Quercetin modulates liver response to circadian rhythm disturbance and γ-radiation in rats: oxidant-antioxidant status and ATP production

Abstract

Background

The mammalian circadian rhythm is an internal biological timekeeping system synchronizing physiology and behavior to light/dark cycles. Several processes throughout the body’s organs, including the liver, are regulated by circadian rhythms and are adversely affected by radiation exposure. This study investigates the effect of circadian rhythms, whether it is regular (light/darkness cycle) or disturbed (permanent darkness or permanent light), under the stress of whole-body irradiation, and the possible protective impact of quercetin on certain liver aspects.

Methods

The animals were divided into three main groups: a normal light/dark group, a permanent darkness group, and a permanent light group. Each group was subdivided into four subgroups: a control subgroup (nonirradiated rats), an irradiated subgroup (exposed to 9-Gy fractionated dose), a quercetin subgroup (received 50 mg/kg b.wt. of quercetin for 1 month), and a quercetin + irradiated subgroup.

Results

The results showed that circadian rhythm disturbance and/or whole-body irradiation-induced oxidative stress, as indicated by elevation of liver MDA and NO, was associated with decreases in antioxidant enzymes’ activities (SOD, GPx, and CAT) and GSH contents, compared to the corresponding control subgroups. This was also accompanied by a disruption in mitochondrial function, as indicated by increased cytochrome P450 reductase and decreased ATP levels. ADP levels were also affected. Histological investigation of liver tissues showed marked alterations. However, oral administration of quercetin pre- and during irradiation attenuated these changes.

Conclusion

Oral administration of quercetin could protect the liver from these damaging effects by modulating the oxidant-antioxidant system and ATP production.

Introduction

Rhythm is a fundamental aspect of life on Earth and the underlying law of the universe. The daily physiological rhythms of waking and sleeping, eating and fasting, and movement and rest are common to most life forms that have evolved under the solar light–dark cycle. The concept of “circadian rhythms” goes back to the mid-twentieth century [1].

The mammalian circadian rhythm is an internal biological timekeeping system that synchronizes physiology and behavior to day-night cycles. This system consists of two interacting components: the central clock located in the suprachiasmatic nucleus (SCN) in the hypothalamus and the peripheral clocks found in various tissues throughout the body. The circadian system is a hierarchical multi-oscillatory network composed of master pacemaker (s) in the brain and oscillators in peripheral organs [2]. The master or central pacemaker is synchronized with the geophysical time by the zeitgeber (time giver) light through the retinohypothalamic tract [3]. In addition to light, temperature, food intake, and exercise can also act as zeitgebers [4].

The peripheral clocks play an essential role in their respective tissues, regulating the circadian expression of specific genes involved in various physiological functions [5, 6]. Adverse physiological sequelae of the multi-oscillator circadian system disruption have been reported [2, 7]. Several processes throughout the body’s organs, including the liver, seem to be regulated by the circadian rhythm. The hepatic clock regulates mitochondrial quality control [8]. Also, it is involved in the regulation of hepatic oxidative stress, inflammation, and triglyceride accumulation, all of which contribute to the development of nonalcoholic fatty liver disease. So, targeting circadian oscillator components may be a potential strategy for treating liver diseases [3]. Maintaining regular sleep schedules and timed meals, minimizing exposure to light at night, and maximizing exposure to bright light during the day may help minimize circadian rhythm disruption, improve patient quality of life, and reduce pain symptoms [9]. Additionally, understanding the regulation of the circadian clock, its disruptions, and the consequences that may affect radiation therapy could lead to developing strategies to enhance treatment efficacy while reducing toxicity to normal tissue [10].

Patients undergoing radiation therapy suffer from its side effects. Although ionizing radiation is utilized to treat different types of cancer, it may cause severe side effects in the normal tissue surrounding the tumor during treatment. This can occur through the production of free radicals, which can result in oxidative stress. Oxidative stress is marked by an overproduction of free radicals (hydroxyl radical, superoxide anion, nitric oxide, and peroxyl radicals), associated with a reduction of endogenous antioxidants (enzymatic and nonenzymatic). The disruption in the oxidant-antioxidant balance is involved in tissue and organ damage. The liver, an essential organ responsible for detoxification, is susceptible to radiation-induced damage[11]. Free radicals generated as a result of ionizing radiation have the potential to damage liver tissue, causing structural damage, metabolic dysfunction, inhibition of the oxidative defense system, mitochondrial dysfunction, and apoptosis [11, 12].

The use of synthetic compounds, such as amosite, is effective in protecting against radiation toxicity. However, their high systemic toxicity limits their practical application. Despite efforts to improve the efficacy of amanozine as a radioprotector for acute radiation syndrome, none of the strategies has successfully addressed its side effects [13]. Therefore, it is urgent to develop less or nontoxic compounds for radioprotection.

Bioactive compounds found in plants have attracted the attention of many researchers for their antioxidant and radioprotective properties [14]. Polyphenols, commonly found in edible plants, have the potential to serve as effective radioprotectors that can protect against the damage caused by ionizing radiation [15]. One of these natural phenolic compounds is quercetin, a bioactive molecule that has been extensively researched due to its various properties, including antioxidant, anti-inflammatory, and anticancer effects, all with low or nontoxicity [16]. Previously, the neuroprotective effect of quercetin has been discussed by Suganthy et al. [17]. Additionally, its beneficial effect against age-related diseases has been demonstrated by Deepika and Maurya [18]. Recently, it was observed that quercetin modulated the pituitary gland and brain damage induced by 10-Gy whole-body gamma irradiation in rats [19]. Moreover, quercetin administration balanced the liver’s circadian rhythm and metabolic disorders in vitamin D-deficient mice [20].

Although many reports suggest its therapeutic efficacy, quercetin has not yet been recognized as an established compound in the pharmaceutical industry, mainly due to the lack of clinical data and a precise understanding of its mechanism of action [21]. Quercetin’s potential impact on circadian rhythm imbalance under irradiation-induced stress is not well-documented. Thus, this research is designed to explore the impact of the circadian clock (light/darkness cycle), whether it is regular or disturbed, on certain liver physiological aspects under the stress of whole-body irradiation in male rats and to study the possible protective impact of quercetin on the oxidant-antioxidant status, ATP production, and the histopathological structure of liver tissue.

Materials and methods

The present study was carried out at the Radiation Biology Research Department, National Center for Radiation Research and Technology (NCRRT), Atomic Energy Authority (AEA), Cairo, Egypt.

Chemicals

Quercetin was purchased from Gamma Trade Company for Scientific and Consultation SAE, Cairo, Egypt. All other chemicals were of the highest commercially available analytical grade and purity.

Experimental animals

Adult male Wistar rats, weighing approximately 150 ± 20 g, were acquired from the animal facility at the NCRRT. Rats were kept at a constant temperature and with adequate ventilation for 2 weeks for acclimatization. Rats were supplied with water and standard granular chow ad libitum. All animal procedures were performed following the international guidelines for animal handling and care of the National Institutes of Health (NIH publication no. 85–23, 1996) and in accordance with regulations and guidelines set by the Research Ethics Committee of the Faculty of Science, Ain Shams University.

Experimental design

Seventy-two rats were divided into 3 main groups, each one including 24 rats as follows:

1. Normal light/dark (L:D) group: Animals in this group were kept in normal day and night periods (12 h day/12 h night) for 1 month.

2. Permanent darkness (D) group: Animals in this group were kept continuously in the dark (day and night 24 h) for 1 month.

3. Permanent light (L) group: Animals in this group were kept continuously in the light (24 h day and night) for 1 month.

Each group was subdivided into four sub-groups; each one included 6 male rats as follows:

1. Control sub-group: Non-irradiated rats received daily 50 mg/kg body weight distilled water for 1 month.

2. Irradiated sub-group: Animals were exposed once weekly to 3 Gy, a dose of γ-irradiation for 3 weeks.

3. Quercetin sub-group: Animals were administered, via gavage, a daily dose of 50 mg/kg body weight quercetin for 1 month (as shown below).

