e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 8 ( 2 0 1 4 ) 774–782

Available online at www.sciencedirect.com

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The protective role of ferulic acid on sepsis-induced oxidative damage in Wistar albino rats Merve Bacanlı a , Sevtap Aydın a , Gökc¸e Taner b , Hatice Gül Göktas¸ a,c , Tolga S¸ahin d , A. Ahmet Bas¸aran e , Nurs¸en Bas¸aran a,∗ a

Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Hacettepe University, 06100 Ankara, Turkey Department of Biology, Faculty of Science, Gazi University, 06500 Ankara, Turkey c Department of Pharmaceutical Toxicology, Faculty of Pharmacy, C ¸ ukurova University, Sarıc¸am, 01330 Adana, Turkey d Department of Surgery, Faculty of Kastamonu Medicine, Hacettepe University, 06100 Ankara, Turkey e Department of Pharmacognosy, Faculty of Pharmacy, Hacettepe University, 06100 Ankara, Turkey b

a r t i c l e

i n f o

a b s t r a c t

Article history:

Oxidative stress has an important role in the development of sepsis-induced multiorgan fail-

Received 19 March 2014

ure. Ferulic acid (FA), a well-established natural antioxidant, has several pharmacological

Received in revised form

activities including anti-inflammatory, anticancer and hepatoprotective. This study aimed

18 August 2014

to investigate the effects of FA on sepsis-induced oxidative damage in Wistar albino rats.

Accepted 25 August 2014

Sepsis-induced DNA damage in the lymphocytes, liver and kidney cells of rats were eval-

Available online 16 September 2014

uated by comet assay with and without formamidopyrimidine DNA glycosylase (Fpg). The oxidative stress parameters such as superoxide dismutase (SOD) and glutathione peroxidase

Keywords:

(GSH-Px) activities and total glutathione (GSH) and malondialdehyde (MDA) levels were also

Sepsis

measured. It is found that DNA damage in sepsis + FA-treated group was significantly lower

DNA damage

than the sepsis group. FA treatment also decreased the MDA levels and increased the GSH

Alkaline single cell electrophoresis

levels and SOD and GSH-Px activities in the sepsis-induced rats. It seems that FA might have

Ferulic acid

ameliorative effects against sepsis-induced oxidative damage. © 2014 Published by Elsevier B.V.

1.

Introduction

Sepsis, the systemic response to an infection, is a common cause of morbidity and mortality in intensive care units that can progress to multiple organ failure which is clinically characterized by the liver, pulmonary, cardiovascular, renal and gastrointestinal dysfunction (Zapelini et al., 2008). Reactive oxygen species (ROS) are believed to be involved in the development of sepsis (Cassol-Jr et al., 2010; Kaymak et al., 2008; Zhou et al., 2012). The pro-inflammatory effects of ROS

∗

Corresponding author. Tel.: +90 312 305 21 78; fax: +90 312 311 47 77. E-mail address: [email protected] (N. Bas¸aran). http://dx.doi.org/10.1016/j.etap.2014.08.018 1382-6689/© 2014 Published by Elsevier B.V.

include endothelial damage, formation of chemotactic factors, neutrophil recruitment, cytokines release, mitochondrial impairment, lipid peroxidation, and DNA damage (Andrades et al., 2009, 2011; Barichello et al., 2006; Hotchkiss and Karl, 2003), all contributing to a free radical overload and to oxidant–antioxidant imbalance (Andrades et al., 2011). It has been demonstrated that ROS lead to the breakage in single and double strands, base modifications, fragmentation of deoxyribose, formation of DNA-protein cross-links as well as abasic sites (Andrades et al., 2009; Barzilai and Yamamoto, 2004; Cooke et al., 2003; Evans and Cooke, 2004).

