N-acetylcysteine and alpha-lipoic acid improve antioxidant defenses and decrease oxidative stress, inflammation and serum lipid levels in ovariectomized rats via estrogen-independent mechanisms

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ScienceDirect Journal of Nutritional Biochemistry 67 (2019) 190 – 200

N-acetylcysteine and alpha-lipoic acid improve antioxidant defenses and decrease oxidative stress, inflammation and serum lipid levels in ovariectomized rats via estrogen-independent mechanisms Marina Delgobo a , Jonathan Paulo Agnes a , Rosângela Mayer Gonçalves a , Vitória Wibbelt dos Santos a , Eduardo Benedetti Parisotto b, c , Ariane Zamoner c , Alfeu Zanotto-Filho a,⁎ a

Laboratório de Farmacologia Bioquímica e Molecular, Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina (UFSC), Florianópolis, SC, Brazil b Faculdade de Ciências Farmacêuticas, Alimentos e Nutrição, Universidade Federal de Mato Grosso do Sul (UFMS), Campo Grande, MS, Brazil c Departamento de Bioquímica, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina (UFSC), Florianópolis, SC, Brazil

Received 9 January 2019; received in revised form 18 February 2019; accepted 28 February 2019

Abstract Sexual hormone deficiency has been associated with metabolic changes, oxidative stress and subclinical inflammation in postmenopausal women. Hormone replacement therapies are effective in many instances, even though some patients either do not respond or are not eligible. The aim of this study was to evaluate the impact of short- (15 days) versus long-term (60 days) sexual hormone depletion and whether antioxidant supplementation with N-acetylcysteine (NAC) and alpha-lipoic acid (LA) improves oxidative stress, metabolic, and inflammatory parameters in ovariectomized (OVX) rats. Short-term OVX rapidly depleted circulating estrogen, causing uterine atrophy and body weight gain without affecting oxidative damage, inflammatory and lipid metabolism markers. In contrast, long-term OVX augmented oxidative damage in serum and peripheral tissues as well as increased serum total cholesterol, TNF-α and IL6 levels. Triglycerides, glucose and HDL cholesterol were not altered. Long-term OVX-induced oxidative stress was associated with depletion of GSH and total non-enzymatic antioxidants as well as decreased activity of Glutathione Peroxidase (GPx) and Glutathione Reductase (GR), but not Superoxide Dismutase (SOD) and Catalase (CAT). NAC and LA supplementation prevented GSH and total non-enzymatic antioxidants depletion as well as restored GPx and GR activities, TNF-α, IL6 and cholesterol in OVX rats. NAC and LA effects appear to be independent on NRF2 activation and estrogen-like activity, since NAC/LA did not promote NRF2 activation and were not able to emulate estrogen effects in OVX rats and estrogen-receptor-positive cells. The herein presented data suggest that NAC and LA may improve some deleterious effects of sexual hormone depletion via estrogen-independent mechanisms. © 2019 Elsevier Inc. All rights reserved. Keywords: Menopause; Antioxidants; Estrogen; Cytokines; Aging

1. Introduction Menopause, the age-related loss of ovarian function, increases risk for various aging-associated conditions, including body weight gain/obesity, dyslipidemia, insulin resistance, osteoporosis, depression and cancer [1–3]. While studying molecular changes associated with menopause and postmenopausal in humans is difficult due to limited access to tissue samples and intrinsic variation among patients, the menopause model induced by bilateral ovariectomy (OVX) in rodents has been widely used to study mechanisms and test new interventions capable of minimizing the deleterious effects associated with menopause [4–6]. Several studies have indicated that lack of sexual hormones in postmenopausal women results in impaired tissue antioxidant potential, ⁎ Corresponding author at: Departamento de Farmacologia, Centro de Ciências Biológicas (CCB) – Bloco D, Universidade Federal de Santa Catarina (UFSC) – Campus Trindade; Florianópolis, 88049-900, Santa Catarina, Brazil. Tel.: +55 48 3721 2474; fax: +55 48 3337 5479. E-mail address: [email protected] (A. Zanotto-Filho). https://doi.org/10.1016/j.jnutbio.2019.02.012 0955-2863/© 2019 Elsevier Inc. All rights reserved.

which has been characterized by decreased activity of non-enzymatic scavengers - such as vitamin E, vitamin A and glutathione - as well as of the antioxidant enzymes Superoxide Dismutase (SOD) and glutathione Peroxidase (GPx). Increased reactive species production associated with decreased antioxidant status contribute to oxidative stress and damage to biomolecules, including lipids, proteins and DNA thereby resulting in chronic progressive loss of tissue function and accelerated aging that typically occurs in postmenopausal women [4,7–9]. In addition to oxidative stress, postmenopausal seems to be associated with low-level chronic inflammation and increased sensitivity to inflammatory stimuli [10,11]. Previous studies have indicated that adipose tissue accumulation triggered by hormonal depletion in OVX models contributes to COX-2, TNF-α and IL1β expression, especially in mice fed high-fat diet [12]. It is well-established that obesity results in pro-inflammatory state, and the latter is particularly exacerbated in postmenopausal obese women as well as in OVX animals, resulting in increased risk for breast cancer [12,13]. However, the cause–effect relationship and crosstalk between hormone depletion, oxidative stress and inflammation are not well understood.

