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Effects of simvastatin on malondialdehyde level and esterase activity in plasma and tissue of normolipidemic rats
Pharmacological Reports 67 (2015) 907–913
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Original research article
Effects of simvastatin on malondialdehyde level and esterase activity in plasma and tissue of normolipidemic rats Marija Macan a,b, Antonija Vuksˇic´ b,c, Suzana Zˇunec d, Pasˇko Konjevoda e, Jasna Lovric´ f, Marta Kelava b, Nikola Sˇtambuk e, Nada Vrkic´ g, Vlasta Bradamante b,* a
Department of Pathology and Cytology, University Hospital Center Zagreb, Zagreb, Croatia Department of Pharmacology, University of Zagreb School of Medicine, Zagreb, Croatia Polyclinic Bonifarm, Zagreb, Croatia d Institute for Medical Research and Occupational Health, Zagreb, Croatia e Rudjer Boskovic Institute, NMR Center, Zagreb, Croatia f Department of Chemistry and Biochemistry, University of Zagreb School of Medicine, Zagreb, Croatia g Faculty of Pharmacy and Biochemistry and Clinical Institute of Chemistry, University Hospital ‘‘Sisters of Charity’’, Zagreb, Croatia b c
A R T I C L E I N F O
Article history: Received 3 November 2014 Received in revised form 28 January 2015 Accepted 13 February 2015 Available online 26 February 2015 Keywords: Simvastatin Paraoxonase 1 Butyrylcholinesterase Malondialdehyde
A B S T R A C T
Background: We investigated the possible non-lipid effects of simvastatin (SIMV) on paraoxonase 1 (PON1) and butyrylcholinesterase (BuChE) activity, as well as on malondialdehyde (MDA) levels in normolipidemic rats. Methods: Two experimental groups of Wistar rats (10 mg/kg/day of SIMV) and two control groups (saline) underwent a 21-day treatment period (TP). On the 22nd day one experimental and one control group of rats were sacrificed. Remaining groups of animals were sacrificied on the 32nd day of the study (10-day after-treatment period (AT)). Blood samples and slices of liver, heart, kidney, and brain tissue were obtained for the measurement of PON1 and BuChE activity and levels of MDA. Data were analyzed by means of t-test for independent samples. p values 0.05 were considered as statistically significant. Results: SIMV caused a significant decrease of serum and liver PON1 activity (18–24%, p 0.05) and MDA concentrations in the plasma, heart, liver, kidney, and brain (9–40%, p 0.05), while plasma and liver BuChE activity increased by 29% (p 0.05) and 18%, respectively. All effects of SIMV were largely diminished following AT. The exception was MDA, which remained significantly decreased in plasma and all tissues analyzed. Conclusion: SIMV significantly decreased PON1 activity and MDA levels and increased BuChE activity. We suggest that the decrease of MDA levels is a beneficial therapeutic effect of SIMV, for example in cardiovascular disorders, while the increase of BuChE activity, especially in brain, may be a potential adverse effect in patients with Alzheimer disease. ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
Introduction Statins represent drugs of first choice for treatment of hypercholesterolemia since they inhibit 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase, a rate-limiting enzyme in mevalonate and cholesterol biosynthesis [1]. Hypercholesterolemia leads to a pronounced increase in reactive oxygen species (ROS) like free oxygen radicals, oxygen ions, and hydrogen
* Corresponding author. E-mail address: [email protected] (V. Bradamante).