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 8 ( 2 0 1 4 ) 774–782

Natural products are widely used as dietary supplements because of their potential antioxidant properties. Plant polyphenols may act as antioxidants by different mechanisms such as free radical scavenging, metal chelation and protein binding (Maurya and Devasagayam, 2010). Ferulic acid (4-hydroxy-3-methoxycinnamic acid, FA), a phenolic compound, arises from the metabolism of phenylalanine and tyrosine by Shikimate pathway in plants (Prasad et al., 2006; Ramar et al., 2012; Sudheer et al., 2008). This phenolic compound is present in fruits, vegetables, rice and wheat and has been suggested to have several properties such as antioxidant, antihyperlipidemic, antimicrobial, anti-inflammatory, antiatherogenic, anticarcinogenic, neuroprotective, and antihypertensive (Balakrishnan et al., 2008; Ramar et al., 2012; Sudheer et al., 2008). It is also shown that FA had protective effects against cardiovascular diseases, Alzheimer’s disease, and ultraviolet radiation (Mancuso and Santangelo, 2014). FA has been used as a food additive and antioxidant in Japan, whereas sodium ferulate is used for treatment of cardiovascular and cerebrovascular diseases in China (Itagaki et al., 2009; Zeni et al., 2012; Zhao and Moghadasian, 2008). Due to its phenolic nucleus and an extended side chain (Fig. 1), FA readily forms a resonance stabilized phenoxy radical which accounts for its free radical-scavenging effects (Graf, 1992; Palacios and Perez, 1990; Zhao and Moghadasian, 2008). This enables FA to protect DNA and lipids against oxidation through ROS (Kanski et al., 2002; Srinivasan et al., 2006; Zhao and Moghadasian, 2008). FA was shown to scavenge both ROS and reactive nitrogen species (RNS) (Trombino et al., 2013). Through its free radical scavenging activity, and enhancing the cell stress response, FA has antioxidant activity (Calabrese et al., 2008; Barone et al., 2009). FA was also shown to be effective as a neuroprotector in several in vitro and ex vivo models of neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease and cerebral ischemia/reperfusion injury. A preclinical study with FA has showed a therapeutic activity in preventing noise-induced auditory loss (Fetoni et al., 2010). The aim of this study was to assess the protective effects of FA on sepsis-induced oxidative damage in the lymphocytes, liver and kidney cells of Wistar albino rats. To determine the oxidative DNA damage, Comet assay was used. The oxidative stress parameters such as superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activities and total glutathione (GSH) and malondialdehyde (MDA) levels in the liver and kidney tissues were also measured to investigate the effects of FA on sepsis-induced oxidative stress.

2.

Materials and methods

2.1.

Chemicals

The chemicals used in the study were purchased from the following suppliers: normal melting agarose (NMA)

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and low melting point agarose (LMA) from Boehringer Manheim (Mannheim, Germany); sodium chloride (NaCl), sodium hydroxide (NaOH), potassium chloride (KCl) and 4-(2hydroxyethyl)-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, trichloroacetic acid, thiobarbituric acid, n-butanol and FA from Sigma–Aldrich Chemicals (St Louis, MO, USA); ethylendiaminetetraacetic acid disodium salt dihydrate (EDTA-Na2 ), natriumlauroylsarcosinate, and Tris from ICN Biomedicals Inc. (Aurora, OH, USA), SOD assay kit, GSH-Px assay kit and GSH assay kit from Cayman Chemicals Co. (Ann Arbor, MI, USA).

2.2.

Animals

Wistar albino rats (3 months old, male, weight range 200–300 g) were used in all experiments. Animals were obtained from Refik Saydam National Public Health Agency, Ankara, Turkey. They were housed in plastic cages with stainless steel grid tops. Rats were maintained on a 12 h light–dark cycle, at controlled temperature (23 ± 2 ◦ C) and humidity (%50). Animals were fed with standard laboratory chow and allowed to access feed and drinking water ad libitum before and after operation. The animals were treated humanely and with regard for alleviation of suffering and the study were approved by Hacettepe University Animal Ethical Committee.

2.3.

Cecal ligation puncture (CLP) model

The animals were subjected to sepsis by cecal ligation puncture (CLP) as previously described (Comim et al., 2009; Ritter et al., 2003; Wichterman et al., 1980). In this model, the rats become bacteremic with Gram-negative enteric organisms (Cassol-Jr et al., 2010), in which caecum is ligated distal to the ileocecal valve and perforated using two needle punctures (Parker and Watkins, 2001). Under aseptic conditions, rats were anesthetized with intraperitoneal (i.p.) injection of 90 mg/kg ketamine hydrochloride (Ketalar, Eczacıbas¸ı Warner-Lambert, Istanbul, Turkey). A midline laparotomy was performed using minimal dissection under the anesthesia and the cecum was ligated just below the ileocecal valve with 3-O silk ligatures so that intestinal continuity was maintained. The cecum was perforated on the antimesentric surface of the cecum at two locations 1 cm apart and was gently squeezed to extrude a small amount of feces. Finally, all rats were resuscitated with saline (5 mL/100 g b.w.) subcutaneously (s.c.). The rats were deprived of food but had free access to water after the operation. The sham operated group underwent laparotomy; the cecum was manipulated but was not ligated or perforated. All animals were maintained under the same conditions after the surgery.

