Amelioration of Doxorubicin-Induced Cognitive Impairment by Quercetin in a Rat Model of Breast Cancer

The management of breast cancer by chemotherapeutic agents has significantly increased the survival rates and, at the same time, raised concerns about the side effects caused by these agents on healthy tissues. Chemotherapy-induced cognitive impairment resulting from non-CNS malignancies like breast cancer has emerged as a significant challenge among cancer survivors due to its negative impact on the quality of day-to-day life activities. Using doxorubicin-based chemotherapy as a preclinical model for chemotherapy-induced cognitive impairment, we assessed the effect of quercetin on behavioral memory alterations in tumor-bearing female rats in vivo and changes in neurite length and apoptosis in PC12 cell lines in vitro. Quercetin is purported to have neuroprotective effects in different preclinical models of human neurological conditions because of its possible anti-inflammatory and antioxidant properties. Mammary carcinoma was induced by intraperitoneal administration of N-methyl-N-nitrosourea followed by doxorubicin administration once in 5 days (50 days). Pre-treatment with quercetin began 1 week before the chemotherapy and continued till the end of the chemotherapy cycle. Mechanistically, quercetin produced protection against doxorubicin-induced neurotoxicity by decreasing apoptosis and had a neurogenic effect evidenced by the prevention of toxicant-induced inhibition of neurite establishment. Quercetin reversed episodic and spatial memory deficits caused by doxorubicin treatment assessed by novel object recognition memory and Morris water maze, respectively. Taken together, our findings suggest that cytotoxic effects of doxorubicin may be a contributor to neurogenic impairment in tumor-bearing animals, leading to memory deficits. Therefore, quercetin could be a promising therapeutic strategy for doxorubicin-related cognitive impairment, thus imparting hope for improved quality of life among cancer survivors.


