Sage Journals: Discover world-class research

Abstract

Reactive oxygen species are believed to be involved in the development of sepsis. Plant-derived phenolic compounds are thought to be possible therapeutic agents against sepsis because of their antioxidant properties. Rosmarinic acid (RA) is a phenolic compound commonly found in various plants, which has many biological activities including antioxidant activity. The aim of this study was to investigate the effects of RA on sepsis-induced DNA damage in the lymphocytes and liver and kidney cells of Wistar albino rats by alkaline comet assay with and without formamidopyrimidine DNA glycosylase protein. 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 and an inflammatory cytokine, tumor necrosis factor α (TNF-α) level in plasma were also evaluated. It is found that DNA damage in the lymphocytes, livers, and kidneys of the RA-treated rats was significantly lower than that in the sepsis-induced rats. RA treatment also decreased the MDA levels and increased the GSH levels and SOD and GSH-Px activities in the livers and kidneys of the sepsis-induced rats. Plasma TNF-α level was found to be decreased in the RA-treated rats. It seems that RA might have a role in the attenuation of sepsis-induced oxidative damage not only by decreasing the DNA damage but also by increasing the antioxidant status and DNA repair capacity of the animals.

Keywords

Rosmarinic acid sepsis DNA damage oxidative stress rats

Introduction

Sepsis is an imbalance between pro and anti-inflammatory responses.^1,2 Multiple organ failure induced by sepsis is associated with mortality and is characterized by liver, renal, cardiovascular, and pulmonary dysfunction.^3
–5

Reactive oxygen species (ROS) are believed to be involved in the development of sepsis.^6
–8 The pro-inflammatory effects of ROS include endothelial damage, formation of chemotactic factors, neutrophil recruitment, cytokine release, mitochondrial impairment, lipid peroxidation, and DNA damage,^1,9
–11 all contributing to a free-radical overload and oxidant–antioxidant imbalance.¹⁰

Plant polyphenols may act as antioxidants by different mechanisms such as free radical scavenging, metal chelation, and protein binding.¹² Literature data indicate possible beneficial effects of plant-derived phenolic compounds against sepsis.^13,14

Rosmarinic acid (α-O-caffeoyl-3,4-dihydroxyphenyllactic acid; RA) is a phenolic compound commonly found in various plants such as Rosmarinus officinalis (rosemary), Origanum vulgare (oregano), Thymus vulgaris (thyme), Mentha spicata (spearmint), Perilla frutescens (perilla), Ocimum basilicum (sweet basil), and several other medicinal plants.¹⁵ RA is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid.^16,17 It has been shown that RA has many biological activities including antioxidant, anti-inflammatory, antiallergic, anticancer, and antimicrobial effects and is widely used in cosmetic and food products.^15

–21

RA was suggested to have ameliorating effects against oxidative stress-related pathological conditions due to its antioxidant activity.¹⁶ This activity of RA could be attributed to its chemical structure, since it has been suggested that hydroxyl groups in the ortho position of the aromatic ring could enhance the antioxidant capacity.^21,22

In a previous study, RA reduced the genotoxic effects of doxorubicin in V79 cell line.²³ In HepG2 cell line, tert-butyl hydroperoxyde (t-BHP)-induced DNA damage was decreased by RA.²⁴ De Oliveira et al.²⁵ showed that RA had antigenotoxic effects against ethanol-induced genotoxicity in mice. Additionally, RA effectively eliminated ultraviolet-A-induced DNA damage on human keratinocytes.²⁶

In the present study, we aimed to determine the protective effects of RA against the oxidative stress parameters and oxidative DNA damage induced by sepsis in Wistar albino rats.

Material and methods

Chemicals

The chemicals used in the study were purchased from the following suppliers: normal melting agarose and low-melting point agarose from Boehringer Manheim (Mannheim, Germany); sodium chloride, sodium hydroxide, potassium chloride and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 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 RA from Sigma-Aldrich Chemicals (St Louis, Missouri, USA); ethylendiaminetetraacetic acid disodium salt dihydrate (EDTA-Na₂), natriumlauroylsarcosinate, and tris(hydroxymethyl)aminomethane from ICN Biomedicals Inc. (Aurora, Ohio, USA), superoxide dismutase (SOD) assay kit, glutathione peroxidase (GSH-Px) assay kit, and total glutathione (GSH) assay kit from Cayman Chemicals Co. (Ann Arbor, MI, USA), and tumor necrosis factor α (TNF-α) assay kit from Invitrogen KRC0011 (Grand Island, Nebraska, USA).

