**1. Introduction**

Exposure to toxic chemicals, environmental changes, and drugs can cause harmful effects and injuries through the metabolic production of reactive oxygen species (ROS). The description of oxidative stress is an imbalance between ROS production and antioxidant defense, which causes cell damage at high levels. The ROS and their pathophysiological effects depend on the concentration, type, and specific production site. When ROS are at a high level, they react with DNA, proteins and cell membrane, and other molecules, causing cellular damage and producing other more reactive radicals [1]. In addition, the formation of ROS leads to DNA strand breaks and oxidative DNA damage that induce changes in mRNA expression of DNA damage responsive genes [2].

Carbon tetrachloride (CCL4), a colorless, transparent, heavy, and non-flammable industrial liquid, is widely used to induce free radical toxicity in various experimental animal tissues such as kidneys, heart, liver, lung, testis, brain, and blood [3]. Exposure to CCL4 initiates a complex process for resistance to toxicity and production of free radicals to metabolize CCL4, leading to further oxidative stress, which participates in the initiation and

**Citation:** Zargar, S.; Wani, T.A. Protective Role of Quercetin in Carbon Tetrachloride Induced Toxicity in Rat Brain: Biochemical, Spectrophotometric Assays and Computational Approach. *Molecules* **2021**, *26*, 7526. https://doi.org/ 10.3390/molecules26247526

Academic Editor: Stefano Castellani

Received: 18 November 2021 Accepted: 9 December 2021 Published: 12 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

progression of brain injury [4]. The resulting oxidative stress leads to DNA fragmentation and produces a significant interconnected change of cellular metabolism and destruction of the cells by lipid peroxidation [4]. Acute and harmful tissue injuries are induced by CCL4 metabolites, reactive metabolic trichloromethyl radicals (CCl3), and peroxy trichloromethyl radicals (OOCCl3). The single hepatotoxic dose produces more intense free-radical stress in the brain than in the liver [5]. These free radicals can covalently bind to macromolecules such as lipids, proteins, and nucleic acids present in the brain [6]. Previous studies showed that CCL4-induction caused an observed reduction in p53, a tumor suppressor gene expression [7]. The antioxidant mechanism prevents cells in the G phase of the cell cycle and gives additional time for DNA repair, while severe DNA damage triggers apoptosis, a life-threatening condition [7].

Flavonoids are polyphenolic compounds that play an essential role in free radical detoxification. These polyphenolic compounds are found in vegetables, fruits, and medicinal plants. Quercetin in plants exists as either a free (aglycone) or conjugated with carbohydrates (quercetin glycosides) and alcohols (quercetin methyl ethers). It was reported that quercetin is an anti-inflammatory, antioxidant, antibacterial, radical-scavenging, antiviral, gastroprotective, and immune-modulator, and is used to treat cardiovascular diseases and obesity [8,9]. Quercetin is abundantly present in apples, berries, onions, capers, broccoli, tea, and red wine. Quercetin is reported to protect against CCL4 induced hepatotoxicity by inhibiting Toll-like receptor 2 (TLR2), TLR4 activation, and mitogen-activated protein kinase (MAP Kinase) phosphorylation. These, in turn, inactivate nuclear factor kappa B (NF-kB) and the inflammatory cytokines in livers of the CCL4-treated animals. Quercetin is reported to protect against brain injury in mice through TLR2/4 and MAPK/NF-kB pathway [10]. A high concentration of quercetin metabolites is present in the brain after several hours of quercetin administration [11].

An intraperitoneal dose of quercetin 10 mg/kg body weight in rats before two hours of acrylamide assault resulted in diminutive acrylamide mediated neurotoxicity. Quercetin treatment leads to decreased dopamine, interferon-γ, and 8-hydroxyguanosine levels and the restoration of serotonin levels [12]. In addition, quercetin can increase the body0 s antioxidant activity by regulating glutathione (GSH) levels. The GSH is a central component of a natural defense mechanism of the body against oxidative stress. The superoxide dismutase (SOD) quickly captures O2<sup>−</sup> and transforms it into hydrogen peroxide H2O2. This enzyme further catalyzes the decomposition of H2O<sup>2</sup> to the non-toxic H2O. This reaction requires GSH as a hydrogen donor. Quercetin in several studies was found to induce reduced glutathione (GSH) synthesis. One study reported that the p53 penetrates in the mechanism of cell response to quercetin through modulation of glutathione-related enzyme expression [13,14]. ROS and reactive nitrogen species (RNS) are produced continuously in the body by oxidative metabolism, mitochondrial bioenergetics, and immune function that can cause potential biological damage [15]. As a flawed anti-oxidative system favors the accumulation of free radicals due to a decrease in the activity of antioxidant enzymes, quercetin may find application in the prevention of neurological disorders due to its neuroprotective effects. The present study evaluated the toxicity of CCL4 in rat brains and investigated whether quercetin can protect against the damage caused by it.

The VirtualToxLab can predict the toxic potential of the tested compound, e.g., endocrine and metabolic disruption, some aspects of carcinogenicity, and cardiotoxicity by simulation and quantification of their interactions towards a series of proteins suspected to trigger mentioned adverse effects [16]. This tool follows an automated protocol and calculates the binding affinity of the investigated compound to selected proteins. It is beneficial to understand that interaction mechanism at the molecular level and estimate the toxic potential of the studied drugs. The VirtualToxLab™ was used to study the interaction between quercetin and various proteins to understand the molecular basis of the protective potential of quercetin against carbon tetrachloride toxicity on rat brains.
