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Effect of broccoli extract enriched diet on liver cholesterol oxidation in rats subjected to exhaustive exercise
Accepted Manuscript Title: Effect of broccoli extract enriched diet on liver cholesterol oxidation in rats subjected to exhaustive exercise Author: Vladimiro Cardenia Maria Teresa Rodriguez-Estrada Antonello Lorenzini Erika Bandini Cristina Angeloni Silvana Hrelia Marco Malaguti PII: DOI: Reference:
S0960-0760(16)30101-7 http://dx.doi.org/doi:10.1016/j.jsbmb.2016.04.005 SBMB 4698
To appear in:
Journal of Steroid Biochemistry & Molecular Biology
Received date: Revised date: Accepted date:
30-11-2015 7-4-2016 11-4-2016
Please cite this article as: Vladimiro Cardenia, Maria Teresa Rodriguez-Estrada, Antonello Lorenzini, Erika Bandini, Cristina Angeloni, Silvana Hrelia, Marco Malaguti, Effect of broccoli extract enriched diet on liver cholesterol oxidation in rats subjected to exhaustive exercise, Journal of Steroid Biochemistry and Molecular Biology http://dx.doi.org/10.1016/j.jsbmb.2016.04.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
EFFECT OF BROCCOLI EXTRACT ENRICHED DIET ON LIVER CHOLESTEROL OXIDATION IN RATS SUBJECTED TO EXHAUSTIVE EXERCISE
Vladimiro Cardenia 1*, Maria Teresa Rodriguez-Estrada 1,2, Antonello Lorenzini 3, Erika Bandini 4, Cristina Angeloni 5, Silvana Hrelia 5, Marco Malaguti5
1
Department of Agricultural and Food Sciences, Alma Mater Studiorum – University of Bologna
(Bologna, Italy) 2
Interdepartmental Centre for Agri-Food Industrial Research, Alma Mater Studiorum – University
of Bologna (Cesena, Italy) 3
Department of Biomedical and Neuromotor Sciences, Alma Mater Studiorum – University of
Bologna (Bologna, Italy) 4
Scientific Institute of Romagna for the Study and Treatment of Cancer (IRST), Unit of Gene
Therapy Meldola-Forlı’, Meldola (FC), Italy 5
Department for Life Quality Studies, Alma Mater Studiorum – University of Bologna (Rimini,
Italy)
*
Corresponding author:
Viale Fanin 40 40127 Bologna (Italy) Tel. +39-051-2096015 Fax: +39-051-2096017 E-mail: [email protected]
Graphical Abstract
Highlights
The broccoli extract reduced cholesterol oxidation after exhaustive exercise The broccoli extract enriched diet increased antioxidant phase 2 enzyme activity Phytochemicals could be useful in the prevention of liver oxidative damage
Abstract The effect of broccoli extract (BE)-enriched diet was studied in order to evaluate its ability to counteract liver cholesterol oxidation products (COPs) induced by acute strenuous exercise in rats. Thirty-two female Wistar rats were randomly divided into four groups: control diet without exercise (C), BE-enriched diet without exercise (B), control diet with acute exhaustive exercise (S) and BEenriched diet with acute exhaustive exercise (BS). The study lasted 45 days and on the last day, rats of S and BS groups were forced to run until exhaustion on a treadmill. Glutathione-S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx), catalase (CAT) and cholesterol oxidation products (COPs) were determined in liver. Exhaustive exercise was clearly responsible for tissue damage, as evidenced by the increase of lactate dehydrogenase (LDH) plasma activity in the S group. Moreover, the exercise protocol reduced CAT activity in liver, while it did not affect GST, GR and GPx. BE-enriched diet raised GST, GR and CAT activities in rats of BS group. The main COPs found were 7α-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol, cholestanetriol, 24-hydroxycholesterol and 27-hydroxycholesterol. The BE-enriched diet led to reduced cholesterol oxidation following exhaustive exercise; the highest level of COPs was found in the S group, whereas the BS rats showed the lowest amount. This study indicates that the BEenriched diet increases antioxidant enzyme activities and exerts an antioxidant effect towards cholesterol oxidation in rat liver, suggesting the use of phytochemicals in the prevention of oxidative damage and in the modulation of the redox environment.
Keywords: Cholesterol oxidation products; broccoli extract; exhaustive exercise; oxidative stress; antioxidant phase 2 enzymes; oxysterols.
1. Introduction The interest on physical exercise has enormously risen in the last decades and a large amount of scientific literature has been published. The success of this topic partly lies on the beneficial effects induced by physical exercise on human health [1, 2]. However, exhaustive exercise can be quite harmful, since it is responsible for a burst of oxidative stress, which is followed by an inflammatory response and a consequent structural damage to muscle fibers; the release of cytosolic enzymes (i.e. lactic dehydrogenase (LDH) and creatine kinase (CK)) in plasma, confirms the occurrence of such events [3]. Exhaustive exercise does not only affect muscle cells homeostasis and viability, but it also exerts deleterious effects on different tissues and organs. At both cardiac and hepatic levels, strenuous activity has been related to functional impairment, oxidative stress, activation of apoptotic signaling pathways and inflammation [4-6]. Due to its deleterious effects, many nutritional interventions have been studied to counteract exhaustive exercise induced damage. In this context, unfortunately, the supplementation with ROS scavengers (such as antioxidant vitamins) seems unable to prevent exhaustive exercise stress [7]. On the other hand, foods rich in indirect antioxidants or molecules able to induce antioxidant/detoxifying systems might represent an alternative nutritional approach to counteract exhaustive exercise induced stress. One example of this type of bioactive compounds is sulforaphane (SF), an isothiocyanate found in cruciferous vegetables (such as broccoli), which can be introduced through the diet. SF was initially studied for its promising chemopreventive activity [8], thanks to its ability to induce phase II enzymes both in vitro and in vivo [9-14]. In a recent study carried out in rats, it was also confirmed that SF treatment induces enzymes with an antioxidant/detoxifying activity (such as NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione-S-transferase (GST), and glutathione reductase (GR)) in muscles, counteracting damages caused by exhaustive exercise [15]. Among biomolecules susceptible to oxidation, cholesterol is able to form a wide range of oxidation products (so-called COPs or oxysterols) in the ring and the side chain of its structure, by means of enzymatic or chemical mechanisms. They can come from exogenous (diet) and endogenous (in
vivo) sources, and are known to be involved in fundamental functions in normal physiologic conditions, including the control of cholesterol homeostasis at cellular level [16]. The interaction with the nuclear receptor LXRα, which is highly expressed in the liver and several tissues, mediates the majority of their biological actions, thus regulating cholesterol metabolism of human body. However, there is a large research-supported evidence on the contribution of COPs to the onset and development of major chronic diseases (such as neurodegenerative processes, atherosclerosis, diabetes, osteoporosis and kidney failure), together with a series of negative biological effects (proinflammatory, pro-apoptotic, cytotoxic, carcinogenic and mutagenic) [17]. Moreover, it seems that COPs may also be involved on non-alcoholic fatty liver disease, thus causing liver damage [12]. Among COPs found in liver, 7α-hydroxycholesterol (7α-HC) is usually one of the most representative, as it is produced by cholesterol 7α-hydroxylase (CYP7/A1) to act as intermediate in the bile acid formation. In addition, cholesterol 27-hydroxylase (CYP27/A1), a mitochondrial P450 enzyme highly expressed in hepatocytes, is able to generate 27-hydroxycholesterol (27-HC). To control in vivo oxidation processes, it would be interesting to evaluate whether exhaustive exercise is related to an increase of lipid peroxidation in liver and whether dietary vegetable extracts containing phytochemicals (such as SF and glucosinolates present in broccoli extract) could represent a strategy to prevent/counteract ROS production and lipid peroxidation, including cholesterol oxidation. Therefore, the aim of the present study was to assess the protective effect of a dietary broccoli extract (BE) against liver cholesterol oxidation in rats subjected to acute exercise, focusing on the activity of antioxidant/detoxification phase 2 enzymes.