2.4.

Experimental design

The rats were divided into four groups:

Fig. 1 – Structure of FA.

Group 1: Sham group (n = 8). This group consisted of animals treated with 0.5 ml i.p. saline alone following laparotomy.

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Group 2: Sepsis group (n = 8). This group consisted of animals in which only CLP was performed and the animals were treated with 0.5 ml i.p. saline following the induction of CLP. Group 3: FA-treated group (n = 8). This group consisted of animals immediately treated with a dose of 100 mg/kg b.w. i.p. FA in 0.5 ml saline following laparotomy. Group 4: FA-treated and sepsis induced group (n = 8). This group consisted of animals immediately treated with a dose of 100 mg/kg b.w. i.p. FA in 0.5 ml saline following the induction of CLP. Considering the results of the other studies of which, the ameliorative activities of FA at a dose of 100 mg/kg b.w. were demonstrated, the same dose of 100 mg/kg b.w. of FA were applied in our study (Kawabata et al., 2000; Maurya et al., 2005). After 24 h following the treatment, all animals were decapitated under the anesthesia. Cardiac blood was collected into preservative-free heparin tubes. Liver and kidneys were removed. The organs were examined for changes in size, color and texture. The samples were kept in the dark at 4 ◦ C and processed within 4 h.

2.5.

Comet assay (single cell gel electrophoresis)

Lymphocytes from whole heparinized blood were separated by Ficoll-Hypaque density gradient and centrifugation (Bøyum, 1976) then the cells were washed with PBS buffer. The concentration of the lymphocytes was adjusted to approximately 2 × 106 cells/ml in PBS buffer. The liver and kidney tissues were carefully dissected from their attachments and totally excised. Preparation of single-cell suspension from the organs was performed according to standard procedures (Bakare et al., 2012; Patel et al., 2006; Tice et al., 2000). Briefly, approximately 0.2 g of each organ was placed in 1 ml chilled mincing solution (HBSS with 20 mM EDTA and 10% DMSO) in a petri dish and chopped into pieces with a pair of scissors. The pieces were allowed to settle and the supernatant containing the single-cell suspension was taken. The concentrations of renal and hepatic tissue cells in the supernatant were adjusted to approximately 2 × 106 cells/ml in HBSS containing 20 mM EDTA/10% DMSO. Alkaline Comet assay technique of Singh et al. (1988), as further described by Anderson et al. (1994) and Collins (2009) was followed. The cells were suspended in 75 ␮l of 0.5% LMA. The suspensions were then 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 immersed in cold lysing solution (2.5 M NaCl, 100 mM EDTA, 100 mM Tris, 1% sodium sarcosinate, pH 10.0 with Triton-X-100 and 10% DMSO) for a minimum of 1 h at 4 ◦ C. Slides were then removed from the lysing solution, drained and were left in the electrophoresis solution (1 mM sodium EDTA and 300 mM NaOH, pH 13.0) for 20 min at 4 ◦ C to allow unwinding of the DNA and expression of alkali-labile damage. The alkaline comet assay using Fpg, a lesion-specific enzyme was used to detect oxidized pyrimidines as a result of oxidative stress-induced DNA damage as described with some modifications (Collins et al., 1993). 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 raising 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 microscopic slides were stained with ethidium bromide (EtBr) (20 ␮g/ml in distilled water, 60 ␮l/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, 100 nuclei per slide were examined at 40× magnification. Results were expressed as the length of the comet (“tail length”), the product of the tail length and the fraction of total DNA in the tail (“tail moment”) and percent of DNA in tail (“tail intensity”).

2.6. Determination of oxidative stress parameters in the liver and kidney tissues The liver and kidney tissues were weighted and extracted following the homogenization and sonication procedure (Sier et al., 1996). The homogenates of the tissue samples were kept −80 ◦ C until the time of analysis. The determination of superoxide dismutase (SOD) and glutathione peroxide (GSH-Px) enzyme activities and the total glutathione (GSH) levels in the liver and kidney tissues were performed spectrophotometrically with SOD, GSH-Px, and GSH assay kits (Cayman Chemicals Co., Ann Arbor, MI, USA) at 440, 340, and 405 nm respectively. Results were expressed as mmol/min/mg tissue. The levels of malondialdehyde (MDA), a biomarker of lipid peroxidation, were determined spectrophotometrically by measuring thiobarbituric acid-reactive substances (TBARS) (Uchiyama and Mihara, 1978). 2.5 ml of 20% trichloroacetic acid and 1.0 ml of 0.67% thiobarbituric acid were added to 0.5 ml of the 10% homogenates of the tissue samples. The mixtures were incubated at 100 ◦ C for 30 min. 4 ml of n-butanol was added after cooling and mixed vigorously. After centrifugation, absorbance of the butanol layer was measured at 535 nm. For standard curve 1,1,3,3-tetraethoxypropane was used. The results were expressed as nmol/g tissue.

