese mammary tumour-bearing mice are highly sensitive to doxorubicin-induced hepatotoxicity Megan Sedeman, Claudia Christowitz, Louis de Jager, Anna-Mart Engelbrecht This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1473640/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background: Breast cancer is a major health burden for women, worldwide. Lifestyle-related risk factors, such as obesity and being overweight, have reached epidemic proportions and contributes to the development of breast cancer. Doxorubicin (DXR) is a chemotherapeutic drug commonly used to treat breast cancer, and although effective, may cause toxicity to other organs. The mechanisms and effects of DXR on hepatic tissue, and the contributing role of obesity, in breast cancer patients are poorly understood. The aim of this study was therefore to investigate the effects of doxorubicin on hepatic tissue in an obese tumour-bearing mouse model. Methods: A diet-induced obesity (DIO) mouse model was established, where seventy-four three-week-old female C57BL/6 mice were divided into two main groups, namely the high fat diet (containing 60% kcal fat) and standard diet (containing 10% kcal fat) groups. After eight weeks on their respective diets, the DIO phenotype was established, and the mice were further divided into tumour and non-tumour groups. Mice were subcutaneously inoculated with E0771 triple negative breast cancer cells in the fourth mammary gland and received three doses of 4 mg/kg DXR (cumulative dosage of 12 mg/kg) or vehicle treatments via intraperitoneal injection. The expression levels of markers involved in apoptosis and alanine aminotransferase (ALT) were compared by means of western blotting. To assess the pathology and morphology of hepatic tissue, haematoxylin and eosin staining was performed. The presence of fibrosis and lipid accumulation in hepatic tissues were assessed with Masson’s trichrome and Oil Red O staining, respectively. Results: Our western blot results indicated that a significant increase in the ratio of cleaved caspase-8 and caspase-8 protein expression was observed in the standard diet tumour-bearing mice treated with DXR compared to the high fat diet tumour-bearing mice treated with DXR. Microscopic examination of liver tissue showed significant changes in the high fat diet tumour-bearing mice treated with DXR, consisting of macrovesicular steatosis, hepatocyte ballooning and lobular inflammation, compared to the standard diet tumour-bearing mice treated with DXR and the control group (standard diet mice). These changes are the hallmarks of non-alcoholic fatty liver disease, associated with obesity. Conclusion: Our results suggest that DXR activated the extrinsic apoptotic pathway in tumour-bearing mice on the standard diet. The histopathological findings indicated that DXR caused significant hepatic parenchymal injury in the obese tumour-bearing mouse model. Hepatotoxicity is aggravated in obesity as an underlying co-morbidity. It has been shown that obesity is associated with poor clinical outcomes in patients receiving neo-adjuvant chemotherapy treatment regimens. Obesity Breast cancer Doxorubicin Apoptosis Non-alcoholic fatty liver disease Hepatotoxicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background Breast cancer is a common malignancy that frequently occurs in women from both developed and developing countries and is therefore a major health burden worldwide (Francies et al. , 2020). Lifestyle-related risk factors, such as obesity and being overweight reached epidemic proportions and is a well-known risk factor that contributes to the development of breast cancer (Mentoor et al. , 2020). In 2018, breast cancer was the most frequently diagnosed cancer and the leading cause of cancer-related deaths among females worldwide (Bray et al. , 2018; Siegel, Miller & Jemal, 2018). Significant progress has been made regarding treatment options for cancer patients, however, these drugs may cause serious multi-organ toxicity, and this remains a major concern. Dysfunctional adipocytes implicated in obesity release metabolic substrates, adipokines, and cytokines, which promote proliferation, progression, invasion, and migration of breast cancer cells. All these factors associated with obesity, mediates tumour initiation, metabolic reprogramming, angiogenesis, progression, and response to treatment (Ando et al., 2019). Doxorubicin (DXR) is an anthracycline glycoside antibiotic that possess antitumour activity and is one of the most effective chemotherapeutic agents used to treat breast cancer (Ren et al. , 2016). Although effective, therapeutic applications of DXR are limited due to its side-effects, such as myelosuppression, chronic cardiotoxicity, and skeletal muscle atrophy (Hiensch et al. , 2020; Zhao & Zhang, 2017). DXR can also be detrimental to other organs, such as the liver, which plays an important physiological role in metabolism, glycogen and triglyceride storage, plasma protein synthesis and detoxification of toxic metabolites (Chiang, 2014). It has been shown that DXR can induce hepatotoxicity, which limits its efficacy in anticancer therapy, resulting in poor patient prognosis and decreased overall survival of cancer patients (Bengaied et al. , 2017; Song et al. , 2019). DXR-induced oxidative stress is characterized by accumulation of reactive oxygen species (ROS), which decrease antioxidant defence systems, resulting in oxidative damage of deoxyribonucleic acid (DNA), therefore, mediating apoptotic cell death in hepatic tissue (Song et al. , 2019). It has been shown that the apoptotic pathway, characterized by the release of cytochrome c from the mitochondria (intrinsic apoptotic pathway) and the Fas ligand (important regulator of the extrinsic apoptotic pathway), were associated with DXR-induced acute toxicity in the liver (Rashid et al. , 2013; Song et al. , 2019). Furthermore, histopathological changes have indicated that DXR can cause structural damage to hepatic tissues which include inflammation, congestion, and necrosis (Mohan et al. , 2011; Rashid et al. , 2013; Song et al. , 2019). Non-alcoholic fatty liver disease (NAFLD), a co-morbidity associated with obesity is characterized by macrovesicular steatosis, triglyceride accumulation in hepatocytes, increased hepatic oxidative stress and increased sensitivity to drug induced liver injury (Li et al ., 2019). A study done by Alghamdi et al. (2015) showed that DXR in the presence of palmitate and oleate (lipid loading) caused a significant accumulation of lipids within the human hepatoma cell line (Huh7 cells) and enhanced acute toxicity in lipid-loaded hepatocytes, which is mediated through increased oxidative stress and ROS (Alghamdi et al. , 2015). The mechanisms of action on hepatic toxicity and the contributing role of obesity in breast cancer patients are not fully elucidated. Therefore, the aim of this study was to investigate the effects of DXR on hepatic tissue in an obese tumour-bearing model. The expression levels of markers involved in the apoptotic pathway was assessed by means of western blotting. Alanine transaminase (ALT) expression was also determined through western blotting. To assess the pathology and morphology of hepatic tissues, haematoxylin and eosin (H&E) staining was performed on formalin fixed paraffin embedded (FFPE) tissue. Furthermore, the presence of fibrosis and steatosis in hepatic tissues were assessed with Masson’s trichrome and Oil Red O staining, respectively. Materials and methods Animal model Ethical clearance was obtained from the Stellenbosch University animal research committee (no. SU-ACUM13-00015 and no. ACU-2020-14751). The experiments involving the use of laboratory animals were carried out in accordance with the Animal Welfare Act and recommendations of the Institutional Animal Care and Committee of Stellenbosch University. This study is reported in accordance with ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) to improve the reporting of research involving animals. Three-week-old female C57BL6 mice (n = 74) were obtained from the Stellenbosch University Central Research Facility and were housed at the Stellenbosch University Animal Unit in individually ventilated cages (IVC) at temperature-controlled conditions (i.e., 22⁰C) and underwent a 12-h light/dark cycle. Mice were allowed an acclimatization period of one week with ad libitum access to mouse pellets and tap water (Fig. 1). The general welfare of the animals was monitored daily. After one week of acclimatization, seventy-four mice were randomly divided into two main groups, namely the high fat diet (HFD, n = 36) and standard diet (SD, n = 38) groups ( Fig. 1 ). To induce obesity, mice were fed a HFD containing 60% kcal fat (D12492, OpenSource Diets ® , Research Diets Inc., New Jersey, USA), whereas a SD containing 10% kcal fat (D12450J, OpenSource Diets ® , Research Diets Inc., New Jersey, USA) were used to generate lean control mice (Additional file 1, Table 1). According to literature, C57BL6 mice are sensitive to diet-induced obesity (DIO) (Chu et al. , 2017). Body weight was monitored weekly over the study period and mice were on their respective diets up until the end point of the study. After eight weeks on their respective diets, the DIO phenotype was established and the two groups were further randomly divided into two groups each, namely the tumour (T) and non-tumour (NT) groups. Mice were subcutaneously inoculated with E0771 triple negative breast cancer cells suspended in Hanks Balanced Salt Solution (HBSS) (Sigma Chemical Co., St Louis, MO, USA) in the fourth mammary pad, using a 23-guage needle syringe. The tumour groups were inoculated with E0771 triple negative breast cancer cells (HFD+T, n = 18; SD+T, n = 18) and the non-tumour groups were not inoculated with cancer cells (HFD+NT, n = 18; SD+NT, n = 20) (Fig. 1). Once the tumours became palpable (200 – 300 mm²), DXR treatment was initiated. The mice were randomly divided into vehicle control (V, isovolumetric intra-peritoneal injection of HBSS) and DXR treatment (D5794, LKTR laboratories, Minnesota, USA) groups. Mice were restrained and three doses of 4 mg/kg DXR were administered (cumulative dosage of 12 mg/kg) via intraperitoneal injection. Each group was then further divided into two groups namely the DXR treatment groups; High fat diet+Tumour+Doxorubicin (HFD+T+DXR, n = 9); High fat diet+Non-Tumour+Doxorubicin (HFD+NT+DXR, n = 10); Standard diet+Tumour+Doxorubicin (SD+T+DXR, n = 10); Standard diet+Non-Tumour+Doxorubicin (SD+NT+DXR, n = 10) and vehicle control groups; High fat diet+Tumour+Vehicle (HFD+T+V, n = 9); High fat diet+Non-Tumour+Vehicle (HFD+NT+V, n = 8); Standard diet+Tumour+Vehicle (SD+T+V, n = 8); Standard diet+Non-Tumour+Vehicle (SD+NT+V, n = 10). In humans, the dosage of 12 mg/kg DXR is equivalent to 36 mg/m² and falls within the relevant dosage range of DXR treatment (15 - 90 mg/m²) administered to cancer patients in the clinical setting (Reagan-Shaw et al ., 2008). The eight experimental groups were assigned as follow: HFD+T+DXR, n = 9; HFD+NT+DXR, n = 10; SD+T+DXR, n = 10; SD+NT+DXR, n = 10) and vehicle control groups (HFD+T+V, n = 9; HFD+NT+V, n = 8; SD+T+V, n = 8; SD+NT+V, n = 10 (Fig. 1). The mice were weighed every second day and the last body weight was recorded on the day of euthanasia. Tumour growth was measured using a Harpenden caliper (mm) and individual tumour volumes were calculated according to the following equation: Tumour voulme(mm 3 ) = (Tomayko & Reynolds, 1989). The mice were euthanized 3 days after the last DXR treatments were administered. The mice were anesthetized with 3% isoflurane (Isofor, Safeline, Pharmaceuticals, Florida, South Africa) and were euthanized by cervical dislocation. Hepatic tissues were excised, where half of the tissues were snap frozen in liquid nitrogen and stored at - 80°C for western blotting and Oil red O staining (n = 4). The other half of the tissues were preserved in 10% formalin for histological analysis (n = 4-5). Western blot Hepatic tissue samples were placed on ice and allowed to thaw. Samples were suspended in 300 μl cold