Introduction
Chemotherapy forms an integral part of cancer treatment. The management of cancer by chemotherapeutic agents has significantly increased the survival rate among breast cancer patients in the last few decades. According to the American Cancer Association, women diagnosed with breast cancer had a survival rate of 91% after 5 years, 84% after 10 years, and 80% after 15 years (Siegel et al. 2020). Cytotoxic chemotherapeutics, which does not discriminate between the cancerous and normal cells, promotes the physiological aging process. The accelerated aging hypothesis proposes that compared to the non-cancer population, cancer/cancer treatment may accelerate early cognitive decline in cancer patients. The different hallmarks of aging include genomic instability, telomere attrition, epigenetic alterations, stem cell exhaustion, mitochondrial dysfunction, cellular senescence, and altered intercellular communication. Consequently, these hallmarks exerted by chemotherapeutics may expose the cells to additional stress and contributes to biochemical and behavioral alterations (Cupit-Link et al. 2017;Chang et al. 2019). Women treated with chemotherapy revealed that the acceleration of aging on an average occurs by 17 years whereas with anthracycline regimen up to 23-27 years (Shachar et al. 2020). Hence, there is a need to understand the correlation of chemotherapy, aging, and cognitive decline among cancer survivors.
The understanding of cognitive impairment in cancer survivors begins from the knowledge that the chemotherapeutic agents are often accompanied by numerous undesirable effects due to their inability to discriminate between healthy and cancerous cells. Among these effects, alterations in the brain due to chemotherapy are seldom studied and reported but have a wide occurrence in cancer survivors. Therefore, dyscognition resulting from chemotherapy is generally termed as chemotherapy-induced cognitive impairment (CICI)/chemobrain, which is manifested mainly as short-and long-term memory problems, delayed mental processing, and impairment in daily function activities (Nguyen and Ehrlich 2020;Peukert et al. 2020). A study performed from the time of diagnosis and after the completion of chemotherapy in breast cancer patients reported that 17-75% of these women showed a cognitive decline (Ahles et al. 2012). A recent study conducted on women with breast cancer showed impairment in processing speed, memory, and attention and was not completely recovered even after the completion of 2 months of chemotherapy (Rodríguez Martín et al. 2020). Neuroinflammation, oxidative stress, and impaired neurogenesis contribute to chemobrain (Wang et al. 2015;Gutmann 2019). Different preclinical studies have elucidated the role of commonly used chemotherapeutic agents in contributing to cognitive impairment and how various inflammatory, oxidative, and neuronal markers are altered as a result of these agents in healthy and tumor-bearing animals (John et al. 2022).
Doxorubicin (DOX) is a widely used chemotherapeutic drug for cancers of the breast, ovary, prostate, thyroid, and Hodgkin's disease. The cytotoxic effects of DOX adversely affect the normal cells resulting in the toxicities of the heart, liver, kidneys, and brain (Carvalho et al. 2009). Although DOX is not permeable to the blood-brain barrier (BBB), the peripheral cytokines produced due to its cell toxicity have an indirect neurotoxic effect. It exerts its anticancer effect mainly by three processes: intercalating into DNA, affecting cancer proliferation, topoisomerase inhibition, and generating reactive oxygen species (El-Agamy et al. 2019). These processes damage vital biomolecules and result in cell death. But on the contrary, different other studies show the presence of DOX in brain tissues after intravenous administration (Nakagawa et al. 1996;Sardi et al. 2013). The accumulation of DOX in the brain results in toxicity and the death of neurons through the hindrance of the antioxidant mechanisms. So, the generation of inflammatory cytokines (TNF-α) within the brain and indirect effects (secondary) contributes to the disruption of synaptic plasticity, impairment of long-term potentiation, and results in difficulties associated with memory functions. Apart from this, neurotransmitter levels, neurogenesis, p38 MAPK signaling pathway, epigenetics, and genetic factors are also altered by the administration of DOX affecting cognitive abilities (El-Agamy et al. 2019). Taking into account the available literature, understanding the role of DOX is still required to elucidate the molecular pathways underlying its dyscognitive effects in cancer patients/survivors.
Cognitive performance of DOX-treated breast cancer patients found in a meta-analysis study showed impaired cognitive function affecting their quality of life compared to healthy controls across different cognitive modalities like executive function, language, processing speed, and memory (Eide and Feng 2020).
Non-availability of proper treatment options for chemobrain raises concerns among cancer survivors. Different epidemiological studies have highlighted the potential of flavonoids in various neurodegenerative disorders (Beking and Vieira 2019; Maher 2019). Flavonoids modulate various signaling pathways like ERK and PI3-kinase/Akt and possess neuroprotective actions. They exhibit several pharmacological effects, including long-term potentiation, neuronal differentiation, and synaptic plasticity, all of which have been demonstrated to increase cognitive function (Bakoyiannis et al. 2019). Quercetin (1) is a naturally available phytochemical and is regarded as an essential flavonoid in our diet. It has a broad spectrum of properties against inflammation and cancer (Ezzati et al. 2019;Vafadar et al. 2020). Several studies have reported the neuroprotective potential of quercetin in animal models (Khan et al. 2019;Zhang et al. 2020). It protects the brain tissues from oxidative stress by preventing hyperoxide formation and free radical scavenging (Costa et al. 2016). It was found to attenuate neuronal damage in different neurodegenerative disorders like Huntington's disease, amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease by suppressing NF-κB and other neuroinflammatory activators and reverse neuronal and behavioral aging (Salehi et al. 2020).
No previous research has looked into quercetin's ability to protect cancer animals from DOX chemotherapy and its effects on episodic and spatial memory paradigms. So, here, we used various behavioral tests and in vitro parameters to investigate the effect of quercetin on memory function in a rat chemobrain model.

Animals
Female Sprague Dawley (SD) rats (180-220 g) (30-35-dayold) were acquired from Central Animal Research Facility (CARF), Manipal Academy of Higher Education, Manipal, Karnataka, India. The animals were housed in a temperature (23 ± 2 °C) and humidity (50 ± 5%) regulated environment in sterile polypropylene cages with sterilized rice husk as bedding and free access to water and food under regular 12-h light-dark cycles. Throughout the experiment period, all behavioral procedures were performed between the hours of 9:00 and 16:00.

Cell Culture and Maintenance
National Centre for Cell Science (NCCS), Pune, Maharashtra, India, provided rat pheochromocytoma-derived (PC12) neuronal cell lines. Cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) in T-25 culture flasks placed in a CO 2 incubator, supplemented with 10% horse serum and gentamycin (100 μg/ ml) in a humidified atmosphere consisting of 95% air and 5% CO 2 . Here, 0.1% v/v DMSO (dimethyl sulfoxide) diluted with DMEM was used to dissolve quercetin, and DOX was prepared as clear red-colored solution in DMEM. While handling and maintaining cell lines, necessary measures and care were followed in accordance with the institution's procedure.