2.7.

Statistical analysis

Statistical analysis was performed by SPSS for Windows 20.0 computer program. Differences between the means of data were compared by the one way variance analysis (ANOVA) test and post hoc analysis of group differences was performed

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A Tail Length

by least significant difference (LSD) test. Kruskal–Wallis K test followed by Mann–Whitney U test was used in comparing the parameters displaying abnormal distribution between groups. The results were given as the mean ± standard deviation. p-Values of less than 0.05 were considered as statistically significant.

50.00

Results

3.1.

Comet assay

c,d,e

30.00 e

20.00

b

d,e

b

Sham group

Sepsis group

FA group Sepsis+FA group

Standard comet assay Fpg-modified comet assay

The DNA damages expressed as tail length, tail intensity and tail moment in the lymphocytes, liver and kidney cells of rats were shown for comet and Fpg-modified comet assays in Figs. 2–4, respectively. There were no statistically significant differences in terms of tail length, tail intensity and tail moment between the sham group and the FA-treated groups (p > 0.05). The DNA damage was found significantly higher in the sepsis-induced group compared to the sham group (p < 0.05). FA treatment in the sepsis-induced group was found to decrease the DNA damage significantly (p < 0.05). Similarly 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 FA-treated group (p > 0.05), and the DNA damage was found significantly higher in the sepsis-induced group compared to the sham group (p < 0.05). FA treatment in the sepsis-induced group was found to decrease the DNA damage significantly (p < 0.05). The enzymesensitive 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.

Tail Intensity

B

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3.2. Oxidative stress parameters in the liver and kidney tissues The SOD and GSH-Px enzyme activities and GSH and MDA levels in the liver and kidney tissues were shown in Tables 1 and 2, respectively. Hepatic and renal SOD, GSH-Px enzyme activities and GSH levels were significantly decreased in the sepsis-induced group compared to the sham group (p < 0.05). SOD, GSHPx enzyme activities and GSH levels in both liver and kidney in the sepsis + FA treated group were found to be significantly lower than the sham group (p < 0.05). However SOD, GSH-Px enzyme activities and GSH levels in both liver and kidney increased significantly in the sepsis + FA treated group compared to sepsis group (p < 0.05). There was no significant increase between sham and sepsis + FA treated group. Hepatic and renal MDA levels were found to significantly increase in both sepsis and sepsis + FA treated groups compared to the sham group (p < 0.05). But the levels were found to significantly decrease in the sepsis + FA treated group compared to the sepsis group (p < 0.05). There was no significant increase between sham and sepsis + FA treated group. In all studied oxidative stress parameters, FA alone did not cause significant changes compared to sham group.

a

10.00 0.00

3.

c,e

40.00

Fig. 2 – DNA damage in the lymphocytes of the experimental groups expressed as (A) tail length; (B) tail intensity; (C) tail moment. The values are expressed as mean ± standard deviation. 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.

4.

Discussion

There is an increasing evidence that oxidative stress has an important role in the development of sepsis-induced multiorgan failure (Prauchner et al., 2011; Sato et al., 2002; Zapelini et al., 2008). 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

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Table 1 – SOD and GSH-Px antioxidant enzyme activities and GSH and MDA levels in the livers of rats. SOD activity (mmol/min/mg tissue) Sham group Sepsis group FA group Sepsis + FA group

164.6 131.6 160.2 138.4

± ± ± ±

GSH-Px activity (nmol/min/ml)

6.9a 5.9b 5.8a 5.4c

86.58 20.34 80.94 33.66

± ± ± ±

4.69a 3.31b 1.63a 3.77c

GSH levels (nmol/mg tissue) 3.52 1.05 3.76 1.85

± ± ± ±

0.40a 0.30b 0.77a 0.34c

MDA levels (nmol/g tissue) 11.32 24.80 10.68 19.32

± ± ± ±

2.73a 4.36b 1.64a 2.58c

The results were given as mean ± standard deviation for eight rats in each group. SOD, superoxide dismutase; GSH-Px, glutathione peroxide; GSH, total glutathione; MDA, malondialdehyde. Sepsis group, cecal ligation and puncture (CLP) performed rats. FA group, FA (100 mg/kg/day, i.p.) treated rats. Sepsis + FA group, CLP performed and FA (100 mg/kg/day, i.p.) treated rats. Superscripts of different letters differ significantly (p < 0.05) from each other within the same column.