modified radio-immunoprecipitation assay buffer (RIPA) containing protease and phosphatase inhibitors. Surgical scissors cleaned with 100% ethanol was used to cut tissues into smaller pieces while on ice. Samples were homogenised (KineMatica PolytronTM PT2100, Fisher Scientific) while on ice. Samples were centrifuged (14, 000 RCF (g), 20 min, 4°C) to yield distinct layers and the supernatant layer was removed and transferred into sterile Eppendorf tubes. Samples were then centrifuged again at 14 000 RCF (g), 20 min, 4°C. The process of removing the supernatant was repeated followed by protein determination using a Bradford assay. Protein samples were prepared with Laemmli’s sample buffer and were loaded onto 4-20% Criterion ™ TGX Stain-Free ™ Precast Gels (mini-PROTEAN ® TGX™ Gels, Bio-Rad), following protein separation at 100 V for 10 min and 120 V for 60 min in Tris/Glycine/SDS running buffer (BioRad, CA, USA). Proteins were transferred onto Polyvinylidene difluoride (PVDF) membranes (Trans-Blot Turbo RTA Midi PVDF transfer kit, BioRad, CA, USA) with the Trans-Blot Turbo Transfer System (BioRad, CA, USA) using mixed molecular weight. The membranes were blocked in 5% milk prepared in tris-buffered saline with tween 20 (TBS-T) for 2 h at room temperature (RT) and then incubated in primary antibody, at 4°C overnight. On the following day, the membranes were incubated with secondary antibody for 1 h at RT. Antibody details are listed in additional file 2. After incubation, the membranes were developed on the ChemiDoc ™ MP System. Specific bands were visualized and detected using enhanced chemiluminescence (ECL) substrate detection (BioRad, CA, USA). Quantification of protein samples were normalized to total protein and expressed as a percentage of the control. Histology In all animals, a small portion of the right lateral lobe of the liver tissue was fixed in 10% neutral formalin buffered solution prior tissue processing (n = 4-5). Hepatic tissues were processed using an automated tissue processor (HistoCore PEARL, Leica Biosystems) on a 12-h cycle followed by infiltration with paraffin embedded wax (Leica EG 1150 H). Tissues were sectioned into 5 μm sections using a microtome (Leica RM 2125 RT) and tissue sections were placed onto positively charged histobond microscope slides. Two histochemical stains were carried out on the sectioned tissue; H&E stain for morphometric and pathological evaluation and Masson’s trichrome stain to evaluate the presence of fibrosis within hepatic tissue. The right median lobe of hepatic tissue was snap frozen in liquid nitrogen and sectioned into 7 μm sections using a cryostat (Leica CM 3050 S Research Cryostat, Leica Biosystems) to evaluate the presence of lipid accumulation using Oil red O staining (n = 4-5). Sections were placed onto positively charged histobond microscope slides and left to defrost. Sections were stained in Oil red O in dextrin staining solution (Sigma-Aldrich, 01391, SA) for 25 min. Coverslips were mounted onto the slides using aqueous mounting media (Sigma-Aldrich, G 0918) prior to imaging under a microscope (Nikon ECLIPSE E400). The relative number of red pixels (lipid droplets) were quantified using Image J software v1.52a. Histopathology Three individuals, blinded to the treatment allocations, scored five images per tissue sample in each treatment group using the non-alcoholic fatty liver disease (NAFLD) activity score (NAS). This is an accredited and validated scoring system frequently used to evaluate hepatic steatosis, inflammation, and hepatocyte-specific pathology (Kleiner et al ., 2005). Each scorer was provided with a grading sheet to familiarize themselves with the scoring system one day prior to analysis. The scores were tabulated and used for inter- and intra-observer analysis. H&E-stained tissues were used to evaluate structural changes that occurs within hepatocytes and Masson’s trichrome-stained tissues were used to assist with the evaluation of liver fibrosis. Steatosis was scored according to its percentage per microscopic field, location (zone, 1, 2 or 3), and the presence of micro- and/or macrovesicular steatosis. With regards to inflammation, the location, lobular and/or portal, were evaluated per 100X microscopic field. For this study, microvesicular steatosis (indicated with black arrows) and macrovesicular steatosis (indicated in red arrows) were observed in hepatic tissue, following H&E staining (Fig. 6). Ten images per sample were taken and analysis was performed from right to left across the liver tissue sections. The H&E-stained and Masson’s trichrome-stained liver tissues were evaluated as follow: To assess the extent of steatosis, the presence of hydropic changes, micro- and macrovesicular steatosis. To determine whether inflammation occurred, the presence of portal mononuclear cell infiltration, haematopoiesis, and Kupffer cell proliferation were evaluated. Characteristics of hepatocytes and the portal system, such as hepatocyte swelling, sinusoidal and central vein dilation, were evaluated. Statistical analysis The western blot experiments were conducted with biological repeats of n = 4 and technical repeats of n = 1. Bio-Rad Image Lab™ software v6.0.1 was used for normalization of the protein specific intensities against total protein intensities. For the Oil Red O staining, ten images per sample were quantified in Image J software v1.52a and the relative number of red pixels were analysed among the different treatment groups. Statistical analysis was performed using GraphPad Prism v7.0. To determine whether the data was normally distributed, a normality test was performed using the Shapiro-Wilks test. To describe the differences between two groups, a Mann Whitney t-test was used. A three-way ANOVA (analysis of variance) followed by Fishers LSD post hoc test was used to describe the differences between three/or more groups and to determine the relationship between the three variables present in this study, namely, diet, disease and treatment. The results were reported as mean ± standard error of the mean (SEM) and p < 0.05 was considered statistically significant. Histological experiments were conducted with biological repeats of n = 4-5 and technical repeats of n = 2. Results Establishing a diet-induced obesity phenotype To establish whether obesity was induced, differences in body weights were determined between mice fed a standard diet (SD) and high fat diet (HFD) for eight weeks, prior to tumour inoculation and DXR treatment (Fig. 2). After eight weeks on their respective diets, mice fed a HFD (19.64 g ± 0.363 g) showed statistically significant higher body weights compared to mice fed a SD (17.76 g ± 0.175 g) ( p < 0.0001) ( Fig. 2). We therefore conclude that the DIO phenotype was established after 8 weeks on their respective diets and continued throughout the study. Hepatic tissue weight and hepatic hypertrophy Mice in the HFD+NT+V group showed a significantly higher hepatic tissue weight compared to the SD+NT+V mice (p = 0.0406) (Fig. 3). The HFD+NT+V mice also showed a significantly higher hepatic tissue weight compared to the SD+T+V mice (p = 0.0218). Mice in the HFD+NT+DXR group showed a significantly higher hepatic tissue weight compared to mice in the SD+T+V group (p = 0.0400) (Fig. 3). Hepatotoxicity: Apoptotic cell death and ALT For the purpose of this study, we focussed on the three objectives namely, 1) the effects of obesity on hepatotoxicity, 2) the effects of DXR treatment on hepatotoxicity, and 3) the effects of obesity on the outcomes of DXR-induced hepatotoxicity. Apoptotic cell death To determine whether apoptosis was induced in the hepatic tissue samples, western blot experiments were performed to compare the protein expression levels of different apoptotic markers between the different groups: caspase-9, caspase-8, cleaved caspase-8, caspase-3, cleaved caspase-3, and cleaved PARP. For the intrinsic apoptotic pathway, no significant differences were observed in caspase-9 protein expression between the different groups (results not shown) (Fig. 4). In the extrinsic pathway, the ratio between cleaved caspase-8 and caspase-8 showed a statistically significant decrease in the HFD+NT+V group compared to the SD+NT+V (control group) (p = 0.0218) (Fig. 5). The ratio between cleaved caspase-8 and caspase-8 showed a highly statistically significant decrease in the HFD+T+DXR compared to the SD+T+DXR group (p = 0.0011) (Fig. 5). We also assessed the effector caspase, caspase-3, where no significant differences were observed between the treatment groups (Fig. 4). Furthermore, no significant differences were observed in cleaved PARP protein expression between the different treatment groups (results not shown) (Fig. 4). Histopathology Histopathology of hepatic tissue (H&E staining) Using the accredited NAFLD activity score (Table 3), macrovesicular steatosis was observed in the HFD+NT+DXR group with >33%-66% steatosis present (grade 2). The HFD+T+DXR group showed extensive hepatocyte ballooning and sinusoidal dilation with >66% steatosis present (grade 3) compared to the SD+NT+V (control group) where areas of steatosis alternated with areas of normal morphology (Fig. 6). Macrovesicular steatosis were therefore observed in the HFD+T+DXR group (indicated in red arrow) accompanied with mild inflammation (indicated in white arrow) and sinusoidal dilation (indicated in yellow arrow) and congestion. Vacuoles of lipid are accumulating in the hepatocytes in the HFD+T+DXR group and are displacing the nucleus to the cell’s periphery (indicated in red arrow). Inflammatory foci (mild inflammation) were observed in the SD+T+V, HFD+NT+V and HFD+T+DXR groups (indicated in white arrow) with <2 foci per 200 x field (grade 1). Table 3: Liver pathology evaluated by the nonalcoholic fatty liver disease activity score. From Y. Takahashi & T. Fukusato, 2014; Kleiner et al ., 2005. Components of nonalcoholic fatty liver disease activity score Item Definition Score Steatosis Lobular inflammation Ballooning 33% - 66% >66% No foci 4 foci per 200 x field None Few balloon cells Many cells/ prominent ballooning 0 1 2 3 0 1 2 3 0 1 2 Macrovesicular steatosis was minor in the HFD+NT+V group (indicated in red arrow) and accompanied with mild inflammation (indicated in white arrow). Macrovesicular steatosis was moderate in the HFD+NT+DXR group (indicating in red arrow), which indicates that DXR aggravates the toxicity in hepatic tissue (Fig. 6). Macrovesicular steatosis was minor in the HFD+T+V group (indicated in red arrow). Microvesicular steatosis with <5% steatosis within hepatocytes occurred in the SD+NT+V control group (grade 0), accompanied with no inflammatory foci (grade 0) and none ballooning present ((grade 0) (indicated in black arrow). Microvesicular steatosis with >33%-66% steatosis within hepatocytes were present in the SD+T+DXR group (grade 2), accompanied with no inflammatory foci (grade 0) and few balloon cells (grade 1) (indicated in black arrow). Macrovesicular steatosis with 5%-33% steatosis within hepatocytes (grade 1), accompanied with inflammatory foci (grade 1) (indicated in white arrow) and non-ballooning were observed in the HFD+NT+V group, where areas of steatosis >33%-66% were observed in the HFD+NT+DXR group with no inflammatory foci present (grade 0). Macrovesicular steatosis 5%-33% were observed within hepatocytes in the HFD+T+V group (grade 1) (indicated in red arrow) (Table 3). To summarise; our results demonstrated that macrovesicular steatosis was observed in mice fed a HFD and microvesicular steatosis was observed in mice fed a SD. Oil Red O staining To evaluate the presence of lipid accumulation within hepatocytes, Oil Red O staining was performed (Fig. 8). To detect the amount of lipid droplets among the different treatment groups, the relative number of red pixels were analysed using Image J (Fig. 7). This method was used by Masone et al ., 2018 & Gojanovich et al ., 2016 to accurately quantify and analyse size distribution of cellular lipid droplets. The HFD+T+V group showed a highly statistically significant increase in the relative number of red pixels (lipid droplets) compared to the SD+T+V group (p = 0.0057). The HFD+T+DXR group indicates a highly statistically significant increase in the relative number of red pixels (lipid droplets) compared to the SD+T+DXR (p = 0.0023) and the HFD+NT+V control group (p = 0.0318). Hepatocytes stain amphophilic and lipid droplets are stained red (indicated in black arrows) (Fig. 8). High levels of lipid accumulation was observed within hepatocytes in the HFD groups. Furthermore, lipid accumulation was significantly increased in the HFD group treated with DXR and the tumour present, which indicates that lipid accumulation was more severe in the HFD+T+DXR group compared to the SD+T+DXR group (Fig. 8). Masson’s trichrome stain To evaluate liver damage, collagen fibres are stained blue using Masson’s trichrome stain to determine the presence of fibrosis. No significant fibrosis was observed within the treatment groups, and therefore, no trends or statistical significance could be determined (Fig. 9). Images were qualitatively represented. Discussion Establishment of an obese model following a high fat diet DXR is an effective chemotherapeutic agent used for the treatment of breast cancer. However, DXR may cause side-effects and induce cardiotoxicity, myelosuppression, and skeletal muscle atrophy (Hiensch et al. , 2020; Zhao & Zhang, 2017). It has been shown that DXR can also induce hepatotoxicity (Bengaied et al. , 2017; Song et al. , 2019). The mechanisms and effects of DXR on hepatic tissue and the contributing role of obesity in breast cancer patients are not fully elucidated. Therefore, the aim of this study was to investigate the effects of doxorubicin on hepatic tissue in an obese tumour-bearing mouse model. In animal models, a HFD is associated with an increase in body mass (BM) (Noeman et al ., 2011; Park et al ., 2012; Schultz et al ., 2012; Layman et al ., 2019) and liver mass, which was observed in the HFD groups in this study. To establish whether obesity was induced, differences in body weight was evaluated for mice fed a SD or HFD for eight weeks prior to tumour inoculation and DXR treatment (Fig. 1). The body weights of mice in the HFD group were significantly higher compared to the SD group (Fig. 1), which confirmed that the obese phenotype was successfully established in our in vivo model. Our results correlate with similar findings of a study done by Lee et al. (2019) and Santander et al . (2015) who indicated that a HFD increased body weight, as a result of increased fat mass in C57BL/6 mice by evaluating body composition. It has been shown that the C57BL/6 mice strain is highly susceptible to the development of DIO and therefore correlates with the findings of our study (Chu et al. , 2017). Furthermore, the DIO phenotype observed in mice fed a HFD was corroborated by a significantly higher hepatic tissue weights observed in the HFD+NT+V and HFD+NT+DXR groups compared to the SD+NT+V and SD+T+DXR groups, respectively (Fig. 3). Our results correlate with a study conducted by Jung et al., 2013 where C57BL/ mice fed a HFD for nine-weeks resulting in an increase in BM and liver mass gain. Hepatotoxicity: Evaluation of apoptotic cell death and ALT DXR induces oxidative stress and is characterized by accumulation of reactive oxygen species (ROS), which decrease antioxidant defence systems, resulting in oxidative damage of DNA and apoptotic cell death (Song et al. , 2019). It has been shown that oxidative stress is a major contributor of DXR-induced hepatotoxicity (Bengaied et al. , 2017; Rashid et al. , 2013). Obesity is associated with low grade chronic inflammation, excessive ROS production and oxidative stress. Together, both the obese state and DXR would be expected to upregulate ROS, which results in the downstream activation of apoptosis in hepatic tissue. To determine whether apoptosis was induced in the hepatic tissue samples, western blot experiments were performed to compare the protein expression levels of different apoptotic markers, such as caspase-9, caspase-8, cleaved caspase-8, caspase-3, cleaved caspase-3 and cleaved PARP in the different experimental groups. To determine whether liver damage occurred, we also assessed the expression levels of ALT in the hepatic tissue samples by performing western blotting. No significant differences in caspase-9 were observed between the different treatment groups (Fig. 4), which indicates that apoptosis was not induced through the intrinsic pathway. The ratio between cleaved caspase-8 and caspase-8 showed a statistically significant decrease in the HFD+NT+V group compared to the SD+NT+V (control group). The ratio between cleaved caspase-8 and caspase-8 showed a highly statistically significant decrease in the HFD+T+DXR compared to the SD+T+DXR group (Fig. 5). No significant differences were observed in caspase-3 and cleaved caspase-3 protein expression between the different treatment groups (Fig. 4). Upregulation of cleaved caspase-8 in mice fed a standard diet does not indicate that apoptotic cell death occurred within the obese tumour-bearing mouse model and could be an indication of protection during liver regeneration. Freimuth et al., 2013 showed that during liver regeneration, caspase-8 consist of a non-apoptotic function by balancing receptor-interacting protein 1 (RIP1), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and Jun N-terminal kinase (JNK) activation. Loss of caspase-8 therefore triggers NF-kB activation and induce liver regeneration. There were no significant differences observed in PARP cleavage between the different groups (Fig. 4). Since PARP cleavage is a down-stream event of caspase-3 cleavage during apoptosis, differences in PARP cleavage might have been observed at a later time point of tissue collection in the obese tumour-bearing mouse model. HFD and DXR promote macrovesicular steatosis in hepatocytes, but not fibrosis As previously mentioned, the first structural changes that occurs when the liver is damaged is hydropic changes, where fluid filled vacuoles accumulates within the hepatocytes. It has been shown that a HFD can induce lipid accumulation via a process known as hepatic steatosis (Layman et al. , 2019; VanSaun et al. , 2009). Microvesicular steatosis was observed in the SD groups since hepatocytes are filled with tiny lipid droplets and the nucleus is located centrally in the cell. Macrovesicular steatosis was observed in the HFD groups where large fat droplets occupy the cytoplasm of hepatocytes, pushing the nucleus to the periphery (Fig. 6). From the non-alcoholic fatty liver disease activity score (table 3), >66% steatosis was present within hepatocytes in the HFD+T+DXR group accompanied with lobular inflammation (score 2) and prominent ballooning (score 2), sinusoidal dilation and congestion, fatty infiltration, and ballooning of hepatocytes compared to the SD+NT+V (control) group where areas of steatosis alternated with areas of normal morphology (Fig. 6). In the SD+T+DXR group, micro-vesicular steatosis was observed, with 5%-33% steatosis in the hepatocytes. In the HFD+NT+DXR group, >33%-66% of the hepatocytes had steatosis compared to the HFD+NT+V group. This indicates that DXR promotes hepatic steatosis in obesity. Our histopathology observations correlate with previous studies conducted where a HFD and DXR treatment induced macrovesicular steatosis, ballooned hepatocytes, inflammatory cell infiltration and sinusoidal dilation, respectively (El-Sayyad et al. , 2009; Layman et al. , 2019; Rashid et al. , 2013; Santander et al. , 2015). No significant fibrosis was observed in our experimental groups (Fig. 9). This indicates that the liver did not progress to cirrhosis, indicative of end-stage liver disease and that the highly metabolic organ had the capacity to regenerate itself or reverse the harmful stimulus in the broad spectrum of liver disease. This could also be attributed to the concentration and frequency of the DXR treatment as observed by other studies (El-Sayyad et al. , 2009). HFD promotes lipid accumulation within hepatocytes and induce hepatotoxicity in combination with DXR treatment The liver plays an important role in lipid metabolism and stores fatty acids in the form of triglycerides. It has been shown that a HFD promotes lipid accumulation within hepatocytes via hepatic steatosis resulting in disturbances in lipid metabolism (Layman et al. , 2019; VanSaun et al. , 2009). Due to image evaluation of Oil Red O-stained fresh tissue sections, our results indicated that the relative number of red pixels are highly significantly increased in the HFD groups compared to mice fed a SD (Fig. 7). In the present study, the relative number of red pixels are highly significantly increased in the HFD+T+DXR compared to the SD+T+DXR group (Fig. 7). Our results correlate with previous studies conducted by Layman et al., 2019 & Tsuru et al., 2020 where a HFD induced hepatic lipid accumulation and inflammation within hepatocytes of mice fed a HFD, resulting in hepatic steatosis. It is evident from our H&E and Oil Red O results that lipid accumulation was elevated in the HFD groups and specifically severe in the HFD+T+DXR group, which indicates that obesity and the tumour present in combination with DXR treatment induced hepatic steatosis, which is a hallmark of NAFL (Fig. 6 & 8). We therefore conclude that hepatic steatosis (>66% steatosis) occurred in the obese tumour-bearing mouse model, resulting in NAFLD. Our findings correlate with a study done by Layman et al., 2019 where mice fed a HFD induced hepatic steatosis, within hepatocytes. Conclusion In conclusion, we have shown that DXR activated the extrinsic pathway of apoptosis in the livers of tumour-bearing mice which were fed a standard diet compared to the mice which were fed a high fat diet, where apoptosis was not initiated. Our histopathology results indicated that hepatic tissues of mice fed a high fat diet and treated with DXR display severe histological damage, such as macrovesicular steatosis, sinusoidal dilation, and lobular inflammation. This indicates that more hepatic toxicity occurred in mice fed a high fat diet and treated with DXR. We therefore conclude that hepatic steatosis, a hallmark of NAFL occurred in the obese tumour-bearing mouse model. There was no evidence of fibrosis which indicates that the livers did not progress to cirrhosis. This study showed that hepatotoxicity is aggravated in obesity as an underlying co-morbidity in breast cancer patients receiving adjuvant chemotherapy, such as DXR. It has been shown that obesity is associated with poor clinical outcomes in patients receiving neo-adjuvant chemotherapy treatment regimens (Iwase et al. , 2016). Lower doses of DXR are prescribed for obese cancer patients to reduce side effects and adverse toxicities, which could compromise drug efficacy and contribute to the development of resistance (Iwase et al. , 2016; Mentoor et al. , 2018; Sarfati, Koczwara & Jackson, 2016) . This study, therefore, produce preliminary safety data on the effects of DXR and the contributing role of obesity on hepatotoxicity. Abbreviations ALT: Alanine transaminase; ANOVA: Analysis of variance; AST: Aspartate aminotransferase; BM: Body Mass; DNA: Deoxyribonucleic acid; DIO: Diet-induced obesity; DXR: Doxorubicin; ECL: Electrochemiluminescence; FFPE: formalin fixed paraffin embedded; HBSS: Hank’s balanced salt solution; H&E: Haematoxylin & eosin; HFD: High fat diet; IVC: Individually ventilated cages; IL-6: Interleukin-6; JNK: Jun N-terminal kinase; MCP-1: Monocyte chemoattractant protein-1; NAFLD: Non-alcoholic fatty liver disease; NAS: Non-alcoholic fatty liver disease activity score; NF-kB: Nuclear factor kappa-light-chain-enhancer of activated B cells; NT: Non-Tumour; PVDF: Polyvinylidene difluoride; RIP1: Receptor-interacting protein 1; RIPA: Radio-immunoprecipitation assay buffer; ROS: Reactive oxygen species; SD: Standard diet; SDS: Sodium dodecyl sulfate; TBS-T: Tris-buffered saline tween; T: Tumour; Top 2: Topoisomerase II Declarations Acknowledgements The authors wish to acknowledge Bianca Bock and Zaakiyah Emjedi for performing the animal model and Mr. Reggie Williams for assistance with the histology staining and Dr. Chantelle Venter for guidance with the histology Oil red O quantification. Author’s contributions All authors have read and approved the manuscript. MS performed the western blot techniques, analysed, and interpreted the results as well as the histological results. MS was also a major contributor in writing the manuscript. CC co-supervised this study and reviewed the final manuscript. LDJ reviewed the final manuscript. AME supervised this study and reviewed the final manuscript. Funding This study was financially supported by research grants from the Cancer Association of South Africa (CANSA); South African Medical Research Council (SAMRC); and the National Research Foundation (NRF). Availability of data and materials The datasets generated and analysed during the current study are not publicly available due datasets being too large to include but are available from the corresponding author on reasonable request. Ethics approval and consent to participate Ethical clearance for the in vivo study was obtained from the Stellenbosch University Ethical Committee (no. SU-ACUM13-00015 and no. ACU-2020-14751). Institutional and international ethical guidelines were applied with respect to the handling of experimental animals. All methods were performed in accordance with the relevant guidelines and regulations of the Animals (Scientific Procedures) Act 1986. Consent for publication Not applicable. Competing interests The authors declare they have no competing interest. Author details 1 Department of Physiological Sciences, Faculty of Science, Stellenbosch University, Stellenbosch 7600, South Africa. 