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 and maintained on a 12-h light/12-h dark cycle at controlled temperature (23 ± 2°C) and humidity (50%). All animals were fed with standard laboratory chow and allowed to access ad libitum feed and drinking water before and after operation. The study was approved by Hacettepe University Animal Ethical Committee (Date and Number: 05/07/2011, 2011/45-14).

CLP model

Wistar rats were subjected to sepsis by cecal ligation puncture (CLP) as previously described.^27
–29 In this model, the rats become bacteremic with Gram-negative enteric organisms, in which cecum is ligated distal to the ileocecal valve and perforated using two needle punctures.^6,30 Under aseptic conditions, rats were anesthetized with intraperitoneal (i.p.) injection of 90 mg/kg ketamine hydrochloride (Ketalar, Eczacıbaşı 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 body weight (b.w.)) subcutaneously. 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.

Experimental design

The rats were divided into four groups:

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

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: RA-treated group (n = 8). This group consisted of animals immediately treated with a dose of 100 mg/kg b.w. i.p. RA in 0.5 mL saline for 24 h following laparatomy.

Group 4: RA-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. RA in 0.5 mL saline for 24 h following the induction of CLP.

Since RA shows very low toxicity with a median lethal dose (LD₅₀) value of 561 mg/kg after intravenous application in mice, the i.p. dose of 100 mg RA/kg b.w. was used in this study and the survival rate of the animals was 100%.^17,31 The RA dose (100 mg/kg) is under the LD₅₀ dose (561 mg/kg) and at the studied dose, the survival rate of the animals was 100%. In our previous studies with some phenolic compounds such as pycnogenol^® and resveratrol, the dosages of 100 mg/kg bw i.p. were also used in the experimental sepsis model.^32,33

After 24 h following the treatment, all animals were decapitated under anesthesia using i.p. injection of 90 mg/kg ketamine hydrochloride. 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. There was no change in these parameters. The samples were kept in the dark at 4°C and processed within 4 h.

Comet assay (single-cell gel electrophoresis)

Lymphocytes from whole heparinized blood were separated by Ficoll–Hypaque density gradient.³⁴ Preparation of single-cell suspensions from the organs was performed according to standard procedures.^35

–38 Briefly, approximately 0.2 g of each organ was placed in 1 mL chilled mincing solution (Hanks’ balanced salt solution with 20 mM ethylenediaminetetraacetic acid 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 used. The concentrations of the lymphocytes in PBS and the renal and hepatic tissue cells in the mincing solution were adjusted to approximately 2 × 10⁶ cells/mL.

The basic alkaline comet assay of Singh et al.,³⁹ as further described by Anderson et al.,⁴⁰ was performed. The Fpg-modified comet assay, as described by Collins,⁴¹ was followed to detect oxidized purines as a result of oxidative stress-induced DNA damage as described with some modifications. The cells were embedded on agarose gel, lysed, and fragmented DNA strands drawn out by electrophoresis to form a comet. After electrophoresis, the slides were neutralized and then 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) with a Leica^® (Germany) 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”).

Determination of oxidative stress parameters in the liver and kidney tissues

The liver and kidney tissues were weighed and extracted following the homogenization and sonication procedure.⁴² The homogenates of the tissue samples were kept at −80°C until the time of analysis.

The determination of SOD and GSH-Px enzyme activities and the GSH levels in the liver and kidney tissues were performed spectrophotometrically with SOD, GSH-Px, and GSH assay kit (Cayman Chemicals Co., Ann Arbor, Michigan, USA) at 440, 340, and 405 nm, respectively. Results were expressed as millimole per minute per milligram tissue.

The levels of MDA, a biomarker of lipid peroxidation, were determined spectrophotometrically by measuring thiobarbituric acid reactive substances.⁴³ Then, 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 in boiling water for 30 min. Four milliliter 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 nanomole per gram tissue.

Determination of plasma TNF-α levels

Whole blood samples were obtained via the intracardiac method. The plasma was immediately separated by centrifugation at 4000 r/min for 10 min at 4°C and stored at −80°C until being assayed. TNF-α from each sample was measured at 30 min according to the manufacturer’s instructions with highly sensitive enzyme-linked immunosorbent assay kits Invitrogen KRC0011 (Waltham, Massachusetts, USA) at 450 nm. The results were expressed as picogram per milliliter.

