Protective effects of resveratrol on sepsis-induced DNA damage in the lymphocytes of rats

Abstract

Sepsis, often initiated by an infection, is a state of disrupted inflammatory homeostasis. There is increasing evidence that oxidative stress has an important role in the development of sepsis-induced multiorgan failure. Resveratrol (RV) is a polyphenolic compound found in the skin of red fruits, such as mulberries and red grapes, and in peanuts. RV has been reported to have an antioxidant, antiproliferative, and anti-inflammatory properties in various models. It has also been found to inhibit the proliferation of a variety of human cancer cell lines, including breast, prostate, colon, pancreatic, and thyroid. This study has been undertaken to assess the role of RV on the sepsis-induced oxidative DNA damage in the lymphocytes of Wistar albino rats by the standard and formamidopyrimidine DNA glycosylase (Fpg)-modified comet assays. The parameters of tail length, tail intensity, and tail moment were evaluated for the determination of DNA damage. According to the study, the DNA damage was found to be significantly higher in the sepsis-induced rats when compared with the control rats (p < 0.05). The parameters were significantly decreased in the RV-treated sepsis-induced group when compared with the sepsis-induced group. The parameters in the sepsis-induced rats were found to be significantly higher in the Fpg-modified comet assay when compared with the standard comet assay (p < 0.05), and RV treatment decreases the DNA damage in the sepsis-induced rats, suggesting that the oxidative stress is likely to be responsible for DNA damage and RV might have a role in the prevention of sepsis-induced oxidative DNA damage.

Keywords

Sepsis resveratrol DNA damage alkaline single cell gel electrophoresis

Introduction

Sepsis is a complex syndrome characterized by an imbalance between proinflammatory and anti-inflammatory response to pathogens, occurs after infectious insults and presents with very high mortality and morbidity in intensive care units.^1

–5 Some of the postulated molecular mechanisms of sepsis progression are linked to the imbalance between reactive oxygen species (ROS) production and its degradation by cellular antioxidants.^5

–8 The proinflammatory effects of ROS include endothelial damage, formation of chemotactic factors, neutrophil recruitment, cytokines release, mitochondrial impairment, lipid peroxidation, and DNA damage.^3,5,8,9 Decrease in the oxidative stress using antioxidants in the cecal ligation and puncture (CLP) model of sepsis limits neutrophil infiltration, attenuates mitochondrial dysfunction, decreases oxidative damage, and thereby improves survival.⁷

Resveratrol (3,4′,5-trihydroxystilbene; RV), a polyphenolic compound, is found naturally in the skin of red grapes, peanuts, and a number of other plants. RV has been suggested to be responsible for the “French paradox”, the observation that the incidence of cardiac disease in France is lower than in other industrialized nations, in spite of the fact that the French consume a diet that is high in cholesterol and saturated fats.¹⁰ It has long been used as a circulatory tonic in herbal medicines.^11
–13

RV has been shown to bind with numerous cell signaling molecules, such as multidrug resistance protein, topoisomerase II, aromatase, DNA polymerase, oestrogen receptors, tubulin, and F1-ATPase. It activates various transcription factors, suppresses the expression of antiapoptotic gene products, inhibits protein kinases, induces antioxidant enzymes, suppresses the expression of inflammatory biomarkers, inhibits the expression of angiogenic and metastatic gene products, and modulates cell cycle regulatory genes. Moreover, numerous studies have demonstrated that this polyphenol holds promise against cancer.¹⁴ Recently, it has been found to prevent and cure cardiovascular diseases and improve microcirculatory disorders by protecting the vascular endothelium, modulation of lipid metabolism, increasing cellular nitric oxide levels, as well as inhibiting platelet aggregation.^12,15
–17 It has an anti-inflammatory effect mainly due to its ability to inhibit cyclooxygenase and hydroperoxidase functions.¹⁸ The observation that RV is an effective radical scavenger has been suggested that it acts as a natural antioxidant against oxidative DNA damage.^19,20 The scope of biological interactions by RV is extensive and has been listed in a recent review.²¹

