*2.8. Ascorbic Acid (AsA)*

Ascorbic acid was determined by the method of Jagota and Dani [24]. First, 0.2 mL of homogenized brain tissue was treated with 0.8 mL of 10% TCA. After vigorous shaking, tubes were kept in an ice-cold bath for 5 min and centrifuged at 1200 g for 5 min. Next, 0.2 to 0.5 mL of the supernatant were diluted to 2 mL with distilled water, and 0.2 mL of Folin reagent (0.2 M) was added. After 10 min, the absorbance was read at 760 nm against a reagent blank. The amount of ascorbic acid was calculated from the standard graph. Values were expressed as µg of ascorbic acid µg protein−<sup>1</sup> .

### *2.9. Reduced Glutathione (GSH)* The estimation was carried out by the method of Beutler et al. [25]. Briefly, 0.4 mL of homogenized brain tissue was mixed with 3.6 mL of double-distilled water and followed

The estimation was carried out by the method of Beutler et al. [25]. Briefly, 0.4 mL of homogenized brain tissue was mixed with 3.6 mL of double-distilled water and followed by treatment with 0.6 mL of precipitating reagent (containing 1.67 g of glacial metaphosphoric acid, 0.2 g of EDTA, and 30.0 g of NaCl and made up to 100 mL with double distilled water). by treatment with 0.6 mL of precipitating reagent (containing 1.67 g of glacial metaphosphoric acid, 0.2 g of EDTA, and 30.0 g of NaCl and made up to 100 mL with double distilled water). The above reaction mixture was centrifuged at 600 g for 10 min. To 0.3 mL of super-

The above reaction mixture was centrifuged at 600 g for 10 min. To 0.3 mL of supernatant, 2 mL of Na2HPO<sup>4</sup> (0.3 M) and 0.25 mL of 5,5<sup>0</sup> dithio-bis-2-nitrobenzoic acid (0.4% in 1% sodium citrate) were added, and the volume was made up to 3 mL with DDW. OD was read at 412 nm against blank. Values were expressed as µg of reduced glutathione µg protein−<sup>1</sup> . natant, 2 mL of Na2HPO<sup>4</sup> (0.3 M) and 0.25 mL of 5,5′ dithio-bis-2-nitrobenzoic acid (0.4% in 1% sodium citrate) were added, and the volume was made up to 3 mL with DDW. OD was read at 412 nm against blank. Values were expressed as μg of reduced glutathione μg protein−1 .

#### *2.10. In Silico Testing of Quercetin Toxicity and Molecular Docking 2.10. In Silico Testing of Quercetin Toxicity and Molecular Docking*

The VirtualToxLab™ is an online platform to estimate the toxicity of drugs, which requires the test compound to be submitted in pdb format. Therefore, the quercetin structure was downloaded from PubChem (PubChem CID: 5280343) in sdf format and was converted to the pdb format using the discovery studio software. The interaction of quercetin with the glucocorticoid, estrogen α, estrogen β, androgen, aryl hydrocarbon, thyroid α, thyroid β, mineralocorticoid, progesterone, hERG, liver X, and PPARγ was evaluated. In addition to these proteins, the enzymes cytochrome P450 1A2, 2C9, 2D6, and 3A4 were assessed for their interactions with quercetin. Flexible molecular docking was conducted for the quercetin and the moderately bound proteins in the VirtualToxLab running on an automated protocol. The low-energy poses are sampled into a dataset. The binding affinities between the quercetin and the target proteins were quantified using the dataset as input for Boltzmann scoring (Software BzScore4D) [16]. The binding mechanisms with these biomolecules were used to assess the protective mechanisms of quercetin to CCL4 toxicity in the brain. The VirtualToxLab™ is an online platform to estimate the toxicity of drugs, which requires the test compound to be submitted in pdb format. Therefore, the quercetin structure was downloaded from PubChem (PubChem CID: 5280343) in sdf format and was converted to the pdb format using the discovery studio software. The interaction of quercetin with the glucocorticoid, estrogen α, estrogen β, androgen, aryl hydrocarbon, thyroid α, thyroid β, mineralocorticoid, progesterone, hERG, liver X, and PPARγ was evaluated. In addition to these proteins, the enzymes cytochrome P450 1A2, 2C9, 2D6, and 3A4 were assessed for their interactions with quercetin. Flexible molecular docking was conducted for the quercetin and the moderately bound proteins in the VirtualToxLab running on an automated protocol. The low-energy poses are sampled into a dataset. The binding affinities between the quercetin and the target proteins were quantified using the dataset as input for Boltzmann scoring (Software BzScore4D) [16]. The binding mechanisms with these biomolecules were used to assess the protective mechanisms of quercetin to CCL4 toxicity in the brain.

