Phase 2 enzyme induction by conjugated linoleic acid improves lupus-associated oxidative stress

Free Radical Biology & Medicine 43 (2007) 71 – 79 www.elsevier.com/locate/freeradbiomed

Original Contribution

Phase 2 enzyme induction by conjugated linoleic acid improves lupus-associated oxidative stress Paolo Bergamo ⁎, Francesco Maurano, Mauro Rossi Istituto di Scienze dell’ Alimentazione, Consiglio Nazionale delle Ricerche (CNR-ISA), via Roma 52, 83100, Avellino, Italy Received 31 October 2006; revised 14 March 2007; accepted 27 March 2007 Available online 12 April 2007

Abstract Conjugated linoleic acid (CLA) exhibits anticancer and anti-inflammatory properties. Its ability to increase total GSH (GSH+GSSG) amount and gamma-glutamylcysteine ligase (γGCL) protein expression was recently associated with the inhibition of typical pathological signs in MRL/ MpJ-Faslpr mice (MRL/lpr). In the present study the ability of CLA to modulate oxidative stress and phase 2 enzyme activity in the same animal model was investigated. Disease severity was associated with age-dependent production of anti-double-stranded DNA antibodies (anti-dsDNA IgGs) and with enhanced extent of oxidative stress markers: reduced total GSH, increased protein 3-nitrotyrosines (3-NT), and protein-bound carbonyl (PC) amounts. To examine the effect of CLA on antioxidant status, CLA or olive oil (as control) was administered to pregnant MRL/lpr mice. Significantly higher total GSH and Trolox equivalent antioxidant capacity (TEAC) levels were measured in serum of CLA-treated dams (and their pups), as compared with controls. Finally, the antioxidant and chemopreventive properties of CLA were investigated in old MRL/lpr mice. Sera of CLA-treated mice contained higher concentrations of total GSH which were negatively correlated with the levels of oxidative stress markers. Moreover, increased GSH, γGCL, glutathione S-transferase (GSTs), and NAD(P)H:quinone oxidoreductase (NQO1) activities were measured in liver and spleen of CLA-treated animals. In conclusion our data indicate that the activation of detoxifying enzymes may be one of the mechanisms whereby dietary CLA down-regulates oxidative stress in MRL/lpr mice. © 2007 Elsevier Inc. All rights reserved. Keywords: Conjugated linoleic acid; Lupus erythematosus; Oxidative stress; Phase 2 enzymes; Chemoprotection

Introduction The alteration of cellular oxidation-reduction (redox) status by a controlled production of reactive oxygen (ROS) or nitrogen species (RNS) plays a signaling role involved in the modulation of cell cycle and gene expression. By contrast, when ROS/RNS Abbreviations: ABTS, 2,2′-azinobis(3-ethylbenzothiazoline 6-sufonate); anti-dsDNA IgGs, anti-double-stranded DNA antibodies; ARE, antioxidantresponse element; CDNB, 1-chloro-2,4-dinitrobenzene; CLA, conjugated linoleic acid; DNPH, 2,4-dinitrophenylhydrazine; DTNB, 5,5′-dithionitrobenzoic acid; PUFA, polyunsaturated fatty acids; γ-GCL, gamma-glutamylcysteinyl ligase; GSH, reduced glutathione; GSSG, oxidized glutathione; GST, glutathione S-transferases; Keap1, Kelch-like ECH-associated protein 1; NQO1, NAD(P)H:quinone oxidoreductase; Nrf2, NF-E2-related factor 2; 3-NT, protein 3-nitrotyrosines; PC, protein-bound carbonyls; ROS, reactive oxygen species; RNS, reactive nitrogen species; TEAC, Trolox equivalent antioxidant capacity; Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; SLE, systemic lupus erythematosus. ⁎ Corresponding author. Fax: +39 0825 781585. E-mail address: [email protected] (P. Bergamo). 0891-5849/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2007.03.023

production prevails over antioxidant defenses, deleterious effects such as damage of macromolecules, membranes, and DNA may occur. This injury, also named oxidative stress, is implicated in alterations occurring with aging and with several degenerative (cancer, atherosclerosis, Alzheimer) or autoimmune diseases such as rheumatoid arthritis or systemic lupus erythematosus (SLE). The MRL/MpJ-Faslpr (MRL/lpr) mouse is a prototypical model for human SLE in which the production of multiple autoantibodies, particularly to dsDNA, is critically involved in tissue damage and disease progression [1]. Moreover, the correlation between the extent of oxidative stress and the activity of SLE disease has been previously evidenced in humans [2,3] and in MRL/MpJ-Faslpr (MRL/lpr) mice, where NO overproduction [4] together with the reduced ability to cope with oxidative stress [5,6] has been implicated in the pathology severity. Under electrophile or oxidative stress, genes coding for highly specialized proteins are upregulated providing a response aimed at the detoxification, and at the reduction of