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Hormone replacement therapy (HRT) for menopause patients has been debated for decades with back-and-forth regarding its safety and benefits. While early data on HRT showed reduction in coronary heart disease and mortality, subsequent clinical trials reported increased risk of coronary disease, venous thromboembolism and breast cancer which, associated with negative and misled information disseminated by the media, led to an abrupt decrease in the use of HRT [14,15]. Although subsequent data analysis indicated more benefits than harm to patients on HRT, a parcel of individuals either do not respond or experience side effects, as well as many physicians and patients remain reluctant on HRT. As an alternative to HRT, prevention strategies involving lifestyle management (i.e. diet and exercise), disease screening and statins have been adopted, although the results are modest, inconclusive and/or difficult to maintain [15]. In this context, natural products and compounds with antioxidant activity are emerging as a new strategy to attenuate symptoms and other menopause-related conditions [4,16]. In this study, we investigated the effect of the supplementation with two thiol-containing antioxidants, namely N-acetylcysteine (NAC) and alpha-Lipoic Acid (LA), upon oxidative, inflammatory and metabolic parameters in ovariectomized (OVX) Wistar rats. Initially, the effects of short- versus long-term OVX (15 and 60 days, respectively) upon these parameters were compared. Then, in the long-term OVX model, the effect of NAC and LA upon oxidative damage and antioxidant enzyme activity in peripheral tissues (liver, kidney and heart), serum lipids, as well as markers of tissue toxicity, cytokines and estrogen levels were determined. 2. Materials and methods 2.1. Materials Phenylmethylsulfonylfluoride (PMSF), reduced glutathione (GSH), sodium dodecyl sulfate (SDS), EDTA, epinephrine, thiobarbituric acid (TBA), 2,4-dinitrophenylhydrazine (DNPH), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), 2,20-azobis[2-amidinopropane] (AAPH), NADPH, guanidine, 1-chloro-2,4-dinitrobenzene (CDNB), MTT (3-(4,5Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide, and cell culture reagents (DMEM, antimycotic:antibiotic and trypsin/EDTA solutions) and MMS (methyl-methane-sulphonate) were purchased from Sigma Chemical Co (St. Louis, USA). Nacetylcysteine (NAC), alpha-Lipoic Acid (LA) and 17-beta-estradiol (estrogen/E2) were purchased from Essential Nutrition/Essentia Pharma (Santa Catarina, Brazil). Biochemical, ELISA assay kits and western blot reagents are described below. 2.2. Bilateral ovariectomy (OVX) All experimental procedures were performed in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals, and were approved by Institutional Animal Care and Use Committee (CEUA-UFSC Protocol #: 2231170317). Female Wistar rats (90 d; 160–230 g) were obtained from our own breeding colony. They were caged in groups of five with food and water ad libitum, and maintained on a 12 h light–dark cycle (7 am/7 pm) in a temperature-controlled room (23±1 °C) throughout the experiments. Rats were allowed 2 weeks to acclimatize before any experimentation to be carried out. For bilateral ovariectomy (OVX), the animals were anesthetized by intraperitoneal injection of ketamine (90 mg/kg) and xylazine (10 mg/kg), and both dorso-lateral sides of the abdomen were shaved and sterilized with 70% ethanol/iodine. For each ovary, one small incision (~1 cm) was made through the skin and muscle, then the ovary was located, and a sterile suture was performed around the uterine horn. The ovary was removed, and then muscle and skin wounds were sutured using Catgute 4.0 suture. Sham animals were equally manipulated but ovaries were not excised. After surgery, the rats were housed for 6 h on heating pads to allow recovery, and then re-grouped into their respective cages. Acetaminophen (150 mg/kg/day, gavage) was administrated for 4 days aiming post-surgery pain management. 2.3. Treatments Five days after surgical procedures, the animals were randomized. For time course experiment, female Wistar rats were divided in two sham-operated (n=8/group), two OVX (n=8/group) and OVX + E2 (E2: 17-beta-estradiol/estrogen, 20 μg/kg/day, dose based on [17]; n=6) groups. The animals were maintained for 15 (short-term OVX) or 60 (long-term OVX) days as schematized in Fig. 1A (upper timeline). For evaluation of NAC/LA impact upon OVX, 47 female Wistar rats were divided as follows: sham (vehicle-treated; n=7); OVX (vehicle-treated, n=8); OVX + NAC 10 (NAC 10 mg/kg;

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n=8); OVX + NAC 25 (NAC 25 mg/kg, n=8); OVX + LA 25 (LA 25 mg/kg, n=8); OVX + LA 50 (LA 50 mg/kg, n=8). Test drugs and vehicle were administrated by oral gavage (0.5 mL/rat) carried out in alternate days for a total of 60 days (Fig. 1A; bottom timeline). The LA and NAC doses used herein were selected based on previous studies in rodents [18–22], and by converting doses found in NAC and LA supplements (typically 100–600 mg/day, assuming a 60 kg human [23]) to rat equivalent doses in accordance with [24]. NAC and LA were prepared by dissolving the compounds in 50 mM phosphate buffered saline and adjusting solubility with 1 N NaOH to a final pH~7. Treatments were prepared fresh every day. Estrogen (17-beta-estradiol) was dissolved in sesame oil and administrated by subcutaneous route. Body weight (BW) was monitored weekly and delta BW gain was expressed as g% (g per 100 g BW) as compared to BW at the day of OVX surgery (Protocol day 0; Fig. 1A). 2.4. Sample collection and processing At the end of treatments, the animals were euthanized by ketamine/xylazine (100/ 10 mg/kg, i.p.) followed by cardiac puncture. Animals were not fasted prior to blood collection. Whole blood samples were collected in EDTA tubes, and part was harvested without anticoagulants. Serum was separated by centrifugation (1300×g, 10 min) and stored at−80 °C. The uterus was cut above the cervical junction, visible fat removed, and the remaining tissue was weighed. Adipose tissue was dissected from the retroperitoneal region and weighted. In addition to blood samples, ~ 300–400 mg of liver, kidney and heart tissues were collected and homogenized in 1.5 mL of 10 mM PBS (pH 7.4) containing 1 mM PMSF, followed by centrifugation at 10,000 g/15 min at 4 °C. The supernatant was separated into different tubes and stored at −80 °C in order to minimize freezing and thawing effects upon enzymes activity. Lowry method was used for protein quantification. 2.5. Serum biochemical parameters Serum cholesterol (total), high-density lipoprotein (HDL), Triglycerides, Alanine aminotransferase (ALT), Alkaline Phosphatase activities (ALP), creatinine and glucose levels were quantified by Labtest liquiform commercial kits per manufacturer's instructions (Labtest, Brazil). Bone-specific ALP (BS-ALP), which is thermo-labile, was estimated by difference between total serum ALP and heat-inactivated (56 °C for 15 min) ALP activities. Haemogram analyses were carried out in fresh blood samples collected in EDTA using an ABX Micros 60 equipment (HORIBA ABX Diagnostics, Montpellier, France). 2.6. ELISA for TNF-α, IL6 and 17-beta-estradiol Serum TNF-α, IL6 and estrogen levels were quantified by ELISA in accordance with manufacturer instructions. The Rat IL6 ELISA Kit (RAB0311) and Rat Tumor Necrosis Factor α ELISA Kit (RAB0479) were from Sigma-Aldrich, and the 17-beta-estradiol ELISA Kit (ab108667) was from Abcam. 2.7. Lipoperoxidation end products (TBARS) The endogenous lipoperoxidation was estimated by determination of thiobarbituric acid-reactive substances (TBARS) according to the method described by Draper and Hadley [25]. Firstly, the samples (~ 5 mg protein) were incubated in the presence of 10% Trichloroacetic acid (TCA) for protein precipitation and then centrifuged at 10,000×g for 15 min. Supernatants were collected and reacted with thiobarbituric acid (TBA) in acid-heating medium thereby producing a pink Schiff base that was measured at 532 nm in a spectrophotometer. TBARS levels were expressed as nmol/mg protein. 2.8. Protein carbonyl The oxidative modification of proteins was measured by means of carbonyl groups quantification as previously described by Levine et al. [26]. Briefly, 2 to 5 mg proteins were incubated in the presence of 5 mM 2,4-dinitrophenylhydrazine (DNPH) for 1 h at room temperature. Afterwards, the proteins were precipitated with TCA, centrifuged and washed thrice with ethanol:ethyl acetate (1:1 v/v). The proteins were solubilized with 6 M guanidine (pH 6.0), and DNPH-carbonyl adducts were read in a microplate reader at 370 nm. Results were expressed as nmol carbonyl/mg protein. 2.9. Total reduced thiol content (R-SH) Briefly, a 100 to 200 μg protein aliquot was diluted in 10 mM PBS (180 μL) plus 35 μL boric acid buffer (100 mM boric acid, 0.2 mM EDTA, pH 8.5), and then 10 μL of 10 mM 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) was added and incubated for 1 h at room temperature. Blank samples and samples incubated with DTNB were read in a spectrophotometer at 412 nm [27]. NAC standard curves were used for calculation of reduced thiol content. The results were expressed as nmol R-SH/mg protein. 2.10. Glutathione quantification Glutathione content (GSH and GSSG) in liver, kidney and heart homogenates was measured fluorimetrically in accordance with Glutathione Fluorimetric Assay Kit protocol