2. Materials and Methods 2.1 Materials Bio-Rad Bradford protein assay was supplied by Bio-Rad (Hercules, CA). NADP, NADPH, NADH, FAD, 5,5’-dithiobis(2-nitrobenzoic) acid (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), oxidized glutathione (GSSG), reduced glutathione (GSH), 4-amino-3-hydrazino-5-mercapto-1,2,4-
triazole (Purpald®), methanol, H2O2, EDTA, tert-butyl hydroperoxide, mammalian protease inhibitor mixture, cholest-5-en-3β-ol (cholesterol) (purity: 99%), β-sitosterol (purity: 60%), campesterol (purity: 37.5%), (24S)-ethylcholest-5,22-dien-3β-ol (stigmasterol) (purity: 95%), betulin (purity: 98%), cholest-5-en-3β,7β-diol (7β-hydroxycholesterol, 7β-HC) (purity: 90%), 5α,6α-epoxy-cholestan-3β-ol (α-epoxycholesterol, α-EC) (purity: 87%), 5β,6β-epoxy-cholestan-3βol (β-epoxycholesterol, β-EC) (purity: 80%), cholestan-3β,5α,6β-triol (cholestanetriol, triol) (purity: 99%) and cholest-5-en-3β-ol-7-one (7-ketocholesterol, 7-KC) (purity: 99%), were purchased from Sigma Chemical (St. Louis, MO). Tecklad 6% Mouse/Rat standard diet was supplied by Harlan Laboratories Inc. (Madison, WI). Broccoli extract capsules (Broccoli-Max® 400 mg) were purchased from Phenix srl. (Italy). n-hexane, chloroform and ethanol were obtained from Merck (Darmstadt,
Germany).
Double
distilled
water
and
the
silylating
agents
(pyridine,
hexamethyldisilazane and trimethylchlorosilane) were purchased from Carlo Erba (Milan, Italy). Potassium hydroxide and anhydrous sodium sulfate were supplied by Prolabo (Fontenay, France) and BDH (Poole, England), respectively. Cholest-5-en-3β,7α-diol (7α-hydroxycholesterol, 7α-HC) (purity: 99%) and cholest-5-en-3β,19-diol (19-hydroxycholesterol, 19-HC) (purity: 99%) were obtained from Steraloids (Newport, Rhode Island, USA). Cholest-5-en-3β,24(S)-diol (24(S)hydroxycholesterol, 24-HC) (purity: 99%) and cholest-5-en-3β,27-diol (27-hydroxycholesterol, 27HC) (purity: 99%) were purchased from Avanti Polar Lipids (Alabaster, Alabama, USA). N°1 filters (70 mm diameter) were supplied by Whatmann (Maidstone, England). Aminopropyl solidphase extraction (SPE) cartridges (Strata NH2-55 mm, 70A, 500 mg/3 mL) from Phenomenex (Torrence, CA, USA) were used for oxysterols purification.
2.2 Animals, diet and exercise protocol The study and its experimental protocol were approved by the Ethics Committee of the University of Bologna (prot. N. 58897-X/10 of November 20th 2008); 32 female Wistar rats (weight: 229±20 g age: 4 months) were caged in controlled conditions (22 °C and 12:12-h light-dark cycle) and
supplied with water and standard chow (Harlan Laboratories Inc.) ad libitum. Data on rats’ food intake were recorded for one week before the beginning of the study, as previously reported [18]; the mean food intake was 22.0±1.7 g/die. Rats were randomly divided into four groups of 8 animals each: group C was fed a standard diet for 45 days and was not subjected to the exhaustive exercise protocol; group B was fed a standard diet enriched with BE for 45 days and was not subjected to the exhaustive exercise protocol; group S was fed a standard diet for 45 days and subjected to a single acute exhaustive exercise session on day 45; group BS was fed a standard diet enriched with BE for 45 days and subjected to a single acute exhaustive exercise session on day 45. Standard diet provided macronutrients in the normal range of adequacy for rats (expressed in g/100 diet): 25 g of proteins, 36.9 g of carbohydrates, 6.2 g of lipids and suitable amounts of vitamins and minerals. BE-enriched diet was prepared by adding BE (see composition in paragraph 2.1) to the standard diet at the ratio of 0.025:1 (w/w; 2.5 mg of supplement/g of diet). As reported in certified product leaflet provided by the supplier, broccoli extract capsule (Broccoli-Max®) contained sulforaphane (1.2 mg, by HPLC), glucosinolates (24 mg, by HPLC), excipients (SiO2, cellulose, Mg stearate; 45 mg) and operculum (95 mg). Considering both the data on food intake and the BE composition provided by the supplement manufacturer, BE-enriched diet provided rats with an average daily intake of 0.15 mg SF (0.55 mg SF/kg bw/day). This dose is justified by the pharmacokinetic studies carried out by Hanlon et al. [19], which demonstrated that, for a wide range of doses of orally administered SF (0.5-5 mg SF/kg bw), its bioavailability decreases with increasing dosages. Moreover, in humans, a 0.55 mg/kg bw SF dose is contained in a 100 g portion of broccoli [20], which could be easily consumed with a regular diet. On day 38, animals were allowed to familiarize with the treadmill; they began walking on the treadmill and then they ran for 10 min at slow speed (7 m/min). On day 45, rats from S and BS groups performed an exhaustive running bout, with the treadmill slope set at +7% and speed risen up to 24 m/min. Animals were forced to run until exhaustion. When they were unable to get off the
treadmill back-wall they were repositioned by hand on the treadmill front for three consecutive times before considering them exhausted [15]. Figure 1 summarises the experimental design.
Figure 1. Experimental design.
2.3 Sample collection Rats of groups S and BS were sacrificed by decapitation as soon as they had completed the exhaustive exercise bout, while rats of groups C and B were sacrificed on day 45 without undergoing any exercise protocol. Blood was quickly withdrawn by the abdominal aorta; plasma samples were obtained by centrifugation (4 °C, 1, 10 min, 500 g) and then stored at -80 °C. Livers were collected, frozen in liquid nitrogen and stored at -80 °C. One hundred mg liver samples were homogenized in 1 mL of lysis buffer (0.1 M PBS pH 7.4, 1% EDTA, and mammalian protease inhibitor mixture) on ice for 90 s with a Teflon pestle homogenizer (Ika-Werke, Staufen, Germany) at 500 rpm. Liver homogenates were centrifuged (15 min, 10,000 g, 4 °C) and supernatants were stored at -80 °C for further enzymatic activity assays.
2.4 LDH activity assay
LDH activity assay was performed on plasma samples according to Korzeniewski et al. [21], as previously reported [15]. The assay monitors NADH oxidation coupled to the reduction of pyruvate to lactic acid. The absorbance variation was measured at 340 nm. Data are reported as U/mL plasma. One unit (U) of LDH activity is the amount of enzyme that reduces one mmol of pyruvate to D-lactate per min at pH 7.0 at 25 °C.
2.5 GST activity assay GST activity was measured according to Habig et al. [22], as previously described [15]. The analysis is based on the conjugation of GSH to CDNB, catalyzed by GST. Briefly, 20 L of liver homogenate were added to 980 L of reaction mix (0.1 M phosphate buffer pH 6.5, 1 mM EDTA, 2 mM GSH and 2 mM CDNB) and the absorbance was spectrophotometrically read at 340 nm. GST activity is expressed as mol of conjugated CDNB per min per milligram of protein.
2.6 GR activity assay GR activity was assayed according to Smith et al. [23], as previously reported [15]. The method is based on the GR-catalyzed reduction of GSSG to GSH, using NADPH as reducing agent; this reaction is coupled to the spontaneous reduction of DTNB to TNB, carried out by GSH. Fifty L of liver homogenate were mixed with 150 L of buffer (0.1 M phosphate buffer pH 7.5, 1 mM EDTA), 250 L of 3 mM DTNB, 50 L of 2 mM NADPH and 500 L of 2 mM GSSG solution. The absorbance was spectrophotometrically monitored at 412 nm. GR activity is expressed as mU/mg of protein. One unit of enzyme activity is defined as the amount of enzyme that reduces 1.0 mol of DTNB to TNB per min at 25 °C and pH 7.5.
2.7 GPx activity assay
GPx activity was spectrophotometrically assayed according to Flohe et al. [24], as previously described [15]. This method is based on the oxidation of GSH to GSSG coupled to its regeneration operated by GR, using NADPH as reducing agent. Fifty L of liver homogenate were diluted with 890 L buffer (0.05 M phosphate buffer pH 8.0, 0.5 mM EDTA), 25 L of reaction mixture (2 mM NADPH, 2mM GSH and GR) and 10 L of 30 mM tert-butyl hydroperoxide as reaction starter. The reduction in NADPH absorbance at 340 nm was recorded for 90 sec. GPx activity is expressed as mU/mg protein. One unit of GPx is defined as the amount of enzyme that reduces 1 mol of NADPH per min at 25 °C.