Oxidative DNA damage may involve the breakage in single and double strands, base modifications, fragmentation of deoxyribose, formation of DNA–protein cross-links as well as abasic sites.^22,23 DNA strand breaks in eukaryotic cells can be detected by single cell gel electrophoresis (comet assay), with and without the addition of the repair enzymes endonuclease-III, formamidopyrimidine DNA glycosylase (Fpg), to characterize DNA lesions. Fpg initiates the repair of oxidized bases by excising them and cutting the sugar–phosphate backbone of the DNA molecule. Thus, additional strand breaks are induced at the location of oxidized base, causing DNA relaxation and migration. The detection of Fpg-sensitive DNA lesions revealed the presence of oxidized purine bases.^24
–26 Comet assay, which is a simple, sensitive, rapid, and versatile assay, is commonly used for the assessment of protective effects of antioxidants on DNA damage in intervention studies.²⁷ It can be virtually applied on any cell type in eukaryotic cells such as lymphocytes, sperm, buccal, nasal, epithelial, and placental cells. In the present study, the aim was to investigate the protective effects of RV on sepsis-induced oxidative DNA damage in the lymphocytes of Wistar albino rats using the standard and Fpg-modified comet assay.

Materials and methods

Chemicals

The chemicals used in the experiments were purchased from the following suppliers: normal melting point agarose (NMA) and low melting point agarose (LMA) from Boehringer Manheim (Mannheim, Germany). Sodium chloride (NaCl), sodium hydroxide (NaOH), potassium chloride (KCl), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) from Merck Chemicals (Darmstadt, Germany); formamidopyrimidine DNA glycosylase (Fpg), bovine serum albumin, dimethyl sulfoxide (DMSO), ethidium bromide (EtBr), Triton X-100, phosphate-buffered saline (PBS) tablets, and RV from Sigma–Aldrich Chemicals (St Louis, Missouri, USA); ethylenediamine tetraacetic acid disodium salt dihydrate (EDTA-Na₂), natrium lauroyl sarcosinate, and Tris from ICN Biomedicals Inc. (Aurora, Ohio, USA).

Animals

A total of 32 Wistar albino rats (3 months old, 200–300 g), from Refik Saydam National Public Health Agency, were used in the study. Animals were housed in plastic cages with stainless steel grid tops. Rats were subjected to a controlled environment regarding temperature (23°C), humidity (50%), and a 12-h light–dark cycle before the experiments. Animals were fed with standard laboratory chow and allowed to access feed and drinking water ad libitum before and after operation. Body weights (bws) were measured. The animals were treated humanely and with regard for alleviation of suffering, and the study was approved by Hacettepe University Animal Ethical Committee.

Experimental procedures

All surgical procedures were performed under anesthesia by intraperitoneal (i.p.) injection of 90 mg/kg ketamine hydrochloride (Ketalar, Eczacıbaşı Warner-Lambert, Istanbul, Turkey).

Sepsis was introduced by CLP technique, as described previously.²⁸ Under the anesthesia, a midline laparotomy was made using minimal dissection and the cecum was ligated just below the ileocecal valve with 3-O silk ligatures so that intestinal continuity was maintained. On the antimesentric surface of the cecum, using an 18-gauge needle, the cecum was perforated at two locations 1 cm apart and the cecum was gently compressed until the feces were extruded. The bowel was then returned to the abdomen and the incision was closed. At the end of the operation, all rats were resuscitated with saline (5 mL/100 g bw) subcutaneously (s.c.). The rats were deprived of food but had free access to water after the operation. The sham-operated control group underwent laparotomy; the cecum was manipulated but was not ligated or perforated.

