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Differential menopause- versus aging-induced changes in oxidative stress and circadian rhythm gene markers
Mechanisms of Ageing and Development 164 (2017) 41–48
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Differential menopause- versus aging-induced changes in oxidative stress and circadian rhythm gene markers
MARK
Oriol A. Rangel-Zuñigaa,b,1, Cristina Cruz-Tenoa,b,1, Carmen Haroa,b, Gracia M. Quintana-Navarroa,b, Fernando Camara-Martosc, Pablo Perez-Martineza,b, Antonio Garcia-Riosa,b, Marta Garauletd, Manuel Tena-Sempereb,e, Jose Lopez-Mirandaa,b, ⁎ Francisco Perez-Jimeneza,b, Antonio Camargoa,b, a
Lipids and Atherosclerosis Unit, Maimonides Biomedical Research Institute of Cordoba (IMIBIC), Reina Sofia University Hospital, University of Cordoba, Spain CIBER Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos III, Cordoba, Spain Dpto de Bromatología y Tecnología de Alimentos, University of Cordoba, Cordoba, Spain d Department of Physiology, University of Murcia, Spain e Department of Cell Biology, Physiology, and Immunology, IMIBIC/Reina Sofia University Hospital/University of Cordoba, Cordoba, Spain b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Biology of aging Gender differences Genomics Oxidative stress Senescence
Menopause is characterized by the depletion of estrogen that has been proposed to cause oxidative stress. Circadian rhythm is an internal biological clock that controls physiological processes. It was analyzed the gene expression in peripheral blood mononuclear cells and the lipids and glucose levels in plasma of a subgroup of 17 pre-menopausal women, 19 men age-matched as control group for the pre-menopausal women, 20 postmenopausal women and 20 men age-matched as control group for the post-menopausal women; all groups were matched by body mass index. Our study showed a decrease in the expression of the oxidative stress-related gene GPX1, and an increase in the expression of SOD1 as consequence of menopause. In addition, we found that the circadian rhythm-related gene PER2 decreased as consequence of menopause. On the other hand, we observed a decrease in the expression of the oxidative stress-related gene GPX4 and an increase in the expression of CAT as a consequence of aging, independently of menopause. Our results suggest that the menopause-induced oxidative stress parallels a disruption in the circadian clock in women, and part of the differences in oxidative stress observed between pre- and post-menopausal women was due to aging, independent of menopause. Clinical Trials.gov.Identifier: NCT00924937
1. Introduction Aging is a complex process. Our current knowledge points towards oxidative stress as the main determinant in the deleterious and cumulative effects in the biology of aging (Sohal and Orr, 2012). In fact, aging increases the risk of cardiovascular diseases mainly by its association with increased oxidative stress and chronic low-grade inflammation (Herrera et al., 2009), which is also related with oxidative stress (Kabe et al., 2005). In women, menopause is a normal consequence of aging. As a major manifestation of female reproductive senescence, it is characterized by the permanent cessation of ovarian follicular activity, which produces an abrupt fall in estrogen levels, leading to the classic signs and symptoms of menopause, as well as an
increased risk of cardiovascular diseases and osteoporosis (Rao et al., 2013; Greendale et al., 1999). Oxidative stress has been implicated in various pathologies such as vasomotor disturbances, osteoporosis and cardiovascular diseases, which significantly correlate with the progressive loss of estrogen and its protective effects, combined with a deficient antioxidant defense which leads to a pronounced redox imbalance. In fact, it has been proposed that the depletion of estrogen in post-menopause could cause oxidative stress, in addition to the known symptoms (Rao et al., 2013; Greendale et al., 1999; Doshi and Agarwal, 2013). In addition, the incidence of metabolic diseases and their co-morbidities, which are sexually dimorphic, increases after menopause in women, at which time sex hormones are thought to play an important role in the development
⁎ Corresponding author at: Lipids and Atherosclerosis Research Unit, GC9 Nutrigenomics, IMIBIC/Reina Sofia University Hospital/University of Cordoba, Av. Menendez Pidal, s/n, 14004 Cordoba, Spain. E-mail address: [email protected] (A. Camargo). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.mad.2017.04.002 Received 25 July 2016; Received in revised form 17 March 2017; Accepted 1 April 2017 Available online 10 April 2017 0047-6374/ © 2017 Elsevier B.V. All rights reserved.
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determined in frozen samples were analyzed centrally by laboratory investigators of the Lipid and Atherosclerosis Unit at the Reina Sofia University Hospital, who were unaware of the interventions. Lipid variables were assessed with a DDPPII Hitachi modular analyzer (Roche) using specific reagents (Boehringer-Mannheim). Plasma triglycerides (TG) and cholesterol concentrations were assayed by enzymatic procedures (Allain et al., 1974; Paisley et al., 1996). High-density lipoprotein-cholesterol (HDL-c) was measured by precipitation of a plasma aliquot with dextran sulphate-Mg2+, as described by Warnick et al. (Warnick et al., 1982). Low-density lipoprotein-cholesterol (LDLc) was calculated by using the following formula: plasma cholesterol − [HDL-C + large TG-rich lipoproteins (TRL-C) + small TRL-C]. Plasma glucose concentrations were measured by using the IL Test Glucose Hexokinase Clinical Chemistry kit (Instrumentation Laboratories, Warrington, United Kingdom).
of cardiovascular disease (Arnlov et al., 2006; Bhupathy et al., 2010; Luczak and Leinwand, 2009). Moreover, it has been reported that postmenopausal women have higher lipoperoxide (Sanchez-Rodriguez et al., 2012), pro-inflammatory cytokines (Vural et al., 2006), and pro-oxidant biomarkers such as malonaldehyde, 4-hydroxynenal, and oxidized LDL (Signorelli et al., 2006) levels than pre-menopausal women, whereas the levels of antioxidant glutathione peroxidase (GPX) are lower in post-menopausal women (Signorelli et al., 2006). Nevertheless, hormone replacement therapies (HRT) have failed to prevent the post-menopausal increase in oxidative stress (Sekhon and Agarwal, 2013). Most of the studies focusing on the physiological changes caused by menopause compare only pre- and post-menopausal women, with an age difference of several years (post-menopausal groups are usually on average at least 10 years older than pre-menopausal groups), without age-matched groups of men to control the potential aging-induced, menopause-independent differences between pre- and post-menopausal women. To fill this gap, the objective of this study was to performer a series of comparative expression analyses in groups of pre- and postmenopausal women, and their corresponding age-matched male control groups. All 4 groups were matched by body mass index (BMI), and in them we analyzed the expression of gene markers of cardiovascular risk-related processes, including endoplasmic reticulum stress, inflammation, oxidative stress, metabolism, and circadian rhythms, in peripheral blood mononuclear cells (PBMC). This sub-set of white blood cells modifies its gene expression profile in response to stimuli and has proved to be useful in distinguishing a disease from a healthy state (Burczynski and Dorner, 2006; Camargo et al., 2014).
2.3. Sex hormone determination Testosterone was determined by the commercial kit: Testosterone Assay (R & D System; Cat. No. KGE010), according to the manufacturer's instructions. Estradiol was determined by the commercial kit: Estradiol EIA kit (Cayman Chemical; Cat. No. 582251), according to the manufacturer instructions. Follicle-Stimulating Hormone was determined by the commercial kit: FSH ELISA (DRG Instruments GmbH; Cat. No. EIA-1288), according to the manufacturer's instructions. 2.4. Isolation of peripheral blood mononuclear cells and RNA extraction Buffy coats were diluted 1:2 in phosphate saline buffer (PBS), and cells were separated in 15 mL Ficoll gradient (lymphocyte isolation solution, Axis-Shield, Oslo, Norway) by centrifugation at 2000 × g for 25 min. PBMC were collected, washed twice with cold PBS and stored in RNA Later Solution (Ambion, Thermo Fisher Scientific, MA, USA). Total RNA was extracted using Tri Reagent (Sigma, St Louis, MO, USA), according to the manufacturers’ instructions. The recovered RNA was quantified using a Nanodrop ND-1000 v3.5.2 spectrophotometer (Nanodrop Technology®, Cambridge, UK), and its integrity was checked on agarose gel electrophoresis and stored at −80 °C. RNA samples were digested with DNAse I (AMPD-1 Kit, Sigma) before RT-PCR.
2. Methods 2.1. Study subjects The current work was conducted in a subgroup of patients as part of the CORDIOPREV study (Clinical Trials.gov.Identifier: NCT00924937), an ongoing prospective, randomized, opened, controlled trial in patients with coronary heart disease (CHD), who had their last coronary event over six months before enrolling in two different dietary models (Mediterranean and Low-fat) over a period of five years, in addition to conventional treatment for CHD (Alcala-Diaz et al., 2014). All the patients gave written informed consent to participate in the study. The trial protocol and all amendments were approved by the local ethics committees, following the Helsinki declaration and good clinical practice. We analyzed a subgroup of 76 patients from the control healthy group included in the CORDIOPREV study (without oncological or cardiovascular diseases): 17 pre-menopausal women (E2 = 109.401 ± 41.46 pg/ mL, FSH = 13.47 ± 2.90 mIU/mL), 19 men matched by age as a control group for the pre-menopausal women (T = 108 ± 14 pg/mL), 20 postmenopausal women (E2 = 11.49 ± 4.47 pg/mL, FSH = 93.54 ± 7.63 mIU/mL) and 20 men (T = 92 ± 9 pg/mL) matched by age as a control group for the post-menopausal women: all 4 groups were matched by BMI. Patient's inclusion was assessed by medical history, biochemical measures and physical examination by clinicians. Exclusion criteria included cardiovascular disease, cancer, and chronic diseases and did not have severe diseases or an estimated life expectancy of less than 5 years. We also performed a diet assessment with a validated 14-item questionnaire (Schroder et al., 2011) to assess the dietary habits of the groups.
