Key findings:
The study investigated the effects of arsenic exposure and high BMI on oxidative stress and health markers in rats, finding increased oxidative stress and immune protein levels, especially in high BMI rats. Quercetin treatment did not mitigate these effects, suggesting a lack of protective effect against arsenic and high BMI-induced health dysfunctions.

What is known and what is new?
Exposure to arsenic and obesity correlate with oxidative stress and health issues, but their severity may vary depending on factors like BMI. This study investigates quercetin's potential to alleviate arsenic-induced health problems in normal and high BMI rats. Results suggest arsenic exacerbates oxidative and immunological dysfunctions, particularly in high BMI rats, with quercetin showing limited effectiveness.

What is the implication, and what should change now?
The study highlights the exacerbated health dysfunctions induced by arsenic exposure and high BMI in rats, despite quercetin administration. These findings underscore the importance of considering multiple factors, such as BMI and oxidative status, in understanding the severity of arsenic poisoning. Future studies should explore alternative interventions to address arsenic and high BMI-induced health complications effectively.
 

Arsenic, the 33rd element of the periodic table is referred to as a heavy metal in the context of toxicology [1], and is classified as human carcinogen (group 1) [2]. Arsenic has become a global health concern, as it is broadly distributed in nature and has been associated with numerous adverse effects which threaten an organism’s health [3], For years, the respiratory, cardiovascular, gastrointestinal, hematological, renal, dermal, reproductive and neurological toxicity of arsenic have been documented [1]. As a natural constituent of the environment, animals are easily exposed to relatively low levels of arsenic through food, air and water [4]. Globally, over 20 million people are chronically exposed to arsenic contaminated water above the safety level of 10µg/l [5]. However, the presence of arsenic is not immediately evident in food, air, or water because arsenic compounds have no colour / smell, hence posing a serious human health hazard due to its toxic nature [6]. Recently, substantial scientific evidence has revealed that low to moderate levels of arsenic from water may lead to the occurrence of a large variety of health dysfunctions, illness, degenerative diseases etc. [4, 7-9]

The primary toxic mechanism of arsenic is not specified, but inflammatory or oxidative dysfunctions have been proposed [10]. Inflammation is involved in the pathogenesis of many degenerative diseases including cardiovascular diseases, metabolic syndrome, chronic kidney and liver diseases and cancer [11,12]. Arsenic has been implicated to induce inflammatory responses and compromise or detrimental to the immune cells [13-15]. This might be the mechanism of arsenic-induced health dysfunction and diseases. Similarly, several in vitro and in vivo studies have revealed that arsenic toxicities were mediated by induction of oxidative stress [15, 16]. Oxidative stress and inflammation are linked in a complex feedback cycle in which reactive oxygen species trigger transcription factors that upregulate the expression of pro-inflammatory cytokines and anti-oxidant enzymes [17].

The extent of arsenic poisoning and susceptibility varies widely from person to person and depends on various factors such as dose, individual susceptibility to arsenic, inter-individual differences in diet, arsenic metabolism, co-exposure, genetics and the age of the individual [6, 18]. However, most of the variability in susceptibility and the influence of lifestyles on arsenic metabolism in humans are not well understood [18] .Although the severity of arsenic toxicity in individual with elevated body mass index (BMI) is not well understood, studies have however revealed that (BMI), an indicator for overall nutritional status, is positively associated with arsenic methylation capacity [19, 20]. and the pathological processes linked to arsenic and obesity such as inflammation, oxidative stress, adipokine expression and insulin resistance are thought to play a role in diseases caused by each. Elevated BMI has been shown to exacerbate oxidative stress and inflammatory responses that has been associated with some types of cancer [21, 22].

Quercetin, a major flavonoid found in edible plants (fruits and vegetables) is one of the most potent antioxidants present in plants and has unique biological properties that may improve mental / physical performances and reduce infection risk [23, 24]. Onion, hot peppers, curly kale, blueberries, apple, tea and broccoli are some of the richest food sources of quercetin [25, 26]. Quercetin is also available and sold as a dietary supplement with daily doses between 200-120000 mg quercetin based on manufacturer’s recommendation [27]. In addition, quercetin has been employed as nutracetical for functional foods within the range of 0.008-0.5% or 10-125 mg/serving [28].