2.8 CAT activity assay CAT activity was measured according to Johansson et al. [25] as previously reported [26]. This method is based on the oxidation, catalyzed by CAT, of methanol in presence of hydrogen peroxide. The formaldehyde produced is measured spectrophotometrically at 540 nm, using 4-amino-3hydrazino-5-mercapto-1,2,4-triazole (Purpald®) as chromogen. CAT activity was expressed as nmol/min/mg protein.
2.9 Protein concentration The protein concentration of the liver samples was determined by using the Bio-Rad Bradford protein assay.
2.10 Determination of sterol composition and cholesterol oxidation products The unsaponifiable matter of livers was first isolated [27] and then analyzed by Fast-GC/MS. Briefly, about 200 mg of liver were added with internal standards (betulin (0.140 mg) and 19hydrohycholesterol (0.012 mg) for sterols and COPs quantification, respectively) and 3 mL of a KOH solution in methanol (4 mol/L) containing BHT (5 mg/mL), to carry out an 18-h cold saponification at room temperature under darkness. Thereafter, 10 mL of dichloromethane and 5
mL of citric acid (0.1% double distilled water) were added to each tube and were mixed thoroughly in a vortex. Samples were then centrifuged at 3,000 rpm for 10 min; after phase separation, the organic phase was isolated and washed with 5 mL citric acid (0.1%, v/v) solution. The dichloromethane phase was evaporated at 40 °C with nitrogen and the residues were dissolved in 5 mL of dichlorometane. One tenth of the extracted unsaponifiable matter was used for the determination of sterol composition, while the 9/10 were used for COPs purification by SPE-NH2 [28]. The silylated sterol and COPs were analyzed by Fast-GC/MS as described by Inchingolo et al. [29] and Cardenia et al. [30], respectively. Briefly, the identification of sterols and COPs was carried out comparing their mass spectra and retention times with those of the corresponding chemical standards. The quantification of sterols and COPs amounts was performed in the SIM acquisition mode, by using calibration curves built for each chemical compound with betulin and 19-hydroxycholesterol as internal standards, respectively.
2.11 Statistical analysis Data are reported as mean ± SD of eight animals (n=8). One-way ANOVA, followed by Bonferroni’s test, was used to compare means in LDH, GST, GPx, GR and CAT activities among groups. Factorial analysis of variance (ANOVA) was carried out to investigate the impact of the different dietary treatments (with or without exhaustive exercise protocol) on sterol composition and the oxidative parameters, as well as their interactions. Tukey's honest significance test was carried out at a 95% confidence level (p ≤ 0.05), to separate means of parameters and interactions that were statistically different. Principal component analysis (PCA) was also performed on all data set, to better understand the data variability. Statistical analysis of the data was carried out by using SPSS 16.0.1 (2007, IBM-SPSS Inc., Chicago, IL, USA) and GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA).
3. Results
3.1 Enzymatic activity Figure 2 shows the effect of BE-enriched diet and acute exhaustive exercise on rats’ plasma LDH activity. The latter significantly raised in the S group with respect to the C, B and BS groups. Moreover, plasma LDH activity in the BS group was similar to that of the C one, suggesting the possibility that a BE-enriched diet is able to counteract damages induced by acute, exhaustive exercise.
Figure 2. Effect of BE-enriched diet and acute exhaustive exercise on plasma LDH activity. LDH activity was measured as described in Materials and Methods. Each bar represents the mean ± SD of 8 rats. Rats were randomly assigned to four groups: controls (C), subjected to physical exercise (S), fed a BE-enriched diet (B), and subjected to physical exercise and BE-enriched diet (BS). The S and BS ran an acute exercise bout, as described in the Materials and Methods section. Data were analyzed by one-way ANOVA, followed by the Bonferroni post-test. Different letters (a and b) indicate statistically different means (p < 0.05).
Figure 3 (A-D) shows the effects of acute exhaustive exercise and BE-enriched diet on GST, GR, GPx, and CAT activity in liver homogenate. Exercise had no influence on GST enzyme activity, while BE dietary treatment significantly increased GST activity in BS group with respect to both C and S groups (Figure 3A).
Likewise, GR activity was not affected by exercise, whereas BE-enriched diet caused a significant rise in GR activity in BS group as compared with S group (Figure 3B). Neither exercise protocol nor dietary treatment showed any effect on GPx activity (Figure 3C). Exhaustive exercise induced a significant reduction of CAT activity with respect to control group, while BE-enriched diet was able to restore CAT activity at a similar level to that observed in control group (Figure 3D).
Figure 3. Effect of BE-enriched diet and acute exhaustive exercise on GST (A), GR (B), GPx (C) and CAT (D) enzymatic activities in liver homogenate. Enzyme activities were measured in liver homogenates as described in the Materials and Methods section. The B and BS groups were fed a diet enriched with BE. The S and BS ran an acute exercise bout, as described in the Materials and Methods section. Each bar represents the mean ± SD of 8 rats. Data were analyzed by one-way ANOVA followed by the Bonferroni post-test. Different letters (a, b and c) indicate statistically different means (p < 0.05).
3.2 Sterols content Table 1 reports the effects of BE-enriched diet and acute exhaustive exercise on sterol content of rats’ livers. Total sterols (0.9–1.2 mg/g liver) were mainly composed by cholesterol (0.8–1.2 mg/g liver), followed by -sitosterol and campesterol. In spite of BE consumption and exhaustive exercise, no significant differences were found among different treatments.
Table 1. Effect of BE-enriched diet and acute exhaustive exercise on sterol content (mg sterol/g liver) of rats’ livers. Treatment
Cholesterol
Campesterol
-sitosterol
Total sterols
Mean
SD
Mean
SD
Mean
SD
Mean
SD
C
0.78
0.34
0.04
0.01
0.06
0.01
0.86
0.37
B
1.04
0.37
0.04
0.01
0.07
0.01
1.17
0.34
S
0.79
0.28
0.04
0.01
0.07
0.01
0.90
0.27
BS
1.19
0.22
0.05
0.01
0.08
0.03
1.31
0.25
Stat. Sign.
ns
ns
ns
ns
Sterol content and composition were obtained as reported in the Materials and Methods section. Each value represents the mean of 8 rats. The B and BS groups were fed a diet enriched with BE. The S and BS ran an acute exercise bout, as described in the Materials and Methods section. Abbreviations: SD, standard deviation; Stat. Sign., statistical significant; ns, not significant. Data were analyzed by Tukey’s test (p < 0.05).
3.3 Cholesterol oxidation products (COPs) Regarding cholesterol oxidation, COPs level ranged from 0.24 to 0.98 g/g liver (Figure 4). Acute exhaustive exercise significantly increased cholesterol oxidation level from 0.50 g of COPs/g liver in the C group to 0.98 g of COPs/g liver in the S group. However, BE was able to significantly
decrease oxysterols, counteracting their accumulation when exhaustive exercise was performed (0.24 g/g liver).
Figure 4. Effect of BE-enriched diet and exhaustive exercise on total COPs contents (g COPs/g liver). The B and BS groups were fed a diet enriched with BE. The S and BS ran an acute exercise bout, as described in the Materials and Methods section. Each bar represents the mean ± SD of 8 rats. Data were analyzed by Tukey’s test. Different letters (a, b and c) indicate statistically different means (p < 0.05). The main oxysterol was 7-HC (0.11–0.53 g/g liver), which significantly increased under exhaustive exercise. A similar behavior was displayed by 7-KC (0.04–0.19 g/g liver) and 27-HC (0.01–0.03 g/g liver). Low amounts of triol and 24-HC were also found (Figure 5). Finally, the cholesterol oxidation ratio (OR %, Table S1) confirmed the antioxidant effect of BE, as supplementation under exhaustive exercise reduced the oxidation ratio (0.02%) as compared to the corresponding non-supplemented treatment (0.22%).
Figure 5. Effect of BE-enriched diet and exhaustive exercise on single cholesterol oxidation product (g COP/g liver) contents. The B and BS groups were fed a diet enriched with BE. The S and BS ran an acute exercise bout, as described in the Materials and Methods section. Each bar represents the mean ± SD of 8 rats. Data were analyzed by Tukey’s test. Different letters (a, b and c) for each COP indicate statistically different means (p < 0.05).