Statistical analysis

Statistical analysis was performed by SPSS for Windows 15.0 computer program. The distribution of the data was checked for normality using the Shapiro–Wilk test. The homogeneity of the variance was verified by the Levene test. The differences among the groups for oxidative stress parameters were evaluated by the Kruskal–Wallis test, and post hoc analysis of group differences was performed by the Conover test using SPSS 15.0. The results of oxidative stress parameters were represented as median and ranges. For comet assay, differences between the means of data were compared by the one-way analysis of variance test, and post hoc analysis of group differences was performed by least significant difference test. Values of p < 0.05 were considered as statistically significant.

Results

Comet assay

The DNA damage expressed as tail length, tail intensity, and tail moment in the lymphocytes, liver, and kidney cells of rats is shown for standard and Fpg-modified comet assay in Figures 1, 2, and 3, respectively.

Figure 1.

DNA damage in the lymphocytes of the experimental groups expressed as (a) tail length, (b) tail intensity, and (c) tail moment. The values are expressed as mean ± standard deviation. ^ap < 0.05: compared with sham group for the standard comet assay; ^bp < 0.05: compared with sepsis group for the standard comet assay; ^cp < 0.05: compared with sham group for the Fpg-modified comet assay; ^dp < 0.05: compared with sepsis group for the Fpg-modified comet assay; ^ep < 0.05: standard comet assay was compared with Fpg-modified comet assay. Fpg: formamidopyrimidine DNA glycosylase.

Figure 2.

DNA damage in the livers of the experimental groups expressed as (a) tail length, (b) tail intensity, and (c) tail moment. The values are expressed as mean ± standard deviation. ^ap < 0.05: compared with sham group for the standard comet assay; ^bp < 0.05: compared with sepsis group for the standard comet assay; ^cp < 0.05: compared with sham group for the Fpg-modified comet assay; ^dp < 0.05: compared with sepsis group for the Fpg-modified comet assay; ^ep < 0.05: standard comet assay was compared with Fpg-modified comet assay. Fpg: formamidopyrimidine DNA glycosylase.

Figure 3.

DNA damage in the kidneys of the experimental groups expressed as (a) tail length, (b) tail intensity, and (c) tail moment. The values are expressed as mean ± standard deviation. ^ap < 0.05: compared with sham group for the standard comet assay; ^bp < 0.05: compared with sepsis group for the standard comet assay; ^cp < 0.05: compared with sham group for the Fpg-modified comet assay; ^dp < 0.05: compared with sepsis group for the Fpg-modified comet assay; ^ep < 0.05: standard comet assay was compared with Fpg-modified comet assay. Fpg: formamidopyrimidine DNA glycosylase.

For the standard comet assay, in the lymphocytes and hepatic and renal cells, there were no statistically significant differences in terms of tail length, tail intensity, and tail moment between the sham group and the RA-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). RA treatment in the sepsis-induced group was found to decrease the DNA damage significantly (p < 0.05). Similarly for the Fpg-modified comet assay, in the lymphocytes and hepatic and renal cells, there were no statistically significant differences in terms of tail length, tail intensity, and tail moment between the sham group and the RA-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). RA treatment in the sepsis-induced group was found to decrease the DNA damage significantly (p < 0.05). 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. Fpg-sensitive sites increased significantly in the sepsis group (p < 0.05) and RA treatment significantly reduced the sepsis-induced Fpg-sensitive sites in the lymphocytes and hepatic and renal cells (p < 0.05; Figures 1 to 3).

Oxidative stress parameters in liver and kidney tissues

The SOD and GSH-Px enzyme activities and GSH and MDA levels in the liver and kidney tissues are 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, GSH-Px enzyme activities, and GSH levels in both liver and kidney in the sepsis + RA-treated group were found to be significantly lower than in the sham group (p < 0.05). However SOD, GSH-Px enzyme activities, and GSH levels in both liver and kidney were increased in the sepsis + RA-treated group compared to sepsis group (p < 0.05). There was no significant difference between sham and RA-treated group. Hepatic and renal MDA levels were found to be significantly increased in both sepsis and sepsis + RA-treated groups compared to the sham group (p < 0.05). But the levels were found to be significantly decreased in the sepsis + RA-treated group compared to the sepsis group (p < 0.05). There was no significant difference between sham and RA-treated group. In all studied oxidative stress parameters, treatment with RA only did not cause significant changes compared to sham group.