Dietary flavonols such as quercetin have been reported to possess physiological effects including antioxidative [29, 30], anti-inflammatory [31-33], anti-pathogenic [34, 35], anti-viral [24, 36, 37] anti-microbial [38], anti-carcinogenic [39], cardio-protective [23], mitochondrial biogenesis activities [40] and thus provide significant potential in the study of improving mammalian health. It is well established that athletes use flavonoids as antioxidants to enhance endurance and physical performance [41]. In addition, epidemiological studies indicated that the risk factors of cardiovascular diseases in subjects who had a high intake of flavonoids were reduced. Given the interplay between the pathological mechanisms of arsenic and elevated BMI and the anti-oxidative and anti-inflammatory potentials of quercetin, this study therefore, assessed the ameliorative capability of quercetin on arsenic induced oxidative stress and inflammatory responses with respect to body mass index (BMI) in apparently healthy male Wistar rats.
 

Reagents Kits and Chemicals
All reagent kits and chemicals used were of analytical grades and purchased from Central Research Laboratory, Ilorin. Arsenic and Quercetin were purchased from Biobridge Laboratory, Ilorin.

Experimental Animals
Forty eight (48) male Wistar rats of 120-150 g and 235-250 g were obtained from the Animal House, Ladoke Akintola University of Technology, Ogbomoso, Oyo State, Nigeria.

Sodium Arsenite
Sodium arsenite was a product of 1/20 B, Narayan Plaza, 26-A, Chandivali Road, Andheri (E), Mumbai-400072, Maharashsta, India.

Experimental Design
The rats were randomly divided into eight groups of six rats per group based on their body mass index (BMI) as illustrated in Table 1. The rats were grouped into normal BMI (N-BMI) and high BMI (H-BMI), depending on the BMI of 0.45-0.60 (N-BMI) and greater than 0.68 as (H-BMI).

Table 1: Groups of Rats and Doses of Arsenic and Quercetin Administered

Conversion from human dose to animal dose was done using the model by Reagan-Shaw et al.,. (2008) [42]. Arsenic was suspended in distilled water at 40 ppm and the rats were allowed access to the water and feed ad libitum, while quercetin was prepared in saline solution and administered orally at 50 mg/kg body weight with the use of oral cannula. Arsenic exposure was for a period of six (6) weeks before the commencement of quercetin administration for the next four (4) weeks. The administration lasted for ten (10) weeks in all, after which all the rats were sacrificed via mild anesthesia.

Table 2: Organs to body weight ratios of rats administered arsenic and quercetin.

Keys: A-NBMI, B HBMI, C- NBMI + Arsenic, D-HBMI + Arsenic, E-NBMI + Quercetin, F-HBMI + Quercetin, G-NBMI + Arsenic + Quercetin, H-HBMI + Arsenic + Quercetin. Values are means ± SEM, n=4 and mean values bearing different alphabets are significantly different (P<0.05). NBMI (normal body mass index) and HBMI (high body mass index).

Serum
After ten (10) weeks of administration, the rats were fasted overnight and anesthetized using diethyl ether. The chest region was quickly opened and blood was drawn by puncturing the heart using a new syringe for each animal.  The blood samples were collected into plain bottles and centrifuged at 4000 revolutions per minute (rpm) for 10 minutes to separate the serum from the whole blood. The serum which is the supernatant was carefully decanted into sample bottles using a micropipette, dropped into a clean bottle labeled and stored in the refrigerator below 40 C immediately for further analysis.

Organs
Organs of interest (brain, heart and liver) were harvested immediately, cleansed of blood and rinsed with normal saline solution and the weights were recorded. The organs were fixed in 10% formal saline solution for histopathology examination.
 

Assessment of Weight of Rats
Changes in body weight of the experimental rats were monitored on a weekly basis and the harvested organs of interest (brain, liver and heart) were weighed to determine the organ-body weight ratio.

Assessment of Oxidative Status of Rats
The serum concentration of reduced glutathione (GSH) was measured using the method of Ellman (1959). Oxidized-low density lipoprotein cholesterol (Ox-LDL-C) and 8-hydroxydeoxyguanosine (8-OHdG) concentrations were determined by ELISA method as described in the ELISA kit manual.