3.4 Principal component analysis of data (PCA) PCA performed with data from enzyme activities and oxidative parameters (Figure 6) was able to distinguish different clusters, explaining 92.22% of total variance in two principal components. The first principal component (F1, 81.85%) clearly separated the single and total COPs from antioxidant enzymes (GST, GR, GPx and CAT), highlighting their indirect correlations. PCA performed with data from different treatments was able to recognize cluster trends of diverse treatments that are well separated on the four different regions of the biplot. S treatment was highly characterized by LDH activity and the presence of single and total COPs, thus evidencing a structural tissue damage as well as an inflammatory process and oxidative stress. On the other hand, BS treatment was more characterized by antioxidant enzymes (GR, GPx and GST), suggesting the protective role of BE supplemented diet against exhaustive exercise-induced stress.
Figure 6. PCA score plot of single and total COPs, LDH, GPx, GST, Catalase, GR activity and crossed treatments.
4. Discussion Skeletal muscles are not the only tissue affected by exercise, since the metabolism of other organs is modulated by physical activity. In particular, liver plays a crucial role during exercise, due to its involvement in maintaining glucose, lipid and amino acid homeostasis; in fact, it has been demonstrated that physical activity activates a series of molecular events leading to the adaptation of liver metabolism to exercise training [31]. On the contrary, exhaustive exercise induces structural damage in liver as reported by Huang et al. [5], who demonstrated that rats undergoing exhaustive exercise showed biomarkers of strong inflammation in liver, such as inflammatory cell infiltration and tissue disruption. Acute exhaustive exercise also induces oxidative stress in liver tissue. Many
authors have reported an increased level of oxidative stress biomarkers (such as malondialdehyde, thiobarbituric acid reactive substances and protein carbonyls), xanthine oxidase activity, a decrease in GSH level, and a reduction of superoxide dismutase (SOD) and CAT activities in livers from rats that underwent exhaustive exercise [32-35]. In this study, as previously reported, plasma LDH activity, a well-known unspecific biomarker of tissue damage, strongly increased after acute exercise, indicating the deleterious effects of this type of activities [15,36-37]. Rats fed the BE-enriched diet showed a significantly reduced LDH plasma level, indicating that BE was able to prevent exercise-induced tissue damage. The cumulative effects of BE treatment on phase 2 antioxidant enzyme activities suggest that BE is able to counteract exercise-promoted oxidative stress, by inducing important antioxidant enzymes. Recently, Yioshida et al. [38] reported that 10 days of a BE supplemented diet induces GST activity in rat liver. Similar results were observed in the present study, since the BE-enriched diet significantly improved GST activity in rats subjected to exhaustive exercise. Many studies [15,32,34,39] found that GR activity is not affected after exhaustive exercise; accordingly, we did not observe any effects of the exercise protocol on GR liver activity, but the BE supplemented diet significantly improved GR in BS with respect to S group. Neither BE supplementation nor exhaustive exercise had any effect on GPx activity. Previous research in endothelial cells demonstrated that SF does not induce GPx [40]; it has been also demonstrated that, in rat skeletal muscle, both SF treatment and exhaustive running do not affect GPx activity [15]. The impact of exercise on CAT expression and activity in liver has been investigated since early ’90s, with poor consensus among different studies [40-41,48-49]. These discrepancies could be ascribed to the diverse exercise protocols and the different elapsed time between the end of the exercise and the sample collection. In the present study, CAT activity significantly decreased after exhaustive exercise and, interestingly, BE-enriched diet completely prevent CAT activity decrease. It is well known that CAT is in charge of hydrogen peroxide detoxification, so a reduction of its activity may result in the accumulation of such ROS. Preventing CAT decrease through BE supplementation,
may represent a key effect of broccoli bioactive compounds in counteracting oxidative stress in liver. According to literature [43], the main phytosterols in broccoli are sitosterol, campesterol and stigmasterol (4-desmethylsterols). It is well known that phytosterols display hypocholesterolemic and anti-inflammatory activities and that sitosterol is also used for treatment of prostatic diseases [44]. As reported in Table 1, cholesterol represented the most abundant sterol (88-91% of total sterols) in liver and, as expected, the main phytosterols were sitosterol and campesterol. However, BE-enriched diet and exhaustive exercise did not significantly (p > 0.05) affect sterol composition and thus they did not favor a differentiated accumulation of sterols. Oxysterols play a critical role in many regulatory processes; in particular, oxysterols activate ligands of liver X receptor and are also involved in diverse human diseases, such as fat induced injury [45] and liver injury in non-alcoholic fatty liver disease [16]. In addition, oxysterols (mainly 7-HC and 27-HC) are intermediates in the bile acid synthesis, which are engaged in the cholesterol homeostasis by suppression of the LDL receptor and the HMG-CoA reductase [46]. However, modifications on the COPs levels in plasma and liver have been observed in both obese mice and patients, which arise to some extent from a higher production of ROS [47]. The results of this study evidence that BE was able to prevent cholesterol oxidation, counteracting COPs increase induced by in vivo auto-oxidation and enzymatic oxidation. Guerrero-Beltràn et al. [48] reported the effect of SF against oxidative stress in liver and other tissues; in particular, the authors found that SF upregulates the expression of GST (π class) in liver cells, inducing the phase 2 detoxifying enzyme system. These observations could explain the lower amount of total COPs detected in rats fed a BE-enriched diet. In the present study, the single COPs displayed a similar behavior. As expected, the main single COP was 7-HC, since it can be generated by CYP7A1-mediated enzymatic oxidation; such enzyme
is,
in
fact,
largely
expressed
in
hepatocytes
and
ovary
cells
[49].
7-
hydroperoxycholesterol originated by auto-oxidation is unstable and isomerizes into 7-
hydroperoxycholesterol, which evolves into 7-HC and 7-KC; 7-hydroperoxyl radicals can also generate /-epoxycholesterol isomers, by reacting with a cholesterol molecule [50]. Exhaustive exercise without any BE dietary supplementation promoted 27-HC accumulation in rat liver; such oxysterol is known to be produced by mitochondrial sterol 27-hydroxylase (CYP27A1). On the other hand, the BE-enriched diet was able to significantly reduce 24-HC production. 24-HC, also called cerebrosterol is generated by 24-hydroxylase (CYP46A1), which is largely expressed in neurons and neural retina [49]. A reduction of 24-HC accumulation at neuronal level might result in the protection of neuronal tissue; however, further studies are needed to clarify whether BE administration exerts a protective role in neurones, which could become an additional neuroprotective effect to be ascribed to broccoli bioactive compounds besides those that had already been reported [14]. Finally, triol was detected in the liver samples analyzed, but no 5,6/-epoxy cholesterol isomers were found; this fact could be due to the total conversion of /-EC isomers into triol, which is a chemical equilibrium favored in specific environmental conditions (presence of water and acid pH) [30]. Finally, the PCA analysis confirmed that BE was able to counteract COPs accumulation promoted by exhaustive exercise in rat liver, by inducing antioxidant phase 2 enzymes (CAT, GST, GR), and to prevent tissue damage. The effect of the BE-enriched diet in the prevention of cholesterol oxidation in rat liver suggests the possibility to utilize phytochemicals in the modulation of the liver redox environment and in the prevention of oxidative damage.
Acknowledgments We thank Basic Research Funding (RFO 0 Italy) for financial support.
The authors have declared no conflict of interest.
,
lma Mater Studiorum-Universit di Bologna,
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Supplementary material Table S1. Effect of BE-enriched diet and exhaustive exercise on the content of single and total COPs (Tot COPs) in rats’ liver (g/g liver), and the cholesterol oxidation ratio (%). Treatment
7α-HC Mean
SD
7β-HC Mean
SD
Triol Mean
SD
24-HC Mean
SD
27-HC Mean
SD
7-KC Mean
SD
Tot COPs Mean
SD
OR Mean
SD
C
0.312 0.006 b
0.070 0.003 b
0.043 0.021 ab
0.013 0.006 ab
0.017 0.000 b
0.098 0.014 b
0.518 0.067 b
0.077 0.031 ab
B
0.236 0.010 c
0.051 0.005 c
0.032 0.005 b
0.010 0.000 b
0.014 0.001 b
0.077 0.006 b
0.410 0.024 bc
0.050 0.010 b
S
0.527 0.027 a
0.111 0.004 a
0.070 0.006 a
0.023 0.006 a
0.029 0.001 a
0.193 0.014 a
1.014 0.159 a
0.221 0.128 a
BS
0.109 0.006 d
0.026 0.001 d
0.014 0.001 b
0.010 0.000 b
0.007 0.001 c
0.041 0.006 c
0.243 0.080 c
0.020 0.010 b
Each value represents the mean and standard deviation (SD) of 8 rats/group. Data were analysed by Tukey’s test. Different letters (a, b and c) indicate statistically different means (p < 0.05).