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the tissue and organism propensity to develop disease or malignancy (chemoprotection). These genes are activated through the antioxidant-response element (ARE) via the dissociation of Kelch-like ECH-associated protein 1 from the NF-E2-related factor 2 (Keap1/Nrf2). This latter represents the sensor of redox status [7] and the oxidant-induced modification of cysteine residues of Keap1 triggers the release of Nrf2 and the translocation to the nucleus in which it binds with ARE and activates the transcription of antioxidant (e.g., catalase, superoxide dismutase, glutathione reductase) and phase 2 enzymes involved in detoxification and chemopreventive mechanisms (glutathione S-transferases, GSTs; NAD(P)H:quinone oxidoreductase, NQO1; heme oxygenase-1, and gamma-glutamylcysteine ligase, γGCL) [8]. This latter enzyme plays a crucial role in GSH synthesis, determining the rate of cellular GSH synthesis [9]. GSH is the main intracellular antioxidant and its central role in xenobiotic or eicosanoid metabolism, the regulation of cell cycle, and gene expression is well established [10–12]. GSH is normally present in cells at millimolar levels and its concentration declines with aging or it may be depleted by oxidant molecules yielded during the progression of many diseases, including cancer [13] and autoimmune diseases [14– 17]. The central role of Nrf2/ARE activation and total thiol status in mammalian response to chemical and oxidative stress has attracted increasing interest during the past few years. In particular, the pathological consequences resulting from the defective functioning of this molecular mechanism have been evidenced in studies on Nrf2–/– mice. In these animals the genetic ablation of the Nrf2 gene resulted in the attenuation of several phase 2 drug-metabolyzing enzyme gene induction (including γGCL and GST), in the early GSH depletion [18] and in lupus-like pathological signs [19,20]. Conjugated linoleic acid (CLA) is the collective term used to describe positional and geometric isomers of linoleic acid (C18:2). Among them cis9 trans11, and trans10 cis12 isomers are the most biologically active in the promotion of numerous health benefits [21] and during the past few years CLA has attracted considerable attention because of its anti-inflammatory and cancer-preventive properties. We recently provided evidence that CLA’s ability to reverse typical pathological signs in MRL/lpr mice paralleled the increased concentration of total GSH (GSH+GSSG) and γGCL expression [22]. The present study was undertaken to determine whether CLA may down-regulate the SLE-associated oxidative stress in the same animal model and to investigate CLA’s ability to induce phase 2 enzyme activation. The obtained results clearly indicate the efficacy of CLA in modulating redox status, both by reducing the extent of age/pathology-associated oxidative stress and by enhancing the animal thiol status and detoxifying enzyme activity (GSTs, NQO1, and γGCS).

t11), 3.4% palmitic acid, 3.8% stearic acid, 15.4% oleic acid, and 1.5% linoleic acid, hereafter called CLA, was used. 5,5′Dithionitrobenzoic acid was purchased from Calbiochem (La Jolla, CA). Nitrated BSA (nitrotyrosine-BSA) and Menadione from Cayman Chemical (Ann Arbor, MI) and sheep antinitrotyrosine IgGs from Oxis (Portland, OR), rabbit polyclonal IgGs against Nrf2 (sc-13032) from Santa Cruz Biotechnology (Santa Cruz, CA,) and goat anti-rabbit biotinylated secondary IgGs (Dako Cytomation, Denmark) were used. Dicumarol, poly-L-lysine, 1-chloro-2-4-dinitrobenzene (CDNB), 2,2′-azinobis(3-ethylbenzothiazoline 6-sufonate) (ABTS), fetal bovine thymus dsDNA, and 6-hydroxy-2,5,7,8-tetramethylchroman-2carboxylic acid (Trolox) were purchased from Hoffman-La Roche. Orthophenylenediamine (OPD), bovine serum albumin fraction V (BSA), and other chemicals of the highest purity were purchased from Sigma-Aldrich (St. Louis, MO). Animals and CLA administration Experimental procedures for the use and care of laboratory animals in this study met the guidelines of the Italian Ministry of Health (DL 116/92). MRL/lpr mice and congeneic control mice (MRL/+), originally obtained from the Jackson Laboratory (Bar Harbor, ME), were bred and maintained at the Institute facility. Mice were fed ad libitum with standard chow. CLA dose used in the present study (30 mg/day) was comparable with that reported in the literature [23]; moreover, the amount of CLA administered and the duration of the treatment were decided on the basis of our recent data indicating that 2 weeks treatment exhibited beneficial effects on MRL/lpr mice autoimmune signs [22]. Individual doses of CLA, diluted in 200 μl of olive oil, were prepared just before administration. Olive oil (200 μl) was given to control animals. Age/disease-dependent increase of oxidative stress markers was preliminarily examined. Eighteen MRL/lpr mice, ranging from 7 to 22 weeks of age, were divided into 3 groups (n = 6): young (7–8 weeks old), adult/prediseased (10–15 weeks old), and old/diseased MRL/lpr mice (20–22 weeks old). Six congeneic MRL/+ mice (20–22 weeks old) were used as control. To evaluate CLA’s ability to modulate animal antioxidant status, primiparous females were gavaged with 30 mg CLA (n = 5) or olive oil (n = 5) for 7 weeks (2 weeks before and 5 weeks after the parturition). At the end of this period animals were sacrificed and blood samples were drawn from dams (18–22 weeks old) and pups (5–7 weeks old; n = 10) of both experimental groups. To investigate the modulatory effect of CLA, MRL/lpr mice were divided in two homogeneous groups (sex and body weight; n = 12 each): one was administered with 30 mg CLA and the other with olive oil (control) for 2 weeks up to 21–22 weeks of age.