2.5. qRT-PCR for gene expression analysis Retrotranscription reaction was performed with 500 ng of total RNA using the Ambion WT Expression Kit (Applied Biosystems, Carlsbad, CA, USA), following the manufacturers’ instructions. Real-time PCR reactions were carried out using the OpenArray™ NT Cycler system (Applied Biosystems, Carlsbad, CA, USA), according to the manufacturers’ instructions. Primer pairs for 53 target genes related to endoplasmic reticulum stress, inflammation, oxidative stress (prooxidants and antioxidants), metabolism and circadian rhythms were selected from the TaqMan Gene Expression assays database (Applied Biosystems, Carlsbad, CA, USA) (Appendix 1 Supplemental Table 1). We used as housekeeping genes: beta-2-microglobulin, glyceraldehyde3-phosphate dehydrogenase and hypoxanthine phosphoribosyltransferase, for which Ct values were combined by the software Bestkeeper (Pfaffl et al., 2004) in order to get a more stable reference value than each one independently. Gene expression values were obtained as a relative expression of the target gene versus the Bestkeeper value (relative expression = 2−(Ct, Targe gene−Ct, Bestkeeper value)). The data set was extracted by using the OpenArray® Real-Time qPCR Analysis Software (Applied Biosystems, Carlsbad, CA, USA).
2.2. Clinical plasma parameters The patients arrived at the clinical centre at 08:00 h. We measured anthropometric (weight, height, waist circumference, BMI and blood pressure) and took a fasting blood sample. Blood was collected in tubes containing ethylenediaminetetraacetic acid (EDTA) to give a final concentration of 0.1% EDTA. The plasma was separated from the red blood cells by centrifugation at 1500 × g for 15 min at 4 °C. Analytes
2.6. Statistical analysis PASW statistical software, version 20.0 (IBM Inc., Chicago, IL, USA) was used for statistical analysis of the data. The normal distribution of 42
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between both groups of women (ANOVA P < 0.001; FDR Q < 0.001). The post hoc Bonferroni's corrected multiple comparison test showed that the expression of PER2 was lower in the post-menopausal group of women than in pre-menopausal group (P = 0.005). In contrast, we did not find any differences in expression between the male-post and malepre groups. In addition, the expression of PER2 was higher in the premenopausal women group than in the corresponding age-matched male-pre group (P = 0.016).
variables was assessed using the Kolmogorov–Smirnov test. When the data distribution did not adjust to the normal values, the values were log10 transformed (XBP1, IL6, MCP1, IL1B, TNF-a, UCP2, NFKB p65, NFKBIA, IKBKA, p91phox, p47phox, p40phox, SOD1, SOD2, GPX1, GPX4, GSR, PU.1, PPARG, MAPK8, MAPK14, RRM2, CDKN1A, MDM2, APEX1, DDB2, GADD45A, GADD45B, IL8, CXCL1, MMP9, MIF, CLOCK, BMAL1) or 1/x transformed (CALR, BiP, IKBKB, p67phox, TXN, TXNRD1, NFE2L2, TP53, OGG1) to achieve this. The statistical differences between the groups were evaluated by One-way ANOVA. Multiple comparisons in the large-scale expression analyses were assessed by False Discovery Rate (FDR) using the Benjamini and Hochberg method. Post hoc statistical analysis was completed by using Bonferroni's comparison tests. A P-value < 0.05 was considered statistically significant.
3.3. Aging-dependent menopause-independent differences in the gene expression We observed differences as function of aging, independently of menopause, in the expression of the oxidative stress-related genes CAT and GPX4 (Fig. 2) and the DNA repair-related genes XPC and POLB (Fig. 3). Whereas no statistically significant differences were found between pre-and post-menopausal women, nor between the corresponding malepre and male-post groups in the expression of CAT, when we merged the pre-menopausal group of women and their corresponding agematched men (mean age 46 y) and post-menopausal women group with their corresponding age-matched men (mean age 56 y), we observed a higher expression in the CAT gene in the 56 y group as compared with the 46 y group (ANOVA P = 0.008; FDR Q = 0.051). Moreover, we found that the expression of GPX4 was lower in post-menopausal women than in the pre-menopausal group (P = 0.005), but we also observed the same profile between the corresponding male-pre and male-post groups (P = 0.018). In addition, we found that the expression of XPC and POLB genes was lower in post-menopausal women than in the pre-menopausal group (P = 0.002 and P < 0.001, respectively), but we also observed the same profile between the corresponding malepre and male-post groups (statistical trend P = 0.073, and P = 0.004, respectively). When we merged pre-menopausal women group and their corresponding age-matched men (mean age 46 y) and postmenopausal women group and their corresponding age-matched men (mean age 56 y), we observed a lower expression of GPX4 (ANOVA P < 0.001; FDR Q < 0.001) and XPC and POLB genes (both, ANOVA P < 0.001; FDR Q < 0.001) in the 56 y group than in the 46 y group, which is a function of aging and independent of menopause.
3. Results 3.1. Baseline characteristic of the study participants No statistically significant differences were found in BMI, waist circumference, LDL-c, TG, Insulin, HbA1C or systolic blood pressure between groups. Pre-menopausal women had higher HDL-c and lower glucose levels (P < 0.05). The pre-menopausal women group and their corresponding age-matched men (male-pre) were younger than postmenopausal women and their age-matched male group counterpart (male-post; P < 0.05) (Table 1). Importantly, no differences were found in the dietary habits between groups that might affect the expression of the genes under analysis (Appendix 2 Supplemental Table 2). 3.2. Menopause-dependent differences in the gene expression In order to assess the impact of aging and menopause on cardiovascular risk, we analyzed the expression of genes related to endoplasmic reticulum stress, inflammation, oxidative stress, metabolism and circadian rhythms in PBMC. (Appendix 1 Supplemental Table 1; only differentially expressed genes are described in the text). We found significant differences in the expression of several genes related with oxidative stress and circadian rhythms in PBMC that were not present in the age-matched groups of men (Fig. 1). Our study also showed differences in the expression of the antioxidant enzyme genes SOD1 and GPX1 between groups (ANOVA, both P = 0.008; FDR, both Q = 0.058). In fact, the post hoc Bonferroni's corrected multiple comparison test showed that the expression of SOD1 gene was higher (P = 0.041) and the expression of GPX1 was lower (P = 0.085, statistical trend) in post-menopausal women than in the pre-menopausal group. Moreover, we observed that the expression of the circadian rhythm-related gene PER2 showed a statistically significant difference
4. Discussion Most of the studies on the impact of menopause compare pre- and post-menopausal women. However, the lack of corresponding studies of age-matched groups of men hampers the identification of agingdependent, menopause-independent changes. Therefore, our experimental design included pre- and post-menopausal women and their agematched male counterparts, which allowed us to distinguish between
Table 1 Metabolic characteristic of the participants in the study. Values correspond to the mean ± SEM. The statistical differences between groups were evaluated by One-way ANOVA. BMI, body mass index; TG, triacylglyceride; HDL-c, high density lipoprotein-cholesterol; LDL-c, low density lipoprotein-cholesterol; HbA1C, glycated hemoglobin; BP, blood pressure. In each row, values with different letters in superscript differs statistically in the Bonferroni's corrected post hoc multiple comparison test (P < 0.05).
Age (years) BMI (kg/m2) Waist circumference (cm) HDL-c (mg/dL) LDL-c (mg/dL) TG (mg/dL) Glucose (mg/dL) Insulin (mU/L) HbA1C (%) Systolic BP (mm Hg) Diastolic BP (mm Hg)
Pre-menopausal women (N = 17)
Men control group pre (N = 19)
Post-menopausal women (N = 20)
Men control group post (N = 20)
P-value
46.12 ± 0.82a 26.31 ± 1.52 88.94 ± 3.15 57.00 ± 3.37a 118.53 ± 6.97 88.88 ± 9.97 87.00 ± 2.75a 5.39 ± 0.79 5.11 ± 0.11 127.96 ± 6.30 77.89 ± 2.43
46.58 ± 0.61a 27.10 ± 0.91 95.68 ± 2.24 41.79 ± 2.14b 132.42 ± 5.97 115.95 ± 14.92 98.21 ± 5.48a,b 6.37 ± 0.90 5.32 ± 0.17 129.53 ± 3.85 81.62 ± 2.54
55.85 ± 0.66b 28.94 ± 1.25 92.65 ± 3.00 54.50 ± 2.82a,c 137.40 ± 7.13 102.95 ± 12.76 92.05 ± 2.38a 6.87 ± 0.92 5.54 ± 0.08 132.83 ± 3.66 77.35 ± 2.39
56.20 ± 0.72b 28.53 ± 1.13 99.30 ± 2.88 45.40 ± 1.92b,c 145.65 ± 7.21 107.95 ± 13.24 108.40 ± 4.29b 8.08 ± 0.99 5.55 ± 0.13 136.18 ± 2.86 85.50 ± 1.56
< 0.001 0.393 0.079 < 0.001 0.057 0.545 0.002 0.233 0.063 0.518 0.041
43
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Fig. 1. Menopause and expression of oxidative stress- and circadian rhythm-related genes. Values represent mean ± SEM of the relative expression of the indicated gene. One-way ANOVA statistical analysis P-value. Q-value, False Discovery Rate. *P < 0.05 and #P < 0.1 in the post hoc Bonferroni's multiple comparison tests. Experimental groups: pre-menopausal women; male-pre: age-matched men as a control group for the pre-menopausal women; post-menopausal women; male-post: age-matched men as a control group for the post-menopausal women. All the four groups are matched by BMI. SOD1, superoxide dismutase 1. GPX1, glutathione peroxidase 1. PER2, period homolog 2.