S0960-0760(16)30101-7 http://dx.doi.org/doi:10.1016/j.jsbmb.2016.04.005 SBMB 4698
To appear in:
Journal of Steroid Biochemistry & Molecular Biology
Received date: Revised date: Accepted date:
30-11-2015 7-4-2016 11-4-2016
Please cite this article as: Vladimiro Cardenia, Maria Teresa Rodriguez-Estrada, Antonello Lorenzini, Erika Bandini, Cristina Angeloni, Silvana Hrelia, Marco Malaguti, Effect of broccoli extract enriched diet on liver cholesterol oxidation in rats subjected to exhaustive exercise, Journal of Steroid Biochemistry and Molecular Biology http://dx.doi.org/10.1016/j.jsbmb.2016.04.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
EFFECT OF BROCCOLI EXTRACT ENRICHED DIET ON LIVER CHOLESTEROL OXIDATION IN RATS SUBJECTED TO EXHAUSTIVE EXERCISE
Vladimiro Cardenia 1*, Maria Teresa Rodriguez-Estrada 1,2, Antonello Lorenzini 3, Erika Bandini 4, Cristina Angeloni 5, Silvana Hrelia 5, Marco Malaguti5
1
Department of Agricultural and Food Sciences, Alma Mater Studiorum – University of Bologna
(Bologna, Italy) 2
Interdepartmental Centre for Agri-Food Industrial Research, Alma Mater Studiorum – University
of Bologna (Cesena, Italy) 3
Department of Biomedical and Neuromotor Sciences, Alma Mater Studiorum – University of
Bologna (Bologna, Italy) 4
Scientific Institute of Romagna for the Study and Treatment of Cancer (IRST), Unit of Gene
Therapy Meldola-Forlı’, Meldola (FC), Italy 5
Department for Life Quality Studies, Alma Mater Studiorum – University of Bologna (Rimini,
Italy)
*
Corresponding author:
Viale Fanin 40 40127 Bologna (Italy) Tel. +39-051-2096015 Fax: +39-051-2096017 E-mail: [email protected]
Graphical Abstract
Highlights
The broccoli extract reduced cholesterol oxidation after exhaustive exercise The broccoli extract enriched diet increased antioxidant phase 2 enzyme activity Phytochemicals could be useful in the prevention of liver oxidative damage
Abstract The effect of broccoli extract (BE)-enriched diet was studied in order to evaluate its ability to counteract liver cholesterol oxidation products (COPs) induced by acute strenuous exercise in rats. Thirty-two female Wistar rats were randomly divided into four groups: control diet without exercise (C), BE-enriched diet without exercise (B), control diet with acute exhaustive exercise (S) and BEenriched diet with acute exhaustive exercise (BS). The study lasted 45 days and on the last day, rats of S and BS groups were forced to run until exhaustion on a treadmill. Glutathione-S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx), catalase (CAT) and cholesterol oxidation products (COPs) were determined in liver. Exhaustive exercise was clearly responsible for tissue damage, as evidenced by the increase of lactate dehydrogenase (LDH) plasma activity in the S group. Moreover, the exercise protocol reduced CAT activity in liver, while it did not affect GST, GR and GPx. BE-enriched diet raised GST, GR and CAT activities in rats of BS group. The main COPs found were 7α-hydroxycholesterol, 7β-hydroxycholesterol, 7-ketocholesterol, cholestanetriol, 24-hydroxycholesterol and 27-hydroxycholesterol. The BE-enriched diet led to reduced cholesterol oxidation following exhaustive exercise; the highest level of COPs was found in the S group, whereas the BS rats showed the lowest amount. This study indicates that the BEenriched diet increases antioxidant enzyme activities and exerts an antioxidant effect towards cholesterol oxidation in rat liver, suggesting the use of phytochemicals in the prevention of oxidative damage and in the modulation of the redox environment.
Keywords: Cholesterol oxidation products; broccoli extract; exhaustive exercise; oxidative stress; antioxidant phase 2 enzymes; oxysterols.
1. Introduction The interest on physical exercise has enormously risen in the last decades and a large amount of scientific literature has been published. The success of this topic partly lies on the beneficial effects induced by physical exercise on human health [1, 2]. However, exhaustive exercise can be quite harmful, since it is responsible for a burst of oxidative stress, which is followed by an inflammatory response and a consequent structural damage to muscle fibers; the release of cytosolic enzymes (i.e. lactic dehydrogenase (LDH) and creatine kinase (CK)) in plasma, confirms the occurrence of such events [3]. Exhaustive exercise does not only affect muscle cells homeostasis and viability, but it also exerts deleterious effects on different tissues and organs. At both cardiac and hepatic levels, strenuous activity has been related to functional impairment, oxidative stress, activation of apoptotic signaling pathways and inflammation [4-6]. Due to its deleterious effects, many nutritional interventions have been studied to counteract exhaustive exercise induced damage. In this context, unfortunately, the supplementation with ROS scavengers (such as antioxidant vitamins) seems unable to prevent exhaustive exercise stress [7]. On the other hand, foods rich in indirect antioxidants or molecules able to induce antioxidant/detoxifying systems might represent an alternative nutritional approach to counteract exhaustive exercise induced stress. One example of this type of bioactive compounds is sulforaphane (SF), an isothiocyanate found in cruciferous vegetables (such as broccoli), which can be introduced through the diet. SF was initially studied for its promising chemopreventive activity [8], thanks to its ability to induce phase II enzymes both in vitro and in vivo [9-14]. In a recent study carried out in rats, it was also confirmed that SF treatment induces enzymes with an antioxidant/detoxifying activity (such as NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione-S-transferase (GST), and glutathione reductase (GR)) in muscles, counteracting damages caused by exhaustive exercise [15]. Among biomolecules susceptible to oxidation, cholesterol is able to form a wide range of oxidation products (so-called COPs or oxysterols) in the ring and the side chain of its structure, by means of enzymatic or chemical mechanisms. They can come from exogenous (diet) and endogenous (in
vivo) sources, and are known to be involved in fundamental functions in normal physiologic conditions, including the control of cholesterol homeostasis at cellular level [16]. The interaction with the nuclear receptor LXRα, which is highly expressed in the liver and several tissues, mediates the majority of their biological actions, thus regulating cholesterol metabolism of human body. However, there is a large research-supported evidence on the contribution of COPs to the onset and development of major chronic diseases (such as neurodegenerative processes, atherosclerosis, diabetes, osteoporosis and kidney failure), together with a series of negative biological effects (proinflammatory, pro-apoptotic, cytotoxic, carcinogenic and mutagenic) [17]. Moreover, it seems that COPs may also be involved on non-alcoholic fatty liver disease, thus causing liver damage [12]. Among COPs found in liver, 7α-hydroxycholesterol (7α-HC) is usually one of the most representative, as it is produced by cholesterol 7α-hydroxylase (CYP7/A1) to act as intermediate in the bile acid formation. In addition, cholesterol 27-hydroxylase (CYP27/A1), a mitochondrial P450 enzyme highly expressed in hepatocytes, is able to generate 27-hydroxycholesterol (27-HC). To control in vivo oxidation processes, it would be interesting to evaluate whether exhaustive exercise is related to an increase of lipid peroxidation in liver and whether dietary vegetable extracts containing phytochemicals (such as SF and glucosinolates present in broccoli extract) could represent a strategy to prevent/counteract ROS production and lipid peroxidation, including cholesterol oxidation. Therefore, the aim of the present study was to assess the protective effect of a dietary broccoli extract (BE) against liver cholesterol oxidation in rats subjected to acute exercise, focusing on the activity of antioxidant/detoxification phase 2 enzymes.