peroxides (H2O2) [1]. Large amounts of H2O2 stimulate the peroxidation of LDL-cholesterol, which causes a significant amount of malondialdehyde (MDA) formation. Therefore MDA is very often used as an index of oxidative status [2]. Individuals with high exposure to oxidative stress, such as those with hypertension [3], have an increased formation of oxidized LDL-cholesterol that is accumulated in the arterial walls and causes endothelial dysfunction. In this situation the endogenous antioxidant defensive effects of superoxid dismutase, catalase, and glutathione peroxidase are very important [3]. Experiments in vitro and in vivo have shown that statins contribute to the protection of organism against oxidative stress
http://dx.doi.org/10.1016/j.pharep.2015.02.005 1734-1140/ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
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through their anti-inflammatory and anti-thrombotic actions, and through the scavenging of superoxide anions and hydroxyl radicals [1,4–6]. All these effects are well-known cholesterol-independent or ‘‘pleiotropic’’ effects of statins. It is also suggested that antioxidative effects of statins depend on patients’ cholesterol levels. Thus, in hypercholesterolemic patients atorvastatin and pravastatin were capable of protecting LDL-cholesterol from oxidation in vivo only in early treatment phase [5] while fluvastatin treatment did not improve oxidative stress or inflammation in patients with arterial hypertension and normal cholesterol levels [6]. Statins may also modulate the activity of two serum esterases, paraoxonase 1 (PON1) [7] and butyrylcholinesterase (BuChE) [8], with unknown physiological function at present. Both enzymes are synthesized in the liver and hypothetically are involved in metabolism of lipids. Paraoxonase 1 (PON1, aryldialkilphosphatase, EC 3.1.8.1, PON1) is secreted into the plasma where it is associated with HDL-cholesterol and apolipoprotein (apo) AI. PON1 is included in the protection of the organism against oxidative stress [7] and it has been suggested that possible antioxidant actions of PON1 are hydrolysis of oxidized lipids formed on LDL- and HDLcholesterol and protection of HDL-cholesterol from peroxidation [9]. The evidence about the effect of statins on PON1 activity remains equivocal [7,10]. The enzyme BuChE (EC 3.1.1.8, pseudocholinesterase, serum cholinesterase) is secreted in the blood after its synthesis in the liver [11]. A known special target in the organism is cleavage of choline esters like acetylcholine (ACh) and butyrylthiocholine. The hypothesis that LDL-cholesterol is formed from very low-density lipoproteins (VLDL-cholesterol) in the presence of BuChE is supported by the fact that increased BuChE activity is associated with an abnormal lipid metabolism in humans [11]. It has been shown that BuChE activity is increased in patients with hypercholesterolemia, hypertension, obesity, and diabetes, where this increased enzyme activity correlates positively with serum levels of LDL-cholesterol and triacylglycerols, and inversely with HDL-C [11,12]. The presence of BuChE has also been detected in various tissues [11], specially in neurons and glial cells of the human brain [13] where it hydrolyses ACh, but less efficiently than acetylcholinesterase (AChE) [13]. It is known that the most striking neurochemical disturbance in Alzheimer disease (AD) is a deficiency of ACh [14,15]. Since both cholinesterases are involved in cholinergic transmission in the brain, cholinesterase inhibitors are used for treatment of AD [14,15]. According to the results in vitro and in vivo, some statins can cause either the inhibition of both enzymes or have no effect on AChE and BuChE activity [16,17]. Since the results of human and animal studies about the effects of statins on BuChE and PON1 activity and oxidative stress vary, our aims were to investigate the effects of multiple administration of lipophilic simvastatin (SIMV) on BuChE and PON1 activities and MDA level in the serum and different tissues of normolipidemic rats, as well as to observe the levels of enzyme activity and MDA 10 days after discontinuation of the treatment. Material and methods Test substances SIMV (CAS-79902-63-9) (Statex1 20) was obtained from Pliva, Croatia. It was suspended in saline and administered daily (9.00– 10.00 a.m.) into the stomach by oral gavage. The daily dose was 10 mg/kg body weight during 21 day.