In Vitro Cell Viability and Dose Assessment Studies
Cell viability was assessed by performing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. After 24 h of adhesion, cells (5×10 3 ) were plated in 96-well plates and treated with quercetin at various doses (ranging from 50 to 500 μM). Cell viability was assessed after 24 h incubation. Two non-toxic concentrations of quercetin (50 and 100 μM) that showed >80% viability were selected for the assessment of the neuroprotective study. And of these two concentrations, the highest effective concentration (100 μM) was selected to determine its effect on neurite length and apoptosis.

In Vitro Neuroprotection Studies
The neuroprotective potential of quercetin against DOXinduced neurotoxicity in PC12 cells was evaluated using the MTT test. After 2 h of incubation with quercetin (50 and 100 μM), DOX (IC 50 1 μM) was added and incubated for additional 24 h. A microplate reader (ELx800; BioTek Instruments Inc., Winooski, VT, USA) was used to quantify the intensity of the purple coloration produced at 540 nm, which directly rates neuronal cell survival.

Assessment of Morphology and Neurite Length in Differentiated PC12 Cells
The neuroprotective ability of quercetin was determined through the examination of morphological features of differentiating and matured neuronal structures. PC12 cells were treated with retinoic acid (10 μM) to induce differentiation into matured neurons. Every 2 days, the media was replaced with fresh media containing 10 μM retinoic acid to induce differentiation into matured neurons for 6 days. After that, the cells were treated for 2 h with 100 μM quercetin before being exposed to DOX and incubated for another 24 h. Neurite length was measured by examining the cells using an inverted microscope (Eclipse TS100F; Nikon Instruments Inc., Melville, NY, USA). Photos were captured at random using ImageJ software (Neuron J plug-in) by the National Institute of Health (NIH).

Detection of Apoptosis in PC12 Cells by AO/EB Staining
Doxorubicin (1 μM) individually and in combination with quercetin (100 μM, 2 h incubation before DOX treatment) was incubated in 6-well plates with differentiated PC12 cells. Wells were washed with phosphate buffer (PBS) after 24 h of incubation, and cells were fixed for 10 min at room temperature with 1 ml ice-cold ethanol (100 %). After another wash with PBS, the cells were stained with 1 ml AO/EB reagent stain and then incubated at 37 °C for 10 min. An inverted fluorescence microscope was used to examine individual cells. The morphology of the cells was assessed using the staining scheme described previously (Ribble et al. 2005). Normal green nuclei were observed in the live cells, and bright green nuclei with fragmented chromatin were observed as early apoptotic cells. Also, orangecolored fragmented chromatin was observed in late apoptotic cells whereas normal, orange-colored nuclei was seen in the necrotic cells.

Experimental Design
Female SD rats were acclimatized in the standard regulated conditions as mentioned earlier. All the animals were subjected to the intraperitoneal treatment of N-methyl-N-nitrosourea (MNU) (50 mg/kg) for the development of mammary cancer. The rats were palpated twice a week for the presence of tumor. Following 8 weeks of injection, the development of breast cancer was established.
After cancer induction, animals were divided into three groups of nine animals per group as follows: Cognitive impairment was induced by the intraperitoneal administration of DOX once every 5 days for 50 days. Quercetin treatment began 1 week before DOX injection and continued until the completion of the chemotherapy cycle. Normal saline was used as the vehicle for dissolving quercetin and MNU. Also, water for injection was used for dissolving doxorubicin throughout the study. The figurative explanation of study design is mentioned in Fig. 1.

Tumor Progression and Assessment
The progression of tumors in the mammary fat pad of the animals was assessed using digital Vernier calipers.