Materials and methods Blood and tissue collection Reagents Isomeric CLA mixture (Tonalin Natural Inc., USA), composed of 76% CLA isomers (38.5% t10,c12; 37.4% c9,

Blood was collected from anesthetized mice, and sera, when not immediately used, were aliquot-stored at 20°C. On animal death, spleen and liver were excised and quickly rinsed in ice-

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cold 10 mM Tris-HCl, 120 mM NaCl (TBS). Organs were cut into pieces and, if not immediately used, were snap-frozen in liquid nitrogen and stored at --80°C. Preparation of cytosolic and nuclear extracts Liver and spleen pieces were chopped into isolation medium (150 mM sucrose, 75 mM KCl, 50 mM Tris-HCl, 1 mM KH2PO4, 5 mM MgCl2, 1 mM EDTA, pH 7.4). Homogenates were centrifuged at 5000g for 20 min at 4°C, and then 0.2 vol of 0.1 M CaCl2 in 0.25 M sucrose was added and incubated on ice for 30 min. At the end of the incubation, samples were centrifuged at 12,000g at 4°C for 20 min and supernatants (cytosolic extracts) were taken. Nuclear protein extracts were prepared from liver tissue accordingly to a published protocol [24]. If not used immediately, cytosolic and nuclear extracts were frozen on dry ice and stored in aliquots at –80°C. Before their use, protein concentration was determined by using the Bradford Protein Assay Kit (Bio-Rad) [25].

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Assay of carbonylated proteins The concentration of protein bound carbonyls (PC) in mouse sera was measured essentially in accordance to the method described by Levine et al. [30]. Briefly, 400 μl of 10 mM 2,4dinitrophenylhydrazine (DNPH) in 2.5 M HCl was added to 100 μl of diluted serum (1:10) and incubated for 1 h. Aliquots treated with 2.5 M HCl were used as blanks. Mixtures were vortexed every 15 min, extracted with 500 μl 20% trichloroacetic acid (TCA), and then spun for 5 min at 10,000g. The precipitates were washed once with 1 ml of of 10% TCA, followed by three washes with 1 ml of ethanol-ethyl acetate (1:1) to remove the free DNPH and lipid contaminants. Precipitates from DNPH-treated samples were dissolved in 1 ml 6 M guanidine hydrochloride and the amount of formed hydrazone was determined by reading the sample absorbance at 370 nm (ε = 22,000 M–1 cm–1) against pellets derived from the HCl-treated samples which were also used to calculate the protein contents. Finally PC concentration was expressed as nanomoles per milligram of protein.