Treatment of animals Male Wistar rats (Department of Pharmacology, School of Medicine, University of Zagreb) weighing 250–350 g were used in this study. Animals were maintained under controlled laboratory conditions. Standard diet in pellet form was available ad libitum. Handling and treatment of the animals were conducted according to international guidelines regulating the use of laboratory animals. The experiments had been approved by the local ethics committee. Study design The study was divided into a 21-day treatment period (TP) and a 10-day after-treatment period (AT). Two experimental groups of Wistar rats were on SIMV treatment (10 mg/kg/day) and two control groups on saline. In each group there were 7–8 animals. SIMV and saline were given for a 21-day TP. After an overnight fast of 12 h, one experimental and one control group of rats were sacrificed under diethyl ether anesthesia on the 22nd day of the study. The remaining groups of animals which received no SIMV or saline for the 10-day ATP were sacrificied after a 12-h overnight fast on the 32nd day of the study. Blood samples for measuring serum PON1 and plasma BuChE activity, plasma lipids, and plasma MDA levels in all groups of rats were obtained by cardiac puncture. Plasma lipids were measured 2–4 h after sampling. All other serum and plasma samples were frozen at 20 8C immediately after sampling, until further processing. Heart, liver, kidney, and brain tissues for determining both PON1 and BuChE activity and MDA level were frozen at 70 8C immediately after sacrifice of the animals, until further processing. Before collecting, the liver had been washed out of blood with saline in situ via the vena cava superior. The brain was also rinsed with saline. Measurement of PON1 activity in serum and liver Serum PON1 activity was measured using synthetic diethyl-pnitrophenyl phosphate (paraoxon, o,o-diethyl-p-nitrophenylphosphate; Sigma Chemical Co., London, UK) and CaCl2 (1 mM in 0.1 M TRIS buffer pH 7.4) as moderator. The activity toward paraoxon was determined by measuring the initial rate of substrate hydrolysis to p-nitrophenol. Slices of liver tissue (0.2 g) were homogenized in four volumes of saline and centrifuged at 3500 g for 15 min. In brief, the reaction for the hydrolysis of paraoxon contained 200 mL of 0.1 M TRIS buffer (pH 7.4)-CaCl2 and 800 mL of 1 mM paraoxon solution, to which 100 mL undiluted serum or 100 mL of supernatant (for measurement of liver PON1 activity) was added to start the reaction. The increase in absorbance at 405 nm was monitored for 3 min [18]. Measurement of BuChE activity in plasma and liver Plasma and liver BuChE activity was measured by spectrophotometric method of Ellman et al. [19], using butyrylthiocholine (0.9 mM) (Sigma ChemCo, St. Louis, USA) as the substrate. Slices of liver tissue (0.2 g) were homogenized in four volumes of saline and centrifuged at 3500 g for 15 min. The reaction for the hydrolysis of butyrythiocholine in plasma and liver contained 1 mL of mixture of 3 mL 0.1 M phosphate buffer and 100 mL 0.38 mM 5.5-dithio-bis(2-nitrobenzoic acid) (DTNB), 100 mL of butyrylthiocholine and 50 mL of plasma or 50 mL of supernatant. The reaction for the measurement of liver BuChE activity was repeated after the addition of 50 mL of etopropazine hydrochloride, which is a specific BuChE inhibitor. The increase in absorbance at 412 nm and a temperature of 25 8C was monitored for 3 min.
M. Macan et al. / Pharmacological Reports 67 (2015) 907–913
During the measurement of PON1 and BuChE activity a blank sample containing the incubation mixture without serum/plasma/ supernatant was run simultaneously to correct the spontaneous substrate breakdown. The activity of serum PON1 and plasma BuChE was expressed as mmol of substrate hydrolyzed per min per ml of serum or plasma. The activity of PON1 and BuChE in the liver was expressed as mmol of substrate hydrolyzed per min per g of tissue. Measurement of MDA-TBA MDA in plasma MDA levels were determined by the MDA-TBA test. The TBA assay was performed according to a previously published method [20,21]. In the first step, an aliquot of plasma (250 mL) was added to 25 mL 0.2% BHT (2.6 di-ter-butyl-4-methylphenol, 99.0%, GC; Sigma–Aldrich Inc. St. Louis, USA), in ethanol (Merck, Darmstadt, Germany) and 1 mL 15% aqueous TCA (trichloracetic acid, 98%; Sigma–Aldrich Inc.). The mixture was then centrifuged (Hettich Universal 32 R Tuttingen Germany) at 4000 g for 15 min at 4 8C. The deproteinized supernatant (stock) was stored at 70 8C. An aliquot of 500 mL of the stoch solution was added to 1 mL TBA (0.375% in 0.25 M) HCl (37%; puriss. p.a., Sigma–Aldrich Inc.). The mixture was then heated at 100 8C for 15 min. After cooling, the solution was analyzed by spectrophotometry [20,21]. MDA in tissue samples The assay was adopted from Angulo et al. [20] and Botsoglou et al. [21] and adjusted for our purposes by Lovric´ et al. [22]. 0.2 g of tissues (liver, heart, kidney, and brain) were used to obtain 10% homogenates in 0.15 M KCl that were further treated with 25 mL 0.2% BHT in 98% ethanol as antioxidant. The homogenates were centrifuged at 18,890 g for 20 min. The separated supernatant was mixed with 5% aqueous TCA in proportion 1:4, and recentrifuged (Hettich Universal 32 R) at 1780 g for 15 min. The deproteinized supernatant was separated and aliquots of 500 mL were mixed with 500 mL TBA 0.375% in 0.25 M HCl. The mixture was heated at 100 8C for 15 min, followed by cooling of the samples to room temperature and measuring the MDA level by UV–vis spectrophotometer (HPV-220, Iskra, Slovenia) using a 1-cm absorption cell. The concentration of MDA was calculated by the absorbance at 532 nm, using a molar extinction coefficient of e = 1.56 105 M/cm [23]. During the whole procedure, until heating, the samples were kept on ice.