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Fig. 1. Time course effect of OVX upon body weight gain, inflammation, lipid metabolism and oxidative stress markers. (A) OVX and treatment protocols used to evaluate the effect of short (15 days) vs. long-term OVX (60 days) (Design 1), and the long-term effect of antioxidant treatment (Design 2); (B) Serum 17-beta-estradiol levels at 15 days post-OVX; (C) Uterine weight at 15 and 60 days post-OVX. (D) Delta Body weight gain curves of sham and OVX rats at 15, 30 and 60 days after OVX/sham surgery. (E) Retroperitoneal fat, (F) serum total cholesterol, (G) serum TNF-α and IL6, and (H-I) serum and hepatic TBARS and carbonyl levels at 15 and 60 days post-OVX. *denotes the statistical difference between sham and OVX groups at same experimental endpoint (15 or 60 days). Data were analyzed by two-way ANOVA (sham/OVX vs. 15/60 days), Pb.05.

(BioVision Incorporated, CA). The fluorescence values were normalized by protein (RFU/ mg protein) and expressed as percentage compared to sham-control (% control).

decay of kinetics was selected, and GPx activity was expressed as mU/mg protein (1 U= 1 μmol of NADPH oxidized per min at 25 °C, pH=7) using the molar extinction coefficient of NADPH at 340 nm (6.22 mM-1 cm-1).

2.11. Non-enzymatic antioxidant defenses measurement (TRAP assay) 2.13. Glutathione reductase (GR) activity Total reactive antioxidant potential assay (TRAP) was evaluated as an estimative of non-enzymatic antioxidants present in the samples [28,29]. This method evaluates the capacity of samples to scavenge peroxyl radicals generated by AAPH (2,20-azobis[2amidinopropane]) decomposition in 0.1 M Glycine buffer (pH=10.2). Unquenched peroxyl radicals react with luminol, and reading is taken by chemiluminescence. Briefly, we prepared an AAPH solution, added luminol and, after 2 h for system stabilization, we added 10, 20 or 50 μg samples and monitored chemiluminescence for 70 min. An “AAPH system” consisting of AAPH, buffer and luminol (without samples) was also carried out. This AAPH system was considered as “100% chemiluminescence = 0% peroxyl scavenging activity” and was used to obtain Relative Luminescence Units (RLU) for each sample along the readouts. For each kinetics time-point, the RLU values were transformed in percentage compared to “AAPH system”, and the area under curve (AUC) was calculated by GraphPad (San Diego, CA, USA) as previously described [30]. These AUC were used to calculate % of peroxyl scavenging. The lower the AUC value, the higher the non-enzymatic antioxidants in the sample. The antioxidant Trolox (100 nM) was used as a positive control. 2.12. Glutathione peroxidase (GPx) activity GPx activity was determined by measuring the rate of NADPH oxidation in a spectrophotometer at 340 nm, as previously described [31]. The enzyme incubation medium consisted of GSH, NADPH, Glutathione Reductase (GR) and tert-butylhydroperoxide in reaction buffer (20 mM potassium phosphate buffer, pH 7, 2 mM EDTA and 0.8 mM sodium azide). The rate of absorbance decrease at 340 nm was monitored over a period of 9 min with 30 s interval in a microplate reader. The linear