Behavioral Assessment
The assessment of cognitive loss was measured by Morris water maze (MWM) and the novel object recognition test (NORT) after ten cycles of DOX therapy.
Novel Object Recognition Task Assessment of episodic memory was performed using NORT (Ramalingayya et al. 2017). Rats were acclimatized to the laboratory conditions. The test consists of 3 phases: habituation, familiarization, and choice. On day 1, animals were individually habituated in square arenas for 20 min. On day 2, animals were subjected to familiarization and choice trials with an intertrial interval (ITI) of 2 h (time interval was chosen based on pilot studies). In familiarization trial, two similar objects were placed at an equal distance from each other, and each animal was allowed to explore the object for 3 min. During the choice trial, each animal was explored for 3 min in the same environment but by placing one familiar and one novel object. The rats' exploration behavior towards the objects was recorded using a camera (Logitech Pro Quick-Cam 9000) mounted at a height from behavioral observation areas, and time spent by the animals on each object was noted using two handheld stopwatches by a blinded observer. After each trial, the objects and arenas were cleaned using 70% v/v ethanol to remove the olfactory clues. Parameters like exploration time, discriminative index, and recognition index were assessed for determining the animal's memory for the objects.
Morris Water Maze Spatial memory was assessed using MWM (Ramalingayya et al. 2019). The maze consists of a circular water tank divided into 4 quadrants. An escape/hidden platform was placed in the target quadrant. The test consists of two parts: acquisition and retention. Animals were trained for 4 days of acquisition trial with one session of four trials, to find the hidden platform placed in the target quadrant. On the 5th day, the hidden platform was removed, and the animals were allowed to swim freely for 60 s. A video camera coupled to a computerized tracking device (ANYmaze, Ugo Basile SRL, Gemonio, VA, Italy) above the center of the pool was used to analyze the data. Different parameters such as swim speed, path length, escape latency, retention time, and target quadrant latency were evaluated in both the acquisition and retention trials.

Histopathological Analysis
Animals were perfused with 10% neutral buffered formalin (NBF) and tumor; cerebral cortex samples were fixed in NBF and processed in gradient alcohol and xylene, followed by paraffin fixation overnight. Sections were cut using a microtome and processed for regular hematoxylin and eosin (H&E) staining. The sections were then mounted in dibutyl phthalate polystyrene xylene and examined under a light microscope.

Statistical Analysis
Statistical analysis of data was performed using Prism 8.0 (GraphPad Software, San Diego, CA, USA). The data were all reported as mean ± SEM, with p ˂ 0.05 being considered statistically significant at a 95% confidence interval. In cell culture studies, for neurite length and percentage of apoptotic cells, we performed each experiment in triplicates for 3 times. For in vitro study and tumor volume analysis, data were analyzed using unpaired Student's t test and Kruskal-Wallis test followed by Dunn' s post hoc test, respectively. Behavioral parameters in NORT and retention trial data from MWM (n=9) were assessed using Student's paired t test and Kruskal-Wallis test followed by Dunn's post hoc test, whereas acquisition trial was analyzed using two-way ANOVA followed by Bonferroni's post hoc test.

Assessment of Morphology and Neurite Length in Differentiated PC12 Cells
Doxorubicin treatment caused morphological changes in neuronal cells, such as nucleus condensation and cell membrane disintegration. Compared to the medium control, DOX administration suppressed neurite outgrowth in differentiated PC12 cells. On the other hand, treatment with quercetin (100 μM) resulted in considerable neurite outgrowth and reduced the inhibitory impact of DOX (Figs. 2 and Fig. 3A).

Detection of Apoptosis in PC12 Cells by Acridine Orange/Ethidium Bromide (AO/EB) Staining
Doxorubicin promoted apoptotic cell death in PC12 cells, as evidenced by increased proportion of apoptotic cells compared to the media control by AO/EB staining. Prior treatment with quercetin reduced the proportion of apoptotic cells considerably, reversing the toxicant-induced increase in apoptotic cell death (Fig. 3B).

Tumor Volume
A significant decrease in the tumor volume was observed in the treated groups compared to the tumor control group. Further, rats treated with quercetin showed no significant difference in tumor volume as compared to the DOXtreated group (Fig. 4).

