Investigation of protective effect of resveratrol and coenzyme Q10 against cyclophosphamide-induced lipid peroxidation, oxidative stress and DNA damage in rats

Erten Akbel; Ismail Kucukkurt; Sinan Ince; Hasan Huseyin Demirel; Damla Arslan Acaroz; Fahriye Zemheri-Navruz; Fahriye Kan

doi:10.1093/toxres/tfad123

. 2023 Dec 30;13(1):tfad123. doi: 10.1093/toxres/tfad123

Investigation of protective effect of resveratrol and coenzyme Q₁₀ against cyclophosphamide-induced lipid peroxidation, oxidative stress and DNA damage in rats

Erten Akbel ¹, Ismail Kucukkurt ^2,^✉, Sinan Ince ³, Hasan Huseyin Demirel ⁴, Damla Arslan Acaroz ⁵, Fahriye Zemheri-Navruz ⁶, Fahriye Kan ⁷

PMCID: PMC10758596 PMID: 38173543

Abstract

It is seen that cyclophosphamide, which is used in treating many diseases, especially cancer, causes toxicity in studies, and its metabolites induce oxidative stress. This study aimed to investigate the protective effects of resveratrol and Coenzyme Q10, known for their antioxidant properties, separately and together, against oxidative stress induced by cyclophosphamide. In this study, 35 Wistar albino male rats were divided into five groups. Groups; Control group, cyclophosphamide (CP) group (CP as 75 mg kg i.p. on day 14), coenzyme Q₁₀ (CoQ₁₀) + CP group (20 mg/kg i.p. CoQ₁₀ + 75 mg kg i.p. CP), resveratrol (Res) + CP group (20 mg/kg i.p. Res + 75 mg/kg i.p. CP), CoQ₁₀ + Res + CP group (20 mg/kg i.p Res + 20 mg/kg i.p CoQ₁₀ and 75 mg/kg i.p.CP). At the end of the experiment, the cholesterol, creatinine and urea levels of the group given CP increased, while a decrease was observed in the groups given Res and CoQ10. Malondialdehyde level was high, glutathione level, superoxide dismutase and catalase activities were decreased in the blood and all tissues (liver, kidney, brain, heart and testis) of the CP given group. DNA damage and histopathological changes were also observed. In contrast, Res and CoQ₁₀, both separately and together, reversed the CP-induced altered level and enzyme activities and ameliorated DNA damage and histopathological changes. In this study, the effects of Res and CoQ₁₀ against CP toxicity were examined both separately and together.

Keywords: cyclophosphamide, resveratrol, coenzyme Q10, rat, oxidative stress, DNA damage

Graphical Abstract

Introduction

Cyclophosphamide (CP), which belongs to the class of nitrogen mustards, is a cytotoxic bifunctional alkylating agent and is a highly reactive compound. While CP is used in chemotherapy of different types of cancer (lung, breast and ovarian, lymphoma, leukemia, Etc.) at high doses, it is widely used in treating autoimmune diseases (rheumatoid arthritis) at low doses. The use of CP in high doses causes oxidative stress in humans and experimental animals and exhibits severe cytotoxicity in normal cells. This may limit the clinical use of CP. The search for alternative solutions against these side effects during treatment continues rapidly today,^1–3 and experimental studies are of great importance in reaching a solution.

Resveratrol, 3,4′, 5-trihydroxystilbene, is an active natural polyphenolic compound in various foods, especially red grapes.⁴ Studies have shown that resveratrol has multiple pharmacological activities, including antitumor, anti-inflammatory, antiaging, anti-diabetic, cardioprotective, and neuroprotective properties. It has been stated that it significantly alleviates hepatic oxidative damage thanks to its antioxidant properties.^5–7 Coenzyme Q₁₀ (CoQ₁₀) was first identified in 1955 as an essential component of the respiratory chain of the mitochondrial membrane and as a vitamin-like substance, and extensive research has been carried out until today. It is a part of the intracellular antioxidant system and is stated to protect phospholipids, membrane proteins, and DNA against free radicals.^8–10 CoQ₁₀ is found in plant and animal tissues that are part of our diet, and especially animal hearts and livers represent the wealthiest source.¹¹ CoQ₁₀ deficiency has been identified in diseases in which oxidative stress plays an important role, such as neurodegenerative disorders, diabetes, cancer, and cardiovascular diseases.¹²

This study aimed to investigate the protective effects of resveratrol and CoQ₁₀, which are known to have antioxidant properties against oxidative stress induced by CP, which are used in the treatment of various diseases, both separately and when given together. For this purpose, antioxidant and oxidative stress parameter values in blood and different tissues were examined. The histopathological changes of the tissue samples were evaluated, and the effect on DNA damage was investigated.