2 Department of Global Health, Faculty of Medicine and Health Sciences, African Cancer Institute, Stellenbosch University, Cape Town 8000, South Africa. 3 Division of Anatomical Pathology, Stellenbosch University and National Health Laboratory Service (NHLS), Tygerberg Hospital, Cape Town 8000, South Africa. 4 Anatomical Pathology, PathCare, Cape Town, South Africa References Ando, S., Gelsomino, L., Panza, S., Giordano, C., Bonofiglio, D., Barone, I & Catalano, S. 2019. Obesity, Leptin and Breast Cancer: Epidemiological Evidence and Proposed Mechanisms. Cancers . 62(11): 1-27. Alghamdi, S., Leoncikas, V., Plant, K.E. & Plant, N.J. 2015. 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Supplementary Files BMCCancerTable1andTable2.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Major revision 10 Jun, 2022 Reviews received at journal 07 Jun, 2022 Reviews received at journal 04 May, 2022 Reviewers agreed at journal 31 Mar, 2022 Reviewers agreed at journal 30 Mar, 2022 Reviewers invited by journal 30 Mar, 2022 Editor assigned by journal 30 Mar, 2022 Editor invited by journal 30 Mar, 2022 Submission checks completed at journal 30 Mar, 2022 First submitted to journal 21 Mar, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1473640","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":94692636,"identity":"dc33efdf-69d9-40dd-926e-b75b8e7aa06f","order_by":0,"name":"Megan Sedeman","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYJACCRDBz3D4AIgtQ7wWycZjCSA2D/FaDA6fMQDRhLXo9h8+eONjjl0ew7Ezn1/dqLHgYWA/fHQDPi1mN9KSLWduSy5m7Dm7zTrnGNBhPGlpN/Br4TGT5t3GnNgscXabcQ4bUIsEjxl+LefPf5P+u60+sU3+zTPjnH/EaDmQwybNuO1wYg/DGebHuW3EaLmRZmzZu+144gyGY2bMuX0SPGwE/XL+8MMbP7dVJ+4/cPjx55xvdXL87IeP4dWCDNjAEcRGrHIQYP5AiupRMApGwSgYOQAA2NdNtHMBLeQAAAAASUVORK5CYII=","orcid":"","institution":"Stellenbosch University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Megan","middleName":"","lastName":"Sedeman","suffix":""},{"id":94692637,"identity":"a704b7c7-db9d-4888-8cc8-9e3388d50d5a","order_by":1,"name":"Claudia Christowitz","email":"","orcid":"","institution":"Stellenbosch University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Claudia","middleName":"","lastName":"Christowitz","suffix":""},{"id":94692638,"identity":"2fd20cc9-b3c0-4b95-8ced-7bcdfa90dd94","order_by":2,"name":"Louis de Jager","email":"","orcid":"","institution":"Stellenbosch University, Tygerberg Hospital","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Louis","middleName":"","lastName":"de Jager","suffix":""},{"id":94692639,"identity":"4fd86121-2465-4f89-9a9b-d6b91215ad1e","order_by":3,"name":"Anna-Mart Engelbrecht","email":"","orcid":"","institution":"Stellenbosch University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Anna-Mart","middleName":"","lastName":"Engelbrecht","suffix":""}],"badges":[],"createdAt":"2022-03-21 11:59:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1473640/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1473640/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":19826415,"identity":"2c26a840-70c5-482f-a3c0-9ff75d7d7cde","added_by":"auto","created_at":"2022-03-31 15:35:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":88882,"visible":true,"origin":"","legend":"\u003cp\u003eSummary of the \u003cem\u003ein vivo\u003c/em\u003e model and respective experimental groups.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-1473640/v1/b98c12759799aaad0114f10d.png"},{"id":19826219,"identity":"25ce6ba7-d7be-4c3f-9c25-af2e3bf4da39","added_by":"auto","created_at":"2022-03-31 15:30:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":13003,"visible":true,"origin":"","legend":"\u003cp\u003eBody weight of female C57BL6 mice from week one to week eight on their respective diets. A Mann Whitney t-test was performed to compare the differences in body weight (grams) between the SD and HFD mice for eight weeks. Values were represented as mean ± SEM and \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 was considered statistically significant. (SD, n = 38; HFD, n = 36). * - statistically significant compared to the SD group (\u003cem\u003ep \u0026lt;\u003c/em\u003e 0.0001). SD: standard diet; HFD: high fat diet.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-1473640/v1/1c980f7d2d60b64a22118283.png"},{"id":19826223,"identity":"cbaee00e-ea66-4313-b9d8-96933d95b506","added_by":"auto","created_at":"2022-03-31 15:30:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":24956,"visible":true,"origin":"","legend":"\u003cp\u003eHepatic tissue weight (grams) between the different groups. Three-way ANOVA with Fishers LSD \u003cem\u003epost hoc \u003c/em\u003ecorrection test was applied and \u003cem\u003ep \u0026lt;\u003c/em\u003e 0.05 was considered statistically significant. Results are presented as mean ± SEM (n = 8-10 per group). * - significantly different compared to the SD+NT+V group (\u003cem\u003ep \u0026lt;\u003c/em\u003e 0.05), # - significantly different compared to the SD+T+V group (\u003cem\u003ep \u0026lt;\u003c/em\u003e 0.05). SD: Standard diet; HFD: High fat diet; T: Tumour; NT: Non-Tumour; DXR: Doxorubicin; V: Vehicle.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-1473640/v1/89f4871d628fc8325c061016.png"},{"id":19826222,"identity":"13a81fdf-2887-4a48-94d8-2d95b007d331","added_by":"auto","created_at":"2022-03-31 15:30:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":89207,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of caspase-9, cleaved caspase-3, caspase-3, cleaved-PARP and ALT between the different treatment groups (n = 4).\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-1473640/v1/227fe92c9857270ac5520912.png"},{"id":19826416,"identity":"28ec9c95-d3db-4293-a46c-8a99b6f7e81a","added_by":"auto","created_at":"2022-03-31 15:35:13","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":126577,"visible":true,"origin":"","legend":"\u003cp\u003eProtein expression of the ratio cleaved caspase-8 and total (uncleaved) caspase-8 between the different groups (n = 4). Results are presented as mean ± SEM. Three-way ANOVA with Fishers LSD \u003cem\u003epost hoc \u003c/em\u003ecorrection test was applied and \u003cem\u003ep \u0026lt;\u003c/em\u003e 0.05 was considered as statistically significant. * - significantly different compared to the SD+NT+V group (\u003cem\u003ep \u0026lt; \u003c/em\u003e0.05). ** - significantly different compared to the SD+T+DXR group (\u003cem\u003ep \u0026lt; \u003c/em\u003e0.01). SD: Standard diet; HFD: High fat diet; T: Tumour; NT: Non-Tumour; DXR: Doxorubicin; V: Vehicle.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-1473640/v1/67410fd79ff69e8834989b58.png"},{"id":19826226,"identity":"6a5aa0fb-1851-4de9-8ba2-8f34a4ec98a1","added_by":"auto","created_at":"2022-03-31 15:30:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":664539,"visible":true,"origin":"","legend":"\u003cp\u003eExamples of pathological lesions observed in H\u0026amp;E stained hepatic tissue in mice fed a HFD evaluated by the non-alcoholic fatty liver disease activity scoring system (NAS) (n = 4-5).\u003cstrong\u003e \u003c/strong\u003eMicrovesicular steatosis (indicated by black arrows) and macrovesicular fatty infiltration in hepatocyte (steatosis) (indicated by red arrows), mild inflammation (inflammatory foci) (indicated by white arrows) and sinusoidal dilation (indicated in yellow arrow) within hepatocytes between the different groups.\u003cstrong\u003e \u003c/strong\u003eImages were taken at 20x magnification, scale bar = 100 μm.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-1473640/v1/2c940cd049883eaa15ac05ae.png"},{"id":19826221,"identity":"71553dcc-e1c1-4259-92fc-83b552320fa1","added_by":"auto","created_at":"2022-03-31 15:30:13","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":20877,"visible":true,"origin":"","legend":"\u003cp\u003eRelative number of red pixels indicating the amount of lipid droplets within hepatocytes (n = 4-5). Results are presented as mean ± SEM. Three-way ANOVA with Fishers LSD \u003cem\u003epost hoc \u003c/em\u003ecorrection test was applied and \u003cem\u003ep \u0026lt;\u003c/em\u003e 0.05 was considered statistically significant. * - Significantly different compared to the Standard diet+Tumour+Doxorubicin (SD+T+DXR) group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05), ** - Significantly different compared to the SD+T+DXR group \u003cem\u003e(p \u0026lt;\u003c/em\u003e 0.01), ## - Significantly different compared to the Standard diet+Tumour+Vehicle (SD+T+V) group (\u003cem\u003ep \u0026lt;\u003c/em\u003e 0.01). SD: Standard diet; HFD: High fat diet; T: Tumour; NT: Non-Tumour; DXR: Doxorubicin; V: Vehicle.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-1473640/v1/93aedf9644d35dc6a68c788a.png"},{"id":19826227,"identity":"ee6bd140-7b92-4ec8-8dd6-d2b56731665b","added_by":"auto","created_at":"2022-03-31 15:30:13","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1039180,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of lipid accumulation in hepatic tissue detected by the Oil Red O stain (n = 4-5).\u003cstrong\u003e \u003c/strong\u003eImages were taken at 20x and 10x magnification, scale bar = 100 μm.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-1473640/v1/6a957288a3b3775dbee1f050.png"},{"id":19826228,"identity":"085ed7d2-f525-4a62-ac72-b3856a49ad62","added_by":"auto","created_at":"2022-03-31 15:30:13","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1276200,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of liver tissue stained by the Masson’s trichrome stain for pathological evaluation of fibrosis. (n = 4-5).\u003cstrong\u003e \u003c/strong\u003eImages were taken at 20x and 40x magnification, scale bar = 100 μm.\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-1473640/v1/d6e9adb97fc5f200e66ead42.png"},{"id":19826418,"identity":"253b0972-ac64-44ee-93be-909890decd96","added_by":"auto","created_at":"2022-03-31 15:35:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":846789,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1473640/v1/ad3b840a-3872-481e-bc1c-617a350b71ec.pdf"},{"id":19826220,"identity":"6c88bdd6-d816-4134-87a4-3faa4ba01ed6","added_by":"auto","created_at":"2022-03-31 15:30:13","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":15007,"visible":true,"origin":"","legend":"","description":"","filename":"BMCCancerTable1andTable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-1473640/v1/53dcf45f597e8d0946a75531.