Assessment of Immunological Proteins in Rats
Interleukin-1 beta (IL-1β), monocyte chemoattractant Protein-1 (MCP-1), and vascular cell Adhesion molecule-1 (VCAM-1) analyses were done using ELISA method as specified in the kit manual based on sandwich immunoassay principle.

Histological Assessment
Histological examination of brain, liver and heart tissues was done according to the method of Avwioro, 2010 [43].

Statistical Analysis
This research work was a completely randomised design (CRD). Results were expressed as mean ± standard error of mean (S.E.M). Data generated were subjected to one way analysis of variance (ANOVA), after which Tukey Test was conducted in order to identify the variation within the treatment group.  P-value <0.05 was regarded as statistically significant and denoted by alphabets.
 

Effect of Arsenic and Quercetin on Weight and Organ to Body Weight Ratio of Rats
Table 1 depicted organ (brain, heart and liver) to body weight ratio of rats administered arsenic and quercetin. There was no significant difference (p>0.05) in the brain to body weight ratio, heart to body weight ratio and liver to body weight ratio across the groups.

Effect of Arsenic and Quercetin on Oxidative Stress Indices
In Figure 1, there were significant increases (p<0.05) in the serum reduced glutathione concentrations in groups D and E rats. In the same vein, the concentration of oxidized-low density lipoprotein cholesterol in the serum was elevated significantly (p<0.05) in groups B, F, G and H (Figure 2). The serum concentrations of 8-hydroxydeoxyguanosine gave significant increases (p<0.05) in rats of group H only (Figure 3).

Effect of Arsenic and Quercetin on Immunological Proteins
The results obtained in the serum interleukin-1β and vascular cell adhesion molecule-1 concentrations gave no significant alterations (p>0.05) across the groups (Figures 4 and 5). However, there was significant increases (p<0.05) in serum the concentrations of monocyte chemoattractant protein-1 in groups B, C, E, F, G and H, but insignificant increase (at p˃0.05) in group D (Figure 6).

Histological Assessment of the Brain, Liver and Heart Tissues
The photomicrographs of the prefrontal cortex of rats administered with arsenic and quercetin in plate 1 (A-H) showed the external granular layer with constituent granular neurons. The normal and intact neuronal cells are depicted with black arrow heads, while the presence of mild vacuoles perceived as histopathological alterations were depicted with red arrows in all the groups (A-H). In Plate 2, the photomicrographs of the heart in rats administered with arsenic and quercetin (A-H) depicted the cardiomyocytes containing the cardiomyocytes nuclei (black arrow). The cellular delineation, cellularity and morphological delineation of groups A,C and E appeared normal, while groups B, D, F, G, and H appeared atypical. The photomicrographs of the liver sections of rats administered arsenic and quercetin is presented in plate 3 (A-H). The central vein, sinusoidal space and hepatocytes are depicted by dotted circles, S, and H respectively. Mild-moderate sinusoidal space dilation and atrophy of the central veins were observed in all the groups (B, E, F, G and H).

In experimental animals, arsenic exposure has been associated with increases in body weight, changes in fat metabolism and deposition, and other related outcomes but the evidence that arsenic is a cause of obesity is not clear [44], hence, the associations between exposure to inorganic arsenic and body mass index (BMI) have been inconsistent. For, instance, Aliyu et al., (2012) [45] showed that arsenic treatment reduced body weight in a concentration dependent manner. In contrast, the findings in this present investigation revealed that arsenic treatment had no effect on the organ (brain, heart and liver) to body weight ratio (Table 2) regardless of the body mass index (BMI) status of the rats or quercetin treatment. The observation in this study is in agreement with the findings of Singh et al., (2017) [46] in which arsenic treatment had no significant effect on body weight and liver to body weight ratio. In addition, certain rodent studies that evaluated lipid lowering effects of quercetin supplementation showed reduction in body weight, serum lipid levels, hepatic lipid accumulation, and/or white adipose tissue mass. However, these effects were not seen in all studies and were sometimes conflicting [47-53]. The trend in the organ (brain, heart and liver) to body weight ratio across the groups (Table 1 and 2) in this present study reflected that quercetin at the administered dose and duration had no effect on the organ (brain, heart and liver) to body weight ratio.