molecular oxygen, are also involved in ROS production (Inoguchi and Nawata, 2005). In response to oxidative stress, PBMC reinforce their antioxidant defences by increasing the expression of antioxidant genes (Camargo et al., 2014). SOD gene expression is induced by %O2− and converts it into H2O2, which is, in turn, converted into water or molecular oxygen by either CAT or GPx. The latter is also involved in the detoxification of lipid hydro-peroxides generated by the action of % O2− (Faraci and Didion, 2004; Rahman et al., 2005). In this context, a higher expression of SOD1 is consistent with an alteration in the ROS detoxification pathway, and depending to the GPx activity, presumably low in post-menopausal women as suggested by the expression levels of GPX1. It may generate a high oxidative stress condition in postmenopausal women (with a higher expression of SOD1 compared with pre-menopausal women). In fact, our results are in line with previous studies in which an increased oxidative stress after menopause was observed (Zitnanova et al., 2011). Moreover, whereas SOD activity has been shown to be reduced with aging in certain tissues (Doonan et al., 2008), our study in PBMC showed that SOD1 gene expression did not decreased with aging in men and was higher in post-menopausal women and no changes were observed in SOD2 gene expression,
menopause-dependent and aging-dependent changes. Furthermore, we performed our large scale gene expression analyses in BMI-matched experimental groups with a similar nutritional background, as both BMI and diet may alter the expression of the genes studied. Our study showed that as consequence of menopause, there is a decrease in the expression of the oxidative stress-related gene GPX1, and an increase in the gene expression of SOD1 in PBMC. In addition, we also found that the circadian rhythm-related gene PER2 decreased as a consequence of menopause. On the other hand, as a consequence of aging (independently of menopause), we observed a decrease in the expression of oxidative stress-related genes such as GPX4 and an increase in the expression of CAT. The changes found in DNA repair-related genes were also specific to aging, and we also observed a decrease in the expression of the XPC and POLB. Oxidative stress occurs when there is an imbalance between the production of reactive oxygen species (ROS) and their elimination by the antioxidant system (Durackova, 2010). ROS are generated byproducts of metabolism, but several enzymes, including NADPHoxidase, an enzyme which transfers an electron from NADPH to 44
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Fig. 2. Aging and expression of oxidative stress-related genes. Values represent mean ± SEM of the relative expression of the indicated gene. One-way ANOVA statistical analysis Pvalue. Q-value, False Discovery Rate. *P < 0.05 and #P < 0.1 in the post hoc Bonferroni's multiple comparison tests. Experimental groups: pre-menopausal women; male-pre: agematched men as a control group for the pre-menopausal women; post-menopausal women; male-post: age-matched men as a control group for the post-menopausal women. All the four groups are matched by BMI. Right panel: Comparison between age groups resulting from merging the group of pre-menopausal women with their age-matched male counterparts (mean age 46 y) and the group of post-menopausal women with their age-matched men male counterparts (mean age 56 y). CAT, catalase. GPX4, glutathione peroxidase 4.
Extensive observations suggest that DNA damage accumulates during aging, as a consequence of an increase in oxidative stress and a decline in DNA repair capacity (Chen et al., 2007). Repair of oxidative DNA damage involves 3 mechanisms: base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (Izumi et al., 2003; Lee et al., 2007). Thus, a lower expression of POLB and XPC (belonging to the BER and NER pathways, respectively) in post-menopausal women and their age-matched male control group is consistent with an aginginduced alteration in DNA repair capacity. All together, these results may explain why hormone replacement therapies have failed to substantially improve the redox status in postmenopausal women, as these therapies cannot counteract the aginginduced increase in oxidative stress. In fact, several studies have documented how hormone replacement therapies only partially restore the antioxidant machinery (Unfer et al., 2006), total antioxidant status (Leal et al., 2000), and nitric oxide (NO) levels (Bednarek-Tupikowska et al., 2008; Kurtay et al., 2006).
suggesting a high complexity of the regulatory mechanism controlling this step of ROS detoxification in post-menopausal women. However, our study showed that at least part of the increase in oxidative stress observed in post-menopausal women is a consequence of aging itself, and is not caused primarily by ovarian senescence. In fact, post-menopausal women and their age-paired group of men displayed higher CAT gene expression than pre-menopausal women and their age-matched men, suggesting that changes in the latter are aging dependent, and independent of menopause and gender. Likewise, the lower expression of GPX isoforms suggests a decrease in the detoxification of hydrogen peroxide in post-menopausal women (GPX1 gene), whereas a reduction of lipid hydroperoxides seems to be a consequence of aging, as suggested by the reduction of GPX4 expression independently of menopause and gender. In addition, no differences in terms of genetic markers of oxidative DNA damage repair were found between pre- and post-menopausal women, as significant differences in these markers are specific to aging. 45
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Fig. 3. Aging and expression of DNA repair-related genes. Values represent mean ± SEM of the relative expression of the indicated gene. One-way ANOVA statistical analysis P-value. Qvalue, False Discovery Rate. *P < 0.05 and #P < 0.1 in the post hoc Bonferroni’s multiple comparison tests. Experimental groups: pre-menopausal women; age-matched men as a control group for the pre-menopausal women; post-menopausal women; age-matched men as a control group for the post-menopausal women. All the four groups are matched by BMI. Right panel: Comparison between age groups resulting from merging the group of pre-menopausal women with their age-matched male counterparts (mean age 46 y) and the group of post-menopausal women with their age-matched male counterparts (mean age 56 y). XPC, xeroderma pigmentosum, complementation group C. POLB, polymerase (DNA directed), beta.
we observed that the expression of PER2 decreased with menopause in PBMC, which suggests that changes in the profile of the expression of clock genes induced by menopause may be tissue-specific. Moreover, with menopause, women exhibit a loss of circadian robustness and an increase in sleep abnormalities – a process that runs parallel with the redistribution of adipose tissue (Gómez-Santos et al., 2016). In turn, the hormonal changes undergone during menopause have been related with an alteration in the circadian rhythms (Joffe et al., 2010), which suggests that the differences observed in PER2 expression between premenopausal women and age-matched men, but not between postmenopausal women and age-paired men, may contribute to the sex differences in the circadian timing system that disappear after menopause (Roenneberg et al., 2007) and phase-advanced endogenous temperature and melatonin rhythms (Cain et al., 2010). This has partly been attributed to a significantly shorter circadian period in women (Duffy et al., 2011). In addition, it has been described that endogenous secretion of melatonin decreases with aging across genders, and, among women, menopause is associated with a significant reduction of
Our study also showed that menopause-induced oxidative stress might be related with a disruption in the circadian rhythms in women. In fact, the processes of oxidative stress and circadian rhythms are intricately involved in a myriad of physiological processes to maintain homeostasis, from blood pressure and sleeping/waking cycles, down to cellular signaling pathways that play critical roles in health and disease (Wilking et al., 2013). When genes regulating circadian functions lose some of their precise orchestration with aging, this leads to impaired homeostasis (Gibson et al., 2009), a situation which is particularly marked in women as they move towards menopause (Chedraui et al., 2010). In fact, menopause changes the profile of the expression of clock genes in adipose tissue (Hernandez-Morante et al., 2011), which may be related with body fat redistribution after menopause, which in turn is characterized by an increase in intra-abdominal visceral fat (Garaulet et al., 2006). In addition, whereas the expression of PER2 does not differ between pre- and post-menopausal women in subcutaneous adipose tissue, it is higher in postmenopausal women in visceral adipose tissue (Hernandez-Morante et al., 2011). In the current study, 46
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melatonin levels, which therefore may also explain the well-known sleep disturbances associated with menopause (Pines, 2016). Our study has limitations. One of the limitations of our study is the reduced sample size of our pilot study, which improves the experimental designs of most of the studies focusing the changes associated to menopause as we included age-matched groups of men to control the potential aging-induced, menopause-independent differences between pre- and post-menopausal women. Another limitation lies in the fact that we performed a genomic approach, as we performed a series of comparative expression analyses in groups of pre- and post-menopausal women, and their corresponding age-matched male control groups. Further studies providing protein data may shed more light in the specific cellular and molecular mechanisms involved in the menopausedependent and aging-dependent menopause-independent differences in the gene expression showed in this study.
informed consent to participate in the study. The trial protocol and all amendments were approved by the local ethics committees, following the Helsinki declaration and good clinical practice.
5. Conclusion
References
Our results suggest that the menopause-induced oxidative stress parallels a disruption in the circadian clock in women, and some of the differences in oxidative stress observed between pre- and post-menopausal women were a consequence of aging and independent of menopause. Moreover, aging per se associates with changes in DNA repair-related genes.