GR activity was determined by monitoring the decrease in NADPH absorbance at 340 nm [32]. The incubation system consisted of 100 mM potassium phosphate buffer, pH 7.6, 1 mM EDTA, oxidized glutathione (GSSG), and NADPH. The decrease in absorbance at 340 nm was monitored with 15 s interval for a total 5 min at 25 °C in a microplate reader. From the linear portion of the curve, the enzyme activity was calculated and expressed as nmol consumed NADPH/min per mg protein (mU GR/mg protein) using the molar extinction coefficient of NADPH at 340 nm (6.22 mM-1 cm-1). 2.14. Catalase (CAT) activity CAT activity was assayed by measuring the rate of decrease in H2O2 absorbance at 240 nm in accordance with Aebi (1984) protocol [33]. Enzyme incubation was performed in 10 mM phosphate buffer (pH 7.0) containing 25 mM H2O2 at 25 °C. The kinetics of H2O2 degradation was monitored with 30 s interval for a total 5 min at 240 nm in an UV/visible spectrophotometer. From the linear portion of the curve, the enzyme activity was calculated and expressed as U/mg protein (1 U CAT degrades of 1 μmol H2O2 per min at 25 °C) using the molar extinction coefficient of H2O2 at 240 nm (43.6M-1 cm-1). 2.15. Superoxide dismutase (SOD) activity Total SOD activity was assessed by means of the inhibition of superoxidedependent adrenaline auto-oxidation as previously described by Misra and Fridovich [34]. The adrenochrome absorbance was monitored with 30 s interval for 5 min at 480

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nm in an UV/visible microplate reader (Spectramax) set at 32 °C. Four protein amounts (from 5 to 30 μg) were tested per sample, and 1 U SOD activity was expressed as the amount of sample required for 50% inhibition of adrenochrome formation. Data were expressed as U SOD/mg protein. 2.16. Cell cultures MCF-7 cell line was from American Type Culture Collection (ATCC) (Rockville, Maryland, USA). The cells were maintained in DMEM containing 10% fetal bovine serum (Cripion Biotecnologia, Brazil) and 1x antibiotic:antimycotic solution (Sigma-Aldrich) at 37 °C in a humidified incubator. For cell culture treatments, NAC and LA were dissolved in sterile 50 mM PBS and pH adjusted to~7. 17-beta-estradiol was dissolved in DMSO. 2.17. Cell viability MCF-7 cells were plated in 96 well plates, treated for 72 h with NAC, LA or 17-betaestradiol, and incubated for 72 h. At the end of treatments, MTT reduction by cellular dehydrogenases was used as an estimative of cellular viability as previously described [35]. 2.18. Immunoblot Protein lysates were prepared using RIPA buffer containing 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM NaF, and protease inhibitors cocktail (Sigma-Aldrich). The proteins (50 μg) were resolved in SDS-PAGE, electro-transferred onto nitrocellulose membranes (Hybond-ECL, GE Healthcare), stained with Ponceau S, and then blocked with 5% BSA for 1 h at room temperature. Primary antibodies included the NRF2 (D1Z9C) from Cell Signaling Technologies for MCF-7 experiments (human) and NRF2 (C-20, sc-722) from Santa Cruz for detection of rat NRF2 protein; beta-actin (Ab8227) was from Abcam. Primary antibodies were incubated overnight at 4 o C (1:1000 dilution) in TBS-T buffer containing 5% BSA. After, secondary HRP-conjugated antibody was incubated at 1:2000 dilution in TBS-T for 2 h at room temperature, and the proteins were detected using 20x Lumiglo substrate (Cell Signaling Technology, CA). Images were captured in a ChemiDoc Image system (Biorad). 2.19. Statistical analysis Most of the results from animal studies are represented as Boxplots. Whiskers denote maximum and minimum values. Results with other graphs are expressed as average ± SD. All analyses were performed using GraphPad Prism (GraphPad Software Inc., San Diego, CA, USA). Statistical differences among groups were inferred by one-way or two-way ANOVA followed by post hoc Tukey's or Bonferroni test, respectively. Differences were considered statistically significant at a Pb.05.

3. Results 3.1. Short- and long-term changes associated with sexual hormone depletion by OVX in rats We initially sought to compare the impact of short- (15 days) versus long-term (60 days) sexual hormone depletion upon various menopause-related dysfunctions in OVX rat model. Serum estradiol levels and uterus weight significantly decreased as soon as 15 days post OVX (Fig. 1B and C). OVX almost doubled the rate of body weight (BW) gain as early as 15 days post OVX and this effect was maintained throughout the 60 days of experimental protocol if compared to Sham group (Fig. 1D). In contrast to these early-onset alterations, adipose fat accumulation (Fig. 1E), serum total cholesterol (Fig. 1F) and the inflammatory cytokines TNF-α and IL6 (Fig. 1G) have only increased in long-term OVX (60 days). Fasting glucose levels and triglycerides showed no difference among the groups and time-points (data not shown). Oxidative damage is another consequence of hormone depletion in OVX. Our data showed that oxidative stress markers increased with 60 days post-OVX whereas no changes were observed at the earlier time-point (15 days) (Fig. 1H-I). Lipoperoxidation (by means of TBARS quantification) increased in both serum and liver while protein carbonylation augmented only in the liver of long-term OVX rats (Fig. 1H-I). Short-term OVX also had no impact on lipoperoxidation and protein carbonylation in kidney and heart tissues (data not shown). Based on these findings, we decided to study more in-depth whether and how supplementation with NAC and LA impacts OVX-related metabolic, oxidative and inflammatory parameters in the long-term OVX model.