Table 1.

SOD and GSH-Px antioxidant enzyme activities and GSH and MDA levels in the livers of rats.^a

	SOD activity (mmol/min/mg tissue)	GSH-Px activity (nmol/min/mL)	GSH levels (nmol/mg tissue)	MDA levels (nmol/g tissue)
Sham group	162.0 ± 3.1 (158.0–175.0)	87.60 ± 2.10 (87.5–90.3)	3.43 ± 0.18 (3.15–4.15)	11.40 ± 1.22 (8.40–15.60)
Sepsis group	134.0 ± 2.62^c (124.0–138.0)	20.30 ± 1.48^c (16.5–175.0)	1.14 ± 0.13^c (0.54 –1.31)	27.50 ± 1.95^c (18.80–28.40)
Sepsis + RA group	142.0 ± 3.1^b,c (128.0–155.0)	45.60 ± 1.78^b,c (38.8–48.9)	1.95 ± 0.15^b,c (1.60–2.40)	16.60 ± 1.59^b,c (12.50–21.40)
RA group	165.0 ± 3.2^c (157.0–175.0)	80.50 ± 0.73^c (78.4–82.6)	3.65 ± 0.18^c (3.12–4.11)	9.50 ± 0.76^c (8.30–12.40)

SOD: superoxide dismutase; GSH-Px: glutathione peroxide; GSH: total glutathione; MDA: malondialdehyde; CLP: cecal ligation puncture; RA: rosmarinic acid; sepsis group: CLP-performed rats; SEM: standard error of mean; RA group: RA (100 mg/kg/day, intraperitoneally)-treated rats; Sepsis + RA group: CLP performed and RA (100 mg/kg/day, intraperitoneally)-treated rats.

^aThe results were given as median ± SEM with minimum and maximum values for eight rats in each group.

^bp < 0.05: statistically different from sham group.

^cp < 0.05: statistically different from sepsis group within the same column.

Table 2.

SOD and GSH-Px antioxidant enzyme activities and GSH and MDA levels in the kidneys of rats.^a

	SOD activity (mmol/min/mg tissue)	GSH-Px activity (nmol/min/mL)	GSH levels (nmol/mg tissue)	MDA levels (nmol/g tissue)
Sham group	151.0 ± 6.9 (128.0–164.0)	123.2 ± 4.9 (110.3–139.3)	2.35 ± 0.21 (1.43–2.55)	13.60 ± 0.36 (12.80–14.65)
Sepsis group	104.0 ± 5.4^b (85.0–118.0)	46.50 ± 2.87^b (38.40–56.30)	0.34 ± 0.11^b (0.24–0.81)	31.50 ± 1.95^b (27.50–37.80)
Sepsis + RA group	127.0 ± 4.8^b,c (109.0–135.0)	65.60 ± 3.07^b,c (53.70–68.90)	1.34 ± 0.09^b,c (0.95–1.45)	21.90 ± 0.72^b,c (18.60–22.50)
RA group	152.0 ± 4.6^c (135.0–163.0)	118.1 ± 2.74^c (113.9–127.0)	2.15 ± 0.13^c (1.96–2.55)	13.30 ± 0.61^c (11.50–14.60)

SOD: superoxide dismutase; GSH-Px: glutathione peroxide; GSH: total glutathione; MDA: malondialdehyde; CLP: cecal ligation puncture; RA: rosmarinic acid; SEM: standard error of mean; sepsis group: CLP-performed rats. RA group: RA (100 mg/kg/day, intraperitoneally)-treated rats; Sepsis + RA group: CLP-performed and RA (100 mg/kg/day, intraperitoneally)-treated rats.

^aThe results were given as median ± SEM with minimum and maximum values for eight rats in each group.

^bp < 0.05: statistically different from sham group.

^cp < 0.05: statistically different from sepsis group within the same column.

Plasma TNF-α levels

Plasma TNF-α levels are shown in Table 3. Plasma TNF-α levels were found to be significantly increased in both sepsis and sepsis + RA-treated groups compared to the sham group (p < 0.05). But the level was found to be significantly decreased in the sepsis + RA-treated group compared to the sepsis group (p < 0.05).

Table 3.