Alcala-Diaz, J.F., et al., 2014. Hypertriglyceridemia influences the degree of postprandial lipemic response in patients with metabolic syndrome and coronary artery disease: from the cordioprev study. PLOS ONE 9 (5), e96297. Allain, C.C., et al., 1974. Enzymatic determination of total serum cholesterol. Clin. Chem. 20 (4), 470–475. Arnlov, J., et al., 2006. Endogenous sex hormones and cardiovascular disease incidence in men. Ann. Intern. Med. 145 (3), 176–184. Bednarek-Tupikowska, G., Tworowska-Bardzinska, U., Tupikowski, K., 2008. Effects of estrogen and estrogen-progesteron on serum nitric oxide metabolite concentrations in post-menopausal women. J. Endocrinol. Invest. 31 (10), 877–881. Bhupathy, P., Haines, C.D., Leinwand, L.A., 2010. Influence of sex hormones and phytoestrogens on heart disease in men and women. Womens Health (Lond. Engl.) 6 (1), 77–95. Burczynski, M.E., Dorner, A.J., 2006. Transcriptional profiling of peripheral blood cells in clinical pharmacogenomic studies. Pharmacogenomics 7 (2), 187–202. Cain, S.W., et al., 2010. Sex differences in phase angle of entrainment and melatonin amplitude in humans. J. Biol. Rhythms 25 (4), 288–296. Camargo, A., et al., 2014. Peripheral blood mononuclear cells as in vivo model for dietary intervention induced systemic oxidative stress. Food Chem. Toxicol. 72, 178–186. Chedraui, P., et al., 2010. Factors related to increased daytime sleepiness during the menopausal transition as evaluated by the Epworth sleepiness scale. Maturitas 65 (1), 75–80. Chen, J.H., Hales, C.N., Ozanne, S.E., 2007. DNA damage, cellular senescence and organismal ageing: causal or correlative? Nucleic Acids Res. 35 (22), 7417–7428. Doonan, R., et al., 2008. Against the oxidative damage theory of aging: superoxide dismutases protect against oxidative stress but have little or no effect on life span in Caenorhabditis elegans. Genes Dev. 22 (23), 3236–3241. Doshi, S.B., Agarwal, A., 2013. The role of oxidative stress in menopause. J. Midlife Health 4 (3), 140–146. Duffy, J.F., et al., 2011. Sex difference in the near-24-hour intrinsic period of the human circadian timing system. Proc. Natl. Acad. Sci. U. S. A. 108 (Suppl. 3), 15602–15608. Durackova, Z., 2010. Some current insights into oxidative stress. Physiol. Res. 59 (4), 459–469. Faraci, F.M., Didion, S.P., 2004. Vascular protection: superoxide dismutase isoforms in the vessel wall. Arterioscler. Thromb. Vasc. Biol. 24 (8), 1367–1373. Garaulet, M., et al., 2006. Anthropometric indexes for visceral fat estimation in overweight/obese women attending to age and menopausal status. J. Physiol. Biochem. 62 (4), 245–252. Gibson, E.M., Williams III, W.P., Kriegsfeld, L.J., 2009. Aging in the circadian system: considerations for health, disease prevention and longevity. Exp. Gerontol. 44 (1–2), 51–56. Gómez-Santos, C., Saura, C.B., Lucas, J.A., Castell, P., Madrid, J.A., Garaulet, M., et al., 2016. Menopausal status is associated with circadian- and sleep-related alterations. Menopause 23 (June (6)), 682–690. http://dx.doi.org/10.1097/GME. 0000000000000612. Greendale, G.A., Lee, N.P., Arriola, E.R., 1999. The menopause. Lancet 353 (9152), 571–580. Hernandez-Morante, J.J., et al., 2011. Influence of menopause on adipose tissue clock gene genotype and its relationship with metabolic syndrome in morbidly obese women. Age (Dordr) 34 (6), 1369–1380. Herrera, M.D., et al., 2009. Endothelial dysfunction and aging: an update. Ageing Res. Rev. 9 (2), 142–152. Inoguchi, T., Nawata, H., 2005. NAD(P)H oxidase activation: a potential target mechanism for diabetic vascular complications, progressive beta-cell dysfunction and metabolic syndrome. Curr. Drug Targets 6 (4), 495–501. Izumi, T., et al., 2003. Mammalian DNA base excision repair proteins: their interactions and role in repair of oxidative DNA damage. Toxicology 193 (1–2), 43–65. Joffe, H., Massler, A., Sharkey, K.M., 2010. Evaluation and management of sleep disturbance during the menopause transition. Semin. Reprod. Med. 28 (5), 404–421. Kabe, Y., et al., 2005. Redox regulation of NF-kappaB activation: distinct redox regulation between the cytoplasm and the nucleus. Antioxid. Redox Signal. 7 (3–4), 395–403. Kurtay, G., Ozmen, B., Erguder, I., 2006. A comparison of effects of sequential
Acknowledgements We would like to thank the EASP (Escuela Andaluza de Salud Publica), Granada, Spain, which performed the randomization process for this study. We would also like to thank José Andrés Morales Martínez for lending technical support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.mad.2017.04.002.
Funding The CIBEROBN is an initiative of the Instituto de Salud Carlos III (ISCIII), Madrid, Spain. The CORDIOPREV study is supported by the Fundación Patrimonio Comunal Olivarero, Junta de Andalucía (Consejería de Salud, Consejería de Agricultura y Pesca, Consejería de Innovación, Ciencia y Empresa), Diputaciones de Jaen y Cordoba, Centro de Excelencia en Investigación sobre Aceite de Oliva y Salud and Ministerio de Medio Ambiente, Medio Rural y Marino, Gobierno de España. It was also partly supported by research grants from the Ministerio de Ciencia e Innovación (AGL2009-122270 to J L-M); Ministerio de Economia y Competitividad (AGL2012/39615, PIE14/ 00005, PIE 14/00031 to J L-M, SAF2014-52480-R to MG, FIS PI13/ 00185 to P P-M, and PI13/00619 to F P-J); Consejería de Economía, Innovación y Ciencia, Proyectos de Investigación de Excelencia, Junta de Andalucía (AGR922 to F P-J); Consejería de Salud, Junta de Andalucía (PI0193/09 to J L-M and PI-0058/10 to P P-M; Consejería de Innovación Ciencia y Empresa (CVI-7450 to J L-M); Fondo Europeo de Desarrollo Regional (FEDER). Antonio Camargo is supported by an ISCIII research contract (Programa Miguel-Servet CP14/00114). Authors’ contributions A.C., J.L-M., P.P-M and F.P-J. contributed to the study conception and design. P.P-M., F.P-J and J.L-M obtained funding. C.C-T., OA.R-Z., C.H., GM. Q-N., F.C-M and A.G-R contributed to acquisition of data. OA.R-Z., C.C-T., M.G., M.T-S and A.C analyzed and interpreted the data. OA. R-Z., C.C-T and A.C drafted the report. All authors have reviewed and approved the final submitted version. Conflict of interest None of the authors has any conflict of interest that could affect the performance of the work or the interpretation of the data. Ethical approval The current work was conducted in a subgroup of patients as part of the CORDIOPREV study (Clinical Trials.gov.Identifier: NCT00924937), an ongoing prospective, randomized, opened, controlled trial in patients with coronary heart disease. All the patients gave written 47
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Menopause 19 (3), 361–367. Schroder, H., et al., 2011. A short screener is valid for assessing Mediterranean diet adherence among older Spanish men and women. J. Nutr. 141 (6), 1140–1145. Sekhon, L.H., Agarwal, A., 2013. The menopause and oxidative stress. Studies on Women's Health, Oxidative Stress in Applied Basic Research and Clinical Practice. Springer Science + Business Media, New York, pp. 180–202. Signorelli, S.S., et al., 2006. Behaviour of some indicators of oxidative stress in postmenopausal and fertile women. Maturitas 53 (1), 77–82. Sohal, R.S., Orr, W.C., 2012. The redox stress hypothesis of aging. Free Radic. Biol. Med. 52 (3), 539–555. Unfer, T.C., et al., 2006. Influence of hormone replacement therapy on blood antioxidant enzymes in menopausal women. Clin. Chim. Acta 369 (1), 73–77. Vural, P., Canbaz, M., Akgul, C., 2006. Effects of menopause and postmenopausal tibolone treatment on plasma TNFalpha, IL-4, IL-10, IL-12 cytokine pattern and some bone turnover markers. Pharmacol. Res. 53 (4), 367–371. Warnick, G.R., Benderson, J., Albers, J.J., 1982. Dextran sulfate-Mg2+ precipitation procedure for quantitation of high-density-lipoprotein cholesterol. Clin. Chem. 28 (6), 1379–1388. Wilking, M., et al., 2013. Circadian rhythm connections to oxidative stress: implications for human health. Antioxid. Redox Signal. 19 (2), 192–208. Zitnanova, I., et al., 2011. Oxidative stress in women with perimenopausal symptoms. Menopause 18 (11), 1249–1255.
transdermal administration versus oral administration of estradiol plus norethisterone acetate on serum NO levels in postmenopausal women. Maturitas 53 (1), 32–38. Leal, M., et al., 2000. Hormone replacement therapy for oxidative stress in postmenopausal women with hot flushes. Obstet. Gynecol. 95 (6 Pt 1), 804–809. Lee, H.W., et al., 2007. Monitoring repair of DNA damage in cell lines and human peripheral blood mononuclear cells. Anal. Biochem. 365 (2), 246–259. Luczak, E.D., Leinwand, L.A., 2009. Sex-based cardiac physiology. Annu. Rev. Physiol. 71, 1–18. Paisley, E.A., et al., 1996. Temporal-regulation of serum lipids and stearoyl CoA desaturase and lipoprotein lipase mRNA in BALB/cHnn mice. J. Nutr. 126 (11), 2730–2737. Pfaffl, M.W., et al., 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pairwise correlations. Biotechnol. Lett. 26 (6), 509–515. Pines, A., 2016. Circadian rhythm and menopause. Climacteric 19 (6), 551–552. Rahman, I., et al., 2005. Glutathione, stress responses, and redox signaling in lung inflammation. Antioxid. Redox Signal. 7 (1–2), 42–59. Rao, P.M., Kelly, D.M., Jones, T.H., 2013. Testosterone and insulin resistance in the metabolic syndrome and T2DM in men. Nat. Rev. Endocrinol. 9 (8), 479–493. Roenneberg, T., et al., 2007. Epidemiology of the human circadian clock. Sleep Med. Rev. 11 (6), 429–438. Sanchez-Rodriguez, M.A., et al., 2012. Menopause as risk factor for oxidative stress.