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3.2. Effects of NAC and LA supplementation upon oxidative damage markers in peripheral tissues of OVX rats. By assessing the levels of some classical markers of oxidative damage to biomolecules, we found that OVX induced oxidative damage mainly in liver and heart, but not kidney, tissues as evaluated by means of TBARS, protein carbonyl and total reduced thiol assays at the end of 60 days post-OVX (Fig. 2A-C). Lipoperoxidation increased in liver, heart and serum, while protein carbonyl augmented in the hepatic tissue but not others (Fig. 2A-B). OVX had none effect upon total reduced thiol content in the different tissues (Fig. 2C). NAC and LA supplementation at both doses decreased TBARS and protein carbonyl groups to levels comparable to sham-operated (Fig. 2A-C). Interestingly, 25 mg/kg NAC and both LA doses augmented reduced thiol content in the serum of OVX rats (Fig. 2C). Despite not investigated herein, this effect could be attributed to either an altered redox status of serum protein thiols or chemical reactivity of DTNB with free thiol groups of LA and NAC molecules present in the serum of NAC/LA-supplemented rats. 3.3. Long-term OVX depletes non-enzymatic antioxidants and affects the GPx/GR/GSH system We next set out to investigate some mechanisms involved in OVXinduced oxidative damage. Firstly, we used TRAP assays to estimate total non-enzymatic antioxidants present in the tissues. The results from TRAP assay showed that liver, heart and kidney of OVX rats were less capable of scavenging peroxyl radicals when compared to shamoperated animals (Fig. 3A-B). NAC and LA supplementation, mainly at the higher doses, restored non-enzymatic antioxidant potential to levels similar to sham-operated group (Fig. 3B). We then hypothesized that OVX might be affecting glutathione levels, which is a major nonenzymatic antioxidant within the cells. Indeed, while long-term OVX did not alter total glutathione levels (GSH + GSSG) in liver, kidney and heart tissues (Fig. 3C), GSH levels decreased (Fig. 3D), and this was counterbalanced by increased GSSG content (data not shown). These data indicate that GSH depletion in OVX rats is likely a consequence of GSH conversion to GSSG, and not to defective de novo GSH synthesis. Supplementing NAC at 25 mg/kg and LA at 25 and 50 mg/kg impeded GSH depletion induced by OVX (Fig. 3D). These OVX-elicited impairments in the GSH/GSSG system led us to test the effects of OVX and NAC/LA upon the main antioxidant enzymes - namely SOD, CAT and GPx - and the GPx pair GR (Glutathione Reductase). While OVX altered neither total SOD nor CAT activities (Fig. 4A-B), GPx decreased in liver, kidney and heart tissues (Fig. 4C). The GPx functional pair, GSSG Reductase (GR), activity was found downregulated in liver and heart, but not kidney, of OVX rats when compared to sham (Fig. 4D). NAC and LA treatment impeded GPx and GR downregulation by OVX, even though with varied dose efficacy across the different tissues (Fig. 4C and D). Taken together, these data indicate that sexual hormone depletion by OVX affects the GPx/GR/ GSH system as well as non-enzymatic antioxidant potential of peripheral tissues, which cumulate in oxidative damage to biomolecules. These changes can be restored/prevented by oral NAC and LA supplementation. 3.4. Effect of NAC and LA supplementation upon lipid profile, inflammatory cytokines and serum markers of tissue toxicity in longterm OVX. Besides attenuating oxidative stress, NAC and LA decreased serum levels of IL6 and TNF-α in OVX rats as determined by ELISA (Fig. 5A). With regard to metabolic markers, total cholesterol levels decreased in NAC- and LA-treated OVX rats, especially at the higher doses (Fig. 5B). Neither OVX nor antioxidants affected HDL, triglycerides or glucose

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Fig. 2. Effect of NAC and LA supplementation upon oxidative damage markers in peripheral tissues of OVX rats. (A) TBARS, (B) Protein carbonyl and (C) total sulfhydryl/R-SH groups in liver, heart, kidney and serum of OVX, OVX treated with differing doses of NAC and LA, and sham-operated rats at 60 days post-OVX. Legends: NAC 10 (NAC 10 mg/kg); NAC 25 (NAC 25 mg/kg); LA 25 (LA 25 mg/kg); LA 50 (LA 50 mg/kg). *denotes statistical difference compared to sham group; # represents the statistical difference between antioxidant-treated group and OVX group (One-way ANOVA, post-hoc Tukey; Pb.05).

levels (Fig. 5C-E). In addition, none evidence of hematological toxicity - such as alterations in leukocyte, platelets or red blood cells parameters -was observed in OVX or antioxidant supplemented groups (data not shown). Also, serum biochemical analysis showed no changes in ALT transaminase, alkaline phosphatase (ALP) (Fig. 5FG) and creatinine (data not shown), indicating absence of major systemic toxicity. Serum activity of Bone-specific ALP (BS-ALP) increased in long-term OVX, and this effect was abrogated by NAC and LA supplementation. Of note, BS-ALP is a serum marker associated with bone loss in OVX models in rodents, and is also useful for monitoring osteoporosis in postmenopausal women. These results altogether suggest that NAC and LA supplementation promoted a better metabolic profile and attenuated chronic low-level inflammation associated with long-term OVX. 3.5. NAC and LA effects are independent on NRF2 activation and estrogen-like activity NRF2 is an important transcription factor involved in antioxidant and xenobiotic responses. Once activated, NRF2 promotes transcription of GSH synthesis genes (GCLC and GCLM), ThioredoxinReductase-1 (TXNRD1), Thioredoxin (TXN) and other components of the detoxification machinery. We were expecting that NAC and LA antioxidant effects were associated with NRF2 activation. By immunoblot, NRF2 activation was detected in none of the tissues evaluated

(Fig. 6A). Our NRF2 immunoblots showed a readily detectable band with ~55–70 kDa. Even though previous studies have described this band as NRF2, two important studies [36,37] reported that this is an unspecific band, given that active NRF2 is expected to run with ~95–110 kDa in SDS/PAGE. We confirmed this evidence by running a positive control consisting of extracts of MDA-MB231 breast cancer cells treated with the alkylating agent MMS (methyl-methanesulphonate), which we previously reported as an inducer of NRF2 gene expression signature as well as Antioxidant Response Element (ARE) activation in reporter gene assays [38]. In fact, MMS induced an NRF2 band detectable with ~95–110 kDa (Fig. 6A, bottom gel, # labeled band). In other to test whether the effects of NAC and LA involve estrogen-like activity, we compared NAC/LA-elicited phenotypes with those of OVX rats treated with estrogen (E2). With regard to uterus, which is a critical estrogen target tissue, estrogen treatment avoided uterus atrophy induced by OVX, while NAC and LA were not capable of emulating this effect (Fig. 6B). In addition, NAC and LA supplementation had no effect upon serum levels of estrogen in OVX, whereas estrogen replacement enhanced serum levels of estrogen as expected (Fig. 6C). In addition, NAC and LA did not impede body weight gain in OVX rats while estrogen exerted preventive effects (Fig. 6D). Interestingly, retroperitoneal adipose tissue deposition induced by OVX was attenuated by both NAC/LA and estrogen treatments (Fig. 6E).