4. Quercetin and γ-irradiated sub-group: Animals were administered via gavage a daily dose of 50 mg/kg body weight quercetin for 1 week and then exposed weekly to a 3-Gy dose of γ-radiation (as group 2) for 3 weeks, plus the same daily dose of quercetin for the rest of the month.

Quercetin treatment

Quercetin powder (dissolved in distilled water) was administered to rats through gastric intubation. The rats administered a daily dose of 50 mg/kg body weight (b.wt.) according to Usadadia et al. [22] for 1 month.

Irradiation process

Total body irradiation was carried out in three doses, each 3 Gy weekly, using a Gamma cell-40 (¹³⁷Cesium) irradiator at the NCRRT. The dose rate was determined at the time of the experiment, per the records of the Dosimeter Department at NCRRT.

Tissue preparation

Upon completion of the experiment, animals were lightly anesthetized using pentobarbital (60 mg/kg b.wt.), and the liver was excised, on ice, in cold Petri dishes, and divided into three parts. The first part was used for the isolation of the mitochondrial fraction from the cell organelles according to Rickwood et al. [23]. The second part was homogenized in saline solution and centrifuged for 10 min at 3000 rpm, and the supernatant was harvested and kept at −20 °C until used for biochemical analyses. The third part was washed in normal saline and fixed in 10% formalin for histopathological studies.

Biochemical assays

Estimation of lipid peroxidation as thiobarbituric acid reactants was described by Stroeve and Makarova [24], based on the determination of malondialdehyde (MDA), an end product of lipid peroxidation, which reacts with thiobarbituric acid (referred to as thiobarbituric acid reactants) to produce a pink-colored trimethylene. The assay kit (MBS480450) used to determine total nitric oxide concentration is based on the enzymatic conversion of nitrate to nitrite by the enzyme nitrate reductase [25]. Reduced glutathione (GSH) content was determined by the method of Foyer et al. [26]. This method is based on protein precipitation using a tungstate-sulfuric acid solution, followed by the development of a stable yellow color through its reaction with Ellman’s reagent (5,5′-dithiobis-2-nitrobenzoic acid). Determination of glutathione peroxidase (GP_X) activity was performed according to the method of Ahmed et al. [27]; the CUPRAC reagent (Cu (Nc)₂²⁺) was added to stop the enzyme’s reaction. The unreacted substrates act to reduce (Cu (Nc)₂²⁺) to a strongly colored complex (Cu (Nc)²⁺), which was measured spectrophotometrically at 450 nm. The activity of superoxide dismutase (SOD) was assayed according to the method of Nishikimi et al. [28]; the assay relies on the enzyme’s capacity to prevent the reduction of nitroblue tetrazolium dye, which is facilitated by phenazine methosulfate. Determination of catalase (CAT) activity was performed according to Kranner and Birtic [29]; this assay measures the amount of hydrogen peroxide remaining after the action of catalase.

Estimation of mitochondrial functions

Cytochrome P450 reductase levels in liver tissues were estimated using an ELISA kit for rat CYP450 reductase (CYP450R), product code: MBS2022373. An ELISA kit (MBS744390) for ADP determination applies to the competitive enzyme immunoassay technique, utilizing a polyclonal anti-ADP antibody and an ADP-HRP conjugate. ATP determination in rat liver tissues employed the competitive inhibition enzyme immunoassay technique using an ELISA kit, product code: MBS166244.

Histological examination

To prepare the liver tissue for histopathological examinations, samples were fixed in 10% formalin solution. After fixation, the tissue samples were dehydrated through a graded alcohol series. They were subsequently cleared with xylene and embedded in paraffin at 60 °C. Sections measuring 5 µm in thickness were prepared using a sled microtome. These sections were then mounted on glass slides, stained with hematoxylin and eosin, and examined under a light microscope [30].

Statistical analysis

The data were analyzed using the Statistical Package for Social Science (SPSS) program (version 23). A one-way analysis of variance (ANOVA) was conducted, followed by a post hoc LSD test. The data was expressed as mean ± standard error (SE). Differences were considered statistically significant at p ≤ 0.05.

Results

Oxidant/antioxidant status

The findings of this study (Figs. 1 & 2) showed that in the normal group, there are significant elevations in liver MDA and total NO levels, with reductions in GSH contents, GPx, SOD, and CAT activities following gamma-radiation exposure, compared to the control sub-group. Administration of quercetin showed no statistically significant change in MDA levels, NO levels, GSH contents, SOD, and CAT activities with an elevation in GPx activity, relative to the matching control sub-group, while administration of quercetin before and during the irradiation period induced significant reductions in the liver MDA and NO levels with marked elevations in GSH contents and GPx, SOD, and CAT activities, relative to the corresponding irradiated sub-groups.

[See PDF for image]

Fig. 1

Influence of the normal (L:D) circadian rhythms, compared to the disturbed conditions of permanent darkness (D) or light (L). ± quercetin (Q) administration (50 mg/kg b.wt.) on liver MDA (nmol/g) (a) and NO (mg/dl) (b) in γ-irradiated rats. Each value is expressed as the mean ± SE for six rats (p < 0.05). ^aSignificantly different from the control subgroup. ^bSignificantly different from the irradiated subgroup. ^cSignificantly different from the normal group. ^dSignificantly different from the darkness group

[See PDF for image]

Fig. 2

Influence of the normal (L:D) circadian rhythms, compared to the disturbed conditions of permanent darkness (D) or light (L). ± quercetin (Q) administration (50 mg/kg b.wt.) on liver GSH (mg/g) contents (a), GP_X (b), SOD (c), and CAT activities (U/g) (d) in γ-irradiated rats. Each value is expressed as the mean ± SE for six rats (p < 0.05). ^aSignificantly different from the control subgroup. ^bSignificantly different from the irradiated subgroup. ^cSignificantly different from the normal group. ^dSignificantly different from the darkness group

In the permanent darkness (D) group, exposure of rats to continuous darkness led to notable reductions in the activities of SOD, GPx, and CAT, accompanied by a marked rise in the NO levels, compared to the normal (L:D) group. Radiation exposure also induced significant increases in liver MDA and NO levels, with reductions in GSH contents, GPx, SOD, and CAT activities, relative to the control sub-group. Quercetin administration did not significantly affect MDA and NO levels, GSH contents, and GPx and CAT activities. However, it significantly increased GPx and SOD activities when compared to the control sub-group. Quercetin administration pre- and during the irradiation period induced significant depletions in MDA and NO levels, with elevations in GSH contents, GPX, SOD, and CAT activities relative to the corresponding irradiated sub-group, but it remained below the levels observed in the control sub-group (Figs. 1 & 2).

In the permanent light (L) group, exposure of rats to continuous light induced a significant reduction in GSH content and SOD, GPx, and CAT activities, associated with a marked increase in the MDA and NO levels, compared to the normal (L:D) group. Exposure of rats to gamma radiation also induced significant elevations in MDA and NO levels, with reductions in GSH contents, GPx, SOD, and CAT activities, compared to the control sub-group. Administration of quercetin induced no significant effects on MDA and NO levels, GSH contents, SOD, and CAT activities, but it did increase GPx activity. While administration of quercetin pre- and during the irradiation period induced reductions in MDA and NO levels, with marked elevations in GSH contents, GPx, SOD, and CAT activities, relative to the corresponding irradiated sub-group, it remained below the levels observed in the control sub-group (Figs. 1 & 2).

Mitochondrial functions

In the normal (L:D) group, radiation exposure led to a marked rise in liver cytochrome P₄₅₀ reductase (Table 1), and in ADP (Table 2) levels, along with a marked reduction in ATP levels (Table 3), compared to the control sub-group. Supplementation of quercetin showed no statistically significant change in the measured mitochondrial parameters, compared to the control sub-group, while its administration before and during the irradiation period induced significant decreases in liver cytochrome P₄₅₀ reductase and ADP levels (but were still higher than those of the control sub-group), associated with a marked rise in ATP level (but was still below the level observed in the control sub-group), compared to the corresponding irradiated sub-group (Tables 1, 2, & 3).