GSH loss observed at 20 min imposes an oxidative stress that can impair other cellular functions, in particular those regulated by the redox mechanism (Ciriolo et al., 1997). Septic patients are reported to have lower plasma antioxidant levels (Barzilai and Yamamoto, 2004). It is also suggested that the most robust and significant alteration in the antioxidant defense is the decrease in GSH concentration (Schulz et al., 2000). In this study we also observed that MDA levels were significantly increased and GSH levels and SOD and GSH-Px enzyme activities were significantly decreased in sepsis induced rats. Antioxidant therapy has found to have preventive effects on sepsis treatment (Goode et al., 1995; Prauchner et al., 2011; Ritter et al., 2003) and in recent years antioxidants derived from natural sources, have attracted special attention (Maurya and Devasagayam, 2010). Plant polyphenols are suggested to exert antioxidant effects through the promotion of some antioxidant enzymes expression. A variety of polyphenols used in the treatment of sepsis are shown in animal studies (Barzilai and Yamamoto, 2004). FA has been suggested to be a very strong antioxidant and to have anticarcinogenic potential against skin, colon, liver, tongue and mammary cancers (Ramar et al., 2012). But the use of FA in ROS-induced damages are limited. In our study we found that FA regulated all of the alterations produced by sepsis. FA treatment was found to significantly decrease MDA levels and significantly increase GSH levels and SOD and GSH-Px enzyme activities in sepsis induced rats.

cytokines that cause oxidative DNA damage (Neviere et al., 1999). The liver is important in the response to sepsis since it is the primary site for the clearance of bacterial endotoxins, and it produces proinflammatory cytokines and acute-phase proteins. Sepsis-induced acute renal failure is also the major problem in intensive care units (Dickson, 2009). On the other hand the peripheral lymphocytes are the early site of intense oxidative processes in the body (Dickson, 2009; Neviere et al., 1999). Excessive free radicals can cause lipid peroxidation and induce damages in the membranes of the cell and mitochondria, which eventually lead to cell apoptosis and necrosis (Mancuso et al., 2007). MDA, a direct product of lipid peroxidation, can reflect the extent of lipid peroxidation in tissues. Several reactive mutagenic and genotoxic lipid peroxidation products, in particular MDA, has been identified to bind and damage DNA (Eder et al., 2006). SOD is an enzyme that converts superoxide radical into hydrogen peroxide (H2 O2 ), which is the substrate of catalase (CAT) and GSH-Px. When a cell has increased levels of SOD without a proportional increase in CAT or GSH-Px, a large amount of H2 O2 becomes available to react with transitional metals and generates the hydroxyl radical (Andrades et al., 2009). There is an imbalance between antioxidant enzymes GSH-Px and SOD that is followed by oxidative damage in the major organ systems after sepsis induction (Andrades et al., 2011, 2005; Ritter et al., 2003). It is usually assumed that GSH depletion reflects an intracellular oxidation. The

Table 2 – SOD and GSH-Px antioxidant enzyme activities and GSH and MDA levels in the kidneys of rats. SOD activity (mmol/min/mg tissue) Sham group Sepsis group FA group Sepsis + FA group

148.0 102.2 149.6 122.8

± ± ± ±

15.6a 12.1b 10.6a 3.5c

GSH-Px activity (nmol/min/ml) 123.78 46.94 124.44 57.04

± ± ± ±

1099a 6.42b 4.51a 5.36c

GSH levels (nmol/mg tissue) 2.13 0.47 2.32 1.27

± ± ± ±

0.47a 0.24b 0.19a 0.30c

The results were given as mean ± standard deviation for eight rats in each group. SOD, superoxide dismutase; GSH-Px, glutathione peroxide; GSH, total glutathione; MDA, malondialdehyde. Sepsis group, cecal ligation and puncture (CLP) performed rats. FA group, FA (100 mg/kg/day, i.p.) treated rats. Sepsis + FA group, CLP performed and FA (100 mg/kg/day, i.p.) treated rats. Superscripts of different letters differ significantly (p < 0.05) from each other within the same column.