Plasma TNF-α levels (pg/mL) in study groups.^a

Sham group	Sepsis group	Sepsis + RA group	RA group
42.60 ± 2.46 (32.80–47.60)	178.8 ± 7.9^b (167.8–209.4)	98.60 ± 4.46^b,c (78.50–104.0)	43.50 ± 2.68^c (32.50–44.70)

CLP: cecal ligation puncture; RA: rosmarinic acid; SEM: standard error of mean; Sepsis group: CLP-performed rats. RA group: RA (100 mg/kg/day, intraperitoneally)-treated rats; Sepsis + RA group: CLP-performed and RA (100 mg/kg/day, intraperitoneally)-treated rats.

^aThe results are given as median ± SEM with minimum and maximum values for eight rats in each group.

^bp < 0.05: statistically different from sham group.

^cp < 0.05: statistically different from sepsis group within the same line.

Discussion

The occurrence of lipid and protein oxidative damage and the involvement of free radicals during sepsis have been well documented.^28,40,42 Plasma antioxidant status in sepsis was reported to be lower than that in healthy subjects.^44,45 Previous studies have demonstrated that ROS play an important role in the development of sepsis-induced multiorgan failure.^{11,28,39,41

–44} The liver is important in the response to sepsis since it is the primary site for the clearance of bacterial endotoxins, and it produces pro-inflammatory cytokines and acute-phase proteins. Sepsis-induced acute renal failure is also the major problem in intensive care units.⁴⁶ On the other hand, the peripheral lymphocytes are the early site of intense oxidative processes in the body.^46,47

The release of endotoxin from bacteria is generally thought to be the initial event in the development of sepsis. Levels of circulating cytokines like TNF-α were found to be higher in healthy volunteers than in patients with sepsis.⁴⁸ In sepsis, TNF-α levels are generally high and correlate with high mortality.^49,50 Endotoxin activates inflammatory cells, which subsequently amplify the inflammatory response via the release of various cytokines that causes oxidative DNA damage.⁴⁷

An imbalance between antioxidant enzymes GSH-Px and SOD, which is followed by oxidative damage in the major organ systems after sepsis induction, has been observed.^10,28,51,52 It is also suggested that the most robust and significant alteration in the antioxidant defense is the decrease in GSH concentration. Rapid depletion of intracellular GSH in human and animal endothelial and epithelial cells occurs in response to TNF-α in vitro.⁵³ Similar to our previous data,^32,33 in this study, we observed that MDA levels were significantly increased and GSH levels and SOD and GSH-Px enzyme activities were significantly decreased in the liver and renal tissues of the sepsis-induced rats. TNF-α levels in the plasma of septic rats were also increased when compared with the controls.

Recent data have indicated that antioxidant therapy could be beneficial against sepsis.^54
–56 RA is a well-known antioxidant suggested to be useful in the treatment of oxidative stress-related disorders.^16,23 Domitrović et al.¹⁵ have previously observed that RA ameliorated acute liver damage in mice through the suppression of oxidative stress, inflammation, and fibrogenesis.

RA has also significantly attenuated the decrease of GSH levels induced by t-BHP.²⁴ Administration of RA to cisplatin-induced mice reduced TNF-α expression, indicating anti-inflammatory effect of RA, and also inhibited gentamicin-induced renal injury.¹⁶ In a previous study, a significant decrease in renal SOD and GSH-Px enzyme activities and GSH levels and an increase in MDA levels in gentamicin-treated rats were observed, but RA treatment has significantly increased renal SOD and GSH-Px enzyme activities and GSH levels and decreased MDA levels.⁵⁷ In Japanese encephalitis virus-infected mice, RA reduced the levels of interleukin 12, TNF-α, interferon γ, monocyte chemoattractant protein 1, and interleukin 6.⁵⁸ Fadel et al.⁵⁹ also demonstrated that RA was an efficient antagonist of lipid peroxidation. Consistent with these studies, we found that RA regulated all of the sepsis-induced alterations in oxidative stress parameters. RA treatment significantly decreased MDA and TNF-α levels and increased GSH levels and SOD and GSH-Px enzyme activities in sepsis-induced rats. RA itself did not induce abnormalities in the oxidative stress parameters. In our previous studies, the protective roles of resveratrol and pycnogenol^® in sepsis-induced rats were also observed. It was found that 100 mg/kg b.w. i.p. doses of both resveratrol and pycnogenol^® ameliorated sepsis-induced oxidative stress parameters and DNA damage.^32,33