Measurement of Trolox equivalent antioxidant activity Measurement of protein thiol concentration Antioxidant activity of mouse sera was measured according to Miller et al. [26]. Briefly, the inhibition of the absorbance of the radical cation formation ABTS was monitored after 20 min in a Dulbecco’s phosphate-buffered saline (PBS) containing 150 μM ABTS, 2.5 μM metmyoglobin, and 75 μM H2O2 at 734 nm. A Trolox solution was used to produce a standard curve for quantification of the Trolox equivalent antioxidant capacity (TEAC) of serum samples. ELISA assay for anti-dsDNA IgG and nitrosylated protein measurement Serum samples were analyzed for their anti-dsDNA IgG content by ELISA, essentially as previously reported [27]. Briefly, 100-μl aliquots of a solution composed of 5 μg/ml poly-L-lysine were added to well plates and incubated overnight at 4°C. Plates were coated (1 h at 37°C) with 100 μl of a Na-carbonate buffer (pH 9.5) containing 10 μg/ml in fetal bovine thymus dsDNA. Mouse sera, diluted 1:400 with PBS containing 0.05% Tween 20, were added to coated wells and incubated at 37°C for 1 h. Wells were next incubated (1 h at 37°C) with rabbit polyclonal primary antibody directed against mouse IgGs conjugated with horseradish peroxidase (dilution 1:10,000). After immunodetection, enzyme substrate (OPD) was added in citrate-phosphate buffer, pH 5, and 0.015% H202 and allowed to react for 30 min. Reaction was stopped by adding 50 μl of 1 M HCl and plate absorbance was read at 490 nm. Each sample was analyzed at least in duplicate and results were expressed as the mean ± standard deviation. The concentration of nitrosylated protein (3-NT) in sera was measured by competitive ELISA assay [28]. A calibration curve prepared with nitrosylated BSA was used for the calculation of 3-NT concentration [29] and its amount was finally expressed as nanomoles per milligram of protein.

Total GSH concentration in sera and in cytosolic tissue extracts was quantified using 5,5′-dithionitrobenzoic acid (DTNB)-GSSG reductase recycling assay as previously described [31]. Briefly, 100 μl of serum (or 1:10 dilution of cytosolic extracts in PBS) was mixed with 1 vol of TCA solution (5% TCA, 0.1 mM HCl, 10 mM EDTA) and incubated for 10 min at 4°C. The mixtures were centrifuged (3 min, 10,000 rpm) and triplicate aliquots of 50 μl of supernatant aliquots were transferred in a 96-well plate. Twenty microliters of 3% NaOH was pipetted to each well to adjust the pH (pH 8.0). One hundred microliters of 125 mM sodium phosphate buffer, pH 7.5, containing 6.3 mM EDTA was added to each well. Finally, 50 μl 2.5 mM DTNB in PBS was added into each well and the absorbance change at 405 nm was monitored for 10 min and calculated as mean V [mean V = (OD at 10 min – OD at 0 min)/10] and converted into total GSH concentration by using a GSH standard curve. TCA protein precipitates were dissolved in 100 μl of 6 M guanidine-HCl, diluted 1:10 in PBS, and used to calculate the protein contents against a BSA standard. After normalization to protein content, total GSH concentration was expressed as nanomole of DTNB-GSH conjugate formed per minute per milligram of protein (nmol GSH/mg protein/min). Chemoprotective enzyme activity NQO1 activity was measured accordingly to Benson et al. [32]. In brief, the freshly prepared reaction mixture contained, in a final volume of 0.9 ml, 25 mM Tris-HC1 (pH 7.4), 0.7 mg of crystalline BSA at pH 7.4, 0.01% Tween 20, 5 μM FAD, 0.2 mM NAD(P)H, 0 or 10 mM dicoumarol, and an appropriate amount of protein. An aliquot of 100 μl of 40 μM menadione (electron acceptor) was added to initiate the reaction and the dicoumarol-inhibitable activity (decrease of NADH absorbance) was calculated by using the extinction coefficient

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ε = 6.27 × 104 M--1 cm--1 and expressed as nanomoles per milligram protein per minute. Cellular glutathione S-transferase (GST) activity was measured using the broad substrate 1-chloro2,4-dinitrobenzene (CDNB) according to a standard procedure [33]. GST activity was calculated using the extinction coefficient of 9.6 mM--1 cm--1 and expressed as nanomole of CDNB-GSH conjugate formed per milligram protein per minute. γGCL activity was measured accordingly to the Seelig and Meister procedure [34] as modified by Lim et al. [35]. The enzyme activity was finally expressed as nanomoles per milligram of protein per minute. Western blotting Aliquots (15 μg of proteins) of hepatic nuclear extracts were fractionated by electrophoresis on a 10% SDS-PAGE and electroblotted onto Immobilon PVDF membranes (Millipore). Membranes were incubated (1 h at 37°C) with rabbit polyclonal primary antibody (dilution, 1:2000) against Nrf 2 (sc-13032; Santa Cruz Biotechnology) and then with goat anti-rabbit biotinylated secondary IgGs. Finally they were incubated (1 h at 37°C) with streptavidin-conjugated peroxidase (dilution, 1:2000; Dako Cytomation) and the formed immunocomplexes were visualized by enhanced chemiluminescence (ECL) and autoradiography according to the manufacturer’s protocol (ECL; Amersham Biosciences). To ensure that comparison between different lanes was valid, at the end of the immunodetection, membranes were stained with Coomassie blue. Statistical analysis Data are expressed as means ± SD and differences were assessed using Student’s t test and the level of significance was designated as follows: *P < 0.001, **P < 0.01, and ***P < 0.05. Correlation analysis was done using the Statistical Package for Social Sciences (SPSS version 8.0; SPSS Inc., Chicago, IL). Results Increased extent of oxidative stress markers and autoantibody levels in MRL/lpr sera associates with murine SLE progression To confirm the relationship between oxidative stress and disease activity, protein oxidation stress markers and antidsDNA IgG levels were measured in serum samples of MRL/lpr mice ranging from 7 to 22 weeks of age. Values measured in old (20–22 weeks of age) MRL/+ mice were used as control. PC and 3-NT concentrations measured in young (7–8 weeks of age) MRL/lpr did not differ significantly from those found in control animals. Significantly higher 3-NT concentrations were found in old MRL/lpr animals as compared with controls (0.18 ± 0.05 and 0.07 ± 0.04, respectively) (P < 0.005) (Fig. 1A). Similarly, PC concentrations were significantly higher in both adult (10– 15 weeks of age) and old mice (0.16 ± 0.02 and 0.28 ± 0.04 nmol/ mg protein, respectively) than in controls (0.16 ± 0.02 nmol/mg protein; P < 0.005) (Fig. 1B). Another aliquot of serum was used to verify the relationship between anti-dsDNA IgG synthesis and