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Lipids and lipoproteins Lipids were determined on the Olympus automatic analyser AU 2700 with the use of original reagents produced by Olympus Diagnostics GmbH (Irish Branch), Lismeehan, Ireland. Plasma concentrations of total cholesterol and triacylglycerols were determined by enzymatic colorimetric methods [24,25]. The HDL-cholesterol level was estimated using a compound method of immuno-inhibition with b-lipoprotein antibody in the first step, and enzymatic colorimetric measuring of accessible cholesterol in the second step [26]. All plasma lipid concentrations were expressed as mmol/L. Data analysis Data were analyzed using an independent two-sample t-test. All applied tests were two-tailed, and p values 0.05 were considered as statistically significant. The data in table are presented as means and standard deviations. In the graphs, all individual data are presented as points, and the horizontal line denotes the mean. All calculations and data plotting were done using GraphPad Prism for Windows, version 5 [27,28]. Results Effects of SIMV on serum and liver PON1 activity SIMV caused a significant decrease of serum and liver PON1 activity, of 18% and 24%, respectively (Table 1). After withdrawal of SIMV treatment and the AT, PON1 activity in the serum was lower by 12% vs. control. On the contrary, PON1 activity in liver was higher by 35% than the activity measured in the control group (Table 1). Effects of SIMV on plasma and liver BuChE activity Both plasma and liver BuChE activity increased by 29% and 18%, respectively, compared to the values in the control group (Table 1). Following the ATP, the activity of BuChE remained higher in the plasma and liver (17% and 15%, respectively) in comparison with the control group (Table 1). Effects of SIMV on MDA levels in plasma, heart, liver, kidney, and brain Application of SIMV resulted in a significant decrease of MDA levels in the plasma (24%), heart (11%), liver (9%), kidney (13%), and brain (40%) (Fig. 1).
Table 1 Effect of simvastatin (10 mg/kg/day for 3 weeks) on paraoxonase 1 and butyrylcholinesterase activity and level of plasma lipids in Wistar rats. Data are presented as means and standard deviations. Variable
Treatment period Controla
Paraoxonase serum (mM/min/mL) Paraoxonase liver (mM/min/g) BuChE plasma (mM/min/mL) BuChE liver (mM/min/g) Total cholesterol (mM) HDL cholesterol (mM) Triacylglyceroles (mM)
0.33 0.07 0.25 0.08 0.07 0.01 0.28 0.06 1.54 0.22 1.08 0.12 0.93 0.22
Bold p values are significant at the 0.05 level. a The values are mean SD. b p value of independent two-sample t-test.
(n = 7) (n = 7) (n = 7) (n = 7) (n = 7) (n = 7) (n = 7)
After treatment Simvastatina (10 mg/kg/day)
pb Value
Controla
0.27 0.03 0.19 0.07 0.09 0.02 0.33 0.08 1.29 0.17 0.93 0.11 0.71 0.18
0.048 0.036 0.033 0.200 0.036 0.035 0.057
0.25 0.03 0.20 0.08 0.06 0.01 0.27 0.06 1.69 0.21 1.18 0.15 0.93 0.17
(n = 8) (n = 7) (n = 8) (n = 8) (n = 7) (n = 7) (n = 7)
(n = 8) (n = 7) (n = 7) (n = 7) (n = 7) (n = 7) (n = 7)
Simvastatina (10 mg/kg/day)
pb Value
0.22 0.04 0.27 0.08 0.07 0.02 0.31 0.08 1.64 0.23 1.13 0.18 1.12 0.36
0.129 0.107 0.260 0.311 0.697 0.607 0.224
(n = 7) (n = 7) (n = 7) (n = 7) (n = 7) (n = 7) (n = 7)
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Fig. 1. MDA levels in plasma and rat tissue (heart, brain, liver, kidney) after 21 days of treatment with simvastatin as well as 10 days after simvastatin discontinuation. Results are expressed as mean 95% confidence intervals. p 0.05 compared to the control group (TP – treatment period, AT – after treatment).