Table 1. Influence of the normal (L:D) circadian rhythms compared to the disturbed conditions of permanent darkness (D) or permanent light (L) and ± quercetin (Q) administration (50 mg/kg b.wt) on liver cytochrome P₄₅₀ reductase activity (ng/ml) in γ-irradiated rats

Parameter	Liver cytochrome P450 reductase (ng/ml)
Sub-groups group	Control	Irrad	Q	Q + irrad
Normal (L:D)	1.03 ± 0.01	4.02 ± 0.35 a	1.02 ± 0.08	2.34 ± 0.15 ab
Darkness (D)	1.62 ± 0.10 c	5.78 ± 0.35 ac	1.29 ± 0.06	2.84 ± 0.22 ab
Light (L)	1.82 ± 0.13 c	5.17 ± 0.20 acd	1.39 ± 0.09	2.65 ± 0.14 ab

Each value is expressed as the mean ± SE for six rats (p < 0.05). ^aSignificantly different from the control subgroup. ^bSignificantly different from the irradiated subgroup. ^cSignificantly different from the normal group. ^dSignificantly different from the darkness group

Table 2. Influence of the normal (L:D) circadian rhythms compared to the disturbed conditions of permanent darkness (D) or permanent light (L) and ± quercetin (Q) administration (50 mg/kg b.wt.) on liver adenosine diphosphate (ADP) concentrations (ng/ml) in γ-irradiated rats

Parameter	Liver ADP (ng/ml)
Sub-group group	Control	Irrad	Q	Q + irrad
Normal (L:D)	7.05 ± 0.49	20.06 ± 1.13 a	7.04 ± 0.55	9.33 ± 0.35 ab
Darkness (D)	8.00 ± 0.64	6.07 ± 0.46 c	8.83 ± 0.94	9.07 ± 0.47
Light (L)	6.67 ± 0.55	18.82 ± 1.72 ad	6.19 ± 0.39 d	9.68 ± 0.76 ab

Each value is expressed as the mean ± SE for six rats (p < 0.05). ^aSignificantly different from the control subgroup. ^bSignificantly different from the irradiated subgroup. ^cSignificantly different from the normal group. ^dSignificantly different from the darkness group

Table 3. Influence of the normal (L:D) circadian rhythms compared to the disturbed conditions of permanent darkness (D) or permanent light (L) and ± quercetin (Q) administration (50 mg/kg b.wt.) on liver adenosine triphosphate (ATP)concentrations (ng/ml) in γ-irradiated rats

Parameter	Liver ATP (ng/ml)
Sub-group group	Control	Irrad	Q	Q + irrad
Normal (L:D)	61.22 ± 2.21	22.30 ± 1.96 a	62.65 ± 2.58	46.20 ± 0.98 ab
Darkness (D)	39.57 ± 1.23 c	13.97 ± 0.62 ac	49.67 ± 1.28 ac	40.07 ± 1.67 b
Light (L)	50.14 ± 4.42 cd	19.72 ± 2.03 a	60.47 ± 3.03 ad	40.91 ± 2.27 ab

Each value is expressed as the mean ± SE for six rats (p < 0.05). ^aSignificantly different from the control subgroup. ^bSignificantly different from the irradiated subgroup. ^cSignificantly different from the normal group. ^dSignificantly different from the darkness group

In the permanent darkness (D) group, exposure to continuous darkness caused a notable elevation in the mitochondrial fractions of liver cytochrome P450 reductase (Table 1), and a marked reduction in ATP (Table 3) levels, relative to the corresponding values in the normal (L:D) group. Radiation exposure also caused a significant elevation in liver cytochrome P₄₅₀ reductase and a significant depletion in ATP levels, compared to the control sub-group. Administration of quercetin showed no significant change in liver cytochrome P₄₅₀ reductase and ADP levels; however, ATP level was increased compared to the control sub-group, while its administration, pre- and during the irradiation period, induced a significant decrease in liver cytochrome P₄₅₀ reductase (yet, it was still elevated compared to the control sub-group), and a notable increase in ATP levels, compared to the corresponding irradiated sub-group (Tables 1, 2, & 3).

In the permanent light (L) group, exposure to continuous light showed a marked increase in the mitochondrial fractions of liver cytochrome P450 reductase (Table 1), along with significant decreases in ATP (Table 3) levels, relative to the values in the normal (L:D) group. Also, radiation exposure led to marked elevations in liver cytochrome P₄₅₀ reductase and ADP levels, relative to the control sub-group. Quercetin administration induced a significant elevation in ATP levels, compared to the control sub-group, while its administration pre- and during the irradiation period showed a marked depletion in cytochrome P₄₅₀ reductase and ADP levels (but were still higher than those of the control sub-groups), associated with a significant increase in ATP levels (yet, its level was still lower than that observed in the control sub-group), compared to the corresponding irradiated sub-group (Tables 1, 2, & 3).

Histological results

In the normal (L:D) group

Histological examinations of liver sections of the normal (L:D) control rats showed normal hepatic architecture, a normal central vein, a normal portal vein, and normal hepatocytes arranged in single-cell cords with normal intervening blood sinusoids (Fig. 3a1 and a2).

Histological examinations of liver sections of the irradiated sub-group showed that exposure to radiation induced marked dilation in the central veins with detached lining. Scattered apoptosis and a mild hydropic change of hepatocytes in the peri-venular areas were observed (Fig. 3b1). Marked dilation in the portal veins and normal portal tracts were also observed (Fig. 3b2).

Histological examinations of liver sections of the quercetin sub-group showed average central veins and average hepatocytes in the peri-venular areas (Fig. 3c1), thus indicating no change compared to the control group. Average portal tracts, average portal veins, and average hepatocytes in the peri-portal area were also observed (Fig. 3c2).

Histological examinations of liver sections of the quercetin-irradiated sub-group showed mildly dilated central veins with a mild hydropic change of hepatocytes in the peri-venular areas (Fig. 3d1). Average portal tracts, mildly dilated congested portal veins, and mild hydropic change of hepatocytes in the peri-portal area were also observed (Fig. 3d2). Thus, the administration of quercetin pre- and during the irradiation period induced an improvement in the dilation of the central and portal veins compared to that in the irradiated group; however, the dilation was still observed compared to the control group.

[See PDF for image]

Fig. 3

A photomicrograph of a section in the liver of normal (L:D). a1 Control rat showing normal hepatic architecture, normal CV, and normal hepatocytes arranged in single-cell cords (black arrow) with normal intervening blood sinusoids (blue arrow). a2 Control rat showing normal portal tract (black arrow) with normal PV and normal hepatocytes in the peri-portal area (blue arrow). b1 Irradiated rat showing markedly dilated CV with detached lining (black arrow) and scattered apoptosis (blue arrow) with mild hydropic change of hepatocytes in the peri-venular area (red arrow). b2 Irradiated rat showing portal tract with markedly dilated PV and scattered apoptosis (black arrow) with mild hydropic change of hepatocytes in the peri-portal area (blue arrow). c1 quercetin-treated rat showing average CV and average hepatocytes in the peri-venular area (black arrow). c2 Quercetin-treated rat showing average portal tract (black arrow) with average portal vein (blue arrow) and average hepatocytes in the peri-portal area (red arrow). d1 Quercetin + irradiated rat showing mildly dilated CV and mild hydropic change of hepatocytes in the peri-venular area (black arrow). d2 Quercetin + irradiated rat showing average portal tract (black arrow) with mildly dilated congested portal vein (PV) and mild hydropic change of hepatocytes in the peri-portal area (blue arrow) (H&E × 400). PV, portal vein; CV, central vein; BD, bile duct

In the permanent darkness (D) group

Histological examinations of the control liver section showed average central veins with a mild hydropic change of hepatocytes in the peri-venular areas (Fig. 4a1). Thus, a change in hepatocytes was observed in the peri-venular areas, compared to the control sub-group of the normal (L:D) group. Average portal tracts, average portal veins, and average hepatocytes in the peri-portal area were also observed (Fig. 4a2).