2. Materials and Methods 2.1 Materials Bio-Rad Bradford protein assay was supplied by Bio-Rad (Hercules, CA). NADP, NADPH, NADH, FAD, 5,5’-dithiobis(2-nitrobenzoic) acid (DTNB), 1-chloro-2,4-dinitrobenzene (CDNB), oxidized glutathione (GSSG), reduced glutathione (GSH), 4-amino-3-hydrazino-5-mercapto-1,2,4-
triazole (Purpald®), methanol, H2O2, EDTA, tert-butyl hydroperoxide, mammalian protease inhibitor mixture, cholest-5-en-3β-ol (cholesterol) (purity: 99%), β-sitosterol (purity: 60%), campesterol (purity: 37.5%), (24S)-ethylcholest-5,22-dien-3β-ol (stigmasterol) (purity: 95%), betulin (purity: 98%), cholest-5-en-3β,7β-diol (7β-hydroxycholesterol, 7β-HC) (purity: 90%), 5α,6α-epoxy-cholestan-3β-ol (α-epoxycholesterol, α-EC) (purity: 87%), 5β,6β-epoxy-cholestan-3βol (β-epoxycholesterol, β-EC) (purity: 80%), cholestan-3β,5α,6β-triol (cholestanetriol, triol) (purity: 99%) and cholest-5-en-3β-ol-7-one (7-ketocholesterol, 7-KC) (purity: 99%), were purchased from Sigma Chemical (St. Louis, MO). Tecklad 6% Mouse/Rat standard diet was supplied by Harlan Laboratories Inc. (Madison, WI). Broccoli extract capsules (Broccoli-Max® 400 mg) were purchased from Phenix srl. (Italy). n-hexane, chloroform and ethanol were obtained from Merck (Darmstadt,
Germany).
Double
distilled
water
and
the
silylating
agents
(pyridine,
hexamethyldisilazane and trimethylchlorosilane) were purchased from Carlo Erba (Milan, Italy). Potassium hydroxide and anhydrous sodium sulfate were supplied by Prolabo (Fontenay, France) and BDH (Poole, England), respectively. Cholest-5-en-3β,7α-diol (7α-hydroxycholesterol, 7α-HC) (purity: 99%) and cholest-5-en-3β,19-diol (19-hydroxycholesterol, 19-HC) (purity: 99%) were obtained from Steraloids (Newport, Rhode Island, USA). Cholest-5-en-3β,24(S)-diol (24(S)hydroxycholesterol, 24-HC) (purity: 99%) and cholest-5-en-3β,27-diol (27-hydroxycholesterol, 27HC) (purity: 99%) were purchased from Avanti Polar Lipids (Alabaster, Alabama, USA). N°1 filters (70 mm diameter) were supplied by Whatmann (Maidstone, England). Aminopropyl solidphase extraction (SPE) cartridges (Strata NH2-55 mm, 70A, 500 mg/3 mL) from Phenomenex (Torrence, CA, USA) were used for oxysterols purification.
2.2 Animals, diet and exercise protocol The study and its experimental protocol were approved by the Ethics Committee of the University of Bologna (prot. N. 58897-X/10 of November 20th 2008); 32 female Wistar rats (weight: 229±20 g age: 4 months) were caged in controlled conditions (22 °C and 12:12-h light-dark cycle) and
supplied with water and standard chow (Harlan Laboratories Inc.) ad libitum. Data on rats’ food intake were recorded for one week before the beginning of the study, as previously reported [18]; the mean food intake was 22.0±1.7 g/die. Rats were randomly divided into four groups of 8 animals each: group C was fed a standard diet for 45 days and was not subjected to the exhaustive exercise protocol; group B was fed a standard diet enriched with BE for 45 days and was not subjected to the exhaustive exercise protocol; group S was fed a standard diet for 45 days and subjected to a single acute exhaustive exercise session on day 45; group BS was fed a standard diet enriched with BE for 45 days and subjected to a single acute exhaustive exercise session on day 45. Standard diet provided macronutrients in the normal range of adequacy for rats (expressed in g/100 diet): 25 g of proteins, 36.9 g of carbohydrates, 6.2 g of lipids and suitable amounts of vitamins and minerals. BE-enriched diet was prepared by adding BE (see composition in paragraph 2.1) to the standard diet at the ratio of 0.025:1 (w/w; 2.5 mg of supplement/g of diet). As reported in certified product leaflet provided by the supplier, broccoli extract capsule (Broccoli-Max®) contained sulforaphane (1.2 mg, by HPLC), glucosinolates (24 mg, by HPLC), excipients (SiO2, cellulose, Mg stearate; 45 mg) and operculum (95 mg). Considering both the data on food intake and the BE composition provided by the supplement manufacturer, BE-enriched diet provided rats with an average daily intake of 0.15 mg SF (0.55 mg SF/kg bw/day). This dose is justified by the pharmacokinetic studies carried out by Hanlon et al. [19], which demonstrated that, for a wide range of doses of orally administered SF (0.5-5 mg SF/kg bw), its bioavailability decreases with increasing dosages. Moreover, in humans, a 0.55 mg/kg bw SF dose is contained in a 100 g portion of broccoli [20], which could be easily consumed with a regular diet. On day 38, animals were allowed to familiarize with the treadmill; they began walking on the treadmill and then they ran for 10 min at slow speed (7 m/min). On day 45, rats from S and BS groups performed an exhaustive running bout, with the treadmill slope set at +7% and speed risen up to 24 m/min. Animals were forced to run until exhaustion. When they were unable to get off the
treadmill back-wall they were repositioned by hand on the treadmill front for three consecutive times before considering them exhausted [15]. Figure 1 summarises the experimental design.
Figure 1. Experimental design.
2.3 Sample collection Rats of groups S and BS were sacrificed by decapitation as soon as they had completed the exhaustive exercise bout, while rats of groups C and B were sacrificed on day 45 without undergoing any exercise protocol. Blood was quickly withdrawn by the abdominal aorta; plasma samples were obtained by centrifugation (4 °C, 1, 10 min, 500 g) and then stored at -80 °C. Livers were collected, frozen in liquid nitrogen and stored at -80 °C. One hundred mg liver samples were homogenized in 1 mL of lysis buffer (0.1 M PBS pH 7.4, 1% EDTA, and mammalian protease inhibitor mixture) on ice for 90 s with a Teflon pestle homogenizer (Ika-Werke, Staufen, Germany) at 500 rpm. Liver homogenates were centrifuged (15 min, 10,000 g, 4 °C) and supernatants were stored at -80 °C for further enzymatic activity assays.
2.4 LDH activity assay
LDH activity assay was performed on plasma samples according to Korzeniewski et al. [21], as previously reported [15]. The assay monitors NADH oxidation coupled to the reduction of pyruvate to lactic acid. The absorbance variation was measured at 340 nm. Data are reported as U/mL plasma. One unit (U) of LDH activity is the amount of enzyme that reduces one mmol of pyruvate to D-lactate per min at pH 7.0 at 25 °C.
2.5 GST activity assay GST activity was measured according to Habig et al. [22], as previously described [15]. The analysis is based on the conjugation of GSH to CDNB, catalyzed by GST. Briefly, 20 L of liver homogenate were added to 980 L of reaction mix (0.1 M phosphate buffer pH 6.5, 1 mM EDTA, 2 mM GSH and 2 mM CDNB) and the absorbance was spectrophotometrically read at 340 nm. GST activity is expressed as mol of conjugated CDNB per min per milligram of protein.
2.6 GR activity assay GR activity was assayed according to Smith et al. [23], as previously reported [15]. The method is based on the GR-catalyzed reduction of GSSG to GSH, using NADPH as reducing agent; this reaction is coupled to the spontaneous reduction of DTNB to TNB, carried out by GSH. Fifty L of liver homogenate were mixed with 150 L of buffer (0.1 M phosphate buffer pH 7.5, 1 mM EDTA), 250 L of 3 mM DTNB, 50 L of 2 mM NADPH and 500 L of 2 mM GSSG solution. The absorbance was spectrophotometrically monitored at 412 nm. GR activity is expressed as mU/mg of protein. One unit of enzyme activity is defined as the amount of enzyme that reduces 1.0 mol of DTNB to TNB per min at 25 °C and pH 7.5.
2.7 GPx activity assay
GPx activity was spectrophotometrically assayed according to Flohe et al. [24], as previously described [15]. This method is based on the oxidation of GSH to GSSG coupled to its regeneration operated by GR, using NADPH as reducing agent. Fifty L of liver homogenate were diluted with 890 L buffer (0.05 M phosphate buffer pH 8.0, 0.5 mM EDTA), 25 L of reaction mixture (2 mM NADPH, 2mM GSH and GR) and 10 L of 30 mM tert-butyl hydroperoxide as reaction starter. The reduction in NADPH absorbance at 340 nm was recorded for 90 sec. GPx activity is expressed as mU/mg protein. One unit of GPx is defined as the amount of enzyme that reduces 1 mol of NADPH per min at 25 °C.
2.8 CAT activity assay CAT activity was measured according to Johansson et al. [25] as previously reported [26]. This method is based on the oxidation, catalyzed by CAT, of methanol in presence of hydrogen peroxide. The formaldehyde produced is measured spectrophotometrically at 540 nm, using 4-amino-3hydrazino-5-mercapto-1,2,4-triazole (Purpald®) as chromogen. CAT activity was expressed as nmol/min/mg protein.