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Obese mammary tumour-bearing mice are highly sensitive to doxorubicin-induced hepatotoxicity","fulltext":[{"header":"Background","content":"\u003cp\u003eBreast cancer is a common malignancy that frequently occurs in women from both developed and developing countries and is therefore a major health burden worldwide (Francies \u003cem\u003eet al.\u003c/em\u003e, 2020). Lifestyle-related risk factors, such as obesity and being overweight reached epidemic proportions and is a well-known risk factor that contributes to the development of breast cancer (Mentoor \u003cem\u003eet al.\u003c/em\u003e, 2020). \u0026nbsp;In 2018, breast cancer was the most frequently diagnosed cancer and the leading cause of cancer-related deaths among females worldwide (Bray \u003cem\u003eet al.\u003c/em\u003e, 2018; Siegel, Miller \u0026amp; Jemal, 2018). Significant progress has been made regarding treatment options for cancer patients, however, these drugs may cause serious multi-organ toxicity, and this remains a major concern.\u003c/p\u003e\n\u003cp\u003eDysfunctional adipocytes implicated in obesity release metabolic substrates, adipokines, and cytokines, which promote proliferation, progression, invasion, and migration of breast cancer cells. All these factors\u0026nbsp;associated with obesity, mediates tumour initiation, metabolic reprogramming, angiogenesis, progression, and response to treatment\u0026nbsp;(Ando \u003cem\u003eet al.,\u003c/em\u003e 2019).\u003c/p\u003e\n\u003cp\u003eDoxorubicin (DXR) is an anthracycline glycoside antibiotic that possess antitumour activity and is one of the most effective chemotherapeutic agents used to treat breast cancer (Ren \u003cem\u003eet al.\u003c/em\u003e, 2016). Although effective, therapeutic applications of DXR are limited due to its side-effects, such as myelosuppression, chronic cardiotoxicity, and skeletal muscle atrophy (Hiensch \u003cem\u003eet al.\u003c/em\u003e, 2020; Zhao \u0026amp; Zhang, 2017). DXR can also be detrimental to other organs, such as the liver, which plays an important physiological role in metabolism, glycogen and triglyceride storage, plasma protein synthesis and detoxification of toxic metabolites (Chiang, 2014).\u003c/p\u003e\n\u003cp\u003eIt has been shown that DXR can induce hepatotoxicity, which limits its efficacy in anticancer therapy, resulting in poor patient prognosis and decreased overall survival of cancer patients (Bengaied \u003cem\u003eet al.\u003c/em\u003e, 2017; Song \u003cem\u003eet al.\u003c/em\u003e, 2019). DXR-induced oxidative stress is characterized by accumulation of reactive oxygen species (ROS), which decrease antioxidant defence systems, resulting in oxidative damage of deoxyribonucleic acid (DNA), therefore, mediating apoptotic cell death in hepatic tissue (Song \u003cem\u003eet al.\u003c/em\u003e, 2019). It has been shown that the apoptotic pathway, characterized by the release of cytochrome c from the mitochondria (intrinsic apoptotic pathway) and the Fas ligand (important regulator of the extrinsic apoptotic pathway), were associated with DXR-induced acute toxicity in the liver (Rashid \u003cem\u003eet al.\u003c/em\u003e, 2013; Song \u003cem\u003eet al.\u003c/em\u003e, 2019). Furthermore, histopathological changes have indicated that DXR can cause structural damage to hepatic tissues which include inflammation, congestion, and necrosis (Mohan \u003cem\u003eet al.\u003c/em\u003e, 2011; Rashid \u003cem\u003eet al.\u003c/em\u003e, 2013; Song \u003cem\u003eet al.\u003c/em\u003e, 2019).\u003c/p\u003e\n\u003cp\u003eNon-alcoholic fatty liver disease (NAFLD), a co-morbidity associated with obesity is characterized by macrovesicular steatosis, triglyceride accumulation in hepatocytes, increased hepatic oxidative stress and increased sensitivity to drug induced liver injury (Li \u003cem\u003eet al\u003c/em\u003e., 2019). A study done by Alghamdi \u003cem\u003eet al.\u003c/em\u003e (2015) showed that DXR in the presence of palmitate and oleate (lipid loading) caused a significant accumulation of lipids within the human hepatoma cell line (Huh7 cells) and enhanced acute toxicity in lipid-loaded hepatocytes, which is mediated through increased oxidative stress and ROS (Alghamdi \u003cem\u003eet al.\u003c/em\u003e, 2015).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe mechanisms of action on hepatic toxicity and the contributing role of obesity in breast cancer patients are not fully elucidated. Therefore, the aim of this study was to investigate the effects of DXR on hepatic tissue in an obese tumour-bearing model. The expression levels of markers involved in the apoptotic pathway was assessed by means of western blotting. Alanine transaminase (ALT) expression was also determined through western blotting. To assess the pathology and morphology of hepatic tissues, haematoxylin and eosin (H\u0026amp;E) staining was performed on formalin fixed paraffin embedded (FFPE) tissue. Furthermore, the presence of fibrosis and steatosis in hepatic tissues were assessed with Masson\u0026rsquo;s trichrome and Oil Red O staining, respectively.\u0026nbsp;\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eAnimal model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical clearance was obtained from the Stellenbosch University animal research committee (no. SU-ACUM13-00015 and no. ACU-2020-14751). The experiments involving the use of laboratory animals were carried out in accordance with the Animal Welfare Act and recommendations of the Institutional Animal Care and Committee of Stellenbosch University. This study is reported in accordance with ARRIVE guidelines (Animal Research: Reporting of \u003cem\u003eIn Vivo\u003c/em\u003e Experiments) to improve the reporting of research involving animals. Three-week-old female C57BL6 mice (n = 74) were obtained from the Stellenbosch University Central Research Facility and were housed at the Stellenbosch University Animal Unit in individually ventilated cages (IVC) at temperature-controlled conditions (i.e., 22⁰C) and underwent a 12-h light/dark cycle. Mice were allowed an acclimatization period of one week with \u003cem\u003ead libitum\u0026nbsp;\u003c/em\u003eaccess to mouse pellets and tap water (Fig. 1). The general welfare of the animals was monitored daily. After one week of acclimatization, seventy-four mice were randomly divided into two main groups, namely the high fat diet (HFD, n = 36) and standard diet (SD, n = 38) groups (\u003cstrong\u003eFig. 1\u003c/strong\u003e). To induce obesity, mice were fed a HFD containing 60% kcal fat (D12492, OpenSource Diets\u003csup\u003e\u0026reg;\u003c/sup\u003e, Research Diets Inc., New Jersey, USA), whereas a SD containing 10% kcal fat (D12450J, OpenSource Diets\u003csup\u003e\u0026reg;\u003c/sup\u003e, Research Diets Inc., New Jersey, USA) were used to generate lean control mice\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(Additional file 1, Table 1). According to literature, C57BL6 mice are sensitive to diet-induced obesity (DIO) (Chu \u003cem\u003eet al.\u003c/em\u003e, 2017). \u0026nbsp;Body weight was monitored weekly over the study period and mice were on their respective diets up until the end point of the study.\u003c/p\u003e\n\u003cp\u003eAfter eight weeks on their respective diets, the DIO phenotype was established and the two groups were further randomly divided into two groups each, namely the tumour (T) and non-tumour (NT) groups. Mice were subcutaneously inoculated with E0771 triple negative breast cancer cells suspended in Hanks Balanced Salt Solution (HBSS) (Sigma Chemical Co., St Louis, MO, USA) in the fourth mammary pad, using a 23-guage needle syringe. The tumour groups were inoculated with E0771 triple negative breast cancer cells (HFD+T, n = 18; SD+T, n = 18) and the non-tumour groups were not inoculated with cancer cells (HFD+NT, n = 18; SD+NT, n = 20) (Fig. 1).\u003c/p\u003e\n\u003cp\u003eOnce the tumours became palpable (200 \u0026ndash; 300 mm\u0026sup2;), DXR treatment was initiated. The mice were randomly divided into vehicle control (V, isovolumetric intra-peritoneal injection of HBSS) and DXR treatment (D5794, LKTR laboratories, Minnesota, USA) groups. Mice were restrained and three doses of 4 mg/kg DXR were administered (cumulative dosage of 12 mg/kg) via intraperitoneal injection. Each group was then further divided into two groups namely the DXR treatment groups; High fat diet+Tumour+Doxorubicin (HFD+T+DXR, n = 9); High fat diet+Non-Tumour+Doxorubicin (HFD+NT+DXR, n = 10); Standard diet+Tumour+Doxorubicin (SD+T+DXR, n = 10); Standard diet+Non-Tumour+Doxorubicin (SD+NT+DXR, n = 10) and vehicle control groups; \u0026nbsp;High fat diet+Tumour+Vehicle (HFD+T+V, n = 9); High fat diet+Non-Tumour+Vehicle (HFD+NT+V, n = 8); Standard diet+Tumour+Vehicle (SD+T+V, n = 8); Standard diet+Non-Tumour+Vehicle (SD+NT+V, n = 10).\u003c/p\u003e\n\u003cp\u003eIn humans, the dosage of 12 mg/kg DXR is equivalent to 36 mg/m\u0026sup2; and falls within the relevant dosage range of DXR treatment (15 - 90 mg/m\u0026sup2;) administered to cancer patients in the clinical setting (Reagan-Shaw \u003cem\u003eet al\u003c/em\u003e., 2008). The eight experimental groups were assigned as follow: HFD+T+DXR, n = 9; HFD+NT+DXR, n = 10; SD+T+DXR, n = 10; SD+NT+DXR, n = 10) and vehicle control groups (HFD+T+V, n = 9; HFD+NT+V, n = 8; SD+T+V, n = 8; SD+NT+V, n = 10 (Fig. 1).\u003c/p\u003e\n\u003cp\u003eThe mice were weighed every second day and the last body weight was recorded on the day of euthanasia. Tumour growth was measured using a Harpenden caliper (mm) and individual tumour volumes were calculated according to the following equation:\u003c/p\u003e\n\u003cp\u003eTumour voulme(mm\u003csup\u003e3\u003c/sup\u003e) = \u003cimg src=\"data:image/png;base64,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\"\u003e\u0026nbsp; (Tomayko \u0026amp; Reynolds, 1989).\u003c/p\u003e\n\u003cp\u003eThe mice were euthanized 3 days after the last DXR treatments were administered. The mice were anesthetized with 3% isoflurane (Isofor, Safeline, Pharmaceuticals, Florida, South Africa) and were euthanized by cervical dislocation. Hepatic tissues were excised, where half of the tissues were snap frozen in liquid nitrogen and stored at - 80\u0026deg;C for western blotting and Oil red O staining (n = 4). The other half of the tissues were preserved in 10% formalin for histological analysis (n = 4-5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHepatic tissue samples were placed on ice and allowed to thaw. Samples were suspended in 300 \u0026mu;l cold modified radio-immunoprecipitation assay buffer (RIPA) containing protease and phosphatase inhibitors. Surgical scissors cleaned with 100% ethanol was used to cut tissues into smaller pieces while on ice. Samples were homogenised (KineMatica PolytronTM PT2100, Fisher Scientific) while on ice. Samples were centrifuged (14, 000 RCF (g), 20 min, 4\u0026deg;C) to yield distinct layers and the supernatant layer was removed and transferred into sterile Eppendorf tubes. Samples were then centrifuged again at 14 000 RCF (g), 20 min, 4\u0026deg;C. The process of removing the supernatant was repeated followed by protein determination using a Bradford assay. Protein samples were prepared with Laemmli\u0026rsquo;s sample buffer and were loaded onto 4-20% Criterion\u003csup\u003e\u0026trade;\u003c/sup\u003e TGX Stain-Free\u003csup\u003e\u0026trade;\u003c/sup\u003e Precast Gels (mini-PROTEAN\u003csup\u003e\u0026reg;\u003c/sup\u003e TGX\u0026trade; Gels, Bio-Rad), following protein separation at 100 V for 10 min and 120 V for 60 min in Tris/Glycine/SDS running buffer (BioRad, CA, USA). Proteins were transferred onto Polyvinylidene difluoride (PVDF) membranes (Trans-Blot Turbo RTA Midi PVDF transfer kit, BioRad, CA, USA) with the Trans-Blot Turbo Transfer System (BioRad, CA, USA) using mixed molecular weight. The membranes were blocked in 5% milk prepared in tris-buffered saline with tween 20 (TBS-T) for 2 h at room temperature (RT) and then incubated in primary antibody, at 4\u0026deg;C overnight. On the following day, the membranes were incubated with secondary antibody for 1 