Behavioral Assessment
Novel Object Recognition Task Tumor control animals spent significantly more time exploring the novel object than the familiar object. Doxorubicin-treated animals could not discern the difference between known and novel objects, spending about the same amount of time exploring both. Rats given quercetin (50 mg/kg, p.o.) with DOX, on the other hand, were able to distinguish between novel and familiar objects, as demonstrated by a substantial increase in exploration time with the novel object. The recognition and discriminative indices of the quercetin and vehicletreated groups enhanced significantly as compared to the DOX-treated group (Fig. 5) Morris Water Maze There was no significant difference in swimming speed between the treatment groups and the tumor + DOX group throughout the first 4 days of the acquisition experiment (Fig. 6A). On days 3 and 4, however, the tumor control and quercetin treatment groups showed a significant reduction in path length compared to the tumor + DOX group (Fig. 6B). Compared to the tumor + DOX group, the escape latency (time taken to reach the hidden platform) in the tumor control and quercetin treatment groups decreased significantly from day 2 to day 4 (Fig. 6C). On the fifth day of the retention trial, the tumor control and quercetin treatment groups had significantly longer retention time (time spent in the target quadrant) than the tumor + DOX-treated group (Fig. 7).

Histopathological Analysis
Mammary Tumor Tumor control animals showed severe mammary gland carcinogenesis with ductal lobular adenocarcinoma along with highly proliferating epithelial and infiltration of lymphocytes. But the treatment of DOX showed diminished tumor progression with moderate ductal carcinoma with reduced infiltration of leukocytes and proliferation of epithelial cells. Also, the treatment with quercetin showed no signs of tumor aggravation when given along with doxorubicin (Fig. 8).

Cerebral Cortex
The cerebral cortex was unaltered in tumor control animals, whereas DOX-treated animals showed severe neurodegeneration with marked gliosis indicating neuroinflammation. But the pre-treatment with quercetin protected the glial cells from DOX-induced neuroinflammation ( Fig. 8)