Several reactive mutagenic and genotoxic lipid peroxidation products have been identified to bind and damage DNA.⁶⁰ Although the protective effects of phenolic compounds on DNA damage have been observed in many in vitro studies, the in vivo data are limited. DNA damage in the lymphocytes and liver and kidney cells in the sepsis-induced rats was found to be higher compared to the sham group. RA (100 mg/kg/day) treatment significantly decreased DNA damage in the lymphocytes, liver, and kidney cells of the sepsis-induced rats. Increases in the tail length, tail intensity, and tail moment in the lymphocytes and liver and kidney cells of sepsis-induced rats treated with RA as measured by the standard comet and Fpg-modified comet assay were much lower than in sepsis-induced rats, showing that RA might reduce oxidative DNA damage and increase DNA repair processes.

In conclusion, RA treatment significantly reduced DNA damage in lymphocytes and liver and kidney tissues of rats, decreased MDA levels, and increased GSH levels, as well as SOD and GSH-Px antioxidant enzyme activities in the liver and kidney cells and increased plasma TNF-α levels in septic rats. RA seems to have a role in the attenuation of sepsis-induced oxidative damage not only by decreasing DNA damage but also increasing the antioxidant status of rats. The antigenotoxic mechanism of RA against sepsis-induced DNA damage can be related to its antioxidant potential to neutralize the toxic effects of ROS generated by sepsis. Although RA and related phenolic compounds have been suggested to inhibit free radical formation, lipid peroxidation, and DNA damage and act as radical scavengers and antioxidants, further investigation should be performed to clarify the possible mechanisms underlying the beneficial effects of RA on several diseases associated with oxidative stress.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

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

References

Hotchkiss

Karl

. The pathophysiology and treatment of sepsis. New Engl J Med 2003; 348: 138–150.

Vandijck

Decruyenaere

Blot

. The value of sepsis definitions in daily ICU-practice. Acta Clin Belg 2006; 61: 220–226.

Bone

Grodzin

Balk

. Sepsis: a new hypothesis for pathogenesis of the disease process. Chest J 1997; 112: 235–243.

Wheeler

Bernard

. Treating patients with severe sepsis. New Engl J Med 1999; 340: 207–214.

Zapelini

Rezin

Cardoso

. Antioxidant treatment reverses mitochondrial dysfunction in a sepsis animal model. Mitochondrion 2008; 8: 211–218.

Cassol-Jr

Comim

Silva

. Treatment with cannabidiol reverses oxidative stress parameters, cognitive impairment and mortality in rats submitted to sepsis by cecal ligation and puncture. Brain Res 2010; 1348: 128–138.

Kaymak

Kadioglu

Ozcagli

. Oxidative DNA damage and total antioxidant status in rats during experimental gram-negative sepsis. Hum Exp Toxicol 2008; 27: 485–491.

Zhou

Chen

Huang

. Hydrogen-rich saline reverses oxidative stress, cognitive impairment, and mortality in rats submitted to sepsis by cecal ligation and puncture. J Surg Res 2012; 178: 390–400.

Andrades

Ritter

Dal-Pizzol

. The role of free radicals in sepsis development. Front Biosci 2009; 1: 277.

10.

Andrades

Ritter

de Oliveira

. Antioxidant treatment reverses organ failure in rat model of sepsis: role of antioxidant enzymes imbalance, neutrophil infiltration, and oxidative stress. J Surg Res 2011; 167: e307–e313.

11.

Barichello

Fortunato

Vitali

ÂM

. Oxidative variables in the rat brain after sepsis induced by cecal ligation and perforation. Crit Care Med 2006; 34: 886–889.

12.

Maurya

Devasagayam

. Antioxidant and prooxidant nature of hydroxycinnamic acid derivatives ferulic and caffeic acids. Food Chem Toxicol 2010; 48: 3369–3373.

13.

Shapiro

Lev

Cohen

. Polyphenols in the prevention and treatment of sepsis syndromes: rationale and pre-clinical evidence. Nutrition 2009; 25: 981–997.

14.

von Dessauer

Bongain

Molina

. Oxidative stress as a novel target in pediatric sepsis management. J Crit Care 2011; 26: 103.e1–103.e7.

15.

Domitrovic

Skoda

Vasiljev Marchesi

. Rosmarinic acid ameliorates acute liver damage and fibrogenesis in carbon tetrachloride-intoxicated mice. Food Chem Toxicol 2013; 51: 370–378.