h at RT. Antibody details are listed in additional file 2. After incubation, the membranes were developed on the ChemiDoc\u003csup\u003e\u0026trade;\u003c/sup\u003e MP System. Specific bands were visualized and detected using enhanced chemiluminescence (ECL) substrate detection (BioRad, CA, USA). Quantification of protein samples were normalized to total protein and expressed as a percentage of the control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn all animals, a small portion of the right lateral lobe of the liver tissue was fixed in 10% neutral formalin buffered solution prior tissue processing (n = 4-5). Hepatic tissues were processed using an automated tissue processor (HistoCore PEARL, Leica Biosystems) on a 12-h cycle followed by infiltration with paraffin embedded wax (Leica EG 1150 H). Tissues were sectioned into 5 \u0026mu;m sections using a microtome (Leica RM 2125 RT) and tissue sections were placed onto positively charged histobond microscope slides. Two histochemical stains were carried out on the sectioned tissue; H\u0026amp;E stain for morphometric and pathological evaluation and Masson\u0026rsquo;s trichrome stain to evaluate the presence of fibrosis within hepatic tissue. The right median lobe of hepatic tissue was snap frozen in liquid nitrogen and sectioned into 7 \u0026mu;m sections using a cryostat (Leica CM 3050 S Research Cryostat, Leica Biosystems) to evaluate the presence of lipid accumulation using Oil red O staining (n = 4-5). Sections were placed onto positively charged histobond microscope slides and left to defrost. Sections were stained in Oil red O in dextrin staining solution (Sigma-Aldrich, 01391, SA) for 25 min. Coverslips were mounted onto the slides using aqueous mounting media (Sigma-Aldrich, G 0918) prior to imaging under a microscope (Nikon ECLIPSE E400). The relative number of red pixels (lipid droplets) were quantified using Image J software v1.52a.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathology\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree individuals, blinded to the treatment allocations, scored five images per tissue sample in each treatment group using the non-alcoholic fatty liver disease (NAFLD) activity score (NAS). This is an accredited and validated scoring system frequently used to evaluate hepatic steatosis, inflammation, and hepatocyte-specific pathology (Kleiner \u003cem\u003eet al\u003c/em\u003e., 2005). Each scorer was provided with a grading sheet to familiarize themselves with the scoring system one day prior to analysis. The scores were tabulated and used for inter- and intra-observer analysis. H\u0026amp;E-stained tissues were used to evaluate structural changes that occurs within hepatocytes and Masson\u0026rsquo;s trichrome-stained tissues were used to assist with the evaluation of liver fibrosis. Steatosis was scored according to its percentage per microscopic field, location (zone, 1, 2 or 3), and the presence of micro- and/or macrovesicular steatosis. With regards to inflammation, the location, lobular and/or portal, were evaluated per 100X microscopic field. For this study, microvesicular steatosis (indicated with black arrows) and macrovesicular steatosis (indicated in red arrows) were observed in hepatic tissue, following H\u0026amp;E staining (Fig. 6). Ten images per sample were taken and analysis was performed from right to left across the liver tissue sections. The H\u0026amp;E-stained and Masson\u0026rsquo;s trichrome-stained liver tissues were evaluated as follow: To assess the extent of steatosis, the presence of hydropic changes, micro- and macrovesicular steatosis. To determine whether inflammation occurred, the presence of portal mononuclear cell infiltration, haematopoiesis, and Kupffer cell proliferation were evaluated. Characteristics of hepatocytes and the portal system, such as hepatocyte swelling, sinusoidal and central vein dilation, were evaluated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe western blot experiments were conducted with biological repeats of n = 4 and technical repeats of n = 1. Bio-Rad Image Lab\u0026trade; software v6.0.1 was used for normalization of the protein specific intensities against total protein intensities. For the Oil Red O staining, ten images per sample were quantified in Image J software v1.52a and the relative number of red pixels were analysed among the different treatment groups.\u0026nbsp;Statistical analysis was performed using GraphPad Prism v7.0. To determine whether the data was normally distributed, a normality test was performed using the Shapiro-Wilks test. To describe the differences between two groups, a Mann Whitney t-test was used. A three-way ANOVA (analysis of variance) followed by Fishers LSD \u003cem\u003epost hoc\u003c/em\u003e test was used to describe the differences between three/or more groups and to determine the relationship between the three variables present in this study, namely, diet, disease and treatment. The results were reported as mean \u0026plusmn; standard error of the mean (SEM) and \u003cem\u003ep \u0026lt;\u003c/em\u003e 0.05 was considered statistically significant. Histological experiments were conducted with biological repeats of n = 4-5 and technical repeats of n = 2.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEstablishing a diet-induced obesity phenotype\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo establish whether obesity was induced, differences in body weights were determined between mice fed a standard diet (SD) and high fat diet (HFD) for eight weeks, prior to tumour inoculation and DXR treatment (Fig. 2). \u0026nbsp; After eight weeks on their respective diets, mice fed a HFD (19.64 g \u0026plusmn; 0.363 g) showed statistically significant higher body weights compared to mice fed a SD (17.76 g \u0026plusmn; 0.175 g) (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001)\u003cstrong\u003e\u0026nbsp;(\u003c/strong\u003eFig. 2). We therefore conclude that the DIO phenotype was established after 8 weeks on their respective diets and continued throughout the study. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHepatic tissue weight and hepatic hypertrophy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice in the HFD+NT+V group showed a significantly higher hepatic tissue weight compared to the SD+NT+V mice (p = 0.0406) (Fig. 3). The HFD+NT+V mice also showed a significantly higher hepatic tissue weight compared to the SD+T+V mice (p = 0.0218). Mice in the HFD+NT+DXR group showed a significantly higher hepatic tissue weight compared to mice in the SD+T+V group (p = 0.0400) (Fig. 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHepatotoxicity: Apoptotic cell death and ALT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the purpose of this study, we focussed on the three objectives namely, 1) the effects of obesity on hepatotoxicity, 2) the effects of DXR treatment on hepatotoxicity, and 3) the effects of obesity on the outcomes of DXR-induced hepatotoxicity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eApoptotic cell death\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether apoptosis was induced in the hepatic tissue samples, western blot experiments were performed to compare the protein expression levels of different apoptotic markers between the different groups: caspase-9, caspase-8, cleaved caspase-8, caspase-3, cleaved caspase-3, and cleaved PARP. For the intrinsic apoptotic pathway, no significant differences were observed in caspase-9 protein expression between the different groups (results not shown) (Fig. 4). In the extrinsic pathway, the ratio between cleaved caspase-8 and caspase-8 showed a statistically significant decrease in the HFD+NT+V group compared to the SD+NT+V (control group) (p = 0.0218) (Fig. 5). The ratio between cleaved caspase-8 and caspase-8 showed a highly statistically significant decrease in the HFD+T+DXR compared to the SD+T+DXR group (p = 0.0011) (Fig. 5). We also assessed the effector caspase, caspase-3, where no significant differences were observed between the treatment groups (Fig. 4). Furthermore, no significant differences were observed in cleaved PARP protein expression between the different treatment groups (results not shown) (Fig. 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHistopathology of hepatic tissue (H\u0026amp;E staining)\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing the accredited NAFLD activity score (Table 3), macrovesicular steatosis was observed in the HFD+NT+DXR group with \u0026gt;33%-66% steatosis present (grade 2). The HFD+T+DXR group showed extensive hepatocyte ballooning and sinusoidal dilation with \u0026gt;66% steatosis present (grade 3) compared to the SD+NT+V (control group) where areas of steatosis alternated with areas of normal morphology (Fig. 6). Macrovesicular steatosis were therefore observed in the HFD+T+DXR group (indicated in red arrow) accompanied with mild inflammation (indicated in white arrow) and sinusoidal dilation (indicated in yellow arrow) and congestion. Vacuoles of lipid are accumulating in the hepatocytes in the HFD+T+DXR group and are displacing the nucleus to the cell\u0026rsquo;s periphery (indicated in red arrow). Inflammatory foci (mild inflammation) were observed in the SD+T+V, HFD+NT+V and HFD+T+DXR groups (indicated in white arrow) with \u0026lt;2 foci per 200 x field (grade 1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3:\u0026nbsp;\u003c/strong\u003eLiver pathology evaluated by the nonalcoholic fatty liver disease activity score. From Y. Takahashi \u0026amp; T. Fukusato, 2014; Kleiner \u003cem\u003eet al\u003c/em\u003e., 2005.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" valign=\"top\" width=\"100%\"\u003e\n \u003cp\u003e\u003cstrong\u003eComponents of nonalcoholic fatty liver disease activity score\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003eItem\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"34.93589743589744%\"\u003e\n \u003cp\u003eDefinition\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"31.73076923076923%\"\u003e\n \u003cp\u003eScore\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"33.333333333333336%\"\u003e\n \u003cp\u003eSteatosis\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eLobular inflammation\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eBallooning\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"34.93589743589744%\"\u003e\n \u003cp\u003e\u0026lt;5%\u003c/p\u003e\n \u003cp\u003e5% - 33%\u003c/p\u003e\n \u003cp\u003e\u0026gt;33% - 66%\u003c/p\u003e\n \u003cp\u003e\u0026gt;66%\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNo foci\u003c/p\u003e\n \u003cp\u003e\u0026lt;2 foci per 200 x field\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e2-4 foci per 200 x field\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026gt;4 foci per 200 x field\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eNone\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eFew balloon cells\u003c/p\u003e\n \u003cp\u003eMany cells/ prominent ballooning\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"31.73076923076923%\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eMacrovesicular steatosis was minor in the HFD+NT+V group (indicated in red arrow) and accompanied with mild inflammation (indicated in white arrow). Macrovesicular steatosis was moderate in the HFD+NT+DXR group (indicating in red arrow), which indicates that DXR aggravates the toxicity in hepatic tissue (Fig. 6). Macrovesicular steatosis was minor in the HFD+T+V group (indicated in red arrow). Microvesicular steatosis with \u0026lt;5% steatosis within hepatocytes occurred in the SD+NT+V control group (grade 0), accompanied with no inflammatory foci (grade 0) and none ballooning present ((grade 0) (indicated in black arrow). Microvesicular steatosis with \u0026gt;33%-66% steatosis within hepatocytes were present in the SD+T+DXR group (grade 2), accompanied with no inflammatory foci (grade 0) and few balloon cells (grade 1) (indicated in black arrow). Macrovesicular steatosis with 5%-33% steatosis within hepatocytes (grade 1), accompanied with inflammatory foci (grade 1) (indicated in white arrow) and non-ballooning were observed in the HFD+NT+V group, where areas of steatosis \u0026gt;33%-66% were observed in the HFD+NT+DXR group with no inflammatory foci present (grade 0). Macrovesicular steatosis 5%-33% were observed within hepatocytes in the HFD+T+V group (grade 1) (indicated in red arrow) (Table 3). To summarise; our results demonstrated that macrovesicular steatosis was observed in mice fed a HFD and microvesicular steatosis was observed in mice fed a SD.