After the completion of the AT, the concentrations of MDA remained significantly lower in plasma (22%), heart (17%), liver (9%), kidney (13%), and brain (29%), as compared with their control values (Fig. 1).
concentration of triacylglycerols increased by 20% vs. control (Table 1).
Effects of SIMV on plasma lipids
The findings of the present study show that SIMV caused a low but significant decrease of PON1 activity in the serum (18%) and liver (24%) (Table 1). At the same time, SIMV induced a parallel significant lowering of HDL and total cholesterol concentration by 14% and 16% respectively, whilst the level of triacylglycerols showed the tendency to decrease by 24% in the plasma of normolipidemic rats (Table 1).
The plasma levels of total and HDL-cholesterol were significantly lower, by 16% and 14% respectively, and the triacyglycerol level decreased by 24% after SIMV administration (Table 1). After the end of AT, the levels of both total and HDL-cholesterol were similar to the values in the control group, while the plasma
Discussion
M. Macan et al. / Pharmacological Reports 67 (2015) 907–913
Several mechanisms might be responsible for the decrease of PON1 activity caused by SIMV. The first potential mechanism is the enzyme’s close structural bond mostly with HDL-cholesterol, but to a lesser extent also with VLDL-cholesterol and chylomicron particles, which stimulate the secretion of the enzyme from liver and ensure the optimal environment for its catalytic activity [29]. Therefore, it seems logical to assume that every change of the structure and/or concentration of these plasma lipoprotein particles, caused by statins, might lead to an alteration of PON1 activity. Because SIMV in our study significantly decreased total and HDL-cholesterol and also showed a tendency to decrease plasma triacylglycerols, these changes of plasma lipid concentration might partially explain the significant decrease of PON1 activity in the serum of rats, although they were normolipidemic [29]. In our study the decrease of PON1 activity in serum and liver was similar. Because of fact that PON1 is synthesized in the liver, it may be supposed that the second potential mechanism responsible for the reduction of PON1 activity is the inhibition of enzyme synthesis caused by SIMV. Our hypothesis is supported by results of Goue´dard et al. [30], who showed that in HuH7 human hepatoma cells pravastatin, fluvastatin, and SIMV caused a significant decrease of PON1 activity and its mRNA level of 25%, 30%, and 40%, respectively. A significant decrease of PON1 gene promoter activity of 40–60% was also obtained [30]. Since liver PON1 activity in our study showed the tendency to increase by 35% 10 days after cessation of SIMV administration (Table 1), we can conclude that decrease of PON1 activity in liver was possibly mediated by its reversible repressive action on the PON1 gene promoter. The third possible mechanism of significant decrease of PON1 activity might be the activation of peroxisome proliferatoractivated receptor a (PPARa) caused by SIMV. According to Beltowski et al. [10] and Berthou et al. [31], in rodents, as opposed to humans, PPARa agonists inhibit the synthesis of apoA-I in the liver. Since apoA-I is the dominant component of HDL-cholesterol that stimulates the secretion of PON1 from the liver into the blood, SIMV as a possible PPARa agonist could cause the inhibition of apoA-I synthesis in the liver and a decrease of PON1 activity. It is important to mention that HDL is the main cholesterol-transporting fraction of plasma lipoproteins in rat. Fourthly, since SIMV is a compound that contains a lactone ring, and in regard of the fact that PON1 hydrolyzes lactone ringcontaining substances [32], the significant decrease of the liver and serum PON1 activity observed in our experiment may simply reflect the consumption of this enzyme in the process of SIMV breakdown. Our results obtained from the observation of PON1 activity and levels of plasma total cholesterol and triacylglycerols are chiefly supported by the results of other authors. The treatment of normolipidemic rats with fluvastatin (2 or 20 mg/kg/day for 3 weeks) reduced PON1 activity in plasma, liver, heart, and kidney, whereas pravastatin (4 or 40 mg/kg/day for 3 weeks) caused a significant decrease of PON1 only in the kidney [10,18]. These statins had no effect on either total or HDL-cholesterol, but significantly reduced plasma triacylglycerols [10]. PON1 activity also showed the tendency to decrease by 16.1% and 11.6% after treatment with cerivastatin (0.03 or 0.3 mg/kg for 3 weeks) [33]. In contrast to these findings, in the study of Bolayirli et al., 0.3 mg/kg/ day atrovastatin for 4 weeks resulted with a significant increase of PON1 activity and HDL-cholesterol, whilst the level of LDLcholesterol decreased significantly in the plasma of hypercholesterolemic rabbits [34]. It is important to emphasize that studies on the effect of SIMV on the activity of PON1 in humans have yielded discordant results as well [7].