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Contents lists available at ScienceDirect
Mechanisms of Ageing and Development journal homepage: www.elsevier.com/locate/mechagedev
Differential menopause- versus aging-induced changes in oxidative stress and circadian rhythm gene markers
MARK
Oriol A. Rangel-Zuñigaa,b,1, Cristina Cruz-Tenoa,b,1, Carmen Haroa,b, Gracia M. Quintana-Navarroa,b, Fernando Camara-Martosc, Pablo Perez-Martineza,b, Antonio Garcia-Riosa,b, Marta Garauletd, Manuel Tena-Sempereb,e, Jose Lopez-Mirandaa,b, ⁎ Francisco Perez-Jimeneza,b, Antonio Camargoa,b, a
Lipids and Atherosclerosis Unit, Maimonides Biomedical Research Institute of Cordoba (IMIBIC), Reina Sofia University Hospital, University of Cordoba, Spain CIBER Fisiopatología de la Obesidad y Nutrición (CIBEROBN), Instituto de Salud Carlos III, Cordoba, Spain Dpto de Bromatología y Tecnología de Alimentos, University of Cordoba, Cordoba, Spain d Department of Physiology, University of Murcia, Spain e Department of Cell Biology, Physiology, and Immunology, IMIBIC/Reina Sofia University Hospital/University of Cordoba, Cordoba, Spain b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Biology of aging Gender differences Genomics Oxidative stress Senescence
Menopause is characterized by the depletion of estrogen that has been proposed to cause oxidative stress. Circadian rhythm is an internal biological clock that controls physiological processes. It was analyzed the gene expression in peripheral blood mononuclear cells and the lipids and glucose levels in plasma of a subgroup of 17 pre-menopausal women, 19 men age-matched as control group for the pre-menopausal women, 20 postmenopausal women and 20 men age-matched as control group for the post-menopausal women; all groups were matched by body mass index. Our study showed a decrease in the expression of the oxidative stress-related gene GPX1, and an increase in the expression of SOD1 as consequence of menopause. In addition, we found that the circadian rhythm-related gene PER2 decreased as consequence of menopause. On the other hand, we observed a decrease in the expression of the oxidative stress-related gene GPX4 and an increase in the expression of CAT as a consequence of aging, independently of menopause. Our results suggest that the menopause-induced oxidative stress parallels a disruption in the circadian clock in women, and part of the differences in oxidative stress observed between pre- and post-menopausal women was due to aging, independent of menopause. Clinical Trials.gov.Identifier: NCT00924937
1. Introduction Aging is a complex process. Our current knowledge points towards oxidative stress as the main determinant in the deleterious and cumulative effects in the biology of aging (Sohal and Orr, 2012). In fact, aging increases the risk of cardiovascular diseases mainly by its association with increased oxidative stress and chronic low-grade inflammation (Herrera et al., 2009), which is also related with oxidative stress (Kabe et al., 2005). In women, menopause is a normal consequence of aging. As a major manifestation of female reproductive senescence, it is characterized by the permanent cessation of ovarian follicular activity, which produces an abrupt fall in estrogen levels, leading to the classic signs and symptoms of menopause, as well as an
increased risk of cardiovascular diseases and osteoporosis (Rao et al., 2013; Greendale et al., 1999). Oxidative stress has been implicated in various pathologies such as vasomotor disturbances, osteoporosis and cardiovascular diseases, which significantly correlate with the progressive loss of estrogen and its protective effects, combined with a deficient antioxidant defense which leads to a pronounced redox imbalance. In fact, it has been proposed that the depletion of estrogen in post-menopause could cause oxidative stress, in addition to the known symptoms (Rao et al., 2013; Greendale et al., 1999; Doshi and Agarwal, 2013). In addition, the incidence of metabolic diseases and their co-morbidities, which are sexually dimorphic, increases after menopause in women, at which time sex hormones are thought to play an important role in the development
⁎ Corresponding author at: Lipids and Atherosclerosis Research Unit, GC9 Nutrigenomics, IMIBIC/Reina Sofia University Hospital/University of Cordoba, Av. Menendez Pidal, s/n, 14004 Cordoba, Spain. E-mail address: [email protected] (A. Camargo). 1 These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.mad.2017.04.002 Received 25 July 2016; Received in revised form 17 March 2017; Accepted 1 April 2017 Available online 10 April 2017 0047-6374/ © 2017 Elsevier B.V. All rights reserved.
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determined in frozen samples were analyzed centrally by laboratory investigators of the Lipid and Atherosclerosis Unit at the Reina Sofia University Hospital, who were unaware of the interventions. Lipid variables were assessed with a DDPPII Hitachi modular analyzer (Roche) using specific reagents (Boehringer-Mannheim). Plasma triglycerides (TG) and cholesterol concentrations were assayed by enzymatic procedures (Allain et al., 1974; Paisley et al., 1996). High-density lipoprotein-cholesterol (HDL-c) was measured by precipitation of a plasma aliquot with dextran sulphate-Mg2+, as described by Warnick et al. (Warnick et al., 1982). Low-density lipoprotein-cholesterol (LDLc) was calculated by using the following formula: plasma cholesterol − [HDL-C + large TG-rich lipoproteins (TRL-C) + small TRL-C]. Plasma glucose concentrations were measured by using the IL Test Glucose Hexokinase Clinical Chemistry kit (Instrumentation Laboratories, Warrington, United Kingdom).
of cardiovascular disease (Arnlov et al., 2006; Bhupathy et al., 2010; Luczak and Leinwand, 2009). Moreover, it has been reported that postmenopausal women have higher lipoperoxide (Sanchez-Rodriguez et al., 2012), pro-inflammatory cytokines (Vural et al., 2006), and pro-oxidant biomarkers such as malonaldehyde, 4-hydroxynenal, and oxidized LDL (Signorelli et al., 2006) levels than pre-menopausal women, whereas the levels of antioxidant glutathione peroxidase (GPX) are lower in post-menopausal women (Signorelli et al., 2006). Nevertheless, hormone replacement therapies (HRT) have failed to prevent the post-menopausal increase in oxidative stress (Sekhon and Agarwal, 2013). Most of the studies focusing on the physiological changes caused by menopause compare only pre- and post-menopausal women, with an age difference of several years (post-menopausal groups are usually on average at least 10 years older than pre-menopausal groups), without age-matched groups of men to control the potential aging-induced, menopause-independent differences between pre- and post-menopausal women. To fill this gap, the objective of this study was to performer a series of comparative expression analyses in groups of pre- and postmenopausal women, and their corresponding age-matched male control groups. All 4 groups were matched by body mass index (BMI), and in them we analyzed the expression of gene markers of cardiovascular risk-related processes, including endoplasmic reticulum stress, inflammation, oxidative stress, metabolism, and circadian rhythms, in peripheral blood mononuclear cells (PBMC). This sub-set of white blood cells modifies its gene expression profile in response to stimuli and has proved to be useful in distinguishing a disease from a healthy state (Burczynski and Dorner, 2006; Camargo et al., 2014).
2.3. Sex hormone determination Testosterone was determined by the commercial kit: Testosterone Assay (R & D System; Cat. No. KGE010), according to the manufacturer's instructions. Estradiol was determined by the commercial kit: Estradiol EIA kit (Cayman Chemical; Cat. No. 582251), according to the manufacturer instructions. Follicle-Stimulating Hormone was determined by the commercial kit: FSH ELISA (DRG Instruments GmbH; Cat. No. EIA-1288), according to the manufacturer's instructions. 2.4. Isolation of peripheral blood mononuclear cells and RNA extraction Buffy coats were diluted 1:2 in phosphate saline buffer (PBS), and cells were separated in 15 mL Ficoll gradient (lymphocyte isolation solution, Axis-Shield, Oslo, Norway) by centrifugation at 2000 × g for 25 min. PBMC were collected, washed twice with cold PBS and stored in RNA Later Solution (Ambion, Thermo Fisher Scientific, MA, USA). Total RNA was extracted using Tri Reagent (Sigma, St Louis, MO, USA), according to the manufacturers’ instructions. The recovered RNA was quantified using a Nanodrop ND-1000 v3.5.2 spectrophotometer (Nanodrop Technology®, Cambridge, UK), and its integrity was checked on agarose gel electrophoresis and stored at −80 °C. RNA samples were digested with DNAse I (AMPD-1 Kit, Sigma) before RT-PCR.
2. Methods 2.1. Study subjects The current work was conducted in a subgroup of patients as part of the CORDIOPREV study (Clinical Trials.gov.Identifier: NCT00924937), an ongoing prospective, randomized, opened, controlled trial in patients with coronary heart disease (CHD), who had their last coronary event over six months before enrolling in two different dietary models (Mediterranean and Low-fat) over a period of five years, in addition to conventional treatment for CHD (Alcala-Diaz et al., 2014). All the patients gave written informed consent to participate in the study. The trial protocol and all amendments were approved by the local ethics committees, following the Helsinki declaration and good clinical practice. We analyzed a subgroup of 76 patients from the control healthy group included in the CORDIOPREV study (without oncological or cardiovascular diseases): 17 pre-menopausal women (E2 = 109.401 ± 41.46 pg/ mL, FSH = 13.47 ± 2.90 mIU/mL), 19 men matched by age as a control group for the pre-menopausal women (T = 108 ± 14 pg/mL), 20 postmenopausal women (E2 = 11.49 ± 4.47 pg/mL, FSH = 93.54 ± 7.63 mIU/mL) and 20 men (T = 92 ± 9 pg/mL) matched by age as a control group for the post-menopausal women: all 4 groups were matched by BMI. Patient's inclusion was assessed by medical history, biochemical measures and physical examination by clinicians. Exclusion criteria included cardiovascular disease, cancer, and chronic diseases and did not have severe diseases or an estimated life expectancy of less than 5 years. We also performed a diet assessment with a validated 14-item questionnaire (Schroder et al., 2011) to assess the dietary habits of the groups.