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOil Red O staining\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the presence of lipid accumulation within hepatocytes, Oil Red O staining was performed (Fig. 8). To detect the amount of lipid droplets among the different treatment groups, the relative number of red pixels were analysed using Image J (Fig. 7). This method was used by Masone \u003cem\u003eet al\u003c/em\u003e., 2018 \u0026amp; Gojanovich \u003cem\u003eet al\u003c/em\u003e., 2016 to accurately quantify and analyse size distribution of cellular lipid droplets. The HFD+T+V group showed a highly statistically significant increase in the relative number of red pixels (lipid droplets) compared to the SD+T+V group (p = 0.0057). The HFD+T+DXR group indicates a highly statistically significant increase in the relative number of red pixels (lipid droplets) compared to the SD+T+DXR (p = 0.0023) and the HFD+NT+V control group (p = 0.0318). Hepatocytes stain amphophilic and lipid droplets are stained red (indicated in black arrows) (Fig. 8). High levels of lipid accumulation was observed within hepatocytes in the HFD groups. Furthermore, lipid accumulation was significantly increased in the HFD group treated with DXR and the tumour present, which indicates that lipid accumulation was more severe in the HFD+T+DXR group compared to the SD+T+DXR group (Fig. 8).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMasson\u0026rsquo;s trichrome stain\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate liver damage, collagen fibres are stained blue using Masson\u0026rsquo;s trichrome stain to determine the presence of fibrosis. No significant fibrosis was observed within the treatment groups, and therefore, no trends or statistical significance could be determined (Fig. 9). Images were qualitatively represented.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003eEstablishment of an obese model following a high fat diet\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDXR is an effective chemotherapeutic agent used for the treatment of breast cancer. However, DXR may cause side-effects and induce cardiotoxicity, myelosuppression, and skeletal muscle atrophy (Hiensch \u003cem\u003eet al.\u003c/em\u003e, 2020; Zhao \u0026amp; Zhang, 2017). It has been shown that DXR can also induce hepatotoxicity (Bengaied \u003cem\u003eet al.\u003c/em\u003e, 2017; Song \u003cem\u003eet al.\u003c/em\u003e, 2019). The mechanisms and effects of DXR on hepatic tissue and the contributing role of obesity in breast cancer patients are not fully elucidated. Therefore, the aim of this study was to investigate the effects of doxorubicin on hepatic tissue in an obese tumour-bearing mouse model.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn animal models, a HFD is associated with an increase in body mass (BM) (Noeman \u003cem\u003eet al\u003c/em\u003e., 2011; Park \u003cem\u003eet al\u003c/em\u003e., 2012; Schultz \u003cem\u003eet al\u003c/em\u003e., 2012; Layman \u003cem\u003eet al\u003c/em\u003e., 2019) and liver mass, which was observed in the HFD groups in this study. To establish whether obesity was induced, differences in body weight was evaluated for mice fed a SD or HFD for eight weeks prior to tumour inoculation and DXR treatment (Fig. 1). The body weights of mice in the HFD group were significantly higher compared to the SD group (Fig. 1), which confirmed that the obese phenotype was successfully established in our \u003cem\u003ein vivo\u003c/em\u003e model. Our results correlate with similar findings of a study done by Lee \u003cem\u003eet al.\u003c/em\u003e (2019) and Santander \u003cem\u003eet al\u003c/em\u003e. (2015) who indicated that a HFD increased body weight, as a result of increased fat mass in C57BL/6 mice by evaluating body composition. It has been shown that the C57BL/6 mice strain is highly susceptible to the development of DIO and therefore correlates with the findings of our study (Chu \u003cem\u003eet al.\u003c/em\u003e, 2017). Furthermore, the DIO phenotype observed in mice fed a HFD was corroborated by a significantly higher hepatic tissue weights observed in the HFD+NT+V and HFD+NT+DXR groups compared to the SD+NT+V and SD+T+DXR groups, respectively (Fig. 3). Our results correlate with a study conducted by Jung et al., 2013 where C57BL/ mice fed a HFD for nine-weeks resulting in an increase in BM and liver mass gain.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHepatotoxicity: Evaluation of apoptotic cell death and ALT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDXR induces oxidative stress and is characterized by accumulation of reactive oxygen species (ROS), which decrease antioxidant defence systems, resulting in oxidative damage of DNA and apoptotic cell death (Song \u003cem\u003eet al.\u003c/em\u003e, 2019). It has been shown that oxidative stress is a major contributor of DXR-induced hepatotoxicity (Bengaied \u003cem\u003eet al.\u003c/em\u003e, 2017; Rashid \u003cem\u003eet al.\u003c/em\u003e, 2013). Obesity is associated with low grade chronic inflammation, excessive ROS production and oxidative stress. Together, both the obese state and DXR would be expected to upregulate ROS, which results in the downstream activation of apoptosis in hepatic tissue.\u0026nbsp;To determine whether apoptosis was induced in the hepatic tissue samples, western blot experiments were performed to compare the protein expression levels of different apoptotic markers, such as caspase-9, caspase-8, cleaved caspase-8, caspase-3, cleaved caspase-3 and cleaved PARP in the different experimental groups. To determine whether liver damage occurred, we also assessed the expression levels of ALT in the hepatic tissue samples by performing western blotting.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNo significant differences in caspase-9 were observed between the different treatment groups (Fig. 4), which indicates that apoptosis was not induced through the intrinsic pathway.\u0026nbsp;The ratio between cleaved caspase-8 and caspase-8 showed a statistically significant decrease in the HFD+NT+V group compared to the SD+NT+V (control group). The ratio between cleaved caspase-8 and caspase-8 showed a highly statistically significant decrease in the HFD+T+DXR compared to the SD+T+DXR group (Fig. 5). No significant differences were observed in caspase-3 and cleaved caspase-3 protein expression between the different treatment groups (Fig. 4). Upregulation of cleaved caspase-8 in mice fed a standard diet does not indicate that apoptotic cell death occurred within the obese tumour-bearing mouse model and could be an indication of protection during liver regeneration. Freimuth et al., 2013 showed that during liver regeneration, caspase-8 consist of a non-apoptotic function by balancing receptor-interacting protein 1 (RIP1), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and Jun N-terminal kinase (JNK) activation. Loss of caspase-8 therefore triggers NF-kB activation and induce liver regeneration. There were no significant differences observed in PARP cleavage between the different groups (Fig. 4).\u0026nbsp;Since PARP cleavage is a down-stream event of caspase-3 cleavage during apoptosis, differences in PARP cleavage might have been observed at a later time point of tissue collection in the obese tumour-bearing mouse model. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHFD and DXR promote macrovesicular steatosis in hepatocytes, but not fibrosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs previously mentioned, the first structural changes that occurs when the liver is damaged is hydropic changes, where fluid filled vacuoles accumulates within the hepatocytes. It has been shown that a HFD can induce lipid accumulation via a process known as hepatic steatosis (Layman \u003cem\u003eet al.\u003c/em\u003e, 2019; VanSaun \u003cem\u003eet al.\u003c/em\u003e, 2009). Microvesicular steatosis was observed in the SD groups since hepatocytes are filled with tiny lipid droplets and the nucleus is located centrally in the cell. Macrovesicular steatosis was observed in the HFD groups where large fat droplets occupy the cytoplasm of hepatocytes, pushing the nucleus to the periphery (Fig. 6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFrom the non-alcoholic fatty liver disease activity score (table 3),\u0026nbsp;\u0026gt;66% steatosis was present within hepatocytes in the HFD+T+DXR group accompanied with lobular inflammation (score 2) and prominent ballooning (score 2), sinusoidal dilation and congestion, fatty infiltration, and ballooning of hepatocytes\u0026nbsp;compared to the SD+NT+V (control) group where areas of steatosis alternated with areas of normal morphology (Fig. 6). In the SD+T+DXR group, micro-vesicular steatosis was observed, with 5%-33% steatosis in the hepatocytes. In the HFD+NT+DXR group, \u0026gt;33%-66% of the hepatocytes had steatosis compared to the HFD+NT+V group. This indicates that DXR promotes hepatic steatosis in obesity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur histopathology observations correlate with previous studies conducted where a HFD and DXR treatment induced macrovesicular steatosis, ballooned hepatocytes, inflammatory cell infiltration and sinusoidal dilation, respectively (El-Sayyad \u003cem\u003eet al.\u003c/em\u003e, 2009; Layman \u003cem\u003eet al.\u003c/em\u003e, 2019; Rashid \u003cem\u003eet al.\u003c/em\u003e, 2013; Santander \u003cem\u003eet al.\u003c/em\u003e, 2015). No significant fibrosis was observed in our experimental groups (Fig. 9). This indicates that the liver did not progress to cirrhosis, indicative of end-stage liver disease and that the highly metabolic organ had the capacity to regenerate itself or reverse the harmful stimulus in the broad spectrum of liver disease. This could also be attributed to the concentration and frequency of the DXR treatment as observed by other studies (El-Sayyad \u003cem\u003eet al.\u003c/em\u003e, 2009).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHFD promotes lipid accumulation within hepatocytes and induce hepatotoxicity in combination with DXR treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe liver plays an important role in lipid metabolism and stores fatty acids in the form of triglycerides. It has been shown that a HFD promotes lipid accumulation within hepatocytes via hepatic steatosis resulting in disturbances in lipid metabolism (Layman \u003cem\u003eet al.\u003c/em\u003e, 2019; VanSaun \u003cem\u003eet al.\u003c/em\u003e, 2009). Due to image evaluation of Oil Red O-stained fresh tissue sections, our results indicated that the relative number of red pixels are highly significantly increased in the HFD groups compared to mice fed a SD (Fig. 7). In the present study, the relative number of red pixels are highly significantly increased in the HFD+T+DXR compared to the SD+T+DXR group (Fig. 7). Our results correlate with previous studies conducted by Layman et al., 2019 \u0026amp; Tsuru et al., 2020 where a HFD induced hepatic lipid accumulation and inflammation within hepatocytes of mice fed a HFD, resulting in hepatic steatosis. It is evident from our H\u0026amp;E and Oil Red O results that lipid accumulation was elevated in the HFD groups and specifically severe in the HFD+T+DXR group, which indicates that obesity and the tumour present in combination with DXR treatment induced hepatic steatosis, which is a hallmark of NAFL (Fig. 6 \u0026amp; 8). We therefore conclude that hepatic steatosis (\u0026gt;66% steatosis) occurred in the obese tumour-bearing mouse model, resulting in NAFLD. Our findings correlate with a study done by Layman et al., 2019 where mice fed a HFD induced hepatic steatosis, within hepatocytes.