murine SLE progression. As expected, autoantibody levels increased by 10–15 weeks of age and were about fourfold higher at 20–22 weeks as compared with young MRL/lpr or controls (Fig. 1C). These results confirm the association of protein oxidation extent and autoantibody yield with SLE disease severity in MRL/lpr mice. As oxidative stress may have a profound effect on thiol balance, total GSH was measured in serum samples of MRL/lpr mice ranging from 7 to 22 weeks of ages. GSH concentration measured in young MRL/lpr did not differ significantly from those found in control animals. Small, but significant, decline in GSH amount was evidenced in adult animals as compared with young animals (P = 0.037) and, as expected, a more marked antioxidant depletion occurred in old mice. In these animals serum protein thiol levels were significantly reduced to about 58% of control values (0.042 ± 0.006 and 0.017 ± 0.004 nmol GSH/mg protein/min, respectively) (P < 0.001) (Fig. 1D). These data confirm the link between SLE progression and oxidative stress extent in MRL/lpr mice. CLA modulates antioxidant status of MRL/lpr mice The concentration of total protein thiols in serum is used as an index of redox status in other less accessible tissues [14]. As dietary intake was reported to influence CLA content in rat’s milk [36], we thus decided to evaluate the effects of dietary CLA on the antioxidant status of both young and old MRL/lpr mice. Total GSH content was measured in sera of CLA-treated dams (20–22 weeks old) and in their pups (5–7 weeks old). Age-matched weaning mice, gavaged with olive oil, and pups were used as control. Notably, the twofold decrease of total GSH concentration in diseased dams (from 0.042 ± 0.006 to 0.017 ± 0.002 nmol GSH/mg protein/min) was significantly recovered by CLA treatment (0.034 ± 0.006 nmol GSH/mg protein/min; P < 0.001). Similarly, serum GSH concentration in pups weaned from CLA-treated dams was significantly higher than in those nursed by control dams (0.059 ± 0.04 versus 0.042 ± 0.062 nmol/ mg protein/min, respectively; P < 0.005) (Fig. 2A). To substantiate CLA’s ability to modulate antioxidant status in MRL/lpr mice, TEAC values were measured in mice sera. Antioxidant capacity of serum from CLA-treated dams was significantly higher as compared with controls (240 ± 99 and 94 ± 48 nmol of TEAC, respectively; P = 0.038). Similarly, increased TEAC values were measured in pups weaned from CLA-treated dams mice as compared with their controls (308 ± 77 and 202 ± 108, respectively; P = 0.025) (Fig. 2B). Taken together, these results indicate CLA’s ability to improve the antioxidant status (thiols and TEAC values) in both young/ healthy and old/diseased MRL/lpr mice. Oxidative stress extent in old MRL/lpr mice is inhibited by CLA To further explore CLA’s effect on SLE-associated oxidative stress, 24 MRL/lpr mice (18–20 weeks old) were divided into two groups: one gavaged for 2 weeks with CLA and the other with olive oil (control). At the end of this period total GSH, PC, 3-NT, and anti-dsDNA IgG contents were measured in serum.