Furthermore, the primary mechanism of arsenic toxicity is not known, but has been associated with oxidative stress and inflammatory response [10, 15], while some other study have also suggested a pathological crosstalk between obesity, oxidative stress, and inflammatory process [54]. Oxidative stress occurs as a result of an imbalance between reactive oxygen species (ROS) and antioxidant defenses, and the inability of the biological system to eliminate free radicals which results in oxidative damage of lipids, proteins and deoxyribonucleic acid (DNA). Consequently, oxidative stress is associated with several pathological implications and might be a major mechanism underlying obesity-related complications [15, 55, 56].

In biological systems, various antioxidant defense systems, including enzymatic and nonenzymatic routes, act to regulate excessive levels of ROS [57]. Reduced glutathione (GSH) which is the most abundant nonprotein sulfhydryl (NPSH) in most cells, acts as a nucleophilic scavenger of free radicals and their metabolites through enzymatic and chemical mechanisms, and plays crucial roles in the protection against oxidative damage caused by ROS either directly as an antioxidant or indirectly by preserving other cellular antioxidants in a functional state [15].  Significant to oxidative stress is that arsenic blocks the generation of glutathione which protects the cell against oxidative damage [58]. Moreover, arsenic is capable of activating the antioxidant system and may increase the expression of antioxidant molecules such as superoxide dismutase, catalase and glutathione that are involved in removal of excess free radicals and peroxides [59]. However, when the level of oxidation overwhelms the capacity of the antioxidant defense system, the level of GSH and other antioxidant molecules will be reduced [15, 60]. Hence, in this present study, the elevation observed in the serum GSH level of rats with high BMI administered arsenic only (group D) (Figure 1) reflected an oxidative progression in which arsenic activated the antioxidant defense system and increased the expression of GSH but the degree of oxidation has not exceeded the antioxidant molecules (buffering capabilities). The oxidative progression might also be an effect of arsenic treatment coupled with the high BMI status of the rats since previous studies implicated elevated BMI in increased oxidative stress conditions [10].

Similarly, various pieces of evidence have revealed that exposure to arsenic increases the morbidity and mortality of cardiovascular diseases [61, 62]. The plasma level of oxidized-low density lipoprotein cholesterol (Ox-LDL-C) has been used as a sensitive marker for oxidative stress in vascular systems [63, 64] and has also been associated with obesity. In 2014, Albuali reported an increase in the level of Ox-LDL-C in obese children, and increased levels of Ox-LDL-C has been linked to increased oxidative stress with lowered antioxidant activities [65]. In this current study, we observed an increase in Ox-LDL-C levels in normal BMI rats administered arsenic and quercetin. This suggests an arsenic-induced oxidative stress progression and might lead to complications in the cardiovascular system. This observation is consistent with the findings of Karim et al., (2013) [66] where it was evident that Ox-LDL-C increased in individuals exposed to arsenic. This opinion is further strengthened by the pattern observed in the levels of 8-hydroxydeoxyguanosine (8-OHdG).

Earlier studies have shown that 8-OHdG is a marker that is often used for evaluation of oxidative deoxyribonucleic acid (DNA) damage and of total systemic oxidative stress in vivo [67]. 8-OHdG is believed to be involved in tissue cell injury through the induction of apoptotic cell death [68] and it is also regarded as a risk factor that can be assessed for pathological conditions like cancer, atherosclerosis, and diabetes [69]. A rise in 8-OHdG levels is considered to reflect an increase in the degree of oxidative stress affecting tissue function and integrity and therefore gives essential information on oxidative stress and disease progression [70]. In this present study, the increase in serum levels of 8-OHdG in High BMI rats administered arsenic and quercetin (group H) (Figure 3) suggests that arsenic exposure might lead to oxidative DNA damage and a systemic oxidative stress state which might be further implicated in various diseases progression. Paradoxically, establishing the relationship between 8-OHdG and BMI has been conflicting, while some studies showed a negative correlation existing between them [71], another showed the opposite [72]. However, in this present investigation, the increase recorded in High BMI rats administered arsenic and quercetin (Figure 3). This increase might be due to the role of arsenic coupled with the high BMI status of the rats. This back-up the reported changes in GSH levels of High BMI rats administered arsenic  (Group D) and is consistent with the findings of [73] who reported a positive correlation between 8-OHdG and BMI. It is also important to note that the oxidative injuries caused by arsenic are dependent on time and dose [60]. Therefore, this might explain the trend observed in High BMI rats administered with arsenic whose level of oxidative stress markers remained unchanged when compared with the control H-BMI rats (Figures 2 and 3).