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, we have shown that DXR activated the extrinsic pathway of apoptosis in the livers of tumour-bearing mice which were fed a standard diet compared to the mice which were fed a high fat diet, where apoptosis was not initiated. Our histopathology results indicated that hepatic tissues of mice fed a high fat diet and treated with DXR display severe histological damage, such as macrovesicular steatosis, sinusoidal dilation, and lobular inflammation. This indicates that more hepatic toxicity occurred in mice fed a high fat diet and treated with DXR. We therefore conclude that hepatic steatosis, a hallmark of NAFL occurred in the obese tumour-bearing mouse model. There was no evidence of fibrosis which indicates that the livers did not progress to cirrhosis. This study showed that hepatotoxicity is aggravated in obesity as an underlying co-morbidity in breast cancer patients receiving adjuvant chemotherapy, such as DXR. It has been shown that obesity is associated with poor clinical outcomes in patients receiving neo-adjuvant chemotherapy treatment regimens (Iwase \u003cem\u003eet al.\u003c/em\u003e, 2016). Lower doses of DXR are prescribed for obese cancer patients to reduce side effects and adverse toxicities, which could compromise drug efficacy and contribute to the development of resistance (Iwase \u003cem\u003eet al.\u003c/em\u003e, 2016; Mentoor \u003cem\u003eet al.\u003c/em\u003e, 2018; Sarfati, Koczwara \u0026amp; Jackson, 2016)\u003cem\u003e.\u0026nbsp;\u003c/em\u003eThis study, therefore, produce preliminary safety data on the effects of DXR and the contributing role of obesity on hepatotoxicity.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eALT: Alanine transaminase; ANOVA: Analysis of variance; AST: Aspartate aminotransferase; BM: Body Mass; DNA: Deoxyribonucleic acid; DIO: Diet-induced obesity; DXR: Doxorubicin; ECL: Electrochemiluminescence; FFPE: formalin fixed paraffin embedded; HBSS: Hank\u0026rsquo;s balanced salt solution; H\u0026amp;E: Haematoxylin \u0026amp; eosin; HFD: High fat diet; IVC: Individually ventilated cages; IL-6: Interleukin-6; JNK: Jun N-terminal kinase; MCP-1: Monocyte chemoattractant protein-1; NAFLD: Non-alcoholic fatty liver disease; NAS: Non-alcoholic fatty liver disease activity score; NF-kB: Nuclear factor kappa-light-chain-enhancer of activated B cells; NT: Non-Tumour; \u0026nbsp;PVDF: Polyvinylidene difluoride; RIP1: Receptor-interacting protein 1; \u0026nbsp;RIPA: Radio-immunoprecipitation assay buffer; ROS: Reactive oxygen species; SD: Standard diet; SDS: Sodium dodecyl sulfate; TBS-T: Tris-buffered saline tween; T: Tumour; Top 2: Topoisomerase II\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to acknowledge Bianca Bock and Zaakiyah Emjedi for performing the animal model and Mr. Reggie Williams for assistance with the histology staining and Dr. Chantelle Venter for guidance with the histology Oil red O quantification.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have read and approved the manuscript. MS performed the western blot techniques, analysed, and interpreted the results as well as the histological results. MS was also a major contributor in writing the manuscript. CC co-supervised this study and reviewed the final manuscript. LDJ reviewed the final manuscript. AME supervised this study and reviewed the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by research grants from the Cancer Association of South Africa (CANSA); South African Medical Research Council (SAMRC); and the National Research Foundation (NRF).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analysed during the current study are not publicly available due datasets being too large to include but are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical clearance for the \u003cem\u003ein vivo\u003c/em\u003e study was obtained from the Stellenbosch University Ethical Committee (no.\u0026nbsp;SU-ACUM13-00015 and no. ACU-2020-14751). Institutional and international ethical guidelines were applied with respect to the handling of experimental animals. All methods were performed in accordance with the relevant guidelines and regulations of the Animals (Scientific Procedures) Act 1986.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare they have no competing interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of Physiological Sciences, Faculty of Science, Stellenbosch University, Stellenbosch 7600, South Africa. \u003csup\u003e2\u003c/sup\u003eDepartment of Global Health, Faculty of Medicine and Health Sciences, African Cancer Institute, Stellenbosch University, Cape Town 8000, South Africa. \u003csup\u003e3\u003c/sup\u003eDivision of Anatomical Pathology, Stellenbosch University and National Health Laboratory Service (NHLS), Tygerberg Hospital, Cape Town 8000, South Africa. \u003csup\u003e4\u003c/sup\u003eAnatomical Pathology, PathCare, Cape Town, South Africa\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAndo, S., Gelsomino, L., Panza, S., Giordano, C., Bonofiglio, D., Barone, I \u0026amp; Catalano, S. 2019. 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Dose translation from animal to human studies revisited. \u003cem\u003eThe FASEB Journal.\u003c/em\u003e 22(3): 659-661.\u003c/li\u003e\n \u003cli\u003eReagan, W.J., Yang, R.Z., Park, S., Goldstein, R., Brees, D. \u0026amp; Gong, D.W. 2012. Metabolic adaptive ALT isoenzyme response in livers of C57/BL6 mice treated with dexamethasone. \u003cem\u003eToxicologic pathology\u003c/em\u003e. 40(8):1117-1127.\u003c/li\u003e\n \u003cli\u003eRen, X., Bo, Y., Fan, J., Chen, M., Xu, D., Dong, Y., He, H., Ren, X., Qu, R., Jin, Y., Zhao, W. \u0026amp; Xu, C. 2016. Dalbergioidin Ameliorates Doxorubicin-Induced Renal Fibrosis by Suppressing the TGF-\u0026beta; Signal Pathway. \u003cem\u003eMediators of inflammation\u003c/em\u003e. 1-30.\u003c/li\u003e\n \u003cli\u003eRob C.M, Van Kruisjsdijk C.M, Van der Wall E, Frank L.J, Visseren L.J. 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Histopathology of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. \u003cem\u003eWorld Journal of Gastroenterology.\u0026nbsp;\u003c/em\u003e20(42): 15539-15548.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eTomayko, M.M. \u0026amp; Reynolds, C.P. 1989. Determination of subcutaneous tumor size in athymic (nude) mice. \u003cem\u003eCancer chemotherapy and pharmacology\u003c/em\u003e. 24(3):148-154.\u003c/li\u003e\n \u003cli\u003eUlman, E.A. 2011. The \u0026ldquo;Original\u0026rdquo; High-Fat Diets for Diet Induced Obesity. \u003cem\u003eProducuct Dat - DIO series diets\u003c/em\u003e. 1-3.\u003c/li\u003e\n \u003cli\u003eVanSaun, M.N., In, K.L., Washington, M.K., Matrisian, L. \u0026amp; Gorden, D.L. 2009. High fat diet induced hepatic steatosis establishes a permissive microenvironment for colorectal metastases and promotes primary dysplasia in a murine model. \u003cem\u003eAmerican journal of pathology\u003c/em\u003e. 175(1):355-364.\u003c/li\u003e\n \u003cli\u003eZhao, L. \u0026amp; Zhang, B. 2017. Doxorubicin induces cardiotoxicity through upregulation of death receptors mediated apoptosis in cardiomyocytes. \u003cem\u003eScientific reports\u003c/em\u003e. 7(October 2016):1-11.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Obesity, Breast cancer, Doxorubicin, Apoptosis, Non-alcoholic fatty liver disease, Hepatotoxicity","lastPublishedDoi":"10.21203/rs.3.rs-1473640/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1473640/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eBreast cancer is a major health burden for women, worldwide. Lifestyle-related risk factors, such as obesity and being overweight, have reached epidemic proportions and contributes to the development of breast cancer. Doxorubicin (DXR) is a chemotherapeutic drug commonly used to treat breast cancer, and although effective, may cause toxicity to other organs. The mechanisms and effects of DXR on hepatic tissue, and the contributing role of obesity, in breast cancer patients are poorly understood. The aim of this study was therefore to investigate the effects of doxorubicin on hepatic tissue in an obese tumour-bearing mouse model. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e A diet-induced obesity (DIO) mouse model was established, where seventy-four three-week-old female C57BL/6 mice were divided into two main groups, namely the high fat diet (containing 60% kcal fat) and standard diet (containing 10% kcal fat) groups. After eight weeks on their respective diets, the DIO phenotype was established, and the mice were further divided into tumour and non-tumour groups. Mice were subcutaneously inoculated with E0771 triple negative breast cancer cells in the fourth mammary gland and received three doses of 4 mg/kg DXR (cumulative dosage of 12 mg/kg) or vehicle treatments via intraperitoneal injection. The expression levels of markers involved in apoptosis and alanine aminotransferase (ALT) were compared by means of western blotting. To assess the pathology and morphology of hepatic tissue, haematoxylin and eosin staining was performed. The presence of fibrosis and lipid accumulation in hepatic tissues were assessed with Masson’s trichrome and Oil Red O staining, respectively. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eOur western blot results indicated that a significant increase in the ratio of cleaved caspase-8 and caspase-8 protein expression was observed in the standard diet tumour-bearing mice treated with DXR compared to the high fat diet tumour-bearing mice treated with DXR. Microscopic examination of liver tissue showed significant changes in the high fat diet tumour-bearing mice treated with DXR, consisting of macrovesicular steatosis, hepatocyte ballooning and lobular inflammation, compared to the standard diet tumour-bearing mice treated with DXR and the control group (standard diet mice). These changes are the hallmarks of non-alcoholic fatty liver disease, associated with obesity.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eOur results suggest that DXR activated the extrinsic apoptotic pathway in tumour-bearing mice on the standard diet. The histopathological findings indicated that DXR caused significant hepatic parenchymal injury in the obese tumour-bearing mouse model. Hepatotoxicity is aggravated in obesity as an underlying co-morbidity. It has been shown that obesity is associated with poor clinical outcomes in patients receiving neo-adjuvant chemotherapy treatment regimens.\u003c/p\u003e","manuscriptTitle":"Obese mammary tumour-bearing mice are highly sensitive to doxorubicin-induced hepatotoxicity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-03-31 15:30:11","doi":"10.21203/rs.3.rs-1473640/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2022-06-10T05:38:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-06-07T16:59:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-05-04T10:37:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"6ef80e95-b268-40ef-abb5-24ff7e26f1ff","date":"2022-03-31T20:16:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2bccbebe-3e8a-4b0c-a8fc-a6fdbbc79e99","date":"2022-03-30T15:37:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-03-30T14:56:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-03-30T13:52:12+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2022-03-30T08:13:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-03-30T08:09:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2022-03-21T11:54:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e130d610-bd8c-48e7-aacc-5881f012b309","owner":[],"postedDate":"March 31st, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-10-07T05:44:24+00:00","versionOfRecord":[],"versionCreatedAt":"2022-03-31 15:30:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1473640","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1473640","identity":"rs-1473640","version":["v1"]},"buildId":"rHA-KDH7Qsr4HCuvH75dn","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}