### **3. Results 3. Results**

CCL4 is lipophilic and passes freely through all biological membranes, including the brain0 s myelin sheath. A marked effect on brain tissue histology was observed with the cortex showing swelling, vacuolar degeneration, and karyopyknosis. All these changes are morphological characteristics of apoptosis. Figure 1a is the normal histology of the cerebral cortex of the brain. Extensive vacuolization was seen in myelin within the white matter, and a few vacuoles were also seen in the gray matter of rats treated with CCL4. CCL4-treated neurons had large nuclei turning from basophilic to pyknotic revealing apoptosis (Figure 1b). However, the normal histology was observed in quercetin-treated rats (Figure 1c). Figure 1d shows a complete reversal of altered histology in rats treated with quercetin two hours before CCL4. CCL4 is lipophilic and passes freely through all biological membranes, including the brain′s myelin sheath. A marked effect on brain tissue histology was observed with the cortex showing swelling, vacuolar degeneration, and karyopyknosis. All these changes are morphological characteristics of apoptosis. Figure 1a is the normal histology of the cerebral cortex of the brain. Extensive vacuolization was seen in myelin within the white matter, and a few vacuoles were also seen in the gray matter of rats treated with CCL4. CCL4-treated neurons had large nuclei turning from basophilic to pyknotic revealing apoptosis (Figure 1b). However, the normal histology was observed in quercetin-treated rats (Figure 1c). Figure 1d shows a complete reversal of altered histology in rats treated with quercetin two hours before CCL4.

**Figure 1.** Cortex sections of normal and treated groups at a scale bar of 100 µm. (**a**) Normal histological appearance of brain tissues with neurocytes having well-defined nuclei. (**b**) CCL4-treated brain cortex section with widespread intracellular vacuolization and infiltration of inflammatory cells (aster). Neurocytes have dark eosinophilic cytoplasm, with cells having heterochromatic nuclei. (**c**) Quercetin-treated brain tissue has fewer vacuoles and inflammation. (**d**) Q + CCL4-treated brain section with mild vacuolization and mild infiltration of inflammatory cells (*n* = 3).

The effect of CCL4 on the brain-to-body weight ratio is presented in Figure 2a. The brain-to-body weight ratio decreased significantly (*p* < 0.0001) in the CCL4 treatment group, showing this group0 s metabolic or growth disorders. Treatments with quercetin and combination groups completely reversed these groups0 metabolic or growth disorders and showed non-significant differences with control group growth patterns. The effect of CCL4 on the brain-to-body weight ratio is presented in Figure 2a. The brain-to-body weight ratio decreased significantly (*p* < 0.0001) in the CCL4 treatment group, showing this group′s metabolic or growth disorders. Treatments with quercetin and combination groups completely reversed these groups′ metabolic or growth disorders and showed non-significant differences with control group growth patterns.

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**Figure 1.** Cortex sections of normal and treated groups at a scale bar of 100 μm. (**a**) Normal histological appearance of brain tissues with neurocytes having well-defined nuclei. (**b**) CCL4-treated brain cortex section with widespread intracel-

treated brain section with mild vacuolization and mild infiltration of inflammatory cells (*n* = 3).