As demonstrated in earlier studies, quercetin has been regarded as a powerful antioxidant and free radical scavenger [74, 75]. In various studies, the administration of quercetin to rodents resulted in increased antioxidant activity [76], decreased lipid peroxidation [77, 78] and inhibition of LDL-C oxidation [79]. Thus, the increase in GSH level in normal BMI rats administered quercetin only (group E) (Figure 1) suggests that quercetin increased antioxidant capacity, and this contradicts the observation of Boots et al.,. (2008) [80] who previously reported that GSH was unaffected by quercetin supplementation. There are controversies on quercetin’s potential antioxidant effects based on results obtained in a few small scale human quercetin supplementation studies. In one of the studies, Egert et al., (2009) [81] reported that 6 weeks of quercetin supplementation decreased Ox-LDL-C, but other human studies reported no effect of quercetin on a variety of measures of antioxidant capacity and oxidative stress [80-86]. In this present study, we recorded no change in levels of Ox-LDL-C as well as 8-OHdG in rats given quercetin only (group E and F) and in rats administered with arsenic and quercetin (group G and H) (Figure 2 and 3). This reflected that at the given dose and duration, quercetin administration did not affect lipid peroxidation and thus indicated no ameliorative effect on elevated BMI and arsenic induced oxidative stress. This opinion agreed with the finding of Shanley et al.,. (2010) [87] where quercetin supplementation in doses of 500 mg or 1000 mg/day did not improve antioxidant capacity or decrease oxidative stress in a large population of subjects ranging widely in age, BMI and disease state.

The overall trend recorded in the levels of GSH, Ox-LDL-C and 8-OHdG in this study implicated high BMI and arsenic in oxidative stress progression. Consequently, enhanced oxidative stress could alter the integrity of biological membranes and contribute to inflammation and increase the secretions of pro- inflammatory cytokines [15, 46, 87]. In humans and rodents, arsenic exposure is said to be involved in the activation of inflammatory cytokines involved in immune related disorders [88]. Arsenic toxicity results in secretion of cytokines including tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β) and interleukin-6 (IL-6) and generates inflammatory responses [15, 46, 89,  90, 91].These pro-inflammatory mediators are involved in the various biological and cellular comebacks including tumor progression, growth factor, transcription factor and activation of proapoptotic proteins [92]. In addition, increasing epidemiologic evidence revealed that arsenic exposure, even at low concentrations, increased the risk of developing cardiovascular diseases such as atherosclerosis [93-95].Initiation of atherosclerosis involves endothelial cell activation by several stimuli, including cytokines, high levels of reactive oxygen species (ROS), and oxidized low density lipoprotein. Atherosclerosis is said to involve chronic inflammation because of the roles of several cytokine, chemokine and immune cells in its progression [96].

Arsenic has been described to participate in endothelial cell activation, and the first adhesion molecule expressed on endothelial cell activation is vascular cell adhesion molecule-1 (VCAM-1), which is virtually absent on the vasculature prior to activation [97]. VCAM-1 expression is uniquely up-regulated upon atherosclerotic stimuli [98] and exacerbation of cellular recruitment to VCAM-1 contributes to atherosclerosis [99]. Similarly, monocyte chemoattractant protein-1 (MCP-1), a monocyte recruiting chemokine is expressed in endothelial cells, foam cells and vascular smooth muscle cells of artheroslerotic lesions [100] and possess a strong chemotactic activity for immune cells [101]. Previous reports have also shown that obesity is linked with alterations in immunity attributed to elevated levels of these circulating proinflammatory cytokines [102] as well as over expression of MCP-1[103].