Histological examinations of liver sections of the irradiated sub-group showed mildly dilated central veins with scattered apoptosis and mild hydropic change of hepatocytes in the peri-venular areas observed (Fig. 4b1). Also, average portal tracts with average portal veins and scattered apoptotic hepatocytes in the peri-portal area are shown (Fig. 4b2).

Histological examinations of liver sections of the quercetin sub-group showed that administration of quercetin exhibited average central veins with marked hydropic change of hepatocytes observed in the peri-venular areas (Fig. 4c1). Also, average portal tracts, average portal veins, and mild hydropic change of hepatocytes were observed in the peri-portal area (Fig. 4c2).

Histological examinations of liver sections of the quercetin + irradiated sub-group (in which quercetin was administered pre- and during the irradiation period) showed average central veins and average hepatocytes in the peri-venular areas and mildly congested blood sinusoids (Fig. 4d1). Average portal tracts, average portal veins, and average hepatocytes in the peri-portal area (Fig. 4d2) were also observed.

[See PDF for image]

Fig. 4

A photomicrograph of a section in the liver of permanent darkness (D). a1 Control rat showing average CV and mild hydropic change of hepatocytes in the peri-venular area (black arrow). a2 Control rat showing average portal tract (black arrow) and average hepatocytes in the peri-portal area (blue arrow). b1 irradiated rat showing mildly dilated CV and scattered apoptotic hepatocytes in the peri-venular area (black arrow). b2 Irradiated rat showing average portal tract (black arrow) with average PV and scattered apoptotic hepatocytes in the peri-portal area (blue arrow). c1 Quercetin-treated rat showing average CV and marked hydropic change of hepatocytes in the peri-venular area (blue arrow). c2 Quercetin-treated rat showing average portal tract (black arrow) with average PV and mild hydropic change of hepatocytes in the peri-portal area (blue arrow). d1 Quercetin + irradiated rat showing average CV and average hepatocytes in the peri-venular area (black arrow) with mildly congested blood sinusoids (red arrow). d2 quercetin + irradiated rat showing average portal tract (black arrow) with average PV and average hepatocytes in the peri-portal area (red arrow) (H&E × 400). PV, portal vein; CV, central vein; BD, bile duct

In the permanent light (L) group

Histological examinations of the control liver sections showed average central veins, with mild hydropic change of hepatocytes in the peri-venular area also observed (Fig. 5a1). Average portal tracts, average portal veins, and scattered apoptotic hepatocytes were also observed in the peri-portal area (Fig. 5a2).

Histological examinations of liver sections of the irradiated sub-group showed markedly dilated central veins with detached lining, mildly dilated blood sinusoids, and scattered apoptotic hepatocytes in the peri-venular area (Fig. 5b1). Average portal tracts with mildly congested portal veins and scattered apoptotic hepatocytes in the peri-portal area were also observed (Fig. 5b2).

Histological examinations of liver sections of the quercetin sub-group showed that administration of quercetin induced markedly dilated central veins with mild micro- and macro-vesicular steatosis of hepatocytes in the peri-venular area. Average portal tracts with mildly congested portal veins and mild micro-vesicular steatosis of hepatocytes in the peri-portal area are also observed (Fig. 5c2).

Histological examinations of liver sections of the quercetin-irradiated sub-group showed that the administration of quercetin before and during the irradiation period induced average central vein and scattered apoptotic hepatocytes in the peri-venular area, as observed (Fig. 5d1). Also, average portal tracts, mildly congested portal veins, and average hepatocytes in the peri-portal area were noted (Fig. 5d2).

[See PDF for image]

Fig. 5

A photomicrograph of a section in the liver of the permanent light (L). a1 Control rat showing average CV and mild hydropic change of hepatocytes in the peri-venular area (black arrow). a2 Control rat showing average portal tracts (black arrow) with average portal vein (PV) and scattered apoptotic hepatocytes in the peri-portal area (blue arrow). b1 Irradiated rat showing markedly dilated CV with detached lining (black arrow) and average hepatocytes in peri-venular area (blue arrow). b2 Irradiated rat showing average portal tracts (black arrow) with mildly congested PV and scattered apoptotic hepatocytes in the periportal area (blue arrow). c1 Quercetin-treated rat showing markedly dilated CV and mild micro- and macro-vesicular steatosis of hepatocytes in peri-venular area (black arrow). c2 Quercetin-treated rat showing average portal tracts (black arrow) with mildly congested PV and mild micro-vesicular steatosis of hepatocytes in the peri-portal area (blue arrow). d1 Quercetin + irradiated rat showing average CV and scattered apoptotic hepatocytes in the peri-venular area (black arrow). d2 Quercetin + irradiated rat showing average portal tracts (black arrow) with mildly congested PV and average hepatocytes in the peri-portal area (blue arrow) (H&E × 400). PV, portal vein; CV, central vein; BD, bile duct

Discussion

All living organisms exhibit circadian rhythms that can be influenced by external cues such as light. Disruption of circadian rhythms, through extrinsic or intrinsic factors, has been associated with physical and psychological disorders. Circadian rhythms may be misaligned when the internal rhythm is misaligned to the light/dark cycle. In the present study, a model of circadian misalignment was performed by keeping the rats in continuous light or darkness for 1 month. We investigated the potential effects of radiation on rats maintained under disrupted cycles (permanent light or permanent darkness), compared to those kept under a normal (L:D) cycle. The liver is a radiosensitive organ vulnerable to oxidative stress and has a vital role in metabolism and detoxification [31], as well as in controlling circadian rhythms [32]. Therefore, we focused on investigating the biomarkers related to the oxidant/antioxidant status and mitochondrial function in the liver, as well as the histopathological changes in liver tissue.

According to the results of this study, exposure of rats to continuous light or continuous darkness induced a state of oxidative stress in liver tissue which appeared as significant declines in SOD, GPx, and CAT activities, associated with marked elevations in the NO levels in both the dark and light groups, compared to the normal (L:D) group. This disturbance was more pronounced in the light group, as there was also a significant reduction in GSH content and a considerable elevation in lipid peroxidation (expressed as MDA) in this group. The results indicate that the disruption of circadian rhythms affects the oxidant-antioxidant status in liver tissue, which is represented here by elevated lipid peroxidation along with reduced levels of both enzymatic and nonenzymatic antioxidants.

These results are somewhat consistent with Mostafa [33], who observed that abnormal light exposure (continuous light or continuous darkness) for 8 weeks affected the antioxidant and immune systems in the rat spleen, leading to elevating NO production and lipid peroxidation, while opposing her results that showed increased antioxidant enzymes and total antioxidant capacity. This difference in the results may be due to the fact that the response to disturbance in circadian rhythms varies according to the type of tissue [34] or to the difference in the exposure period to light. A study by Mezhnina et al. [35] demonstrated that animal models of a disrupted circadian clock exhibit oxidative stress and impaired antioxidant systems that cause many diseases, pointing out that oxidative stress is a contributing factor to these diseases. However, the researchers illustrated that the functional circadian clock controls the rhythmic activity and expression of nuclear factor erythroid-2-related factor 2 (NRF2), a key regulator of the antioxidant defense pathway that promotes the transcription of numerous antioxidant enzymes [36] and also regulates rhythmic release of melatonin, a powerful free radical scavenger and antioxidant system up-regulator [37]. Melatonin, and its metabolites, has antioxidant actions and act together as a cascade for the elimination of reactive oxygen species (ROS) and reactive nitrogen species (RNS) [38].

Among the negative consequences of circadian rhythm disruption is mitochondrial dysfunction. The circadian clock is closely linked with mitochondrial function by coordinating the generation of mitochondrial ROS and the activity of antioxidant defense by controlling the expression and activity of both mitochondrial antioxidant enzymes and metabolic enzymes [35]. Cytochrome P450 reductase (CPR) is a membrane-bound enzyme required for electron transfer from NADPH to various heme proteins, including cytochrome P450 enzymes. The cytochrome P450 is an important drug-metabolizing enzyme in the body, primarily in the liver, and is mainly distributed in the endoplasmic reticulum and mitochondrial inner membrane. The disruption in cytochrome P450 expression leads to β-oxidation impairment and mitochondrial dysfunction, stimulating oxidative stress [39]. Accumulation of lipid peroxides in cellular membranes induces ferroptotic cell death. In this regard, Koppula et al. [40] suggested that cytochrome P450 reductase promotes lipid peroxidation and ferroptosis through the generation of H₂O₂.