MDA levels (nmol/g tissue) 13.81 32.20 12.60 26.12

± ± ± ±

0.80a 4.35b 1.28a 4.33c

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Fig. 3 – DNA damage in the livers of the experimental groups expressed as (A) tail length; (B) tail intensity; (C) tail moment. The values are expressed as mean ± standard deviation. 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.

Fig. 4 – DNA damage in the kidneys of the experimental groups expressed as (A) tail length; (B) tail intensity; (C) tail moment. The values are expressed as mean ± standard deviation. 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.

FA itself did not induce abnormalities in the oxidative stress parameters. Diets and nutrients enriched with FA are suggested to be beneficial for people who are at risk for UV-B devastating complications related to over-production of oxygen free radicals. The protective effect of FA against UV radiation was evaluated in human skin cells and lymphocytes (Prasad et al., 2007). FA has been found to have strong UV absorptive ability which is important in skin protection (Zhao and Moghadasian, 2008). In alcoholic abusive and diabetic rats, FA induced antioxidant

mechanisms such as SOD, CAT and GSH-Px activities (Prasad et al., 2006). Balasubashini et al. showed that FA decreased the oxidative stress caused during diabetes. The levels of GSH and activities of antioxidant enzymes like GSH-Px, SOD and CAT were elevated in the liver and the effect was more pronounced with the low dose than the high dose of FA (Balasubashini et al., 2004). In nicotine-treated rats, FA has reduced the extent of MDA, DNA damage and restored the endogenous antioxidant status with increased SOD, CAT, GSHPx and GSH levels (Sudheer et al., 2007). Our study is consistent

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with limited data of antioxidant activity of FA against oxidative damage. Sudheer et al. also showed that there was a significant increase in the levels of comet parameters (tail length, tail moment, %DNA in tail and olive tail moment) in nicotinetreated lymphocytes, which is the indicative of cellular DNA strand breaks. FA treatment produced no DNA damaging effects in the normal lymphocytes (Sudheer et al., 2007). Pretreatment with FA has protected cells from ␥-radiation induced DNA damage. Hence, FA administration prior to radiation therapy may also be useful to cancer patients to prevent normal cell damage (Karthikeyan et al., 2011; Srinivasan et al., 2006; Zeni et al., 2012). In our study, the DNA damage in the lymphocytes, liver and kidney cells in the sepsis induced rats have been found to be higher compared to the sham group. FA (100 mg/kg/day) treatment was found to significantly decrease the DNA damage in the lymphocytes and the liver and kidney cells of the sepsis induced rats. Increases in the tail length, tail intensity and tail moment in the lymphocytes, livers and kidneys of sepsis-induced rats treated with FA as measured by the standard comet and Fpg-modified comet assay are much lower than in sepsisinduced rats, showing that FA prevents the oxidative DNA damage and increases the DNA repair processes. The findings of this study are consistent with our previous data with the protective roles of lycopene and curcumin in obstructive jaundice (Aydin et al., 2013; Tokac et al., 2013). In this study, the DNA damage and oxidative stress parameters were evaluated in the lymphocytes, the liver and kidney tissues of the sepsis induced rats. We determined that the antioxidant hepatic and renal SOD and GSH-Px enzyme activities and GSH levels were significantly decreased and hepatic and renal MDA levels were significantly increased in the sepsis group. Taken together, these results showed that sepsis caused oxidative stress because of the increased liver and kidney MDA levels and decreased GSH levels and SOD and GSH-Px enzyme activities. Our results are in accordance with previous reports about sepsis and oxidative stress parameters. In conclusion, FA treatment significantly recovered the reduced DNA damage in lymphocytes, liver and kidney tissues of rats, increased MDA levels and decreased GSH levels, as well as SOD and GSH-Px antioxidant enzyme activities in the liver and kidney of rats with sepsis. FA seems to have a role in the prevention of sepsis-induced oxidative damage not only by decreasing the DNA damage but also increasing the antioxidant status of the animals. The antigenotoxic mechanism of FA against sepsis induced DNA damage can be related to its antioxidant potential to neutralize the toxic effects of ROS generated by sepsis. Although FA and related phenolic compounds have been suggested to inhibit free radical formation, lipid peroxidation, and DNA damage and act as radical scavengers and antioxidant, further investigation should be performed for clarifying the possible mechanisms underlying FA’s beneficial effects on several diseases associated with oxidative stress.

Conflicts of interest The authors declare that there are no conflicts of interest.

Transparency document The Transparency document associated with this article can be found in the online version.

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

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