Exposure to arsenic in an earlier study significantly increased the levels of serum IL-1β in mice [104] and significant increases were observed in VCAM-1 levels in plasma of individuals exposed to arsenic [66]. However, arsenic is a potent immunotoxicant which modulates non-specific immune responses and alters the expression of cytokines in time and dose dependent manners [105]. This explained the insignificant effect of arsenic at the administered dose and duration on serum levels of IL-1β as well as VCAM-1, even in rats with high BMI in this study (Figure 4 and 5). Conversely, the reasons for the recorded increase in serum monocyte chemoattractant protein (MCP-1) concentrations in rats administered arsenic only (group C) and rats administered arsenic and quercetin (group G and H) (Figure 6). These were probably reflection of a possible progression of inflammatory events that might result in atherosclerosis if the duration of exposure to arsenic was longer, because significant increase of chemokines and cytokines were reported as atherosclerotic plaques started to form [106-108]. This is consistent with the findings of Wu et al., (2003) [109] who reported a positive correlation between arsenic exposure and expression of MCP-1 in human subjects. Similarly, in previous studies, MCP-1 increased in obese mice when compared with the lean control [110, 111]. Thus, the increase we observed in serum MCP-1 levels in groups B, F and G in this present study suggested that high BMI could enhance the risk of arsenic-induced inflammatory responses.

In several studies, quercetin has been recognized as a long lasting anti-inflammatory substance which has previously been shown to possess and exert its anti-inflammatory activities [112-114]. Stewart et al.,. (2008) [115] reported that quercetin was effective in reducing circulating markers of inflammation including IL-1β after 8 weeks of administration. However, the administration of quercetin did not have such an effect on the levels of IL-1β, MCP-1 and VCAM-1 across the groups administered quercetin (groups E, F, G, H) (Figure 4, 5 and 6) in this present study. This showed that quercetin, at the administered doses and duration of administration was not sufficient to exert any noticeable anti-inflammatory activity and did not regulate arsenic and high BMI induced inflammatory responses. Thus, we opined that the anti-inflammatory roles of quercetin might be significantly expressed at increased doses or if the duration of administration was extended.

Figure 1: Concentration of reduced glutathione serum of rats following administration of arsenic and quercetin
Values are means ± SEM, n=4 and mean values bearing different alphabets are significantly different (P<0.05).
BMI (body mass index)

Figure 2: Serum concentration of oxidized-density lipoprotein cholesterol in rats administered arsenic and quercetin. Values are means ± SEM, n=4 and mean values bearing different alphabets are significantly different (P<0.05). BMI (body mass index)

Figure 3: 8-hydroxydeoxyguanosine levels in serum of rats administered arsenic and quercetin
Values are means ± SEM, n=4 and mean values bearing different alphabets are significantly different (P<0.05). BMI (body mass index).

Figure 4: Concentration of Interleukin-1 β in serum of rats administered with arsenic and 
quercetin. Values are means ± SEM, n=4 and mean values bearing different alphabets are significantly 
different (P<0.05). BMI (body mass index).

Figure 5: Concentration of Vascular cell adhesion molecule-1 in serum of rats following administration with arsenic and quercetin. Values are means ± SEM, n=4 and mean values bearing different alphabets are significantly different (P<0.05). BMI (body mass index)

Figure 6: Monocyte chemoattractant protein-1 concentrations in serum of rats administered with arsenic and quercetin. Values are means ± SEM, n=4 and mean values bearing different alphabets are significantly different (P<0.05). BMI (body mass index)

Plate 1: Photomicrographs of the brain section of rats administered with arsenic and quercetin (H&E). Keys: A-NBMI, B HBMI, C- NBMI + Arsenic, D-HBMI + Arsenic, E-NBMI + Quercetin, F-HBMI + Quercetin, G-NBMI + Arsenic + Quercetin, H-HBMI + Arsenic + Quercetin. NBMI (normal body mass index) and HBMI (high body mass index)

Plate 2: Photomicrograph of the heart of rats administered with arsenic and quercetin (H&E).

Keys: A-NBMI, B HBMI, C- NBMI + Arsenic, D-HBMI + Arsenic, E-NBMI + Quercetin, F-HBMI + Quercetin, G-NBMI + Arsenic + Quercetin, H-HBMI + Arsenic + Quercetin. NBMI (normal body mass index) and HBMI (high body mass index)

Plate 3: Photomicrograph of the liver of rats administered with arsenic and quercetin (H&E,).