**Figure 2.** (**a**–**e**). Effect of CCL4 (1 mg/kg) and Q (100 mg/kg b.w) on the brain-to-body weight ratio, ALT, AST, brain urea, and brain sodium (*n* = 6). Data were analyzed by (one-way ANOVA), and Tukey's test was used for multiple comparisons. Treated groups are compared to the control group. \*\*\*\* *p* < 0.0001, \*\*\* *p* < 0.001, \*\* *p* < 0.01, \* *p* < 0.1, ns is non-significant. **Figure 2.** (**a**–**e**). Effect of CCL4 (1 mg/kg) and Q (100 mg/kg b.w) on the brain-to-body weight ratio, ALT, AST, brain urea, and brain sodium (*n* = 6). Data were analyzed by (one-way ANOVA), and Tukey's test was used for multiple comparisons. Treated groups are compared to the control group. \*\*\*\* *p* < 0.0001, \*\* *p* < 0.01, ns is non-significant.

Figure 2b shows significantly decreased levels of ALT in group CCL4-treated rats (*p* < 0.0001) when compared to Group C (control). Quercetin treatment alone (Group Q) caused a non-significant decrease in the ALT levels (*p* > 0.05) compared to controls. Pretreatment of quercetin in Group IV reversed the decreased ALT levels and showed significant protection when compared with Group CCL4. The decreased ALT levels in the group Q + CCL4 were non-significant (*p* > 0.05) compared to the control group. Figure 2b shows significantly decreased levels of ALT in group CCL4-treated rats (*p* < 0.0001) when compared to Group C (control). Quercetin treatment alone (Group Q) caused a non-significant decrease in the ALT levels (*p* > 0.05) compared to controls. Pretreatment of quercetin in Group IV reversed the decreased ALT levels and showed significant protection when compared with Group CCL4. The decreased ALT levels in the group Q + CCL4 were non-significant (*p* > 0.05) compared to the control group.

Figure 2c shows significantly increased levels of AST in Group CCL4 rats (*p* < 0.0001) when compared to control, indicating the induction of brain damage as AST catalytic activities in the CSF are linked to high risk. Quercetin treatment alone was relatively similar to control in the AST levels (*p* > 0.05) compared to control. However, pretreatment of quercetin in the Q + CCL4 group could not reverse the increased levels of AST. Figure 2c shows significantly increased levels of AST in Group CCL4 rats (*p* < 0.0001) when compared to control, indicating the induction of brain damage as AST catalytic activities in the CSF are linked to high risk. Quercetin treatment alone was relatively similar to control in the AST levels (*p* > 0.05) compared to control. However, pretreatment of quercetin in the Q + CCL4 group could not reverse the increased levels of AST.

Figure 2d shows significantly increased levels of urea in Group CCL4 rats (*p* < 0.0001) when compared to control, indicating brain damage and increased metabolic activity. Quercetin treatment alone caused a non-significant increase in urea levels compared to Figure 2d shows significantly increased levels of urea in Group CCL4 rats (*p* < 0.0001) when compared to control, indicating brain damage and increased metabolic activity. Quercetin treatment alone caused a non-significant increase in urea levels compared to controls. However, pretreatment of Quercetin in the Q + CCL4 group decreased the increased urea levels significantly.

Figure 2e shows significantly increased sodium levels in the group Q rats (*p* < 0.0001) compared to controls, which is another indication of brain damage due to the voltage potential. Quercetin treatment alone was relatively similar to control; non-significant change in the sodium levels (*p* > 0.05) compared to controls. Pretreatment of quercetin in the Group Q + CCL4 reversed the increased sodium levels and showed significant protection compared

with the group CCL4. The increase of sodium levels in group Q + CCL4 was significantly decreased (*p* < 0.001) compared to the control group, relatively similar to controls.