2.5. qRT-PCR for gene expression analysis Retrotranscription reaction was performed with 500 ng of total RNA using the Ambion WT Expression Kit (Applied Biosystems, Carlsbad, CA, USA), following the manufacturers’ instructions. Real-time PCR reactions were carried out using the OpenArray™ NT Cycler system (Applied Biosystems, Carlsbad, CA, USA), according to the manufacturers’ instructions. Primer pairs for 53 target genes related to endoplasmic reticulum stress, inflammation, oxidative stress (prooxidants and antioxidants), metabolism and circadian rhythms were selected from the TaqMan Gene Expression assays database (Applied Biosystems, Carlsbad, CA, USA) (Appendix 1 Supplemental Table 1). We used as housekeeping genes: beta-2-microglobulin, glyceraldehyde3-phosphate dehydrogenase and hypoxanthine phosphoribosyltransferase, for which Ct values were combined by the software Bestkeeper (Pfaffl et al., 2004) in order to get a more stable reference value than each one independently. Gene expression values were obtained as a relative expression of the target gene versus the Bestkeeper value (relative expression = 2−(Ct, Targe gene−Ct, Bestkeeper value)). The data set was extracted by using the OpenArray® Real-Time qPCR Analysis Software (Applied Biosystems, Carlsbad, CA, USA).
2.2. Clinical plasma parameters The patients arrived at the clinical centre at 08:00 h. We measured anthropometric (weight, height, waist circumference, BMI and blood pressure) and took a fasting blood sample. Blood was collected in tubes containing ethylenediaminetetraacetic acid (EDTA) to give a final concentration of 0.1% EDTA. The plasma was separated from the red blood cells by centrifugation at 1500 × g for 15 min at 4 °C. Analytes
2.6. Statistical analysis PASW statistical software, version 20.0 (IBM Inc., Chicago, IL, USA) was used for statistical analysis of the data. The normal distribution of 42
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between both groups of women (ANOVA P < 0.001; FDR Q < 0.001). The post hoc Bonferroni's corrected multiple comparison test showed that the expression of PER2 was lower in the post-menopausal group of women than in pre-menopausal group (P = 0.005). In contrast, we did not find any differences in expression between the male-post and malepre groups. In addition, the expression of PER2 was higher in the premenopausal women group than in the corresponding age-matched male-pre group (P = 0.016).
variables was assessed using the Kolmogorov–Smirnov test. When the data distribution did not adjust to the normal values, the values were log10 transformed (XBP1, IL6, MCP1, IL1B, TNF-a, UCP2, NFKB p65, NFKBIA, IKBKA, p91phox, p47phox, p40phox, SOD1, SOD2, GPX1, GPX4, GSR, PU.1, PPARG, MAPK8, MAPK14, RRM2, CDKN1A, MDM2, APEX1, DDB2, GADD45A, GADD45B, IL8, CXCL1, MMP9, MIF, CLOCK, BMAL1) or 1/x transformed (CALR, BiP, IKBKB, p67phox, TXN, TXNRD1, NFE2L2, TP53, OGG1) to achieve this. The statistical differences between the groups were evaluated by One-way ANOVA. Multiple comparisons in the large-scale expression analyses were assessed by False Discovery Rate (FDR) using the Benjamini and Hochberg method. Post hoc statistical analysis was completed by using Bonferroni's comparison tests. A P-value < 0.05 was considered statistically significant.
3.3. Aging-dependent menopause-independent differences in the gene expression We observed differences as function of aging, independently of menopause, in the expression of the oxidative stress-related genes CAT and GPX4 (Fig. 2) and the DNA repair-related genes XPC and POLB (Fig. 3). Whereas no statistically significant differences were found between pre-and post-menopausal women, nor between the corresponding malepre and male-post groups in the expression of CAT, when we merged the pre-menopausal group of women and their corresponding agematched men (mean age 46 y) and post-menopausal women group with their corresponding age-matched men (mean age 56 y), we observed a higher expression in the CAT gene in the 56 y group as compared with the 46 y group (ANOVA P = 0.008; FDR Q = 0.051). Moreover, we found that the expression of GPX4 was lower in post-menopausal women than in the pre-menopausal group (P = 0.005), but we also observed the same profile between the corresponding male-pre and male-post groups (P = 0.018). In addition, we found that the expression of XPC and POLB genes was lower in post-menopausal women than in the pre-menopausal group (P = 0.002 and P < 0.001, respectively), but we also observed the same profile between the corresponding malepre and male-post groups (statistical trend P = 0.073, and P = 0.004, respectively). When we merged pre-menopausal women group and their corresponding age-matched men (mean age 46 y) and postmenopausal women group and their corresponding age-matched men (mean age 56 y), we observed a lower expression of GPX4 (ANOVA P < 0.001; FDR Q < 0.001) and XPC and POLB genes (both, ANOVA P < 0.001; FDR Q < 0.001) in the 56 y group than in the 46 y group, which is a function of aging and independent of menopause.
3. Results 3.1. Baseline characteristic of the study participants No statistically significant differences were found in BMI, waist circumference, LDL-c, TG, Insulin, HbA1C or systolic blood pressure between groups. Pre-menopausal women had higher HDL-c and lower glucose levels (P < 0.05). The pre-menopausal women group and their corresponding age-matched men (male-pre) were younger than postmenopausal women and their age-matched male group counterpart (male-post; P < 0.05) (Table 1). Importantly, no differences were found in the dietary habits between groups that might affect the expression of the genes under analysis (Appendix 2 Supplemental Table 2). 3.2. Menopause-dependent differences in the gene expression In order to assess the impact of aging and menopause on cardiovascular risk, we analyzed the expression of genes related to endoplasmic reticulum stress, inflammation, oxidative stress, metabolism and circadian rhythms in PBMC. (Appendix 1 Supplemental Table 1; only differentially expressed genes are described in the text). We found significant differences in the expression of several genes related with oxidative stress and circadian rhythms in PBMC that were not present in the age-matched groups of men (Fig. 1). Our study also showed differences in the expression of the antioxidant enzyme genes SOD1 and GPX1 between groups (ANOVA, both P = 0.008; FDR, both Q = 0.058). In fact, the post hoc Bonferroni's corrected multiple comparison test showed that the expression of SOD1 gene was higher (P = 0.041) and the expression of GPX1 was lower (P = 0.085, statistical trend) in post-menopausal women than in the pre-menopausal group. Moreover, we observed that the expression of the circadian rhythm-related gene PER2 showed a statistically significant difference
4. Discussion Most of the studies on the impact of menopause compare pre- and post-menopausal women. However, the lack of corresponding studies of age-matched groups of men hampers the identification of agingdependent, menopause-independent changes. Therefore, our experimental design included pre- and post-menopausal women and their agematched male counterparts, which allowed us to distinguish between
Table 1 Metabolic characteristic of the participants in the study. Values correspond to the mean ± SEM. The statistical differences between groups were evaluated by One-way ANOVA. BMI, body mass index; TG, triacylglyceride; HDL-c, high density lipoprotein-cholesterol; LDL-c, low density lipoprotein-cholesterol; HbA1C, glycated hemoglobin; BP, blood pressure. In each row, values with different letters in superscript differs statistically in the Bonferroni's corrected post hoc multiple comparison test (P < 0.05).
Age (years) BMI (kg/m2) Waist circumference (cm) HDL-c (mg/dL) LDL-c (mg/dL) TG (mg/dL) Glucose (mg/dL) Insulin (mU/L) HbA1C (%) Systolic BP (mm Hg) Diastolic BP (mm Hg)
Pre-menopausal women (N = 17)
Men control group pre (N = 19)
Post-menopausal women (N = 20)
Men control group post (N = 20)
P-value
46.12 ± 0.82a 26.31 ± 1.52 88.94 ± 3.15 57.00 ± 3.37a 118.53 ± 6.97 88.88 ± 9.97 87.00 ± 2.75a 5.39 ± 0.79 5.11 ± 0.11 127.96 ± 6.30 77.89 ± 2.43
46.58 ± 0.61a 27.10 ± 0.91 95.68 ± 2.24 41.79 ± 2.14b 132.42 ± 5.97 115.95 ± 14.92 98.21 ± 5.48a,b 6.37 ± 0.90 5.32 ± 0.17 129.53 ± 3.85 81.62 ± 2.54
55.85 ± 0.66b 28.94 ± 1.25 92.65 ± 3.00 54.50 ± 2.82a,c 137.40 ± 7.13 102.95 ± 12.76 92.05 ± 2.38a 6.87 ± 0.92 5.54 ± 0.08 132.83 ± 3.66 77.35 ± 2.39
56.20 ± 0.72b 28.53 ± 1.13 99.30 ± 2.88 45.40 ± 1.92b,c 145.65 ± 7.21 107.95 ± 13.24 108.40 ± 4.29b 8.08 ± 0.99 5.55 ± 0.13 136.18 ± 2.86 85.50 ± 1.56
< 0.001 0.393 0.079 < 0.001 0.057 0.545 0.002 0.233 0.063 0.518 0.041
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Fig. 1. Menopause and expression of oxidative stress- and circadian rhythm-related genes. Values represent mean ± SEM of the relative expression of the indicated gene. One-way ANOVA statistical analysis P-value. Q-value, False Discovery Rate. *P < 0.05 and #P < 0.1 in the post hoc Bonferroni's multiple comparison tests. Experimental groups: pre-menopausal women; male-pre: age-matched men as a control group for the pre-menopausal women; post-menopausal women; male-post: age-matched men as a control group for the post-menopausal women. All the four groups are matched by BMI. SOD1, superoxide dismutase 1. GPX1, glutathione peroxidase 1. PER2, period homolog 2.