16.

Domitrovic

Potocnjak

Crncevic-Orlic

. Nephroprotective activities of rosmarinic acid against cisplatin-induced kidney injury in mice. Food Chem Toxicol 2014; 66: 321–328.

17.

Petersen

Simmonds

. Rosmarinic acid. Phytochemistry 2003; 62: 121–125.

18.

Chu

. Effects of a natural prolyl oligopeptidase inhibitor, rosmarinic acid, on lipopolysaccharide-induced acute lung injury in mice. Molecules 2012; 17: 3586–3598.

19.

Moreno

Scheyer

Romano

. Antioxidant and antimicrobial activities of rosemary extracts linked to their polyphenol composition. Free Radical Res 2006; 40: 223–231.

20.

Osakabe

Takano

Sanbongi

. Anti-inflammatory and anti-allergic effect of rosmarinic acid (RA); inhibition of seasonal allergic rhinoconjunctivitis (SAR) and its mechanism. Biofactors 2004; 21: 127–131.

21.

Tepe

Eminagaoglu

Akpulat

. Antioxidant potentials and rosmarinic acid levels of the methanolic extracts of Salvia verticillata (L.) verticillata and S. verticillata (L.) amasiaca (Freyn & Bornm.) Bornm. Food Chem 2007; 100: 985–989.

22.

Sroka

Cisowski

. Hydrogen peroxide scavenging, antioxidant and anti-radical activity of some phenolic acids. Food Chem Toxicol 2003; 41: 753–758.

23.

Furtado

de Araujo

Resende

. Protective effect of rosmarinic acid on V79 cells evaluated by the micronucleus and comet assays. J Appl Toxicol 2010; 30: 254–259.

24.

Lima

Fernandes-Ferreira

Pereira-Wilson

. Phenolic compounds protect HepG2 cells from oxidative damage: relevance of glutathione levels. Life Sci 2006; 79: 2056–2068.

25.

De Oliveira

Sarmento

Nunes

. Rosmarinic acid as a protective agent against genotoxicity of ethanol in mice. Food Chem Toxicol 2012; 50: 1208–1214.

26.

Psotova

Svobodova

Kolarova

. Photoprotective properties of Prunella vulgaris and rosmarinic acid on human keratinocytes. J Photoch Photobio B 2006; 84: 167–174.

27.

Collins

Duthie

Dobson

. Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA. Carcinogenesis 1993; 14: 1733–1735.

28.

Comim

Pereira

Steckert

. Rivastigmine reverses habituation memory impairment observed in sepsis survivor rats. Shock 2009; 32: 270–271.

29.

Ritter

Andrades

Frota

MLC

Jr . Oxidative parameters and mortality in sepsis induced by cecal ligation and perforation. Intens Care Med 2003; 29: 1782–1789.

30.

Wichterman

Baue

Chaudry

. Sepsis and septic shock—a review of laboratory models and a proposal. J Surg Res 1980; 29: 189–201.

31.

Parker

Watkins

. Experimental models of Gram-negative sepsis. Brit J Surg 2001; 88: 22–30.

32.

Parnham

Kesselring

. Rosmarinic acid. Drugs Future 1985; 10: 756–757.

33.

Aydin

Bacanli

Taner

. Protective effects of resveratrol on sepsis-induced DNA damage in the lymphocytes of rats. Hum Exp Toxicol 2013; 32: 1048–1057.

34.

Taner

Aydin

Bacanli

. Modulating effects of pycnogenol^® on oxidative stress and DNA damage induced by sepsis in rats. Phytother Res 2014; 28: 1692–1700.

35.

Bøyum

. Isolation of lymphocytes, granulocytes and macrophages. Scand J Immunol 1976; 5: 9–15.

36.

Patel

Pandey

Bajpayee

. Cypermethrin-induced DNA damage in organs and tissues of the mouse: evidence from the comet assay. Mutat Res-Gen Tox En 2006; 607: 176–183.

37.

Tice

Agurell

Anderson

. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Molecular Mutagen 2000; 35: 206–221.

38.

Bakare

Patel

Pandey

. DNA and oxidative damage induced in somatic organs and tissues of mouse by municipal sludge leachate. Toxicol Ind Health 2012; 28: 614–623.

39.

Singh

McCoy

Tice

. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 1988; 175: 184–191.

40.