Table 1 shows the levels of biochemical oxidative stress biomarkers lipid hydroperoxides, ascorbic acid, and reduced glutathione catalase and superoxide dismutase in control and experimental rats (Table 1). The levels of lipid hydroperoxides and AsA were significantly (*p* < 0.05) increased in CCL4- treated rats compared to control and quercetin groups. The treatment of rats with quercetin resulted in significant recovery of these free radicals generated in brain tissues of group Q + CCL4. Exposure with CCL4 significantly decreased the reduced glutathione (*p* < 0.05) that was reversed considerably in group Q + CCL4 (*p* < 0.05). Catalase activity was reduced substantially with CCL4 treatment that was significantly recovered by treatment with quercetin in group Q + CCL4 (Table 1).

**Table 1.** Effect of quercetin on CCL4 induced oxidative stress in the brains of control and experimental rats.


Querectin (100 mg/kg b.w) was administered 2 h before CCL4 assault. Data are representative of mean ± SD of three independent experiments, each group containing six mice. <sup>a</sup> significant (*p* < 0.05) when compared to control; <sup>b</sup> significant (*p* < 0.05) when compared to CCL4 (1 mg/kg) group; <sup>c</sup> significant (*p* < 0.05) when compared to Q (100 mg/kg) group; <sup>d</sup> significant (*p* < 0.05) when compared to Q + CCL4 group.

> The results from the VirtualToxLab™ are presented in Table 2. Quercetin was found to have a strong binding affinity to the androgens and had moderate binding to glucocorticoid and estrogen beta receptors. In addition, the quercetin was found to have weak binding to Estrogen receptor α (ERα), hERG, Peroxisome Proliferator-Activated receptor γ (PPARγ) and Thyroid receptor β (TRβ). Furthermore, quercetin did not bind to Aryl hydrocarbon receptor (AhR), Thyroid receptor α, Mineralocorticoid receptor (MR), progesterone receptor (PR), hERG, and Liver X receptor (LXR), nor to any of the cytochrome P450 enzymes. Figure 3 shows real-time 3D/4D visualization of binding modes of quercetin with glucocorticoids, estrogen beta, and androgen receptors in concomitance with binding results from BzScore4D. Based on the results, a possible mechanism of quercetin protection against CCL4 toxicity in the rat brain was hypothesized.

**Table 2.** Binding of quercetin to various proteins.



**Target Binding Type Binding Affinity** 

Androgens moderate binding 948 nM Aryl hydrocarbon negligible >100 μM

**(VirtualToxLab)**

**Table 2.** *Cont.*

Overall toxic potential was found to be 0.418 [16].

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**Figure 3.** Docking conformation of quercetin with different targets. (**a**) Predicted bonded interactions (blue dashed lines) between quercetin and glucocorticoid; (**b**) binding interaction between quercetin and androgen; (**c**) binding interaction of quercetin and estrogen alpha. The ligand is based on atom type and the protein-based on amino acid residue type coloring. **Figure 3.** Docking conformation of quercetin with different targets. (**a**) Predicted bonded interactions (blue dashed lines) between quercetin and glucocorticoid; (**b**) binding interaction between quercetin and androgen; (**c**) binding interaction of quercetin and estrogen alpha. The ligand is based on atom type and the protein-based on amino acid residue type coloring.