molecular oxygen, are also involved in ROS production (Inoguchi and Nawata, 2005). In response to oxidative stress, PBMC reinforce their antioxidant defences by increasing the expression of antioxidant genes (Camargo et al., 2014). SOD gene expression is induced by %O2− and converts it into H2O2, which is, in turn, converted into water or molecular oxygen by either CAT or GPx. The latter is also involved in the detoxification of lipid hydro-peroxides generated by the action of % O2− (Faraci and Didion, 2004; Rahman et al., 2005). In this context, a higher expression of SOD1 is consistent with an alteration in the ROS detoxification pathway, and depending to the GPx activity, presumably low in post-menopausal women as suggested by the expression levels of GPX1. It may generate a high oxidative stress condition in postmenopausal women (with a higher expression of SOD1 compared with pre-menopausal women). In fact, our results are in line with previous studies in which an increased oxidative stress after menopause was observed (Zitnanova et al., 2011). Moreover, whereas SOD activity has been shown to be reduced with aging in certain tissues (Doonan et al., 2008), our study in PBMC showed that SOD1 gene expression did not decreased with aging in men and was higher in post-menopausal women and no changes were observed in SOD2 gene expression,
menopause-dependent and aging-dependent changes. Furthermore, we performed our large scale gene expression analyses in BMI-matched experimental groups with a similar nutritional background, as both BMI and diet may alter the expression of the genes studied. Our study showed that as consequence of menopause, there is a decrease in the expression of the oxidative stress-related gene GPX1, and an increase in the gene expression of SOD1 in PBMC. In addition, we also found that the circadian rhythm-related gene PER2 decreased as a consequence of menopause. On the other hand, as a consequence of aging (independently of menopause), we observed a decrease in the expression of oxidative stress-related genes such as GPX4 and an increase in the expression of CAT. The changes found in DNA repair-related genes were also specific to aging, and we also observed a decrease in the expression of the XPC and POLB. Oxidative stress occurs when there is an imbalance between the production of reactive oxygen species (ROS) and their elimination by the antioxidant system (Durackova, 2010). ROS are generated byproducts of metabolism, but several enzymes, including NADPHoxidase, an enzyme which transfers an electron from NADPH to 44
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Fig. 2. Aging and expression of oxidative stress-related genes. Values represent mean ± SEM of the relative expression of the indicated gene. One-way ANOVA statistical analysis Pvalue. Q-value, False Discovery Rate. *P < 0.05 and #P < 0.1 in the post hoc Bonferroni's multiple comparison tests. Experimental groups: pre-menopausal women; male-pre: agematched men as a control group for the pre-menopausal women; post-menopausal women; male-post: age-matched men as a control group for the post-menopausal women. All the four groups are matched by BMI. Right panel: Comparison between age groups resulting from merging the group of pre-menopausal women with their age-matched male counterparts (mean age 46 y) and the group of post-menopausal women with their age-matched men male counterparts (mean age 56 y). CAT, catalase. GPX4, glutathione peroxidase 4.
Extensive observations suggest that DNA damage accumulates during aging, as a consequence of an increase in oxidative stress and a decline in DNA repair capacity (Chen et al., 2007). Repair of oxidative DNA damage involves 3 mechanisms: base excision repair (BER), nucleotide excision repair (NER) and mismatch repair (Izumi et al., 2003; Lee et al., 2007). Thus, a lower expression of POLB and XPC (belonging to the BER and NER pathways, respectively) in post-menopausal women and their age-matched male control group is consistent with an aginginduced alteration in DNA repair capacity. All together, these results may explain why hormone replacement therapies have failed to substantially improve the redox status in postmenopausal women, as these therapies cannot counteract the aginginduced increase in oxidative stress. In fact, several studies have documented how hormone replacement therapies only partially restore the antioxidant machinery (Unfer et al., 2006), total antioxidant status (Leal et al., 2000), and nitric oxide (NO) levels (Bednarek-Tupikowska et al., 2008; Kurtay et al., 2006).
suggesting a high complexity of the regulatory mechanism controlling this step of ROS detoxification in post-menopausal women. However, our study showed that at least part of the increase in oxidative stress observed in post-menopausal women is a consequence of aging itself, and is not caused primarily by ovarian senescence. In fact, post-menopausal women and their age-paired group of men displayed higher CAT gene expression than pre-menopausal women and their age-matched men, suggesting that changes in the latter are aging dependent, and independent of menopause and gender. Likewise, the lower expression of GPX isoforms suggests a decrease in the detoxification of hydrogen peroxide in post-menopausal women (GPX1 gene), whereas a reduction of lipid hydroperoxides seems to be a consequence of aging, as suggested by the reduction of GPX4 expression independently of menopause and gender. In addition, no differences in terms of genetic markers of oxidative DNA damage repair were found between pre- and post-menopausal women, as significant differences in these markers are specific to aging. 45
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Fig. 3. Aging and expression of DNA repair-related genes. Values represent mean ± SEM of the relative expression of the indicated gene. One-way ANOVA statistical analysis P-value. Qvalue, False Discovery Rate. *P < 0.05 and #P < 0.1 in the post hoc Bonferroni’s multiple comparison tests. Experimental groups: pre-menopausal women; age-matched men as a control group for the pre-menopausal women; post-menopausal women; age-matched men as a control group for the post-menopausal women. All the four groups are matched by BMI. Right panel: Comparison between age groups resulting from merging the group of pre-menopausal women with their age-matched male counterparts (mean age 46 y) and the group of post-menopausal women with their age-matched male counterparts (mean age 56 y). XPC, xeroderma pigmentosum, complementation group C. POLB, polymerase (DNA directed), beta.
we observed that the expression of PER2 decreased with menopause in PBMC, which suggests that changes in the profile of the expression of clock genes induced by menopause may be tissue-specific. Moreover, with menopause, women exhibit a loss of circadian robustness and an increase in sleep abnormalities – a process that runs parallel with the redistribution of adipose tissue (Gómez-Santos et al., 2016). In turn, the hormonal changes undergone during menopause have been related with an alteration in the circadian rhythms (Joffe et al., 2010), which suggests that the differences observed in PER2 expression between premenopausal women and age-matched men, but not between postmenopausal women and age-paired men, may contribute to the sex differences in the circadian timing system that disappear after menopause (Roenneberg et al., 2007) and phase-advanced endogenous temperature and melatonin rhythms (Cain et al., 2010). This has partly been attributed to a significantly shorter circadian period in women (Duffy et al., 2011). In addition, it has been described that endogenous secretion of melatonin decreases with aging across genders, and, among women, menopause is associated with a significant reduction of
Our study also showed that menopause-induced oxidative stress might be related with a disruption in the circadian rhythms in women. In fact, the processes of oxidative stress and circadian rhythms are intricately involved in a myriad of physiological processes to maintain homeostasis, from blood pressure and sleeping/waking cycles, down to cellular signaling pathways that play critical roles in health and disease (Wilking et al., 2013). When genes regulating circadian functions lose some of their precise orchestration with aging, this leads to impaired homeostasis (Gibson et al., 2009), a situation which is particularly marked in women as they move towards menopause (Chedraui et al., 2010). In fact, menopause changes the profile of the expression of clock genes in adipose tissue (Hernandez-Morante et al., 2011), which may be related with body fat redistribution after menopause, which in turn is characterized by an increase in intra-abdominal visceral fat (Garaulet et al., 2006). In addition, whereas the expression of PER2 does not differ between pre- and post-menopausal women in subcutaneous adipose tissue, it is higher in postmenopausal women in visceral adipose tissue (Hernandez-Morante et al., 2011). In the current study, 46
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melatonin levels, which therefore may also explain the well-known sleep disturbances associated with menopause (Pines, 2016). Our study has limitations. One of the limitations of our study is the reduced sample size of our pilot study, which improves the experimental designs of most of the studies focusing the changes associated to menopause as we included age-matched groups of men to control the potential aging-induced, menopause-independent differences between pre- and post-menopausal women. Another limitation lies in the fact that we performed a genomic approach, as we performed a series of comparative expression analyses in groups of pre- and post-menopausal women, and their corresponding age-matched male control groups. Further studies providing protein data may shed more light in the specific cellular and molecular mechanisms involved in the menopausedependent and aging-dependent menopause-independent differences in the gene expression showed in this study.
informed consent to participate in the study. The trial protocol and all amendments were approved by the local ethics committees, following the Helsinki declaration and good clinical practice.
5. Conclusion
References
Our results suggest that the menopause-induced oxidative stress parallels a disruption in the circadian clock in women, and some of the differences in oxidative stress observed between pre- and post-menopausal women were a consequence of aging and independent of menopause. Moreover, aging per se associates with changes in DNA repair-related genes.