The findings of this work revealed that continuous light or continuous darkness caused a notable increase in the mitochondrial fractions of liver cytochrome P450 reductase level, along with a marked reduction in ATP level, relative to the corresponding values in the normal (L:D) group. Previously, it is reported that the production of cytochrome P450 reductase is coordinated with that of cytochrome P450 enzymes, concerning both daytime-dependent and inducible expression [41].

Thus, dysregulation of the circadian clock, often caused by modern lifestyles, has a critical impact on cellular metabolism and energy production and may lead to the progression and worsening of metabolic diseases. Therefore, intervention strategies that target circadian oscillator components could potentially be effective in treating liver metabolic diseases [3, 4]. Restoring a normal circadian rhythm may also modulate the endogenous metabolic processes to their proper functions.

On the other hand, exposure to ionizing radiation is commonly known to induce cellular injury due to its direct and indirect effects, leading to the generation of ROS, which react rapidly with macromolecules such as proteins, lipids, and nucleic acids. The overproduction of ROS disrupts the oxidant/antioxidant status of various body organs [42]. The present findings show that exposure of rats to 9-Gy gamma radiation decreased significantly SOD, GPx, and CAT activities and GSH contents associated with a marked increase in MDA and NO levels in liver tissue of all irradiated subgroups (in normal, dark, and light groups) compared to the corresponding control subgroups, indicating an imbalance in the oxidants and the antioxidant capacity. The excessive production of ROS and/or insufficient neutralization by endogenous antioxidants leads to cellular injury. In this context, the progression of cellular radiation damage involves lipid peroxidation reactions that may affect protein activity.

Ionizing radiation can also activate the inducible form of nitric oxide synthase and increase NO synthesis. The NO may react with the superoxide anion, forming the peroxynitrite anion, a stronger oxidant, with a rate constant greater than that for the SOD-catalyzed dismutation of the superoxide anion. Peroxynitrite anion reacts with other molecules to generate other RNS leading to a disturbed redox homeostasis [43]. Again, the present results show that the effect of radiation on the antioxidant system is more pronounced in the light group than in the normal and dark groups, indicating circadian oscillations of oxidative stress responses.

Previously, it was reported that the oscillation of oxidative stress is related to the rhythm of expression and activity of antioxidant enzymes [44]. The study of Budkowska et al. provided evidence for circadian control over antioxidant enzymes: SOD, CAT, GPx, and R-GSSG [45]. In this regard, Çiftel et al. [46] observed a significant decrease in the oxidative stress-related DNA damage marker (8-OHdG) and apoptotic spermatocytes in the testicular tissue of rats exposed to ionizing radiation at night, compared to those exposed during the day.

Moreover, an excess of free radicals has been correlated with disordered mitochondrial components and, consequently, mitochondrial function [47]. Dong et al. [48] indicated that ionizing radiation triggers the production of ROS in mitochondria, causing mitochondrial damage. The damaged mitochondrial remnants release more ROS, leading to programmed cell death. This study demonstrated that 9-Gy gamma irradiation caused a marked elevation in cytochrome P450 reductase along with significant decreases in ATP levels in the mitochondrial fractions of liver tissues of all irradiated subgroups (normal, dark, and light groups) in relation to the respective values observed in the control subgroups. However, ADP levels were significantly increased in the irradiated subgroups (in normal and light groups) in relation to the respective levels observed in the control subgroups. The disruption of mitochondrial components may be correlated with reduced mitochondrial energy. Mitochondria are susceptible to radiation injury, which interferes with oxidative phosphorylation, a key metabolic pathway vital for energy production.

The impairment of mitochondrial function and the reduction of ATP production after radiation exposure and a disturbed light/darkness cycle may be related to the reduction of thyroid hormone levels. This can be explained by considering the liver as a major site of thyroid hormone action and the mitochondria being the primary site of thyroid hormone accumulation in the cell. Triiodothyronine has been reported to promote mitochondrial biogenesis and as a contributor to the enhancement of mitochondrial activity and their energy process [49]. A significant depletion in circulating thyroid hormones and a nonsignificant elevation in TSH values were observed in rats exposed to 6-Gy whole-body irradiation [50]. Interestingly, although the relationship between circadian clocks and thyroid function has been described [51], the thyroid state interaction with the circadian regulation of physiological processes in the body remains largely unknown [52].

In addition, the impairment of the hepatic antioxidant defense system, and the consequent decline in mitochondrial metabolic activity and ATP synthesis also, may be related to the inhibition of melatonin secretion. Amer et al. [53] observed a significant decrease in rat serum melatonin levels, 3 and 14 days after irradiation, indicating disruptions of pineal gland function and melatonin synthesis. However, it was anticipated that the mitochondria would utilize its melatonin-forming ability to harness its benefits as a potent free radical scavenger, thereby reducing injury induced by mitochondrial ROS and boosting ATP production [54]. In this respect, it was reported that mitochondrial melatonin level was not related to circulating melatonin (derived from the pineal gland), as its concentration was not decreased in pinealectomized rats, in which circulating melatonin levels were close to zero [55].

The oxidative stress and metabolic disturbance caused by exposure to ionizing radiation, and/or disruption of circadian rhythms, call for the search for safe agents capable of neutralizing or eliminating free radicals, which cause harmful effects. The use of exogenous antioxidants or protective agents, before or during exposure, may be a potential means of reducing these effects. These exogenous antioxidants act synergistically with the endogenous antioxidants. Their synergistic mechanisms should be reducing the detrimental effects associated with free radicals, particularly lipid peroxidation, by neutralizing free radicals, interacting with prooxidants, and promoting the reduction of ROS [56]. Previous experimental studies on various animal models have demonstrated that quercetin, a flavonoid found in fruits and vegetables, has protective effects against radiation or xenobiotics. Baran et al. [19] observed that quercetin oral supplementation for 5 days, before or after irradiation, modulated radiation-induced brain and pituitary damage in rats. The beneficial action of quercetin against aflatoxin B1-caused toxicity in both in vitro studies and animal models was described by Dai et al. [57]. Moreover, the study of [20] indicated that quercetin improved liver circadian rhythm and metabolism disorders in vitamin D-deficient mice.

Findings from this study indicated that quercetin administered for 1 month in nonirradiated rats, kept under normal or disturbed circadian rhythms (normal, light, and darkness groups), significantly increased GP_X activity, SOD (in the darkness group) activity, and ATP levels (in the darkness and light groups), compared to the corresponding control subgroups. However, its administration for 1 week before and 3 weeks during irradiation significantly decreased MDA and NO levels and increased SOD, CAT, and GP_X activities, as well as GSH contents in all the quercetin-irradiated subgroups (normal, light, and darkness), compared to the corresponding values in the irradiated subgroups. These findings indicated that quercetin could safeguard the liver from oxidative damage, caused by gamma irradiation, through its antioxidant properties.

Quercetin administration before and during the irradiation period also modulated liver mitochondrial function in the irradiated treated subgroups (normal, light, and darkness). It significantly decreased cytochrome P450 reductase activities (in all groups) and the levels of ADP (in the normal and light groups); however, it increased ATP levels in all the groups, compared to the irradiated subgroups. The obvious improvement in the oxidant-antioxidant status and mitochondrial markers in liver tissue indicated the protective effect of quercetin against the adverse effects of gamma irradiation and/or circadian rhythm disturbance. These results agree with the former study of Ji et al. [58] who observed that quercetin administration alleviated oxidative stress, inflammation, mitochondrial dynamics imbalance, and mitochondrial apoptosis induced by a fungicide difenoconazole in carp. Also, quercetin prevented ROS accumulation and restored redox homeostasis induced by exposure to avermectin [59].

The mechanisms of antioxidant action of quercetin include the following:

1. A direct mechanism to eliminate the surplus ROS. This mechanism is related to the chemical configuration of quercetin, which contains a pair of benzene rings (A and B); the B ring is prone to react with ROS [60]. Also, quercetin contains five active hydroxyl groups that serve as electron donors and are responsible for scavenging free radicals [61].