Material and method

Chemicals

Chemicals used in this study: cyclophosphamide (97% purity) (Alfa Aesar, USA), Resveratrol (95%) abcr (abcr GmbH, Germany), Coenzyme Q_10—Ubiquinone (Acros, 98%) and other chemicals of analytical purity were obtained from commercial companies.

Animal material

Ethics Committee approval was obtained from Afyon Kocatepe University Animal Experiments Ethics Committee (AKUHAYDEK) for the study with the number 495337021/82 dated 24.05.2016. The study used 35 male Wistar rats weighing 250–300 g. The experimental stage of the rats obtained from the Afyon Kocatepe University Experimental Animals Unit was also carried out in this center. The rats were first divided into groups and allowed to acclimate for seven days before being included in the experimental process. The rats were given standard rat food and ad libitum water by keeping the ambient conditions at 50–55% humidity and room temperature (25 °C).

Experimental protocol

For the experimental phase of the study, five experimental groups were formed, and the study took place in 15 days. Groups and applications;

Control Group: The male rats in the control group were given standard rat food and drinking water.
CP Group: After giving distilled water intraperitoneally (i.p.) for 14 days, CP i.p. at a 75 mg/kg dose dissolved in distilled water on the 14th day given as.¹³
CoQ₁₀ Group: 20 mg/kg i.p given for 14 days¹⁴ and CP 75 mg/kg i.p. on day 14. given as.
Resveratrol (Res) Group: 20 mg/kg i.p. given for 14 days,¹⁵ and on day 14, CP 75 mg/kg i.p. given as.
Res + CoQ₁₀ Group: 20 mg/kg, i.p. was given for 14 days, and on the 14th day, CP 75 mg/kg i.p. was presented as.

Blood and tissues (liver, kidney, brain, heart, and testis) were collected from animals under xylazine and ketamine anesthesia 24 h after the last application. The plasmas of the blood samples were separated by centrifugation at 3,000 rpm for 10 min; the plasmas were taken into 1.5 mL Eppendorf tubes and stored at −80 °C until analysis. While one part of each tissue was stored at −80 °C for biochemical examination, the other parts were followed for histopathological examination.

Preparation of whole blood, erythrocyte lysate, and tissue homogenates

Removed liver, kidney, brain, heart, and testis tissues were first thoroughly washed with cold 0.9% NaCl, then homogenized at a ratio of 1:40 w/v in 0.1 M phosphate buffer (PH = 7.4). The homogenate was centrifuged at 5,000 rpm for 15 min.¹⁶ The obtained supernatant was used for the determination of superoxide dismutase (SOD) and catalase (CAT) activities by the measurements of malondialdehyde (MDA) and glutathione (GSH).

Blood samples taken into heparinized tubes were divided into two parts. A portion of the blood was prepared for the measurement of SOD and CAT activities. For this purpose, erythrocytes and plasma were separated by centrifugation at 3,500 g for 15 min at 4 °C for 30 min. The precipitated erythrocytes were washed three times with isotonic saline and the fluffy layer was removed. Then, isotonic saline and erythrocytes were added in the same volume and stored at −20C.¹⁷

Biochemical parameters measured in the study

MDA, a lipid peroxidation marker, was measured in whole blood¹⁸ and tissue homogenate.¹⁹ In determining GSH concentration in whole blood and tissue homogenates, Beutler et al.,²⁰ the method described by was used. The method described by Sun et al.²¹ was used for SOD enzyme activity in erythrocyte lysate and tissue homogenate. For CAT enzyme activity, Luck²² in erythrocyte lysate; in tissue homogenate, Aebi et al.²³ methods were used. For the determination of hemoglobin, the tissue protein content was determined using a colorimetric cyanomethemoglobin method according to Drabkin and Austin,²⁴ and the tissue protein content was determined by Lowry et al.²⁵ was analyzed according to the colorimetric method.

Other biochemical parameters; aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), triglyceride, total cholesterol, protein, urea, creatinine (HUMAN, Wiesbaden, Germany), and glucose (BIOLABO, Maizy, France) levels were measured spectrophotometrically (Thermo Fisher Scientific Oy Ratastie 2, FI-01620 Vantaa, Finland).

Histopathological evaluation

The liver, kidney, brain, heart, and testis tissues taken were prepared for histological tissue follow-up. These prepared samples were first fixed in 10% formalin solution, then dehydrated with graded alcohol solutions (70%–100%). Tissues were kept in xylene, then blocked with paraffin, then 5-micron thick sections were taken using a microtome (Leica, RM 2245). The sections taken were stained with Hematoxylin-Eosin staining to evaluate their general histological properties, and each section was examined under a light microscope (Nikon Eclipse CI, Tokyo, Japan).