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Fig. 1. Oxidative stress extent associates with murine SLE progression. Protein oxidation stress markers (3-NT and PC), pathogenetic autoantibodies (anti-dsDNA IgGs), and total protein thiol levels (serum total GSH) were evaluated in serum samples of young (7–8 weeks old), adult (10–15 weeks), and old (20–22 weeks) MRL/ lpr mice and in old congeneic control animals (MRL/+) (n = 6/group). 3-NT (A) and anti-dsDNA IgG levels (C) were evaluated by ELISA. PC (B) and total GSH concentration (D) were measured spectrophotometrically. Samples were analyzed at least in duplicate and results are given as mean ± SD. *, **, *** Significantly different (P < 0.005; <0.01 or <0.05) from control samples.

As expected, significantly higher total GSH concentrations were measured in sera of CLA-treated mice (0.04 ± 0.01) than in controls (0.02 ± 0.00 nmol/mg protein/min) (P < 0.001). Conversely, lower PC and 3-NT concentrations were measured in CLA-treated animals (0.07 ± 0.04 and 0.12 nmol/mg protein, respectively) as compared with controls (0.13 ± 0.02 and 0.3 ± 0.03 nmol/mg protein; P = 0.002 and < 0.001, respectively). Similarly, anti-dsDNA IgG yield was markedly inhibited in animals gavaged with CLA as compared with olive oil-treated animals (0.32 ± 0.20 and 0.67 ± 0.21, respectively; P = 0.003). To investigate CLA-induced modulation of animal redox status, individual values of serum protein thiol amounts were plotted against protein oxidation markers (PC and 3-NT) and pathogenic autoantibody levels. Interestingly, total GSH content negatively correlated with protein oxidation extent (PC and 3NT) (r = –0.43; P = 0.036 and r = –0.70; P = 0.0001, respectively) (Figs. 3A and 3B) and with anti-dsDNA IgG level (r = –0.547; P = 0.006) (Fig. 3C). The results indicate that CLA treatment ameliorates SLE-associated oxidative stress in old MRL/lpr mice through the improvement of animal redox status. CLA enhances GSH content and γGCL activity in liver and spleen CLA’s ability to reduce typical pathological SLE signs was recently associated with enhanced intracellular total GSH

content and γGCL protein expression in splenic cells [22]; thus, in this study, we further investigated CLA’s ability to improve GSH content and γGCL activity in spleen of MRL/lpr mice. In addition, as liver is commonly studied in research aimed at the evaluation of phase 2 enzyme induction by chemoprotective agents, GSH amount and γGCL activity were also measured in liver cytoplasmic extracts. The presented results are evidence that both liver and spleen from CLA-treated mice contained significantly higher total GSH concentrations (40.1 ± 11.5 and 4.8 ± 9.07 nmol/mg protein/min, respectively) than controls (27.5 ± 9.2 and 20.5 ± 0.6 nmol/mg protein/min) (P < 0.001 and < 0.01, respectively) (Fig. 4, upper panels). Moreover, as predicted by GSH levels, SLE-associated decline of γGCL activity was reversed by CLA administration in both spleen and liver cytoplasmic extracts. In particular, γGCL activity was about twofold higher in organs (liver and spleen) of CLA-treated animals (4.3 ± 0.6 and 2.4 ± 1.1, respectively) as compared with those gavaged with olive oil (2.2 ± 0.5 and 1.7 ± 0.9, respectively) (Fig. 4, lower panels). These results demonstrate CLA’s ability to improve animal thiol status through the activation of γGCL activity. CLA enhances GST and NQO1 activity in liver and spleen As NQO1 and GST enzymes are typically involved in the detoxification of xenobiotics as well as ROS, [37,38], their

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Discussion The current study confirms and extends previous findings indicating the beneficial effect of CLA supplement on an animal model of autoimmune disease. Our data demonstrate the inhibitory activity of CLA on SLE-associated oxidative stress through the enhancement of animal redox status and the activation of detoxifying enzymes. To our knowledge, this is first time that CLA’s ability to activate phase 2 enzymes in vivo is reported, and the presented results provide a new insight into the molecular mechanism responsible for its anticancer activity. Oxidative damage, consequential to defective antioxidant defenses and increased generation of ROS, has been implicated in the pathogenesis of both normal aging and of autoimmune disease. In particular, NO overproduction played a critical role in SLE development (3), and our data evidencing the diseasedependent increase of 3-NT concentration in MRL/lpr mice are consistent with previous studies showing the correlation of

Fig. 2. CLA enhances antioxidant status of MRL/lpr mice. MRL/lpr female mice were gavaged with 30 mg CLA (black bars) or with olive oil as control (white bars) for 7 weeks (2 weeks before and 5 weeks after the parturition). At the end of this period animals were sacrificed and total GSH (A) and TEAC values (B) levels were measured in dams and pups sera (n = 5/group). Samples were analyzed at least in duplicate and results are given as mean ± SD. *, ** Significantly different (P < 0.005; or <0.01) from control animals.