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Fig. 3. Effect of the antioxidant supplementation upon non-enzymatic antioxidant potential and glutathione levels in peripheral tissues of OVX rats. (A) Representative TRAP assay curves showing the peroxyl radical scavenging activity of liver tissues from sham, OVX, and OVX + NAC treated rats. Trolox (100 nM) positive control curve is also shown. Relative light units in each condition/treatment are expressed as % compared to luminescence of the AAPH system in the absence of samples (100% RLU). The smaller the area under curve the more antioxidant is the sample. (B) Peroxyl radical scavenging activity of liver, heart and kidney homogenates obtained from sham, OVX and OVX + NAC/LA-treated rats as evaluated by TRAP assay. Area under curve with each sample was converted to “% peroxyl radical scavenging”. (C) Total glutathione levels (GSH + GSSG) and (D) GSH levels in different tissues of sham-, OVX- and OVX + antioxidant-treated rats at 60 days post-OVX. Legends: NAC 10 (NAC 10 mg/kg); NAC 25 (NAC 25 mg/kg); LA 25 (LA 25 mg/kg); LA 50 (LA 50 mg/kg). *denotes statistical difference compared to sham group; # represents the statistical difference between antioxidant-treated group and OVX group (One-way ANOVA, post-hoc Tukey; Pb.05).

We also evaluated the estrogen-like effect of LA and NAC in the ER+ (estrogen receptor positive) breast cancer cell line, MCF-7. In the presence of estrogenic agents, these cells are expected to proliferate. Indeed, estrogen treatment promoted MCF-7 proliferation at low concentration (10 nM) whereas NAC and LA, even in the millimolar range, had no stimulatory effect upon MCF-7 cells proliferation as evaluated by MTT assay after 72 h treatment. In contrast, 5 mM LA caused cytotoxicity (Fig. 6F). We also evaluated the effect of NAC and LA upon NRF2 activation in MCF-7 cells in vitro. MCF-7 immunoblots showed a two-band pattern, with one band at~70 KDa which was considered as non-specific (ns) and a NRF2 band at~100 KDa. We found that LA, but not NAC, promoted NRF2 activation after 8 h treatment. This LA effect was not altered in the presence of estrogen or MMS treatments (Fig. 6G). 4. Discussion There is compelling evidence that antioxidant, metabolic and inflammatory homeostasis are impaired in postmenopausal women as well as in animal models of OVX. Alterations in these biological processes are shared with various aging-related conditions such as cardiovascular disease, osteoporosis, depression, diabetes and cancer amidst others which, in fact, increase incidence in postmenopausal [2,3,39,40]. Dealing with physiological changes and symptoms during menopause is a major challenge for women and, while HRT can provide benefits, there still are several concerns and misconceptions with regard to pros and cons of HRT [4,14–16]. In this context, we hypothesized that oxidative stress caused by sexual hormone depletion could play a role in the physiological changes associated with menopause, and supplementation with antioxidants could provide benefits. NAC and LA are described as antioxidants and have been shown as safe in clinical trials even at high doses [41–46]. N-acetylcysteine

(NAC) is a derivative of L-cysteine, which is rapidly absorbed via oral, though its oral bioavailability is low (~9–19%) [46–48]. In the cell, NAC forms cysteine which is ultimately involved in regulation of extracellular glutamate levels and maintenance of intracellular GSH pools. NAC has been used for decades in treatment of conditions such as acetaminophen overdose, and as a renal protectant in contrastinduced nephropathy [49,50]. LA, in turn, is a cofactor for the mitochondrial enzymes pyruvate dehydrogenase and αketoglutarate dehydrogenase, and plays an important role in Krebs cycle phase of the mitochondrial energy metabolism. LA also interacts both in the hydrophilic and hydrophobic portion of cell membranes, and has the ability of quenching superoxide, hydrogen peroxide and hydroxyl radicals as well as recycling non-enzymatic antioxidants such as GSH, vitamin E and ascorbic acid. LA also may acts as an antioxidant via activation of NRF2 [51–53]. In our model, NAC and LA supplementation ameliorated oxidative stress markers induced by long-term OVX. OVX-induced oxidative stress appears to be mild, given that the magnitude increase in damage markers was ~1.5–2-fold compared to sham group. Lipoperoxidation and depletion of GSH and other non-enzymatic defenses were the most consistent changes across the tissues studied; protein carbonylation and protein thiol oxidation showed minor alterations. OVX also impaired the GPx/GR enzymes with minor changes in SOD and CAT. NAC and LA supplementation prevented OVX-induced impairment in GPx/GR/GSH homeostasis, and improved total non-enzymatic potential in OVX rats. Moreover, neither OVX nor antioxidants altered total glutathione (GSH + GSSG) levels, indicating that NAC/LA improved GSH metabolism by restoring GPx/GR pair activity, but not via stimulation of de novo GSH biosynthesis. However, it is difficult to ascertain whether NAC and LA effects are attributed to either their direct scavenger activity upon oxidants or via modulation of antioxidant gene expression, or both. Our data did not show activation of NRF2 across the different tissues and treatments. However, it is

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Fig. 4. SOD, CAT, GPx and GR activities in peripheral tissues of OVX rats treated with NAC and LA. (A) Total SOD; (B) Catalase (CAT); (C) Glutathione Peroxidase (GPx) and (D) Glutathione Reductase (GR) activities in liver, kidney and heart tissues of sham-, OVX- and OVX + antioxidant-treated rats at 60 days post-OVX. Legends: NAC 10 (NAC 10 mg/kg); NAC 25 (NAC 25 mg/kg); LA 25 (LA 25 mg/kg); LA 50 (LA 50 mg/kg). *indicates statistical difference compared to sham group; # represents the statistical difference between antioxidant-treated group and OVX group (One-way ANOVA, post-hoc Tukey; Pb.05).