Comet analysis

The comet assay is known as single-cell gel electrophoresis and is a method used for the qualitative and quantitative detection of DNA damage in single-celled eukaryotic organisms. It is the most common and most popular technique used in in vivo genotoxicity studies.²⁶ At the end of the experiment, 500 μL of non-hemolyzed rat blood was taken three times for each group and centrifuged at 300 g at room temperature, the supernatant was discarded, and the pellet was washed with 500 μL of PBS (Phosphate Buffered Saline). 0.5% agarose (LMA) with a low melting temperature was melted and kept in a 40 °C heater to prevent freezing. 1% agarose (NMA) with a normal melting point was prepared, and the slides were coated. 20 μL of the washed blood samples were mixed with 100 μL of 0.5% LMA, added to the slide coated with NMA, and allowed to freeze. It was incubated at 4 °C for 5 min. The prepared preparations were kept in lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris base, pH adjusted to 10 and 1% Triton X-100, 10% DMSO freshly added) at 4 °C for 1 h. After standing in electrophoresis buffer (10 N NaOH, 200 mM EDTA, pH> 13.0) for 15 min at 4 °C, alkaline electrophoresis was applied at 24 V and 300 mA for 40 min. After soaking in cold distilled water, the preparations were neutralized in 0.4 M Tris buffer (pH 7.5) for 5 min. This process was repeated three times.²⁷ The preparations were then stained with 100 μL ethidium bromide (10 μL/mL), and fluorescence microscopy (Zeiss, Germany) was used to examine. While examining the acquired images, 100 (50–100) randomly selected cells were evaluated and recorded according to the damage range between 0 and 4. (0 undamaged, 4 significantly damaged).

Statistical analyzes

SPSS 22.0 (IBM Corporation, Armonk, New York, United States) program was used to analyze the data. Shapiro-Wilk test was used for the normal distribution of the data, and the Leneve test was used for the homogeneity of variance. When comparing multiple independent groups with each other, One-Way ANOVA (Brown-Forsythe) from parametric methods was used with Bootstrap results, while Games Howell and LSD tests were used for post hoc analysis. Kruskal-Wallis H Test, one of the nonparametric tests, was used with the results of Monte Carlo simulation technique and was used for Post Hoc analysis. Quantitative data are mean ± std. (standard deviation) and median ± IQR (Interquartile Range) in the tables). The data were analyzed at a 95% confidence level, and a P-value less than 0.05 was accepted as significant.

Results

Biochemical parameters

The effects of CoQ₁₀ and resveratrol on some biochemical parameters of oxidative stress induced by CP in rats are shown in Table 1. Compared to the control, it was determined that CP application caused an increase in cholesterol (P < 0.05), creatinine, and urea amount (P < 0.001), whereas CoQ₁₀ and resveratrol application decreased these values. In addition, it was determined that these values decreased in CP application when CoQ₁₀ and resveratrol were given together compared to when they were given alone, and this decrease was not statistically significant. In addition, no significant difference was observed between the groups in glucose, protein, and triglyceride levels.

Table 1.

Effect of CoQ₁₀ and resveratrol on glucose, cholesterol, protein, triglyceride, creatinine, and urea in oxidative stress induced by CP in rats.

Groups	Glucose (mg/dL)	Cholesterol (mg/dL)	Protein (g/L)	Triglyceride (mg/dL)	Creatinine (mg/dL)	Urea (mg/dL)
Control	99,79 ± 9,34	79,89 ± 20,65^ab	6,41 ± 0,26	115,14 ± 27,99	0,68 ± 0,07^d	33,87 ± 5,74^bc
CP	108,02 ± 9,05	99,79 ± 29,77^a	6,88 ± 0,54	135,20 ± 30,71	1,05 ± 0,19^a	49,63 ± 10,13^a
CoQ₁₀ + CP	101,18 ± 3,87	71,83 ± 11,02^b	6,60 ± 0,36	125,76 ± 18,81	0,95 ± 0,14^ab	31,07 ± 4,31^c
Res + CP	103,26 ± 5,87	73,06 ± 21,11^b	6,95 ± 0,56	104,84 ± 10,01	0,88 ± 0,10^bc	40,38 ± 11,01^b
CoQ₁₀ + Res + CP	102,96 ± 5,98	61,02 ± 5,19^b	6,71 ± 0,47	108,80 ± 15,98	0,75 ± 0,10^cd	29,53 ± 6,34^c
p	0,280	0,013	0,195	0,089	0,000	0,000

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