### **4. Discussion 4. Discussion**

CCL4 is a lipophilic colorless liquid and readily crosses cell membranes, including the blood–brain barrier. All body tissues rapidly take up CCL4, but the toxicity on the brain remains poorly understood. Some studies reported that adverse events of CCL4 in rodents are mainly based on systemic effects in the liver (centrilobular necrosis) and some impact on the kidney in inducing free radical toxicity and some tissues injuries [26]. However, in this study, we evaluated all the parameters in the brain tissue itself. In this study, the elevation of ROS was reported in brain cells on exposures to a single dose of CCL4 that is scavenged by antioxidant quercetin during normal cell metabolism. Histopathological analysis on brain tissues indicated a CCL4 induced karyopyknosis, apoptosis, and swelling. The toxic effect of CCL4 is believed to be due to trichloromethyl radicals produced during oxidative stress, as per previous literature [27]. The results of our study are in corroboration with Ritesh et al., 2015, who reported that a single hepatotoxic dose of CCL4 is equally neurotoxic to rats as many consecutive doses [5]. Organ and body weights showed significantly decreased growth changes in the brain-to-body weight ratios. The brain/bw ratio is one of the most sensitive parameters measured for detecting the effect of exposure to toxins on growth and development [28]. CCL4 significantly altered AST and CCL4 is a lipophilic colorless liquid and readily crosses cell membranes, including the blood–brain barrier. All body tissues rapidly take up CCL4, but the toxicity on the brain remains poorly understood. Some studies reported that adverse events of CCL4 in rodents are mainly based on systemic effects in the liver (centrilobular necrosis) and some impact on the kidney in inducing free radical toxicity and some tissues injuries [26]. However, in this study, we evaluated all the parameters in the brain tissue itself. In this study, the elevation of ROS was reported in brain cells on exposures to a single dose of CCL4 that is scavenged by antioxidant quercetin during normal cell metabolism. Histopathological analysis on brain tissues indicated a CCL4 induced karyopyknosis, apoptosis, and swelling. The toxic effect of CCL4 is believed to be due to trichloromethyl radicals produced during oxidative stress, as per previous literature [27]. The results of our study are in corroboration with Ritesh et al., 2015, who reported that a single hepatotoxic dose of CCL4 is equally neurotoxic to rats as many consecutive doses [5]. Organ and body weights showed significantly decreased growth changes in the brain-to-body weight ratios. The brain/bw ratio is one of the most sensitive parameters measured for detecting the effect of exposure to toxins on growth and development [28]. CCL4 significantly altered AST and ALT activity in brain tissue, and no modified ALT levels were observed in the prophylactically quercetin-treated group. CCL4 treatment causes brain tissue damage followed by the release of AST molecules into the extracellular spaces of brain tissue. The function of this enzyme is the reversible transport of amines from aspartate to α-ketoglutarate [29]. In concomitance to this study,

Kelbich et al. reported an increase in AST catalytic activities in the cerebrospinal fluids of cerebral ischemia patients [30].

Furthermore, the increased AST activity could be connected with increased transport of NADH from the cytosol to mitochondria. In contrast, the increased ALT activity would represent more transformation of pyruvate to alanine due to increased glycolysis and hence increased pyruvate [31]. Increased brain urea by CCL4 was reversed by quercetin. The 'reverse urea effect causes brain edema', i.e., the significant urea gradient between blood and brain causes an inflow of water into the brains of induced animals [32]. For the first time, this study has evaluated brain sodium levels with CCL4 exposure. Brain sodium is increased with sympathoexcitatory and pressor responses [33], which further destroys brain potential and leads to brain inflammation. In addition, increased sodium levels hyperactivate Na/K channels that trigger excitotoxic neuron death [34]. In concomitance to sodium levels, the Herg k<sup>+</sup> channel (Pottasium channel) was found to show weak binding with quercetin in silico analysis. Hence, it can be predicted that quercetin protected via the action of Na+/K<sup>+</sup> pump. However, the action of protection by quercetin with respect to Na+/K<sup>+</sup> pump must be verified in future studies. Glutathione eliminates reactive oxygen species (ROS) produced in oxidative stress [35]. GSH, a ubiquitous intracellular cytosolic tripeptide at millimolar concentrations, is the primary non-enzymatic biomarker of redox homeostasis. The reduced GSH in brains after the exposure to CCL4 could result from increased GSH-peroxidase activity in the exposed rat. The decreased GSH was further associated with ascorbic acid and lipid peroxidation [19,36,37]

Additionally, glutathione depletion induces glycogenolysis-dependent ascorbic acid synthesis in murine hepatocytes in vitro [38]. The significant decreased levels of antioxidant enzymes such as CAT and SOD were reversed to normal by quercetin supplementation (Table 1). Based on our results, we conclude that quercetin shows significant prophylactic effects against CCL4 with respect to the studied parameters. Thus, it was indicated that quercetin prevents alterations in oxidative stress parameters and neurotransmitters parameters [39]. Our results highlight the importance of understanding the potential prophylactic effects of quercetin against neurotoxicity.