important to note that we have only evaluated NRF2 in the study endpoint (Day-60 and 24 h after the last NAC/LA dose), thus we cannot exclude that NAC and LA promoted a transient NRF2 activation missed in our protocol. The herein proposed mechanisms for NAC and LA in OVX-induced oxidative damage are presented in Fig. 7. It is also important to note that while NAC and LA restored GSH levels in OVX rats, total thiol content (which comprises both protein and nonprotein thiol residues) showed no differences among treatments. This may be explained by technical characteristics of the DTNB assay which does not comprise the acidification step required to prevent GSH oxidation, thereby limiting the detection of GSH – as well as by a lack of effect of NAC and LA upon protein thiol levels. With regard to the LA and NAC doses used herein, in Sprague–Dawley rats, an oral dose of 50 mg/kg LA achieved a Cmax and C120 min of 133 and 13 μM, respectively, and restored conduction velocity of the sciatic and digital nerves in a model of neuropathy [54]. At 20 mg/kg LA, Uchida et al. found a Cmax of ~9.7 μM [21]. In addition, patients taking 1200 mg of LA achieved serum Cmax and bioavailability comparable to that observed in mice receiving 50 mg/kg LA, which is a highly effective dose in experimental

autoimmune encephalomyelitis [22]. In patients, after an oral dose of 200 and 400 mg NAC (rat equivalent dose of ~20 and 40 mg/kg; based on [24]), the peak plasma concentration of 0.35 to 4 mg/L (~ 2 to 25 μM) is achieved within 1 to 2 h [55]. In rats, oral administration of radiolabeled NAC showed that NAC distributes to most of the tissues, with peak radioactivity and increased GSH levels occurring with 1 to 4 h [56]. Even though we were unable to measure tissue levels of NAC and LA in our model, the doses used herein seem to be within the pharmacological range for both compounds. There is compelling evidence for oxidative alterations in OVX murine models [4,8,9], although the results diverge among the models, especially with regard to antioxidant enzymes modulation. For instance, Hermoso et al. (2006) reported increased TBARS and carbonyl, and decreased levels of GSH and GPx without alterations in SOD and CAT in liver of Wistar rats at ~50 days post-OVX [8]. Rodriguez et al. (2013) demonstrated that OVX decreased GPx, GSH and Vitamin E levels, while SOD and CAT activities, and TBARS were not altered [9]. On the other hand, Schuller et al. (2018) showed lower levels of SOD1 and SOD2 in liver of OVX rats [4]. In other tissues, it has been demonstrated that OVX

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Fig. 5. Effect of antioxidant supplementation upon inflammatory, lipid profile and tissue damage markers in OVX rats. Serum levels of (A) TNF-α and IL6, (B) total cholesterol, (C) HDL, (D) Triglycerides, (E) glucose, (F) ALT, (G) ALP and (H) BS-ALP in sham-, OVX- and OVX + antioxidant-treated rats as evaluated at 60 days post-OVX. Serum was harvested from nonfasted rats. Legends: NAC 10 (NAC 10 mg/kg); NAC 25 (NAC 25 mg/kg); LA 25 (LA 25 mg/kg); LA 50 (LA 50 mg/kg). For comparison, the grid-lines within graphs indicate serum levels for each biomarker obtained from biochemical monitoring of our Wistar rat colony. *denotes statistical difference compared to sham group; # represents the statistical difference between antioxidant-treated group and OVX group (One-way ANOVA, post-hoc Tukey; Pb.05).

promotes lipoperoxidation/TBARS and H2O2 production in mouse bone, and NAC administration (100 mg/kg, i.p.) attenuated abnormal osteogenesis and bone turnover defects caused by OVX [57]. LA at 50 and 100 mg/kg also stimulated bone formation in the OVX model [18]. Although OVX-induced bone loss was not a primary endpoint of our study, NAC and LA prevented OVX-induced serum BS-ALP activity, which is in keeping with improved bone metabolism reported in the aforementioned studies. In brain cortex and liver, OVX enhanced lipoperoxidation and decreased GSH and GPx, CAT and SOD activities. Estrogen and combined estrogen/vitamin E attenuated these alterations [1]; an effect corroborated by Behling et al. [58]. Despite some differences with regard SOD and CAT modulations across the various studies and tissues evaluated, there is a clear evidende of downregulation of the GPx/GR/GSH system in OVX models. Taken together, these data suggest that lack of sexual hormones in postmenopausal contributes to systemic oxidative imbalance, which can confer tissue susceptibility to both physiological and environmental oxidants. In our model, body weight gain and uterus atrophy were early events that paralleled estrogen depletion whereas oxidative, inflam-

matory and cholesterol alterations occurred later. It has been postulated that activated estrogen receptors modulate body weight and energy consumption by multiple mechanisms, including regulation of fat metabolism, central nucleus of appetite and satiety, and adipocyte differentiation [59]. In our model, estrogen replacement prevented uterus atrophy and body weight gain whereas NAC and LA had no effects upon these parameters. NAC and LA had no impact upon estrogen levels, and showed none evidence of estrogen-like effects in the MCF-7 cell model. These results suggest that benefits associated with NAC and LA supplementation are not dictated by estrogen-like activity. In addition to body weight gain, various studies have reported an augmented serum total cholesterol, insulin resistance, adipose tissue deposition and inflammation in OVX models [1,60–63]. We only detected alterations in total cholesterol, but not HDL, indicating that LDL is likely high in OVX rats; serum triglycerides and blood glucose levels were not altered, agreeing with [60,62,63]. NAC and LA were capable of reducing OVX-induced total cholesterol, although we cannot confirm this metabolic effect is related to improved oxidative balance associated with NAC and LA supplementation.

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Fig. 6. NAC and LA effects are independent on NRF2 activation and estrogenic activity. (A) NRF2 immunoblots of liver, heart and kidney tissues from sham, OVX and OVX + antioxidanttreated rats. Three rats each group are shown for each tissue. In the bottom panel (liver tissue), an additional line consisting of 40 μg/mL MMS-treated MDA-MB231 cells extracts is also shown as a positive control for NRF2 activation. With the bottom panel, the arrow indicates the NRF2 expected molecular weight. (B) Uterus weight, (C) 17-beta-estradiol in serum, (D) delta body weight (BW) gain, and (E) retroperitoneal fat in sham, OVX and OVX + antioxidant-treated rats. Hormone reposition with 17-beta-estradiol/estrogen (E2 group) was used as a positive control for estrogenic activity. (F) Cell viability of MCF-7 cells after 72 h treatment with NAC, LA and estrogen (E2, 100 nM); (G) Immunoblot showing the effect of NAC and LA - alone or combined with MMS or E2 - upon NRF2 activation in MCF-7 cells treated for 8 h. LC: loading control. *denotes statistical difference compared to sham/control groups; # different from OVX group (One-way ANOVA, post-hoc Tukey; Pb.05).