2. A regulatory influence on GSH level, as it stimulates the synthesis of GSH [62, 63]

3. A modulatory effect on the Nrf2 signaling pathways that promote the expression of antioxidant enzymes such as SOD, CAT, and GP_X and inhibit lipid peroxidation. In addition, the activation of Nrf2 stimulates cellular respiration and mitochondrial biosynthesis and reduces DNA damage and apoptosis [64]. In this respect, quercetin reduces the formation of ROS in microglia cells and increases a variety of antioxidants [65].

The data of the present study showed that the histopathological examination of liver sections of irradiated rats revealed marked dilation in the central and portal veins, with scattered apoptosis; also, mild hydropic change of hepatocytes in the peri-portal and peri-venular areas was observed in the three main groups. These findings are in harmony with Abdou et al. [66], who observed liver injury in rats subjected to 6 Gy of gamma rays, coupled with oxidative stress. Also, Abd El-Hady et al. [67] reported that rats exposed to 8-Gy (2 Gy every 3 days) whole-body irradiation caused loss of the normal hepatic architecture with dilatation of the central vein and hepatic blood sinusoids. Some degenerated hepatocytes appeared with pyknotic nuclei and vacuolated cytoplasm. These alterations may be attributed to the damaging effect of γ-radiation, resulting in the overproduction of ROS within cells and the depletion of cellular antioxidants.

The present study showed that disturbance of circadian rhythms (exposure to continuous darkness or continuous light) induced scattered apoptotic hepatocytes in the peri-portal area and mildly dilated central veins with scattered apoptosis and mild hydropic change of hepatocytes in the peri-venular areas. This may be due to the surplus generation of ROS. Padilla et al. [68] stated that circadian irregularities are associated with the development of hepatic diseases, including hepatic fat accumulation, inflammation, and fibrosis, hepatitis, and hepatocellular carcinoma. Furthermore, these disorders disrupt circadian clock function. However, quercetin administration for 1 month improved the histopathological changes in liver tissues in the irradiated and disturbed circadian subgroups. These data are in harmony with those of Abd El-Hady et al. [67], who reported that administration of quercetin following irradiation showed signs of recovery in rat liver tissue, as a result of quercetin’s antioxidative and anti-inflammatory properties.

In conclusion, the results of the current study revealed that gamma irradiation and/or circadian rhythm disturbance induces oxidative stress and mitochondrial metabolic disturbance in liver tissue. Rats exposed to permanent light are more affected by γ-radiation than those exposed to permanent darkness. Oral administration of quercetin could protect the liver from these damaging effects by modulating the oxidant-antioxidant system and mitochondrial function. The histopathological investigation supported the biochemical results and confirmed the protective effect of quercetin against ionizing radiation and/or circadian disturbance.

Acknowledgements

Not applicable

Authors’ contributions

O. A. K. performed the practical procedures of this study, followed by the statistical analysis of the data, and writing up the preliminary results section. A. N., H. G. H., and S. A.A. El. Continuously supervised the progress of the work, analyzed, interpreted, discussed, and jointly wrote the results obtained. All authors read and approved the final manuscript.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

All animal procedures were performed following the international guidelines for animal handling and care of the National Institutes of Health (NIH publication No. 85- following 23, 1996), and in accordance with regulations and guidelines set by the Research Ethics Committee of the Faculty of Science, Ain Shams University and Ethics Committee of the NCCRT, Cairo, Egypt.

Consent for publication.

Not applicable

Competing interests

The authors declare no competing interests.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

1. Hussey, KD. Rhythmic history: towards a new research agenda for the history of health and medicine. Endeavour; 2022; 46, 4 [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36521301]100846.

2. Brown, AJ; Pendergast, JS; Yamazaki, S. Peripheral circadian oscillators. Yale J Biol Med; 2019; 92, 2 pp. 327-335.1:CAS:528:DC%2BB3cXjsFWmsrk%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31249493][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6585520]

3. Reinke, H; Asher, G. Circadian clock control of liver metabolic functions. Gastroenterology; 2016; 150, 3 pp. 574-580. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26657326]

4. Sadria, M; Layton, AT. Aging affects circadian clock and metabolism and modulates timing of medication. iScience; 2021; 24, 41:CAS:528:DC%2BB3MXhs1OiurfO [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33796837][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7995490]102245.

5. Saran, AR; Dave, S; Zarrinpar, A. Circadian rhythms in the pathogenesis and treatment of fatty liver disease. Gastroenterology; 2020; 158, 7 pp. 1948-1966.1:CAS:528:DC%2BB3cXosVaisb8%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32061597]

6. Ayyar, VS; Sukumaran, S. Circadian rhythms: influence on physiology, pharmacology, and therapeutic interventions. J Pharmacokinet Pharmacodyn; 2021; 48, 3 pp. 321-338. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33797011][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8015932]

7. Sletten TL, Cappuccio FP, Davidson AJ, Van Cauter E, Rajaratnam SMW, Scheer FA (2020) Health consequences of circadian disruption. Sleep 43(1):zsz194.

8. Jacobi, D; Liu, S; Burkewitz, K; Kory, N; Knudsen, NH; Alexander, RK; Unluturk, U; Li, X; Kong, X; Hyde, AL; Gangl, MR; Mair, WB; Lee, CH. Hepatic Bmal1 regulates rhythmic mitochondrial dynamics and promotes metabolic fitness. Cell Metab; 2015; 22, 4 pp. 709-720.1:CAS:528:DC%2BC2MXhsV2ls7bO [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/26365180][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4598294]

9. Bumgarner, JR; Nelson, RJ. Implications of disrupted circadian rhythms on pain. J Psychiatry Brain Sci; 2021; 6, 4 e210014.

10. Nelson, N; Lombardo, J; Matlack, L; Smith, A; Hines, K; Shi, W; Simone, NL. Chronoradiobiology of breast cancer: the time is now to link circadian rhythm and radiation biology. Int J Mol Sci; 2022; 23, 31:CAS:528:DC%2BB38XktFWhurg%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35163264][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8836288]1331.

11. Hassan, AA; Abdelgayed, SS; Mansour, SZ. Liver and ovarian toxicities boosted by bisphenol and gamma radiation in female albino rats. Hum Exp Toxicol; 2024; 43, 9603271231219264.1:CAS:528:DC%2BB2cXivVKnurnP [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/38263794]

12. Abdel-Aziz, N; Haroun, RA; Mohamed, HE. Low-dose gamma radiation modulates liver and testis tissues response to acute whole body irradiation. Dose Response; 2022; 20, 2 15593258221092364.

13. Singh, VK; Seed, TM. The efficacy and safety of amifostine for the acute radiation syndrome. Expert Opin Drug Saf; 2019; 18, 11 pp. 1077-1090.1:CAS:528:DC%2BC1MXhsl2gtrvM [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31526195]

14. Zhang, L; Xu, LY; Tang, F; Liu, D; Zhao, XL; Zhang, JN; Xia, J; Wu, JJ; Yang, Y; Peng, C; Ao, H. New perspectives on the therapeutic potential of quercetin in non-communicable diseases: targeting Nrf2 to counteract oxidative stress and inflammation. J Pharm Anal; 2023; 14, 6 100930.