activity was measured in cytoplasmic extract of MRL/lpr mice to confirm CLA’s ability as an inducer of phase 2 activator. A remarkable increase (1.7-fold) of NQO1 and GST activity was measured in liver of CLA-treated animals (7600 ± 1330 and 8.14 ± 2.1, respectively) as compared with controls (4327 ± 1176 and 4.8 ± 1.7, respectively; P < 0.005) (Fig. 5, left panels). Similarly, significantly higher GST and NQO1 activities (1.4and 1.2-fold higher) were measured in spleen cytoplasm of CLA-treated animals as compared with those measured in control animals (Fig. 5, right panels). These results further substantiate CLA’s activity as an inducer of detoxification enzymes in MRL/lpr mice. Finally, to confirm the involvement of the Nrf2/ARE pathway in CLA-induced activation of phase 2 detoxifying enzymes, the translocation into the nucleus of Nrf2 was visualized by Western blotting. Increased Nrf2 amount (57 kDa) in nuclear extracts was evidenced in liver of MRL/ lpr liver mice treated with CLA, as compared with controls (Fig. 6). This result further supports the involvement of the Nrf2/ARE mechanism in the CLA-mediated activation of phase 2 enzymes.

Fig. 3. Total GSH concentration negatively correlates with anti-dsDNA IgG, 3-NT, and PC amounts. Twenty-four MRL/lpr mice were divided into two experimental groups (n = 12 animals/each). One was gavaged with 30 mg CLA and the other with olive oil (as control) for 2 weeks. At the end of the treatment, total GSH, 3-NT, PC, and anti-dsDNA IgG levels were measured in serum samples of CLA- (filled circles) and olive oil-treated animals (open circles). Each sample was analyzed at least in triplicate and average values are shown. Individual GSH concentrations were plotted against PC (A), 3-NT concentration (B), or anti-dsDNA IgG level (C). Deviations over 5% from the average were not found.

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Fig. 6. CLA enhances nuclear translocation of Nrf2 in mouse liver. Protein aliquots (15 μg) of hepatic nuclear extracts from CLA- or olive oil-treated mice were fractionated by SDS-PAGE and nuclear translocation of Nrf 2 was immunochemically visualized (upper panel). On the immunodetection, membrane was stained with Coomassie blue as internal standard (lower panel).

Fig. 4. CLA improves GSH content and γGCL activity in liver and spleen tissue. CLA-induced activation of phase 2 enzymes was evaluated in cellular extracts prepared from liver and spleen tissue from animals receiving CLA (black bars) or olive oil as control (white bars). Total GSH content (A, B) and γGCL activity (C, D) were measured in cytoplasmic extract. Samples were analyzed at least in duplicate and results are given as mean ± SD. *, **, Significantly different (P < 0.005 or <0.01) from control.

enhanced NO production with both human [2,3] and murine SLE disease [6,39]. Similarly, the finding of the age-associated enhancement of carbonyls concentration in serum protein of MRL/lpr mice is in good accordance with data showing the association of increased PC with the human SLE activity [3]. As oxidatively damaged protein and DNA were typically associated with the yield of pathogenic autoantibodies [40], the ageassociated increase of anti-dsDNA IgGs in MRL/lpr mice

Fig. 5. CLA improves GST and NQO1 activity in mouse liver and spleen tissue. CLA-induced activation of phase 2 enzymes was evaluated in cellular extracts prepared from liver and spleen of animals receiving CLA (black bars) or olive oil as control (white bars). GST (A, B) and NQO1 (C, D) activities were measured in cytoplasmic extracts. Samples were analyzed at least in duplicate and results are given as mean ± SD. *, ** Significantly different (P < 0.005 or <0.01) from control.