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The significant decrease of HDL-cholesterol (Table 1) obtained in our results is an exception in comparison with the results mentioned above. These decrease of HDL-cholesterol could be the consequence of the inhibition of apoA-I synthesis in the rat liver caused by SIMV due to its agonistic action on PPARa receptors [10]. The lowering of plasma triacylglycerols in our experiments is also partially mediated by activation of PPARa receptors [35], causing in rodents the induction of genes that decrease the availability of triacylglycerols for hepatic VLDL secretion and induction of genes that promote lipoprotein lipase-mediated lipolysis of triacylglycerol-rich plasma lipoproteins [31,35]. According to our results, SIMV caused the significant increase of plasma BuChE activity of 29% (Table 1), while liver BuChE activity showed a tendency to be increased by 18% (Table 1). Following the AT, BuChE activity remained higher in the plasma and liver (17% and 15%, respectively) vs. control, which implies that the effect of SIMV does not cease 10 days after discontinuation (Table 1). Our results show a negative correlation between plasma and liver BuChE activity and the levels of total and HDLcholesterol and triacylglycerols in the plasma of normolipidemic rats (Table 1) which is in the contrast with results of Rustemeijer et al. [12]. This difference can partially be explained by the fact that our results were obtained in a model of normolipidemic rats, whereas in clinical studies changes in BuChE activity and lipid profiles were observed under pathological conditions. It can be assumed that in our experiment the increase in BuChE activity represents a direct consequence of SIMV on the PPARa receptors as well [10]. We suggest that in rodents statins cause induction of beta-oxidation of fatty acids that is followed by an increased production of butyrylcholine as its metabolite [11]. Therefore, the increased BuChE activity in our experiments might actually result from the increased demand for butyrylcholine degradation. If not metabolized, butyrylcholine could cause unwanted toxic effects [11]. Our recent results obtained in hyperlipidemic rats show an increase of BuChE activity in rat brain after SIMV treatment [36]. We suggest that as an adverse effect, especially when the role of cholinesterase in the development of AD is being considered. Our results are completely in contrast with the results of other investigators [16,17], who found that statins cause either the inhibition of both enzymes or have no effect on AChE and BuChE activity. Although it is claimed that statins with high lipid solubility, such as SIMV, suppress AChE activity [37] our results obtained in normolipidemic and hyperlipidemic rats abolish their protective effect against AD, due to the inhibition of cholinesterase activity. In our investigations SIMV treatment resulted in a significant decrease of MDA levels in the plasma (24%), heart (11%), liver (9%), kidney (13%), and brain (40%) (Fig. 1). Our results obtained from the observation of MDA plasma levels are chiefly supported by the results of Beltowski et al. [10,33], who proved a statistically significant decrease in MDA (46.6%) and oxidized lipids (59.3%) in rat plasma after administration of cerivastatin (0.3 mg/kg/day for 3 weeks) and fluvastatin (19.8% and 30.9%, respectively, at doses of 2 and 20 mg/kg/day for 3 weeks), and also a tendency for MDA decrease after pravastatin (25.9%, at a dose of 40 mg/kg/day for 3 weeks). Similar results were confirmed by experiments of Bolayirli et al., who showed the antioxidant activity of 0.3 mg/kg/ day atrovastatin for 4 weeks in rabbits with hypercholesterolemia [34]. Investigations of the effects of statins on the MDA level in dyslipidemic type 2 diabetic patients gave the same results [38]. We have shown very clearly that the significant MDAlowering effect of SIMV established in the plasma, heart, kidney, liver, and brain at the end of the treatment was maintained even 10 days after the treatment period (Fig. 1). These results are a