15. Masiala, A; Vingadassalon, A; Aurore, G. Polyphenols in edible plant leaves: an overview of their occurrence and health properties. Food Funct; 2024; 15, 13 pp. 6847-6882.1:CAS:528:DC%2BB2cXht1Ohu77M [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/38853513]

16. Aghababaei, F; Hadidi, M. Recent advances in potential health benefits of quercetin. Pharmaceuticals (Basel); 2023; 16, 1020.1:CAS:528:DC%2BB3sXhs1WlurbE [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37513932]

17. Suganthy, N; Devi, KP; Nabavi, SF; Braidy, N; Nabavi, SM. Bioactive effects of quercetin in the central nervous system: focusing on the mechanisms of actions. Biomed Pharmacother; 2016; 84, pp. 892-908.1:CAS:528:DC%2BC28XhslSnsbrE [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27756054]

18. Deepika, MPK. Health benefits of quercetin in age-related diseases. Molecules; 2022; 27, 8 2498.1:CAS:528:DC%2BB38XhtFCjsLbP [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35458696][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9032170]

19. Baran, M; Başaran, KE; Gergin, OO; Cengız, O; Yıldız, O; Yay, A. Evaluation of the neuroprotective effect of quercetin against damage caused by gamma radiation. J Cancer Res Ther; 2024; 20, 1 pp. 261-267.1:CAS:528:DC%2BB2cXhsV2rurzJ [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/38554331]

20. Li, R; Wang, G; Liu, R; Luo, L; Zhang, Y; Wan, Z. Quercetin improved hepatic circadian rhythm dysfunction in middle-aged mice fed with vitamin D-deficient diet. J Physiol Biochem; 2024; 80, 1 pp. 137-147.1:CAS:528:DC%2BB3sXitlOisr%2FE [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37948027]

21. Hagde, P; Pingle, P; Mourya, A; Katta, CB; Srivastava, S; Sharma, R; Singh, KK; Sodhi, RK; Madan, J. Therapeutic potential of quercetin in diabetic foot ulcer: mechanistic insight, challenges, nanotechnology driven strategies and future prospects. J Drug Deliv Sci Technol; 2022; 74, 1:CAS:528:DC%2BB38Xit1OnsLvJ 103575.

22. Usadadia, SV; Patel, J; Vihol, P; Urkude, AP. Protective effect of quercetin on azithromycin induced hepatotoxicity and nephrotoxicity in Wistar rats. Int J Curr Microbiol App Sci; 2020; 9, 9 pp. 2926-2934.1:CAS:528:DC%2BB3cXisFyhsbzM

23. Rickwood D, Wilson MT, Darley-Usmar VM (1987) Isolation and characteristics of intact mitochondria. In: Darley-Usmar VM, Rickwood D, Wilson MT (eds) Mitochondria: a practical approach. IRL Press, Washington, D.C., pp 4–5

24. Stroev E A, Makarova A G (1989) Study in peroxide oxidation of biological membrane lipids (Chapter 15, pp. 251–255). In Laboratory Manual in Biochemistry (English translation of Praktikum po biologicheskoi khimii; translated edition). Moscow: MIR Publishing.

25. Miles, AM; Wink, DA; Cook, JC; Grisham, MB. Determination of nitric oxide using fluorescence spectroscopy. Methods Enzymol; 1996; 268, pp. 105-120.1:CAS:528:DyaK28XlvFeju70%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/8782577]

26. Foyer, CH; Souriau, N; Perret, S; Lelandais, M; Kunert, K; Pruvost, C; Jouanin, LL. Overexpression of glutathione reductase but not glutathione synthetase leads to increases in antioxidant capacity and resistance to photoinhibition in poplar trees. Plant Physiol; 1994; 109, pp. 1047-1057.

27. Ahmed, AY; Aowda, SA; Hadwan, MH. A validated method to assess glutathione peroxidase enzyme activity. Chem Pap; 2021; 75, 11 pp. 6625-6637.1:CAS:528:DC%2BB3MXitVWgsLbJ

28. Nishikimi, M; Roa, NA; Yogi, K. The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem Bioph Res Common; 1972; 46, pp. 849-854.1:CAS:528:DyaE38Xps1Wisw%3D%3D

29. Kranner, I; Birtic, S. A modulating role for the antioxidants in desiccation tolerance. Integr Comp Biol; 2005; 45, pp. 734-740.1:CAS:528:DC%2BD28XntlSktQ%3D%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/21676824]

30. Bancroft J D, & Stevens A, Turner DR (1996) Theory and practice of histological techniques (4th ed.). Edinburgh: Churchill Livingstone. ISBN 978‑0443047602.

31. Burraco, P; Salla, RF; Orizaola, G. Exposure to ionizing radiation and liver histopathology in the tree frogs of Chornobyl (Ukraine). Chemosphere; 2023; 315, 1:CAS:528:DC%2BB3sXmtF2htQ%3D%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36608893]137753.

32. Guan, D; Lazar, MA. Circadian regulation of gene expression and metabolism in the liver. Semin Liver Dis; 2022; 42, 2 pp. 113-121.1:CAS:528:DC%2BB3sXhs1ejt7fN [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35263797][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9806798]

33. Moustafa, A. Chronic exposure to continuous brightness or darkness modulates immune responses and ameliorates the antioxidant enzyme system in male rats. Front Vet Sci; 2021; 8, [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33937367][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8081841]621188.

34. Koritala, BSC; Dakup, PP; Porter, KI; Gaddameedhi, S. The impact of shift-work light conditions on tissue-specific circadian rhythms of canonical clock genes: insights from a mouse model study. F1000Res; 2023; 12, 1:CAS:528:DC%2BB2cXisFGhu74%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37576540][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10422053]762.

35. Mezhnina, V; Ebeigbe, OP; Poe, A; Kondratov, RV. Circadian control of mitochondria in reactive oxygen species homeostasis. Antioxid Redox Signal; 2022; 37, 10–12 pp. 647-663.1:CAS:528:DC%2BB38XisFCgsrfE [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/35072523][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9587791]

36. Pekovic-Vaughan, V; Gibbs, J; Yoshitane, H; Yang, N; Pathiranage, D; Guo, B; Sagami, A; Taguchi, K; Bechtold, D; Loudon, A; Yamamoto, M; Chan, J; van der Horst, GT; Fukada, Y; Meng, QJ. The circadian clock regulates rhythmic activation of the NRF2/glutathione-mediated antioxidant defense pathway to modulate pulmonary fibrosis. Genes Dev; 2014; 28, 6 pp. 548-560.1:CAS:528:DC%2BC2cXlsF2nsb4%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/24637114][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3967045]

37. Potter, GD; Skene, DJ; Arendt, J; Cade, JE; Grant, PJ; Hardie, LJ. Circadian rhythm and sleep disruption: causes, metabolic consequences, and countermeasures. Endocr Rev; 2016; 37, 6 pp. 584-608.1:CAS:528:DC%2BC1cXlvV2kt78%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/27763782][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5142605]

38. Monteiro, KKAC; Shiroma, ME; Damous, LL; Simões, MJ; Simões, RDS; Cipolla-Neto, J; Baracat, EC; Soares-Jr, JM. Antioxidant actions of melatonin: a systematic review of animal studies. Antioxidants; 2024; 13, 4 439. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/38671887][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11047453]

39. Jiang, YJ; Cao, YM; Cao, YB; Yan, TH; Jia, CL; He, P. A review: cytochrome P450 in alcoholic and non-alcoholic fatty liver disease. Diabetes Metab Syndr Obes; 2024; 17, pp. 1511-1521. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/38586542][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10997053]

40. Koppula, P; Zhuang, L; Gan, B. Cytochrome P450 reductase (POR) as a ferroptosis fuel. Protein Cell; 2021; 12, 9 pp. 675-679.1:CAS:528:DC%2BB3MXhtVakurnP [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/33539003][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8403093]

41. Gachon, F; Olela, FF; Schaad, O; Descombes, P; Schibler, U. The circadian PAR-domain basic leucine zipper transcription factors DBP, TEF, and HLF modulate basal and inducible xenobiotic detoxification. Cell Metab; 2006; 4, 1 pp. 25-36.1:CAS:528:DC%2BD28XmvFyiu7k%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/16814730]

42. Abdel-Aziz, N; Elkady, AA; Elgazzar, EM. Choline glycerophosphate and silymarin modulate brain and intestinal injuries in rats exposed to gamma -radiation. Int J Radiat Res; 2022; 20, 4 pp. 829-837.

43. Azzam, EI; Jay-Gerin, J; Pain, D. Ionizing radiation-induced metabolic oxidative stress and prolonged cell injury. Cancer Lett; 2020; 327, 1–2 pp. 48-60.