It has been very well-established that adipose tissue promotes subclinical (low-level chronic) inflammation, which is correlated with breast cancer, heart disease and neurodegeneration risk [64]. Increased serum IL6 has been reported in obese postmenopausal women compared to age matched normal weight group [39,65]. Prior studies also correlated menopause and older age with increased serum IL6, TNF-α and IL1β in women [39,65]. In animal models, C57BL/6J OVX mice fed a high-fat diet showed increased expression of COX-2, TNF-α and IL1β in mammary adipose tissue, and supplementation with estrogen protected against body weight gain and mammary adipose tissue inflammation; ERα knockout in ovary intact mice emulated OVX effects [12]. In keeping with the role of adipose tissue as a source of inflammatory mediators, our model showed that OVX-induced body weight gain preceded, and retroperitoneal fat accumulation paralleled, the increase in TNF-α and IL6. It is important to note that cytokine levels were detected at a low ng/mL range, which are very low compared to more aggressive inflammatory

conditions; thus agreeing with the concept of subclinical inflammation. Attenuating OVX-induced oxidative imbalance with NAC and LA also improved inflammation markers in our model. Corroborating to our results, plasma TBARS levels increased, neutrophil GSH decreased, and inflammatory mediators augmented in post-menopausal compared to 30–49 years-old women; NAC supplementation (600 mg/day) for 2 and 4 months reversed these alterations [23]. In summary, our data suggest that NAC and LA improve oxidative stress, cholesterol and inflammatory components associated with sexual hormone depletion. However, it is difficult to establish how the oxidative imbalance elicited by OVX, adiposity/lipid changes and inflammation crosstalk in vivo. It has been reported that inflammation induces oxidative stress and, vice-versa, oxidative stress and adiposity promote inflammation. Despite these gaps of knowledge, our data and data from others [23] indicate a rational for testing antioxidants as a potential means to improve health in menopause.

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Fig. 7. Schematic representation and working model of NAC/LA effects in OVX rats. In this study, we found that OVX is associated with increased damage to biomolecules, which is likely attributed to either increased reactive species production – hypothetically generated by mitochondria or pro-oxidant enzymes such as xanthine oxidase (XOD) or NADPH oxidase (NOX) - or decreased non-enzymatic and enzymatic antioxidants, or both. In the tissues evaluated herein (liver, heart and kidney), OVX depleted non-enzymatic antioxidants such as GSH as well as decreased the GSH-dependent H2O2 detoxifying enzyme GPx, as well as the GSH recycling enzyme GR. On the other hand, SOD and CAT activities were not altered. NAC and LA supplementation prevented GPx/GR/GSH system impairments and impeded depletion of total non-enzymatic antioxidants thereby attenuating oxidative damage induced by OVX. Because NAC and LA also restored cholesterol and decreased inflammatory cytokines levels, we also hypothesize that pro-oxidant environments induced by OVX may play a role in metabolic alterations and chronic-low level inflammation associated with sexual hormone depletion (see working model).

Acknowledgements We acknowledge the Brazilian funding agencies CNPq and CAPES for providing PhD fellowship to RMG, Master's fellowship to MD and JPA, Scientific Initiation fellowship to VWS, and Principal Investigator Fellowship (CNPq Research Productivity II) to AZF. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. We also acknowledge LAMEBUFSC facility (Laboratório Multiusuário de Estudos em Biologia da UFSC), Dr. Luciana Honorato (CCB, UFSC) and Dr. Moreira's Lab (UFRGS, Brazil) for technical support. Declarations of interest None. References [1] Abbas AM, Elsamanoudy AZ. Effects of 17beta-estradiol and antioxidant administration on oxidative stress and insulin resistance in ovariectomized rats. Can J Physiol Pharmacol 2011;89:497–504. https://doi.org/10.1139/y11-053. [2] Chang CJ, Wu CH, Yao WJ, Yang YC, Wu JS, Lu FH. Relationships of age, menopause and central obesity on cardiovascular disease risk factors in Chinese women. Int J Obes Relat Metab Disord 2000;24:1699–704. [3] Uppoor RB, Rajesh A, Srinivasan MP, Unnikrishnan B, Holla R. Oxidative stress in obese postmenopausal women: an additive burden for atherosclerosis. J Clin Diagn Res 2015;9:OC03–5. https://doi.org/10.7860/JCDR/2015/16467.6868. [4] Schuller AK, Mena Canata DA, Hackenhaar FS, Engers VK, Heemann FM, Putti JS, et al. Effects of lipoic acid and n-3 long-chain polyunsaturated fatty acid on the liver ovariectomized rat model of menopause. Pharmacol Rep 2018;70:263–9. https://doi.org/10.1016/j.pharep.2017.10.006. [5] Siebert C, Kolling J, Scherer EBS, Schmitz F, da Cunha MJ, Mackedanz V, et al. Effect of physical exercise on changes in activities of creatine kinase, cytochrome c oxidase and ATP levels caused by ovariectomy. Metab Brain Dis 2014;29:825–35. https://doi.org/10.1007/s11011-014-9564-x. [6] Munoz-Castaneda JR, Muntane J, Herencia C, Munoz MC, Bujalance I, Montilla P, et al. Ovariectomy exacerbates oxidative stress and cardiopathy induced by adriamycin. Gynecol Endocrinol 2006;22:74–9. https://doi.org/10.1080/ 09513590500490249. [7] Muthusami S, Ramachandran I, Muthusamy B, Vasudevan G, Prabhu V, Subramaniam V, et al. Ovariectomy induces oxidative stress and impairs bone antioxidant system in adult rats. Clin Chim Acta 2005;360:81–6. https://doi.org/ 10.1016/j.cccn.2005.04.014.

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