confirms previous results obtained by us and by others [22,27] and also validates their suitable use as a predictor of SLE disease progression [41]. The significant negative correlation of total GSH concentration with oxidative stress markers or antidsDNA IgG levels demonstrates CLA-induced reversal of SLEassociated oxidative stress in MRL/lpr mice. In particular, the strong association of decreased GSH concentration with antidsDNA IgGs is consistent with the reported oxidative stress involvement in autoantibody yield [42,43]. Interestingly, the negative correlation between total GSH and 3-NT concentration, beside the confirmation of the important role played by thiol status in the down-regulation of SLE-induced oxidative stress, also provides an indication of the biochemical mechanism involved. Indeed, 3-NT production, a specific indicator of RNS yield [44,45], is known to be modulated by nuclear factor κB (NFκB) activity [46] which is one of the more prominent regulators of the cellular response to oxidative stress. Therefore, as NFκB activity was triggered by ROS or specifically inhibited by diverse thiol antioxidants [47], it is thus likely that enhanced GSH status in CLA-treated mice might be accountable for NFκB down-regulation in this [22] and other animal models [48,49]. The main hypothesis of our study was that CLA treatment would reverse SLE-associated oxidative stress in diseased MRL/lpr mice. In these animals the genetic background was implicated in the defective functioning of antioxidant defenses [5,6]; thus, data showing the marked decline of total GSH and TEAC value in olive oil-treated dams are consistent with studies showing the association of decreased thiol status with the progression of autoimmune disease [3,18]. Similarly, thiol status improvement in CLA-treated mice is consistent with our recent data indicating the beneficial effect of dietary CLA on total GSH content in spleen cells [22]. Interestingly, the significantly higher antioxidant status in animals weaned by CLA-treated dams suggests the involvement of the Nrf2/ARE mechanism in the reported defective antioxidant defenses [5,6] and CLA’s ability to improve basal γGCL activity. Intriguingly, these data are similar to those reported in a recent study in which n-3 PUFA administration exerted their beneficial effects on the oxidative status of both adult rats and their offspring [50]. It is widely accepted that dietary n-3 PUFAs and CLA exhibited beneficial effects on autoimmune and chronic antiinflammatory diseases [23,51]; although the exact mechanisms are not known, they were reported to display similar biological

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effects [52]. Interestingly, n-3 PUFA–induced amelioration of murine SLE pathology was associated with antioxidant enzyme activity (SOD, catalase) [53–55] or antioxidant status enhancement [56]. Similarly, CLA-mediated enhancement of antioxidant enzyme activity in in vitro experiments first indicates their ability to modulate Nrf2/ARE gene expression [52]. More interestingly, our results showing CLA’s ability as a phase 2 enzyme activator (as evidenced by enhanced NQO1, GST, and γGCL enzyme activity) are consistent with recent data showing n-3 PUFAs (docosahexaenoic acid) ability to enhance intracellular GSH concentration and γGCL expression in vitro [57,58]. The presented data, together with our previous finding, demonstrate that Nrf2/ARE activation by CLA may be implicated in anti-inflammatory and inhibitory effects on SLE-associated oxidative stress [22]. The induction of detoxifying enzymes by chemicals or by dietary factors has been recently indicated as a novel therapeutic approach for the treatment of inflammatory disease [8] and the beneficial role of detoxifying enzyme activation on the amelioration of pathology-associated oxidative stress was previously evidenced in murine [59] or human autoimmune disease [60]. The exact mechanism whereby CLA activates phase 2 enzymes remains to be elucidated. However, it is likely that the modulatory effects of CLA-induced prooxidant activity [61] on several cytosolic kinase activities (including PKC, MAPK, and PI3k) participating in the Nrf2/ARE signal transduction [62] may be involved in the activation of detoxifying and chemoprotective enzymes. In conclusion, the presented results demonstrate for the first time the inhibitory effects of CLA on pathology-associated oxidative stress in MRL/lpr mice and strongly indicate that the activation of phase 2 enzymes by CLA may contribute to the treatment of autoimmune disease in this animal model. In addition, as the Nrf2/ARE pathway is one of the main chemopreventive mechanisms against carcinogens [63] this could likely be implicated in the anticancer activity of CLA. Investigations aimed at a better understanding of the biochemical pathways underlying the CLA-induced activation of detoxifying enzymes are in progress. In addition, owing to the general limitations inherent in animal experiments, CLA doses used in mice cannot be directly extrapolated to humans; thus further studies, to set up the minimal dose producing similar effects, are required to confirm the feasibility of this therapeutic strategy in humans. References [1] Kotzin, B. L. Systemic lupus erythematosus. Cell 85:303–306; 1996. [2] Gilkeson, G.; Cannon, C.; Oates, J.; Reilly, C.; Goldman, D.; Petri, M. Correlation of serum measures of nitric oxide production with lupus disease activity. J. Rheumatol. 26:318–324; 1999. [3] Morgan, P. E.; Sturgess, A. D.; Davies, M. J. Increased levels of serum protein oxidation and correlation with disease activity in systemic lupus erythematosus. Arthritis Rheum. 52:2069–2079; 2005. [4] Weinberg, J. B.; Gilkeson, G. S.; Mason, R. P.; Chamulitrat, W. Nitrosylation of blood hemoglobin and renal nonheme proteins in autoimmune MRL-lpr/lpr mice. Free Radic. Biol. Med. 24:191–196; 1998.

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