The rats were divided into four experimental groups and each group consisted of eight animals. Sham group consisted of animals treated i.p. with 0.5 mL of saline alone following laparatomy. Abdominal layers were closed with appropriate suture materials. All animals were maintained under the same conditions after surgery. Sepsis group consisted of animals in which only CLP was performed and the animals were treated with 0.5 mL saline i.p. following the induction of CLP. RV-treated group consisted of animals immediately treated with a dose of 100 mg/kg i.p. RV in 0.5 mL saline following laparatomy. The RV-treated and sepsis-induced group consisted of animals immediately treated with 0.5 mL of a solution containing 100 mg/kg i.p. RV following the induction of CLP.

After 24 h following the treatment, all rats were decapitated under the anesthesia and cardiac blood was collected into preservative-free heparin tubes for DNA damage analysis. The samples were kept in the dark at 4°C and processed within 4 h.

Single cell gel electrophoresis (comet assay) and Fpg-modified comet assay

The basic alkaline technique of Singh et al.,²⁹ as further described by Anderson et al.³⁰ and Collins et al.,²⁶ was followed. The concentrations of heparinized whole blood cells were adjusted to approximately 2 × 10⁶ cells/mL in PBS buffer. A total of 5 μL of the cells (2 × 10⁶ cells/mL) suspended in 75 μL of 0.5% LMA was embedded on slides precoated with a layer of 1% NMA. Slides were allowed to solidify on ice for 5 min. Coverslips were then removed. The slides were immersed in cold lysing solution (2.5 M NaCl,100 mM EDTA, 100 mM Tris, 1% sodium sarcosinate, and pH: 10), with 1% Triton X-100 and 10% DMSO added just before use for a minimum of 1 h at 4°C. Then they were removed from the lysing solution, drained and were left in the electrophoresis solution (1 mM sodium EDTA and 300 mM NaOH, pH = 13) for 20 min at 4°C to allow unwinding of the DNA and expression of alkali-labile damage.

The alkaline comet assay using Fpg, lesion-specific enzyme was used to detect oxidized pyrimidines as a result of oxidative stress-induced DNA damage as described with some modifications.²⁴ The cell–agarose suspension slides were prepared as described earlier for the standard comet assay. After lysing, the slides were washed three times for 5 min with the enzyme buffer (40 mM HEPES, 100 mM KCl, 0.5 mM EDTA, and 0.2 mg/mL bovine serum albumin) at room temperature and were incubated at 37°C for 30 min with Fpg (1:500) and with enzyme buffer (control). Then they were left in the electrophoresis solution (1 mM sodium EDTA and 300 mM NaOH, pH = 13) for 20 min at 4°C to allow unwinding of the DNA and expression of alkali-labile damage.

Electrophoresis was also conducted at a low temperature (4°C) for 20 min using 24 V and adjusting the current to 300 mA by rising or lowering the buffer level. The slides were neutralized by washing three times in 0.4 M Tris–HCL (pH = 7.5) for 5 min at room temperature. After neutralization, the slides were incubated in 50%, 75%, and 98% of alcohol for 5 min each.

The dried microscopic slides were stained with EtBr (20 μg/mL in distilled water, 60 μL/slide), covered with a cover-glass prior to analysis with a Leica® fluorescence microscope under green light. The microscope was connected to a charge-coupled device camera and a personal computer-based analysis system (Comet Analysis Software, version 3.0, Kinetic Imaging Ltd, Liverpool, UK) to determine the extent of DNA damage after electrophoretic migration of the DNA fragments in the agarose gel. In order to visualize DNA damage, slides were examined at 40× magnification.

Results were expressed as tail length, tail intensity, and tail moment. From each of the two replicate slides, 100 cells were assayed.

For each animal, the differences in the tail length, tail intensity, and tail moment between the samples obtained with the Fpg-modified comet assay (total DNA damage) and standard alkaline comet assay (basic DNA damage) were considered as oxidative DNA damage in a single cell.

Statistical analysis

Statistical analysis was performed by SPSS for Windows 11.5 computer program. The results are expressed as mean ± SD. Differences between the means of data were compared by the one-way analysis of variance test, and the post hoc analysis of group differences was performed by least significant difference test. p < 0.05 were considered as statistically significant.