2.9 Protein concentration The protein concentration of the liver samples was determined by using the Bio-Rad Bradford protein assay.
2.10 Determination of sterol composition and cholesterol oxidation products The unsaponifiable matter of livers was first isolated [27] and then analyzed by Fast-GC/MS. Briefly, about 200 mg of liver were added with internal standards (betulin (0.140 mg) and 19hydrohycholesterol (0.012 mg) for sterols and COPs quantification, respectively) and 3 mL of a KOH solution in methanol (4 mol/L) containing BHT (5 mg/mL), to carry out an 18-h cold saponification at room temperature under darkness. Thereafter, 10 mL of dichloromethane and 5
mL of citric acid (0.1% double distilled water) were added to each tube and were mixed thoroughly in a vortex. Samples were then centrifuged at 3,000 rpm for 10 min; after phase separation, the organic phase was isolated and washed with 5 mL citric acid (0.1%, v/v) solution. The dichloromethane phase was evaporated at 40 °C with nitrogen and the residues were dissolved in 5 mL of dichlorometane. One tenth of the extracted unsaponifiable matter was used for the determination of sterol composition, while the 9/10 were used for COPs purification by SPE-NH2 [28]. The silylated sterol and COPs were analyzed by Fast-GC/MS as described by Inchingolo et al. [29] and Cardenia et al. [30], respectively. Briefly, the identification of sterols and COPs was carried out comparing their mass spectra and retention times with those of the corresponding chemical standards. The quantification of sterols and COPs amounts was performed in the SIM acquisition mode, by using calibration curves built for each chemical compound with betulin and 19-hydroxycholesterol as internal standards, respectively.
2.11 Statistical analysis Data are reported as mean ± SD of eight animals (n=8). One-way ANOVA, followed by Bonferroni’s test, was used to compare means in LDH, GST, GPx, GR and CAT activities among groups. Factorial analysis of variance (ANOVA) was carried out to investigate the impact of the different dietary treatments (with or without exhaustive exercise protocol) on sterol composition and the oxidative parameters, as well as their interactions. Tukey's honest significance test was carried out at a 95% confidence level (p ≤ 0.05), to separate means of parameters and interactions that were statistically different. Principal component analysis (PCA) was also performed on all data set, to better understand the data variability. Statistical analysis of the data was carried out by using SPSS 16.0.1 (2007, IBM-SPSS Inc., Chicago, IL, USA) and GraphPad Prism 6.0 (GraphPad Software Inc., San Diego, CA, USA).
3. Results
3.1 Enzymatic activity Figure 2 shows the effect of BE-enriched diet and acute exhaustive exercise on rats’ plasma LDH activity. The latter significantly raised in the S group with respect to the C, B and BS groups. Moreover, plasma LDH activity in the BS group was similar to that of the C one, suggesting the possibility that a BE-enriched diet is able to counteract damages induced by acute, exhaustive exercise.
Figure 2. Effect of BE-enriched diet and acute exhaustive exercise on plasma LDH activity. LDH activity was measured as described in Materials and Methods. Each bar represents the mean ± SD of 8 rats. Rats were randomly assigned to four groups: controls (C), subjected to physical exercise (S), fed a BE-enriched diet (B), and subjected to physical exercise and BE-enriched diet (BS). The S and BS ran an acute exercise bout, as described in the Materials and Methods section. Data were analyzed by one-way ANOVA, followed by the Bonferroni post-test. Different letters (a and b) indicate statistically different means (p < 0.05).
Figure 3 (A-D) shows the effects of acute exhaustive exercise and BE-enriched diet on GST, GR, GPx, and CAT activity in liver homogenate. Exercise had no influence on GST enzyme activity, while BE dietary treatment significantly increased GST activity in BS group with respect to both C and S groups (Figure 3A).
Likewise, GR activity was not affected by exercise, whereas BE-enriched diet caused a significant rise in GR activity in BS group as compared with S group (Figure 3B). Neither exercise protocol nor dietary treatment showed any effect on GPx activity (Figure 3C). Exhaustive exercise induced a significant reduction of CAT activity with respect to control group, while BE-enriched diet was able to restore CAT activity at a similar level to that observed in control group (Figure 3D).
Figure 3. Effect of BE-enriched diet and acute exhaustive exercise on GST (A), GR (B), GPx (C) and CAT (D) enzymatic activities in liver homogenate. Enzyme activities were measured in liver homogenates as described in the Materials and Methods section. The B and BS groups were fed a diet enriched with BE. The S and BS ran an acute exercise bout, as described in the Materials and Methods section. Each bar represents the mean ± SD of 8 rats. Data were analyzed by one-way ANOVA followed by the Bonferroni post-test. Different letters (a, b and c) indicate statistically different means (p < 0.05).
3.2 Sterols content Table 1 reports the effects of BE-enriched diet and acute exhaustive exercise on sterol content of rats’ livers. Total sterols (0.9–1.2 mg/g liver) were mainly composed by cholesterol (0.8–1.2 mg/g liver), followed by -sitosterol and campesterol. In spite of BE consumption and exhaustive exercise, no significant differences were found among different treatments.
Table 1. Effect of BE-enriched diet and acute exhaustive exercise on sterol content (mg sterol/g liver) of rats’ livers. Treatment
Cholesterol
Campesterol
-sitosterol
Total sterols
Mean
SD
Mean
SD
Mean
SD
Mean
SD
C
0.78
0.34
0.04
0.01
0.06
0.01
0.86
0.37
B
1.04
0.37
0.04
0.01
0.07
0.01
1.17
0.34
S
0.79
0.28
0.04
0.01
0.07
0.01
0.90
0.27
BS
1.19
0.22
0.05
0.01
0.08
0.03
1.31
0.25
Stat. Sign.
ns
ns
ns
ns
Sterol content and composition were obtained as reported in the Materials and Methods section. Each value represents the mean of 8 rats. The B and BS groups were fed a diet enriched with BE. The S and BS ran an acute exercise bout, as described in the Materials and Methods section. Abbreviations: SD, standard deviation; Stat. Sign., statistical significant; ns, not significant. Data were analyzed by Tukey’s test (p < 0.05).
3.3 Cholesterol oxidation products (COPs) Regarding cholesterol oxidation, COPs level ranged from 0.24 to 0.98 g/g liver (Figure 4). Acute exhaustive exercise significantly increased cholesterol oxidation level from 0.50 g of COPs/g liver in the C group to 0.98 g of COPs/g liver in the S group. However, BE was able to significantly
decrease oxysterols, counteracting their accumulation when exhaustive exercise was performed (0.24 g/g liver).
Figure 4. Effect of BE-enriched diet and exhaustive exercise on total COPs contents (g COPs/g liver). The B and BS groups were fed a diet enriched with BE. The S and BS ran an acute exercise bout, as described in the Materials and Methods section. Each bar represents the mean ± SD of 8 rats. Data were analyzed by Tukey’s test. Different letters (a, b and c) indicate statistically different means (p < 0.05). The main oxysterol was 7-HC (0.11–0.53 g/g liver), which significantly increased under exhaustive exercise. A similar behavior was displayed by 7-KC (0.04–0.19 g/g liver) and 27-HC (0.01–0.03 g/g liver). Low amounts of triol and 24-HC were also found (Figure 5). Finally, the cholesterol oxidation ratio (OR %, Table S1) confirmed the antioxidant effect of BE, as supplementation under exhaustive exercise reduced the oxidation ratio (0.02%) as compared to the corresponding non-supplemented treatment (0.22%).
Figure 5. Effect of BE-enriched diet and exhaustive exercise on single cholesterol oxidation product (g COP/g liver) contents. The B and BS groups were fed a diet enriched with BE. The S and BS ran an acute exercise bout, as described in the Materials and Methods section. Each bar represents the mean ± SD of 8 rats. Data were analyzed by Tukey’s test. Different letters (a, b and c) for each COP indicate statistically different means (p < 0.05).