Anderson

T-W

Phillips

. The effect of various antioxidants and other modifying agents on oxygen-radical-generated DNA damage in human lymphocytes in the COMET assay. Mutat Res-Fund Mol M 1994; 307: 261–271.

41.

Collins

. Investigating oxidative DNA damage and its repair using the comet assay. Mutat Res 2009; 681: 24–32.

42.

Sier

Kubben

Ganesh

. Tissue levels of matrix metalloproteinases MMP-2 and MMP-9 are related to the overall survival of patients with gastric carcinoma. Br J Cancer 1996; 74: 413.

43.

Uchiyama

Mihara

. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 1978; 86: 271–278.

44.

Doise

Aho

Quenot

. Plasma antioxidant status in septic critically ill patients: a decrease over time. Fund Clin Pharmacol 2008; 22: 203–209.

45.

Cowley

Bacon

Goode

. Plasma antioxidant potential in severe sepsis: a comparison of survivors and nonsurvivors. Crit Care Med 1996; 24: 1179–1183.

46.

Dickson

. Sepsis and multiple organ failure. Anaesth Intens Care Med 2009; 10: 165–168.

47.

Neviere

Cepinskas

Madorin

. LPS pretreatment ameliorates peritonitis-induced myocardial inflammation and dysfunction: role of myocytes. Am J Physiol-Heart C 1999; 277: H885–H892.

48.

Deitch

. Animal models of sepsis and shock: a review and lessons learned. Shock 1998; 9: 1–11.

49.

Girardin

Grau

Dayer

J-M

. Tumor necrosis factor and interleuktn-1 in the serum of children with severe infectious purpura. New Engl J Med 1988; 319: 397–400.

50.

Hatherill

Tibby

Turner

. Procalcitonin and cytokine levels: relationship to organ failure and mortality in pediatric septic shock. Crit Care Med 2000; 28: 2591–2594.

51.

Prauchner

Prestes Ade

da Rocha

. Effects of diphenyl diselenide on oxidative stress induced by sepsis in rats. Pathol Research Pract 2011; 207: 554–558.

52.

Andrades

Ritter

Moreira

JCF

. Oxidative parameters differences during non-lethal and lethal sepsis development. J Surg Res 2005; 125: 68–72.

53.

Schulz

Lindenau

Seyfried

. Glutathione, oxidative stress and neurodegeneration. Eur J Biochem 2000; 267: 4904–4911.

54.

Kolgazi

Şener

Çetinel

. Resveratrol reduces renal and lung injury caused by sepsis in rats. J Surg Res 2006; 134: 315–321.

55.

Ritter

Andrades

Reinke

. Treatment with N-acetylcysteine plus deferoxamine protects rats against oxidative stress and improves survival in sepsis. Crit Care Med 2004; 32: 342–349.

56.

Galley

Howdle

Walker

. The effects of intravenous antioxidants in patients with septic shock. Free Radical Bio Med 1997; 23: 768–774.

57.

Tavafi

Ahmadvand

. Effect of rosmarinic acid on inhibition of gentamicin induced nephrotoxicity in rats. Tissue Cell 2011; 43: 392–397.

58.

Swarup

Ghosh

. Antiviral and anti-inflammatory effects of rosmarinic acid in an experimental murine model of Japanese encephalitis. Antimicrob Agents Ch 2007; 51: 3367–3370.

59.

Fadel

El Kirat

Morandat

. The natural antioxidant rosmarinic acid spontaneously penetrates membranes to inhibit lipid peroxidation in situ. BBA-Biomembranes 2011; 1808: 2973–2980.

60.

Eder

Wacker

Lutz

. Oxidative stress related DNA adducts in the liver of female rats fed with sunflower-, rapeseed-, olive-or coconut oil supplemented diets. Chem-Biol Interact 2006; 159: 81–89.

Does rosmarinic acid treatment have protective role against sepsis-induced oxidative damage in Wistar Albino rats?

Abstract

Keywords

Introduction

Material and methods

Chemicals

Animals

CLP model

Experimental design

Comet assay (single-cell gel electrophoresis)

Determination of oxidative stress parameters in the liver and kidney tissues

Determination of plasma TNF-α levels

Statistical analysis

Results

Comet assay

Oxidative stress parameters in liver and kidney tissues

Plasma TNF-α levels

Discussion

Footnotes

Declaration of Conflicting Interests

Funding

References