Keys: A-NBMI, B HBMI, C- NBMI + Arsenic, D-HBMI + Arsenic, E-NBMI + Quercetin, F-HBMI + Quercetin, G-NBMI + Arsenic + Quercetin, H-HBMI + Arsenic + Quercetin. NBMI (normal body mass index) and HBMI (high body mass index)

In addition, the trend observed in levels of the assessed oxidative stress markers were reflected in the histological examinations of the organs of interest (brain, heart and liver). Degenerations in various organs have been shown to be indicators of arsenic induced oxidative stress during exposure [116], thus, previous studies suggested that histological changes that occur as a result of arsenic mediated oxidative stress might be due to the association of chronic arsenic exposure with methyl insufficiency and loss of DNA methylation in animals [117, 118]. Samuel and Adewale (2019) [119] showed that arsenic exposure is toxic to various organs in animals including the brain and the heart in animals. The brain is known to contain a high level of polyunsaturated fatty acids and relatively low levels of antioxidant defenses, thereby making it vulnerable to arsenic mediated oxidative damage [120, 121]. Consequently, the unregulated production of reactive oxygen species in the brain and alteration in balance of the antioxidants are associated with several pathological changes in neurodegenerative diseases [122]. Histological examination of the prefrontal cortex of rat in this present study revealed the presence of mild vacuoles in all the normal BMI and high BMI rats administered arsenic with or without subsequent quercetin treatment (Groups C, D, G, H) (Plate 1). This suggested that arsenic is potentially toxic to the brain and might result in severe oxidative damage and neurodegenerative disorders if the duration of administration was extended. This observation was corroborated by the findings of Ghosh, (2011) [123] who implicated  chronic exposure to arsenic in oxidative stress induction,  Koehler et al., (2014) [124] who indicated the accumulation inorganic arsenicals in brain astrocytes and Noman et al., (2015) [116] that reported alterations such as edema, intracellular space, edematous changes in arsenic exposed brain tissue.

The liver has long been identified as a target organ of arsenic toxicity due to its unique metabolic functions and its association with the gastrointestinal tract [116]. Several histopathological changes such as mild sinusoidal dilation and atrophy of the central vein were observed across the groups (A-H) (Plate 3) in this present study and supported the hepatotoxic potential of arsenic. Our findings in this study were consistent with a previous study where mild to moderate sinusoidal dilation is usually indicated by several features such as widening of hepatic capillaries [125, 127]. In the same study, Al-forkan et al.,. (2016) [126] reported that the heart had lesser amount of arsenic and least histopathological injury, and explained that it might be due to the short half-life of arsenic in the blood which makes its chance of  accumulation in the heart low when compared with other organs [128]. However, in this present study, the cellular delineation, cellularity and morphological delineation of the heart in normal BMI rats (A, C and E) appeared normal, while that of high BMI rats administered arsenic only or with subsequently quercetin treatment (B, D, F, G, and H) appeared atypical (Plate 2) and reflected the possible effects of arsenic induced toxicity in heart tissues.

The overall trend of the results in this study indicated alterations in the health indices that could result in dysfunctions in immune functions and health following arsenic exposure irrespective of the body mass index. However, a high body mass index increased the risk of arsenic-induced dysfunctions in the health indices and quercetin did not ameliorate the dysfunction nor improved the quality of life in high body mass index subjects.

Ethical Approval and Funding
This study was carried out in accordance with national ethical laws on animal handling. We did not receive any grant for the conduct of this study. The study was funded personally.

Authors’ Contributions
EBO conceived, designed and supervised the study, and drafted the manuscript. AGE did the statistical analyses and provided some materials. AAL and OBT provided some reagents and performed some of the experimental procedures. AJO and FJO provided some materials and reagents used in the study. All authors read and approved the final manuscript with the order of author’s names.

Acknowledgements
We acknowledge the immense efforts and laboratory assistance of Dr. R. A. Ajani (LAUTECH), the technical inputs of Biolab (Ogbomoso) and Messr Olabanji Debo of Bridge Scientifik Limited (Ilorin).

Consent for Publication and Competing Interests
All the authors gave their consents for the publication of the study and there were no competing interests among the authors.

Funding: No funding sources.

Conflict of interest: None declared.

Ethical approval: The study was approved by the Institutional Ethics Committee of Ladoke Akintola University of Technology, Ogbomoso.

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