3.4 Principal component analysis of data (PCA) PCA performed with data from enzyme activities and oxidative parameters (Figure 6) was able to distinguish different clusters, explaining 92.22% of total variance in two principal components. The first principal component (F1, 81.85%) clearly separated the single and total COPs from antioxidant enzymes (GST, GR, GPx and CAT), highlighting their indirect correlations. PCA performed with data from different treatments was able to recognize cluster trends of diverse treatments that are well separated on the four different regions of the biplot. S treatment was highly characterized by LDH activity and the presence of single and total COPs, thus evidencing a structural tissue damage as well as an inflammatory process and oxidative stress. On the other hand, BS treatment was more characterized by antioxidant enzymes (GR, GPx and GST), suggesting the protective role of BE supplemented diet against exhaustive exercise-induced stress.
Figure 6. PCA score plot of single and total COPs, LDH, GPx, GST, Catalase, GR activity and crossed treatments.
4. Discussion Skeletal muscles are not the only tissue affected by exercise, since the metabolism of other organs is modulated by physical activity. In particular, liver plays a crucial role during exercise, due to its involvement in maintaining glucose, lipid and amino acid homeostasis; in fact, it has been demonstrated that physical activity activates a series of molecular events leading to the adaptation of liver metabolism to exercise training [31]. On the contrary, exhaustive exercise induces structural damage in liver as reported by Huang et al. [5], who demonstrated that rats undergoing exhaustive exercise showed biomarkers of strong inflammation in liver, such as inflammatory cell infiltration and tissue disruption. Acute exhaustive exercise also induces oxidative stress in liver tissue. Many
authors have reported an increased level of oxidative stress biomarkers (such as malondialdehyde, thiobarbituric acid reactive substances and protein carbonyls), xanthine oxidase activity, a decrease in GSH level, and a reduction of superoxide dismutase (SOD) and CAT activities in livers from rats that underwent exhaustive exercise [32-35]. In this study, as previously reported, plasma LDH activity, a well-known unspecific biomarker of tissue damage, strongly increased after acute exercise, indicating the deleterious effects of this type of activities [15,36-37]. Rats fed the BE-enriched diet showed a significantly reduced LDH plasma level, indicating that BE was able to prevent exercise-induced tissue damage. The cumulative effects of BE treatment on phase 2 antioxidant enzyme activities suggest that BE is able to counteract exercise-promoted oxidative stress, by inducing important antioxidant enzymes. Recently, Yioshida et al. [38] reported that 10 days of a BE supplemented diet induces GST activity in rat liver. Similar results were observed in the present study, since the BE-enriched diet significantly improved GST activity in rats subjected to exhaustive exercise. Many studies [15,32,34,39] found that GR activity is not affected after exhaustive exercise; accordingly, we did not observe any effects of the exercise protocol on GR liver activity, but the BE supplemented diet significantly improved GR in BS with respect to S group. Neither BE supplementation nor exhaustive exercise had any effect on GPx activity. Previous research in endothelial cells demonstrated that SF does not induce GPx [40]; it has been also demonstrated that, in rat skeletal muscle, both SF treatment and exhaustive running do not affect GPx activity [15]. The impact of exercise on CAT expression and activity in liver has been investigated since early ’90s, with poor consensus among different studies [40-41,48-49]. These discrepancies could be ascribed to the diverse exercise protocols and the different elapsed time between the end of the exercise and the sample collection. In the present study, CAT activity significantly decreased after exhaustive exercise and, interestingly, BE-enriched diet completely prevent CAT activity decrease. It is well known that CAT is in charge of hydrogen peroxide detoxification, so a reduction of its activity may result in the accumulation of such ROS. Preventing CAT decrease through BE supplementation,
may represent a key effect of broccoli bioactive compounds in counteracting oxidative stress in liver. According to literature [43], the main phytosterols in broccoli are sitosterol, campesterol and stigmasterol (4-desmethylsterols). It is well known that phytosterols display hypocholesterolemic and anti-inflammatory activities and that sitosterol is also used for treatment of prostatic diseases [44]. As reported in Table 1, cholesterol represented the most abundant sterol (88-91% of total sterols) in liver and, as expected, the main phytosterols were sitosterol and campesterol. However, BE-enriched diet and exhaustive exercise did not significantly (p > 0.05) affect sterol composition and thus they did not favor a differentiated accumulation of sterols. Oxysterols play a critical role in many regulatory processes; in particular, oxysterols activate ligands of liver X receptor and are also involved in diverse human diseases, such as fat induced injury [45] and liver injury in non-alcoholic fatty liver disease [16]. In addition, oxysterols (mainly 7-HC and 27-HC) are intermediates in the bile acid synthesis, which are engaged in the cholesterol homeostasis by suppression of the LDL receptor and the HMG-CoA reductase [46]. However, modifications on the COPs levels in plasma and liver have been observed in both obese mice and patients, which arise to some extent from a higher production of ROS [47]. The results of this study evidence that BE was able to prevent cholesterol oxidation, counteracting COPs increase induced by in vivo auto-oxidation and enzymatic oxidation. Guerrero-Beltràn et al. [48] reported the effect of SF against oxidative stress in liver and other tissues; in particular, the authors found that SF upregulates the expression of GST (π class) in liver cells, inducing the phase 2 detoxifying enzyme system. These observations could explain the lower amount of total COPs detected in rats fed a BE-enriched diet. In the present study, the single COPs displayed a similar behavior. As expected, the main single COP was 7-HC, since it can be generated by CYP7A1-mediated enzymatic oxidation; such enzyme
is,
in
fact,
largely
expressed
in
hepatocytes
and
ovary
cells
[49].
7-
hydroperoxycholesterol originated by auto-oxidation is unstable and isomerizes into 7-
hydroperoxycholesterol, which evolves into 7-HC and 7-KC; 7-hydroperoxyl radicals can also generate /-epoxycholesterol isomers, by reacting with a cholesterol molecule [50]. Exhaustive exercise without any BE dietary supplementation promoted 27-HC accumulation in rat liver; such oxysterol is known to be produced by mitochondrial sterol 27-hydroxylase (CYP27A1). On the other hand, the BE-enriched diet was able to significantly reduce 24-HC production. 24-HC, also called cerebrosterol is generated by 24-hydroxylase (CYP46A1), which is largely expressed in neurons and neural retina [49]. A reduction of 24-HC accumulation at neuronal level might result in the protection of neuronal tissue; however, further studies are needed to clarify whether BE administration exerts a protective role in neurones, which could become an additional neuroprotective effect to be ascribed to broccoli bioactive compounds besides those that had already been reported [14]. Finally, triol was detected in the liver samples analyzed, but no 5,6/-epoxy cholesterol isomers were found; this fact could be due to the total conversion of /-EC isomers into triol, which is a chemical equilibrium favored in specific environmental conditions (presence of water and acid pH) [30]. Finally, the PCA analysis confirmed that BE was able to counteract COPs accumulation promoted by exhaustive exercise in rat liver, by inducing antioxidant phase 2 enzymes (CAT, GST, GR), and to prevent tissue damage. The effect of the BE-enriched diet in the prevention of cholesterol oxidation in rat liver suggests the possibility to utilize phytochemicals in the modulation of the liver redox environment and in the prevention of oxidative damage.
Acknowledgments We thank Basic Research Funding (RFO 0 Italy) for financial support.
The authors have declared no conflict of interest.
,
lma Mater Studiorum-Universit di Bologna,
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Supplementary material Table S1. Effect of BE-enriched diet and exhaustive exercise on the content of single and total COPs (Tot COPs) in rats’ liver (g/g liver), and the cholesterol oxidation ratio (%). Treatment
7α-HC Mean
SD
7β-HC Mean
SD
Triol Mean
SD
24-HC Mean
SD
27-HC Mean
SD
7-KC Mean
SD
Tot COPs Mean
SD
OR Mean
SD
C
0.312 0.006 b
0.070 0.003 b
0.043 0.021 ab
0.013 0.006 ab
0.017 0.000 b
0.098 0.014 b
0.518 0.067 b
0.077 0.031 ab
B
0.236 0.010 c
0.051 0.005 c
0.032 0.005 b
0.010 0.000 b
0.014 0.001 b
0.077 0.006 b
0.410 0.024 bc
0.050 0.010 b
S
0.527 0.027 a
0.111 0.004 a
0.070 0.006 a
0.023 0.006 a
0.029 0.001 a
0.193 0.014 a
1.014 0.159 a
0.221 0.128 a
BS
0.109 0.006 d
0.026 0.001 d
0.014 0.001 b
0.010 0.000 b
0.007 0.001 c
0.041 0.006 c
0.243 0.080 c
0.020 0.010 b
Each value represents the mean and standard deviation (SD) of 8 rats/group. Data were analysed by Tukey’s test. Different letters (a, b and c) indicate statistically different means (p < 0.05).