Based on the binding affinities of quercetin to various proteins, the normalized binding affinity of quercetin to the studied proteins was found to be 0.418 (average). It should be noted that the toxicity values can be overestimated since binding to a particular protein may or may not lead to any adverse event. The software used for the flexible docking in VirtualToxLab is Alignator and Cheetah. All the binding modes between the quercetin and the target proteins were identified during the study. The interaction between quercetin and the target proteins, which showed moderate binding, is given in Figure 3. The binding affinity are defined between >100 µM (not binding) to <1.0 nM (strong binding). The overall binding potential ranged from 0.0 (benign) to 1.0 (extreme) [16].

Moderate binding between quercetin and glucocorticoids, estrogen beta, and androgens was evaluated. Previous studies suggested that oxidative stress increases glucocorticoid (GC) hormones; hence, the hippocampus, which has a high concentration of GC receptors, is especially vulnerable to elevated levels of GCs. The GCs have been suggested to endanger hippocampal neurons by exacerbating the excitotoxic glutamate-calciumreactive oxygen species (ROS) cascade [40–42]. Our binding results suggest quercetin binding to glucocorticoids as a preventing mechanism for preventing ROS cascade generated by CCL4 neurotoxicity.

Previous studies demonstrated that estrogen receptor β (ERβ) signaling alleviates systemic inflammation in animal models, and suggested that ERβ-selective agonists may deactivate microglia and suppress T cell activity via the downregulation of nuclear factor k-light-chain-enhancer of activated B cells (NF-kB) [43]. The estrogen receptor agonists play an essential role in protecting the central nervous system against neuroinflammation and neurodegeneration. Quercetin aglycone and its glucuronide possess estrogenic activity, and quercetin is also classified as a phytoestrogen [8,44]. Thus, the binding of quercetin to estrogen alpha and beta might have caused the protection against the stress induced by

carbon tetrachloride [8]. Future studies are needed to verify this mechanism for protection. Another possible mechanism for the protection of quercetin can be the suppression of stress-induced plasma corticosterone and adenocorticosterone levels. The DNA binding activity of the glucocorticoid receptor is modulated on binding to quercetin and is one of the possible reasons for suppressing plasma corticosterone and adrenocorticotropic hormone levels [45,46]. The effect of androgens on the cerebral vasculature is a complex mechanism. They have both protective and detrimental effects, depending on several factors, such as age, drug dose, and state of disease. Chronically elevated androgens are pro-angiogenic, promote vasoconstriction, and influence blood–brain barrier permeability. In addition to these, androgens have been shown to affect the cerebral vasculature [44] directly. This study found moderate binding with androgens; hence, we propose that elevated androgen levels by CCL4, which could otherwise be harmful to the brain, were probably prevented by quercetin by binding with androgen receptors. Future studies are needed to verify our hypothesis.

**Author Contributions:** Conceptualization: S.Z.; Methodology: S.Z.; Software: T.A.W.; Formal analysis: T.A.W.; S.Z.; Investigation: T.A.W.; S.Z.; Resources: S.Z.; Writing: S.Z.; T.A.W.; Review & Editing: S.Z.; Project administration: T.A.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** Research Groups, Deanship of Scientific Research, King Saud University, Grant Number: RG-1438-042.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institutional Review Board of King Saud University approval no. KSU-SE-21-05 dated 06-12-2020.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data will be available on request to corresponding author.

**Acknowledgments:** This work was supported by the Deanship of Scientific Research, King Saud University; Research group No. RG-1438-042.

**Conflicts of Interest:** The authors declare no conflict of interest.