Alcala-Diaz, J.F., et al., 2014. Hypertriglyceridemia influences the degree of postprandial lipemic response in patients with metabolic syndrome and coronary artery disease: from the cordioprev study. PLOS ONE 9 (5), e96297. Allain, C.C., et al., 1974. Enzymatic determination of total serum cholesterol. Clin. Chem. 20 (4), 470–475. Arnlov, J., et al., 2006. Endogenous sex hormones and cardiovascular disease incidence in men. Ann. Intern. Med. 145 (3), 176–184. Bednarek-Tupikowska, G., Tworowska-Bardzinska, U., Tupikowski, K., 2008. Effects of estrogen and estrogen-progesteron on serum nitric oxide metabolite concentrations in post-menopausal women. J. Endocrinol. Invest. 31 (10), 877–881. Bhupathy, P., Haines, C.D., Leinwand, L.A., 2010. Influence of sex hormones and phytoestrogens on heart disease in men and women. Womens Health (Lond. Engl.) 6 (1), 77–95. Burczynski, M.E., Dorner, A.J., 2006. Transcriptional profiling of peripheral blood cells in clinical pharmacogenomic studies. Pharmacogenomics 7 (2), 187–202. Cain, S.W., et al., 2010. Sex differences in phase angle of entrainment and melatonin amplitude in humans. J. Biol. Rhythms 25 (4), 288–296. Camargo, A., et al., 2014. Peripheral blood mononuclear cells as in vivo model for dietary intervention induced systemic oxidative stress. Food Chem. Toxicol. 72, 178–186. Chedraui, P., et al., 2010. Factors related to increased daytime sleepiness during the menopausal transition as evaluated by the Epworth sleepiness scale. Maturitas 65 (1), 75–80. Chen, J.H., Hales, C.N., Ozanne, S.E., 2007. DNA damage, cellular senescence and organismal ageing: causal or correlative? Nucleic Acids Res. 35 (22), 7417–7428. Doonan, R., et al., 2008. Against the oxidative damage theory of aging: superoxide dismutases protect against oxidative stress but have little or no effect on life span in Caenorhabditis elegans. Genes Dev. 22 (23), 3236–3241. Doshi, S.B., Agarwal, A., 2013. The role of oxidative stress in menopause. J. Midlife Health 4 (3), 140–146. Duffy, J.F., et al., 2011. Sex difference in the near-24-hour intrinsic period of the human circadian timing system. Proc. Natl. Acad. Sci. U. S. A. 108 (Suppl. 3), 15602–15608. Durackova, Z., 2010. Some current insights into oxidative stress. Physiol. Res. 59 (4), 459–469. Faraci, F.M., Didion, S.P., 2004. Vascular protection: superoxide dismutase isoforms in the vessel wall. Arterioscler. Thromb. Vasc. Biol. 24 (8), 1367–1373. Garaulet, M., et al., 2006. Anthropometric indexes for visceral fat estimation in overweight/obese women attending to age and menopausal status. J. Physiol. Biochem. 62 (4), 245–252. Gibson, E.M., Williams III, W.P., Kriegsfeld, L.J., 2009. Aging in the circadian system: considerations for health, disease prevention and longevity. Exp. Gerontol. 44 (1–2), 51–56. Gómez-Santos, C., Saura, C.B., Lucas, J.A., Castell, P., Madrid, J.A., Garaulet, M., et al., 2016. Menopausal status is associated with circadian- and sleep-related alterations. Menopause 23 (June (6)), 682–690. http://dx.doi.org/10.1097/GME. 0000000000000612. Greendale, G.A., Lee, N.P., Arriola, E.R., 1999. The menopause. Lancet 353 (9152), 571–580. Hernandez-Morante, J.J., et al., 2011. Influence of menopause on adipose tissue clock gene genotype and its relationship with metabolic syndrome in morbidly obese women. Age (Dordr) 34 (6), 1369–1380. Herrera, M.D., et al., 2009. Endothelial dysfunction and aging: an update. Ageing Res. Rev. 9 (2), 142–152. Inoguchi, T., Nawata, H., 2005. NAD(P)H oxidase activation: a potential target mechanism for diabetic vascular complications, progressive beta-cell dysfunction and metabolic syndrome. Curr. Drug Targets 6 (4), 495–501. Izumi, T., et al., 2003. Mammalian DNA base excision repair proteins: their interactions and role in repair of oxidative DNA damage. Toxicology 193 (1–2), 43–65. Joffe, H., Massler, A., Sharkey, K.M., 2010. Evaluation and management of sleep disturbance during the menopause transition. Semin. Reprod. Med. 28 (5), 404–421. Kabe, Y., et al., 2005. Redox regulation of NF-kappaB activation: distinct redox regulation between the cytoplasm and the nucleus. Antioxid. Redox Signal. 7 (3–4), 395–403. Kurtay, G., Ozmen, B., Erguder, I., 2006. A comparison of effects of sequential
Acknowledgements We would like to thank the EASP (Escuela Andaluza de Salud Publica), Granada, Spain, which performed the randomization process for this study. We would also like to thank José Andrés Morales Martínez for lending technical support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.mad.2017.04.002.
Funding The CIBEROBN is an initiative of the Instituto de Salud Carlos III (ISCIII), Madrid, Spain. The CORDIOPREV study is supported by the Fundación Patrimonio Comunal Olivarero, Junta de Andalucía (Consejería de Salud, Consejería de Agricultura y Pesca, Consejería de Innovación, Ciencia y Empresa), Diputaciones de Jaen y Cordoba, Centro de Excelencia en Investigación sobre Aceite de Oliva y Salud and Ministerio de Medio Ambiente, Medio Rural y Marino, Gobierno de España. It was also partly supported by research grants from the Ministerio de Ciencia e Innovación (AGL2009-122270 to J L-M); Ministerio de Economia y Competitividad (AGL2012/39615, PIE14/ 00005, PIE 14/00031 to J L-M, SAF2014-52480-R to MG, FIS PI13/ 00185 to P P-M, and PI13/00619 to F P-J); Consejería de Economía, Innovación y Ciencia, Proyectos de Investigación de Excelencia, Junta de Andalucía (AGR922 to F P-J); Consejería de Salud, Junta de Andalucía (PI0193/09 to J L-M and PI-0058/10 to P P-M; Consejería de Innovación Ciencia y Empresa (CVI-7450 to J L-M); Fondo Europeo de Desarrollo Regional (FEDER). Antonio Camargo is supported by an ISCIII research contract (Programa Miguel-Servet CP14/00114). Authors’ contributions A.C., J.L-M., P.P-M and F.P-J. contributed to the study conception and design. P.P-M., F.P-J and J.L-M obtained funding. C.C-T., OA.R-Z., C.H., GM. Q-N., F.C-M and A.G-R contributed to acquisition of data. OA.R-Z., C.C-T., M.G., M.T-S and A.C analyzed and interpreted the data. OA. R-Z., C.C-T and A.C drafted the report. All authors have reviewed and approved the final submitted version. Conflict of interest None of the authors has any conflict of interest that could affect the performance of the work or the interpretation of the data. Ethical approval The current work was conducted in a subgroup of patients as part of the CORDIOPREV study (Clinical Trials.gov.Identifier: NCT00924937), an ongoing prospective, randomized, opened, controlled trial in patients with coronary heart disease. All the patients gave written 47
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Menopause 19 (3), 361–367. Schroder, H., et al., 2011. A short screener is valid for assessing Mediterranean diet adherence among older Spanish men and women. J. Nutr. 141 (6), 1140–1145. Sekhon, L.H., Agarwal, A., 2013. The menopause and oxidative stress. Studies on Women's Health, Oxidative Stress in Applied Basic Research and Clinical Practice. Springer Science + Business Media, New York, pp. 180–202. Signorelli, S.S., et al., 2006. Behaviour of some indicators of oxidative stress in postmenopausal and fertile women. Maturitas 53 (1), 77–82. Sohal, R.S., Orr, W.C., 2012. The redox stress hypothesis of aging. Free Radic. Biol. Med. 52 (3), 539–555. Unfer, T.C., et al., 2006. Influence of hormone replacement therapy on blood antioxidant enzymes in menopausal women. Clin. Chim. Acta 369 (1), 73–77. Vural, P., Canbaz, M., Akgul, C., 2006. Effects of menopause and postmenopausal tibolone treatment on plasma TNFalpha, IL-4, IL-10, IL-12 cytokine pattern and some bone turnover markers. Pharmacol. Res. 53 (4), 367–371. Warnick, G.R., Benderson, J., Albers, J.J., 1982. Dextran sulfate-Mg2+ precipitation procedure for quantitation of high-density-lipoprotein cholesterol. Clin. Chem. 28 (6), 1379–1388. Wilking, M., et al., 2013. Circadian rhythm connections to oxidative stress: implications for human health. Antioxid. Redox Signal. 19 (2), 192–208. Zitnanova, I., et al., 2011. Oxidative stress in women with perimenopausal symptoms. Menopause 18 (11), 1249–1255.
transdermal administration versus oral administration of estradiol plus norethisterone acetate on serum NO levels in postmenopausal women. Maturitas 53 (1), 32–38. Leal, M., et al., 2000. Hormone replacement therapy for oxidative stress in postmenopausal women with hot flushes. Obstet. Gynecol. 95 (6 Pt 1), 804–809. Lee, H.W., et al., 2007. Monitoring repair of DNA damage in cell lines and human peripheral blood mononuclear cells. Anal. Biochem. 365 (2), 246–259. Luczak, E.D., Leinwand, L.A., 2009. Sex-based cardiac physiology. Annu. Rev. Physiol. 71, 1–18. Paisley, E.A., et al., 1996. Temporal-regulation of serum lipids and stearoyl CoA desaturase and lipoprotein lipase mRNA in BALB/cHnn mice. J. Nutr. 126 (11), 2730–2737. Pfaffl, M.W., et al., 2004. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pairwise correlations. Biotechnol. Lett. 26 (6), 509–515. Pines, A., 2016. Circadian rhythm and menopause. Climacteric 19 (6), 551–552. Rahman, I., et al., 2005. Glutathione, stress responses, and redox signaling in lung inflammation. Antioxid. Redox Signal. 7 (1–2), 42–59. Rao, P.M., Kelly, D.M., Jones, T.H., 2013. Testosterone and insulin resistance in the metabolic syndrome and T2DM in men. Nat. Rev. Endocrinol. 9 (8), 479–493. Roenneberg, T., et al., 2007. Epidemiology of the human circadian clock. Sleep Med. Rev. 11 (6), 429–438. Sanchez-Rodriguez, M.A., et al., 2012. Menopause as risk factor for oxidative stress.
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