44. Wilking, M; Ndiaye, M; Mukhtar, H; Ahmad, N. Circadian rhythm connections to oxidative stress: implications for human health. Antioxid Redox Signal; 2013; 19, 2 pp. 192-208.1:CAS:528:DC%2BC3sXpsFKit70%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/23198849][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3689169]

45. Budkowska, M; Cecerska-Heryć, E; Marcinowska, Z; Siennicka, A; Dołęgowska, B. The influence of circadian rhythm on the activity of oxidative stress enzymes. Int J Mol Sci; 2022; 23, 221:CAS:528:DC%2BB38XjtVahurfI [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36430753][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9697911]14275.

46. Çiftel, S; Mercantepe, F; Tümkaya, L; Mercantepe, T; Yılmaz Rakıcı, S; Çiftel, E. Circadian clock on ionizing radiation-induced testicular injury. Endocrinol Res Pract; 2024; 28, 2 pp. 113-120.

47. Martemucci, G; Costagliola, C; Mariano, M; D’andrea, L; Napolitano, P; D’Alessandro, AG. Free radical properties, source and targets, antioxidant consumption and health. Oxygen; 2022; 2, 2 pp. 48-78.1:CAS:528:DC%2BB3sXpvVCjtbs%3D

48. Dong, S; Lyu, X; Yuan, S; Wang, S; Li, W; Chen, Z; Yu, H; Li, F; Jiang, Q. Oxidative stress: a critical hint in ionizing radiation induced pyroptosis. Radiat Med Prot; 2020; 1, 4 pp. 179-185.

49. Ramanathan, R; Patwa, SA; Ali, AH; Ibdah, JA. Thyroid hormone and mitochondrial dysfunction: therapeutic implications for metabolic dysfunction-associated steatotic liver disease (MASLD). Cells; 2023; 12, 24 2806.1:CAS:528:DC%2BB2cXhtlGrsro%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/38132126][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10741470]

50. Abdel-Aziz, N. Disturbances in thyroid hormone metabolism in rats exposed to γ- radiation and carbon tetrachloride: the role of N- acetyl cysteine and sodium selenite. Egypt J Rad Sci Applic; 2020; 33, 2 pp. 119-131.

51. Ikegami, K; Refetoff, S; Van Cauter, E; Yoshimura, T. Interconnection between circadian clocks and thyroid function. Nat Rev Endocrinol; 2019; 15, 10 pp. 590-600. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/31406343][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7288350]

52. de Assis, LVM; Harder, L; Lacerda, JT; Parsons, R; Kaehler, M; Cascorbi, I; Nagel, I; Rawashdeh, O; Mittag, J; Oster, H. Tuning of liver circadian transcriptome rhythms by thyroid hormone state in male mice. Sci Rep; 2024; 14, 1 640. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/38182610][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10770409]

53. Amer, ME; Othman, AI; Abozaid, HM; El-Missiry, MA. Utility of melatonin in mitigating ionizing radiation-induced testis injury through synergistic interdependence of its biological properties. Biol Res; 2022; 55, 1 33.1:CAS:528:DC%2BB38XivVaqsrjP [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/36333811][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9636653]

54. Suzen, S; Saso, L. Melatonin as mitochondria-targeted drug, chapter eight. Adv Protein Chem Struct Biol; 2023; 136, pp. 249-276. [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37437980]

55. Reiter, RJ; Ma, Q; Sharma, R. Melatonin in mitochondria: mitigating clear and present dangers. Physiology; 2020; 35, pp. 86-95.1:CAS:528:DC%2BB3cXitV2iu7rK [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/32024428]

56. Al-Zharani, M; Mubarak, M; Rudayni, HA; Al-Doaiss, AA; Abd-Elwahab, MM; Al-Eissa, MS. Quercetin as a dietary supplementary flavonoid alleviates the oxidative stress induced by lead toxicity in male wistar rats. Nutrients; 2023; 15, 1888.1:CAS:528:DC%2BB3sXpsFSqsr0%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37111107][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10143896]

57. Dai, C; Sharma, G; Liu, G; Shen, J; Shao, B; Hao, Z. Therapeutic detoxification of quercetin for aflatoxin B1-related toxicity: roles of oxidative stress, inflammation, and metabolic enzymes. Environ Pollut; 2024; 345, 1:CAS:528:DC%2BB2cXjs12ntbg%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/38309422]123474.

58. Ji, X; Guo, J; Ma, Y; Zhang, S; Yang, Z; Li, Y; Ping, K; Xin, Y; Dong, Z. Quercetin alleviates the toxicity of difenoconazole to the respiratory system of carp by reducing ROS accumulation and maintaining mitochondrial dynamic balance. Toxicol Appl Pharmacol; 2024; 484, 1:CAS:528:DC%2BB2cXjsFCgtbk%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/38342444]116860.

59. Pan, E; Chen, H; Wu, X; He, N; Gan, J; Feng, H; Sun, Y; Dong, J. Protective effect of quercetin on avermectin induced splenic toxicity in carp: resistance to inflammatory response and oxidative damage. Pestic Biochem Physiol; 2023; 193, 1:CAS:528:DC%2BB3sXhtVKis7bF [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37248014]105445.

60. Tang J, Diao P, Shu X, Li L, Xiong L (2019) Quercetin and quercitrin attenuates the inflammatory response and oxidative stress in LPS-Induced RAW264.7 cells: in vitro assessment and a theoretical model. BioMed Res Int 2019:7039802.

61. Carrillo-Martinez, EJ; Flores-Hernández, FY; Salazar-Montes, AM; Nario-Chaidez, HF; Hernández-Ortega, LD. Quercetin, a flavonoid with great pharmacological capacity. Molecules; 2024; 29, 1000.1:CAS:528:DC%2BB2cXmtV2ru74%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/38474512][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10935205]

62. Granado-Serrano, AB; Martín, MA; Bravo, L; Goya, L; Ramos, S. Quercetin modulates Nrf2 and glutathione-related defenses in HepG2 cells: involvement of p38. Chem Biol Interact; 2012; 195, 2 pp. 154-164.1:CAS:528:DC%2BC38XotVCrtw%3D%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/22197970]

63. Xu, D; Hu, MJ; Wang, YQ; Cui, YL. Antioxidant activities of quercetin and its complexes for medicinal application. Molecules; 2019; 24, 6 1123.1:CAS:528:DC%2BC1MXpt12hsLs%3D [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/30901869][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6470739]

64. Zhang, L; Xu, LY; Tang, F; Liu, D; Zhao, XL; Zhang, JN; Xia, J; Wu, JJ; Yang, Y; Peng, C; Ao, H. New perspectives on the therapeutic potential of quercetin in non-communicable diseases: targeting Nrf2 to counteract oxidative stress and inflammation. J Pharm Anal; 2024; 14, 6 [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/39005843][PubMedCentral: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11245930]100930.

65. Tsai, CF; Chen, GW; Chen, YC; Shen, CK; Lu, DY; Yang, LY; Chen, JH; Yeh, WL. Regulatory effects of quercetin on M1/M2 macrophage polarization and oxidative/antioxidative balance. Nutrients; 2022; 14, 1 67.1:CAS:528:DC%2BB38XhsFyjtLk%3D

66. Abdou, FY; Fahmy, HA; Mohamed, MA. Amlodipine repurposing in amending liver toxicity induced by gamma irradiation. Egypt J Rad Sci Applic; 2023; 36, 1–2 pp. 99-109.

67. Abd El-Hady, AM; Azzoz, RM; Soliman, SM; Abdelrahman, IY; Khalil, WM; Ali, SA. Studies on the effect of curcumin and quercetin in the liver of male albino rats exposed to gamma irradiation. Histochem Cell Biol; 2024; 162, 4 pp. 299-309.

68. Padilla, J; Osman, NM; Bissig-Choisat, B; Bissig, K; Moore, DD; Fu, L. Circadian dysfunction induces NAFLD-related human liver cancer in a mouse model. J Hepatol; 2024; 80, 2 pp. 282-292.1:CAS:528:DC%2BB3sXitlKitbzN [PubMed: https://www.ncbi.nlm.nih.gov/pubmed/37890720]