Results

The DNA damage expressed as tail length, tail intensity, and tail moment in the lymphocytes was given in Table 1 and Figures 1 and 2.

Figure 1.

DNA damage in whole blood cells of the experimental groups expressed as (a) DNA tail length; (b) DNA tail intensity; (c) DNA tail moment. The values are expressed as mean ± SD (n = 8). ^a p < 0.05, compared with sham group for the standard comet assay; ^b p < 0.05, compared with sepsis group for the standard comet assay; ^c p < 0.05, compared with sham group for the Fpg-modified comet assay; ^d p < 0.05, compared with sepsis group for the Fpg-modified comet assay; ^e p < 0.05, standard comet assay was compared with Fpg-modified comet assay. Fpg: formamidopyrimidine DNA glycosylase.

Figure 2.

Comet assay images of lymphocyte DNA in the study groups. Sham group for standard comet assay (a) and Fpg-modified comet assay (b); sepsis-induced group for standard comet assay (c) and Fpg-modified comet assay (d); RV-treated group for standard comet assay (e) and Fpg-modified comet assay (f); RV-treated sepsis-induced group for standard comet assay (g) and Fpg-modified comet assay (h). Fpg: formamidopyrimidine DNA glycosylase; RV: resveratrol.

Table 1.

DNA damage in the experimental groups expressed as tail length, tail intensity, and tail moment in the standard and Fpg-modified comet assay^a

	Sham group	Sepsis-induced group	RV-treated group	RV-treated sepsis-induced group
Tail length
Standard comet assay	12.46 ± 0.80 (11.19–13.41)	15.88 ± 1.56^b (13.45–18.48)	12.58 ± 0.63^c (11.92–13.74)	13.76 ± 1.50^c (12.35–15.76)
Fpg-modified comet assay	15.57 ± 1.36^d (14.24–17.48)	32.21 ± 8.71^d,e (16.85–41.88)	16.58 ± 1.72^d,f (14.84–18.58)	21.04 ± 3.26^d,e,f (18.40–27.32)
Tail intensity
Standard comet assay	5.19 ± 0.69 (4.27–6.05)	8.86 ± 1.43^b (7.03–11.43)	5.16 ± 1.07^c (3.76–6.56)	5.53 ± 1.90^c (2.98–8.56)
Fpg-modified comet assay	6.14 ± 2.95^d (4.11–11.30)	27.18 ± 9.26^d,e (11.60–36.29)	6.90 ± 3.30^d,f (3.76–11.88)	15.29 ± 4.65^d,e,f (10.01–22.65)
Tail moment
Standard comet assay	0.47 ± 0.07 (0.35–0.53)	0.96 ± 0.22^b (0.72–1.42)	0.47 ± 0.12^c (0.33–0.63)	0.53 ± 0.19^c (0.27–0.84)
Fpg-modified comet assay	0.68 ± 0.52^d (0.34–1.60)	5.70 ± 3.02^d,e (1.04–9.82)	0.85 ± 0.57^d,f (0.31–1.71)	2.31 ± 0.99^d,e,f (1.34–4.04)

Fpg: formamidopyrimidine N-glycosylase; RV: resveratrol.

^aThe results are given as mean ± SD (minimum–maximum).

^b p < 0.05, compared with sham group for the standard comet assay.

^c p < 0.05, compared with sepsis group for the standard comet assay.

^d p < 0.05, standard comet assay was compared with Fpg-modified comet assay.

^e p < 0.05, compared with sham group for the Fpg-modified comet assay.

^f p < 0.05, compared with sepsis group for the Fpg-modified comet assay.

In the standard comet assay, there were no statistically significant differences in terms of tail length, tail intensity, and tail moment between the sham group and the RV-treated group (p > 0.05). The parameters of tail length, tail intensity, and tail moment were found to be significantly higher in the sepsis-induced group in comparison with the sham group (p < 0.05). RV treatment in the sepsis-induced group was found to decrease the DNA damage significantly (p < 0.05).