Discussion
Cognitive changes related to treatments for breast cancer have long been recognized (El-Agamy et al. 2019;Janelsins et al. 2017). Over the past few decades, studies have proposed that chemotherapy for non-CNS malignancies can result in adverse effects on cognitive abilities, which can finally affect the quality of life among cancer survivors (Ahles et al. 2012;Koppelmans et al. 2012;Eide and Feng 2020). A recent meta-analysis study performed in breast cancer patients for assessing the prevalence of cognitive impairment revealed that chemotherapy appeared to have a more negative influence on cognitive functioning than endocrine therapy and radiotherapy (Dijkshoorn et al. 2021).
The robust anticancer effect of DOX is responsible for its cytotoxic actions in healthy tissues, especially neuronal cells causing cognitive abnormalities. In a clinical setting, rather than a single chemotherapeutic agent, the combination of chemotherapeutic agents is often employed for cancer treatment (Ongnok et al. 2020). So, to assess the role of individual agents in contributing to cognitive impairment, the development of animal models gives a broader picture in understanding the mechanism of action and to verify different treatment strategies (Matsos and Johnston 2019). It is critical to understand the cognitive changes associated with chemotherapy and its impact on cancer patients because of the dramatic increase in the number of long-term breast cancer survivors. We suggest that quercetin, through its neuroprotective actions, may prevent chemotherapy-induced cognitive dysfunction. Quercetin is widely assessed for its neuroprotective and anticancer properties. But its effect on neuronal functions in a cancer environment is not studied before. Even though we have extensively evaluated the effect of rutin against DOX-induced cognitive impairment in our previous study (Ramalingayya et al. 2017(Ramalingayya et al. , 2019, the role of quercetin aglycone alone was not explored. Also, rutin is a derivative of quercetin, and studies comparing the absorption of quercetin and rutin show that the rutin was absorbed more slowly than quercetin. So, this study is a preliminary attempt to understand the impact of quercetin on the chemo brain. Also, we tried to unravel the possible in vitro neuroprotective mechanisms of quercetin in CICI using PC12 neuronal cells. PC12 neuronal cell lines are commonly used in neuroscience research for studies involving neurosecretion, neuroprotection, neurotoxicity, and neuroinflammation (Hu et al. 2018). It was revealed from the in vitro studies that treatment with DOX induced cellular death, whereas the pre-treatment with quercetin produced significant protection against DOXinduced neurotoxicity shown by a decreased percentage of apoptotic cells. In addition, the neuritogenic effect of quercetin was evident by the prevention of DOX-induced inhibition of neurite establishment.
So, with the data obtained from in vitro neuroprotection studies, quercetin was further assessed to determine its efficacy in alleviating DOX-induced cognitive dysfunction in vivo. Quercetin (50 mg/kg, p.o.) was selected based on our previous studies on rutin in the same animal model where we could find that the drug at this dose could produce neuroprotection against DOX-induced cognitive impairment (Ramalingayya et al. 2017). Similarly, other studies also reported the effectiveness of quercetin for its neuroprotective actions in the similar dose (50 mg/kg, p.o.) (Dong et al. 2014;Costa et al. 2016).
We evaluated the extent of episodic and spatial memory deficits induced by DOX in the presence of a tumor and the protective role of quercetin in slowing these cognitive impairments using NORT and MWM, respectively.
Data from the tumor parameters showed no substantial difference in the tumor volumes of DOX and quercetintreated groups. This clearly signifies that the pre-treatment with quercetin did not compromise the anticancer potential of DOX with quercetin. Also, histological analysis of the tumor samples revealed the formation of ductal and lobular carcinomas with leukocyte infiltration, which was reduced in doxorubicin alone and combination treatment with quercetin treatment groups. In addition, the pathological analysis of the cerebral cortex showed that quercetin reversed DOX-induced structural alterations and neuroinflammation revealed by a marked reduction in the gliosis. This is in accordance with the literature evidence which reveals that DOX causes the elevation of pro-inflammatory cytokines and disturbs the BBB and initiating the inflammatory cascade leading to neuroinflammation. The infiltration of inflammatory markers through BBB disrupts the antioxidant defense mechanism of the brain shown by the death of neurons, impaired long-term potentiation, and synaptic plasticity which together contributes to the cognitive dysfunction (Tangpong et al. 2011;John et al. 2021). Moreover, this interlinks the involvement of DOX in the hippocampal-and cortex-dependent dyscognition resulting in reduced neurogenesis and continuous microglial activation (Allen et al. 2019).
Morris water maze assessed spatial learning and memory consisting of acquisition and retention trials. The DOXtreated group's path length grew significantly on days 3 and 4 of the acquisition trial, indicating that animals were unable to acquire the spatial orientation of the escape platform. Rats treated with quercetin demonstrated a substantial improvement in learning behavior, as seen by a decrease in the distance travelled and time taken by the animals to reach the escape platform. In the retention trial, a considerable increase in retention time in the tumor control and quercetin groups compared to the DOX group indicates the searching behavior and the restoration of spatial memory, which was found to be damaged in the DOX group.
Episodic memory was assessed using NORT. It exploits the innate exploratory behavior of the animals. Animals treated with DOX spent nearly equal time exploring both novel and familiar objects during choice trial, indicating that they showed no preference for the novel object and could not discriminate between the objects. Tumor control animals and those pre-treated with quercetin preferred novel objects, indicating that familiar objects already exist in their memory.
The tumor control group did not show any spatial impairment and episodic memory deficit in MWM and NOR tasks. At the same time, they performed similar to quercetintreated group, which indicates that the presence of tumor may not be a contributing factor to cognitive impairment due to chemotherapy. Several studies performed in tumorbearing animals have reported similar results showing that chemotherapy is a major contributing factor for cognitive dysfunction (Ramalingayya et al. 2016(Ramalingayya et al. , 2019.
From the in vitro studies and animal behavioral assessments, it is evident that quercetin ameliorates DOXinduced cognitive impairment. The antioxidant and antiinflammatory properties of quercetin may be responsible for counteracting the cytotoxic action of doxorubicin which disrupts the redox balance leading to oxidative stress. So, adjuvant therapy of quercetin and chemotherapy may alleviate cognitive deterioration implicated by spatial and episodic memory deficits induced by chemotherapeutic agents. In this regard, additional studies are required to assess different biochemical parameters and biomarkers to unravel the molecular pathways underlying chemobrain with special emphasis on accelerated aging and dyscognition and the role of quercetin in reversing chemotherapy-induced cognitive dysfunction in animal models of mammary carcinoma.
Despite the negative effects caused by chemotherapy, it is necessary to acknowledge the countless benefits imparted by the chemotherapeutic agents to cancer patients. At the same time, cancer patients/survivors also deserve better followup and treatment for long-term side effects, which are often unrecognized.