In the Fpg-modified comet assay, there were no statistically significant differences in terms of tail length, tail intensity, and tail moment between the sham group and the RV-treated group (p > 0.05). The parameters of tail length, tail intensity, and tail moment were found to be significantly higher in the sepsis-induced group in comparison with the sham group (p < 0.05). RV treatment in the sepsis-induced group was found to decrease the DNA damage significantly (p < 0.05).

Furthermore, the enzyme-sensitive sites are more prone for the strand breaks, which showed significantly higher DNA damage in the Fpg-modified comet assay when compared with the standard comet assay.

The tail length obtained from the Fpg-modified comet assay was found to be 1.2-, 2.0-, 1.3-, and 1.5-fold higher than that from standard comet assay for the sham, sepsis, RV, and sepsis + RV groups, respectively. The tail intensity obtained from the Fpg-modified comet assay was found to be 1.2-, 3.1-, 1.3-, and 2.8-fold higher than that from standard comet assay for the sham, sepsis RV, and sepsis + RV groups, respectively. The tail moment obtained from the Fpg-modified comet assay was found to be 1.5-, 5.9-, 1.8-, and 4.3-fold higher than that from standard comet assay for the sham, sepsis, RV, and sepsis + RV groups, respectively.

Fpg-sensitive sites were found to increase significantly in the sepsis-induced group (p < 0.05), which suggest that sepsis-induced oxidative stress is likely to be responsible for DNA damage.

RV treatment in the sepsis-induced group was found to decrease the Fpg-sensitive sites, which suggests that RV might have a role in the prevention of sepsis-induced oxidative DNA damage.

Discussion

Sepsis is associated with the development of progressive damage in multiple organ systems remote from the locus of infection.³¹ There is increasing evidence that oxidative stress has an important role in the development of sepsis-induced multiorgan failure.³² The release of endotoxin from bacteria is generally thought to be the initial event in the development of sepsis. Endotoxin activates inflammatory cells, which subsequently amplify the inflammatory response via the release of various cytokines that causes oxidative nuclear damage. This inflammatory cascade results in the infiltration of leukocytes in various organs leading to vascular as well as parenchymal cell dysfunctions.³¹ In addition, reactive nitrogen species resulting from lipid peroxidation of biological membranes also cause further DNA damage and nuclear lysis of the cells.^1,33 The peripheral lympocytes are an early site of intense oxidative processes in the body. The most easily formed DNA lesions induced by ROS are 8-oxo-7,8-dihydro-2-deoxyguanosines (8-oxodG), but the background levels of altered purines are usually efficiently repaired.^34,35

It has been shown that RV has extensive effects, including protection against cardiovascular diseases and cancer. There is growing evidence that RV may provide an alternative (and early) intervention approach and prevent or delay the onset of heart diseases, ischemic and chemically induced injuries, shock and multiple organ dysfunction syndrome, pathological inflammation, and viral infections via inhibiting oxidation, leukocyte priming, and expression of inflammatory mediators as well as by regulating microcirculation.^36

–40

RV has been proposed as a potential chemopreventive agent and its antimutagenic and anticarcinogenic activity has been demonstrated in several models. It has been suggested to be chemopreventive by its ability to protect DNA as well as to induce DNA repair.⁴¹ Recent studies demonstrate that RV can directly target signaling cascades involved in inflammation and the development of cancer.^39,42 Low-density lipoprotein and tumor necrosis factor-alpha oxidation eliciting significant increase in caspase 3/7 activity in endothelial cells and cultured rat aortas, which were prevented by RV treatment 1–100 μM. Thus, RV seems to prevent oxidative stress-induced endothelial cell death.⁴³

It has been reported that the anticancer mechanism of plant polyphenols, such as RV, might be due to the pro-oxidant action induced by the mobilization of endogenous copper.^44,45 It has been shown that 100 μM RV, depending on the individual basal DNA status, could switch from antioxidant to pro-oxidant in the voluntary runner.⁴⁶ Antiproliferative, DNA damaging, and apoptotic effects of RV in colorectal cancer cells, head, and neck squamous cell carcinoma cells have already been reported in vitro.^47,48 It has been shown that RV, by which following DNA damage induced, caused to apoptotic cell death in rat hepatoma cells but necrosis in C6 glioma cells.⁴⁹ It has been suggested that RV caused the induction of apoptosis and a block of cell cycle progression at an early step S-phase. RV alleviates the apoptotic clearance of irradiated cells and prevents the G′ phase cell cycle arrest induced by x-rays in lymphoblastoid cells.⁵⁰ RV of 100 and 200 μM tested in androgen-resistant prostate cancer cells has shown anticancer activity exerting cytotoxicity via necrosis, which might be useful in chemotherapy.⁵¹ It has been reported that long-term exposure of moderate to high doses of RV (1–100 mg/kg diet) in male F344 rats could damage lymphocytes DNA, induce low levels of preneoplastic liver lesions and via pro-oxidative and nonoxidative mechanisms might modulate carcinogenesis.⁵²

RV was also efficient at quenching the chemotherapy drugs cisplatin and selenium–cisplatin and the alkylating carcinogen1-methyl-2-nitro-1-nitrosoguanidine (MNNG) and 1-methyl-1-nitrosourea (MNU).^41,53
–55 It has been reported that the treatment regimen of RV of 8 mg/kg bw orally everyday with alkylating agent 1,2-dimethlyhydrazine (DMH) of 20 mg/kg bw given s.c. as four injections for 16 days reduced the DMH-induced leukocytes DNA damage in Wistar male rats.⁵⁶

Our study is consistent with the findings of antioxidant activity of RV against oxidative DNA damage.^{20,41,56

–62} We demonstrated that sepsis induced the oxidative DNA damage in the peripheral lymphocytes. The increase in Fpg sites in sepsis-induced group indicates that sepsis may result in elevated levels of DNA damage. Kaymak et al.⁶³ also reported the elevated levels of oxidized purine and pyrimidine bases, which were correlated with different stages in sepsis-induced rats. Increase in the tail length, tail intensity, and tail moment in the lymphocytes of sepsis-induced rats treated with RV as measured by the standard comet and the Fpg-modified comet assay is much lower than in sepsis-induced rats, which shows that RV prevents the oxidative DNA damage.

Kolgazi et al.⁶⁴ reported that RV reduced sepsis-induced remote organ injury and the protective effect of RV could be attributed to balance oxidant–antioxidant status, to inhibit neutrophil infiltration, and to regulate the inflammatory mediators. The antioxidant properties of RV have already been described elsewhere.^65,66 RV inhibits free-radical formation and has an antimutagenic activity.⁶⁷ The observation that RV is an effective radical scavenger suggested that it acts as a natural antioxidant against oxidative DNA damage.^20,21 It has been reported that at the concentration of 30 μM, RV reduced oxidative DNA damage and at 30 μM and 90 μM, RV could prevent the increase in ROS levels induced by hydrogen peroxide (H₂O₂) and tobacco-smoke condensate in normal rat fibroblasts, mouse mammary epithelial cells, human breast, colon, and prostate cancer cells.⁵⁸ Yen et al.⁵⁹ also revealed that 10–100 μM of RV in a concentration-dependent decrease in the oxidative DNA damage induced by H₂O₂ induced activity of glutathione peroxidase and glutathione reductase, glutathione-S-transferase, inhibited the catalase and induced an increase in glutathione levels induced by H₂O₂ in human lymphocytes.

In conclusion, the protective effect of RV on the sepsis has been demonstrated in our study and the observations from the study might light the way for the possible beneficial outcomes of its use against sepsis related disorders.

Footnotes

Conflict of interest

The authors declared no conflicts of interest.

Funding

This research received no specific grant from any funding agency in the public, commercial, or not for-profit sectors.

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