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strong proof for the positive ‘‘pleiotropic’’ effect of SIMV and for the justified statin therapy of some acute cardiovascular diseases. In addition, the antioxidative effect of SIMV on MDA levels in the brain proven in our experiments could also explain its positive effects in patients with AD. The antioxidant effects of statins may be attributed to their antiinflammatory and lipid-lowering action, i.e., that in animals and humans with hyperlipidemia these effects might be the consequence of their lipid-lowering action and thus reduced availability of plasma lipids for the process of peroxidation [32]. Our results show that antioxidative effects of SIMV are also present in normolipidemic rats and that they are completely independent of its other lipid and non-lipid effects. In conclusion, our results have clearly demonstrated that SIMV in normolipidemic rats caused the decrease of PON1 activity, while BuChE activity was increased. Our results also gave proof for the antioxidant action of SIMV, because the MDA level was significantly lower in the plasma, heart, kidney, and brain after SIMV treatment, and was maintained even 10 days after the treatment period. Therefore, we suggest that the positive effects of SIMV in patients with cardiovascular disorders and Alzheimer disease are the consequence of its antioxidative action. The effect of SIMV on BuChE activity represents an adverse event in the context of its action on the brain and possible therapy of Alzheimer disease. Authors’ contribution Marija Macan, Antonija Vuksˇic´, Suzana Zˇunec, Jasna Lovric´, Marta Kelava, Nikola Sˇtambuk, and Vlasta Bradamante participated in the research and the article preparation. Pasˇko Konjevoda participated in the article preparation and conduction of statistical analysis. Funding This study was supported by the Ministry of Science, Education, and Sports of the Republic of Croatia, Grant No. 108-00000000013. Conflict of interest All authors declare no conflict of interest. Acknowledgments We gratefully acknowledge the expert technical assistance of Mrs Jelka Roca. We also wish to thank Ms Aleksandra Zˇmegacˇ Horvat for language editing the text. References [1] Parizadeh SM, Azarpazhooh MR, Moohebati M, Nematy M, Ghayour-Mobarhan M, Tavallaie S, et al. Simvastatin therapy reduces prooxidant-antioxidant balance: results of a placebo-controlled cross-over trial. Lipids 2011;46: 333–40. [2] Janero DR. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic Biol Med 1990;9:515–40. [3] Giugliano D, Ceriello A, Paolisso G. Diabetes mellitus, hypertension, and cardiovascular disease: which role for oxidative stress? Metabolism 1995; 44:363–8. [4] Endres M. Statins: potential new indications in inflammatory conditions. Atheroscler Suppl 2006;7:31–5. [5] Thallinger C, Urbauer E, Lackner E, Graselli U, Kostner K, Wolzt M, et al. The ability of statins to protect low density lipoprotein from oxidation in hypercholesterolemic patients. Int J Clin Pharmacol Ther 2005;43:551–7. [6] Schneider MP, Schmidt BM, John S, Schmieder RE. Effects of statin treatment on endothelial function, oxidative stress and inflammation in patients with arterial hypertension and normal cholesterol levels. J Hypertens 2011;29: 1757–64.
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Pharmacological Reports journal homepage: www.elsevier.com/locate/pharep
Original research article
Effects of simvastatin on malondialdehyde level and esterase activity in plasma and tissue of normolipidemic rats Marija Macan a,b, Antonija Vuksˇic´ b,c, Suzana Zˇunec d, Pasˇko Konjevoda e, Jasna Lovric´ f, Marta Kelava b, Nikola Sˇtambuk e, Nada Vrkic´ g, Vlasta Bradamante b,* a
Department of Pathology and Cytology, University Hospital Center Zagreb, Zagreb, Croatia Department of Pharmacology, University of Zagreb School of Medicine, Zagreb, Croatia Polyclinic Bonifarm, Zagreb, Croatia d Institute for Medical Research and Occupational Health, Zagreb, Croatia e Rudjer Boskovic Institute, NMR Center, Zagreb, Croatia f Department of Chemistry and Biochemistry, University of Zagreb School of Medicine, Zagreb, Croatia g Faculty of Pharmacy and Biochemistry and Clinical Institute of Chemistry, University Hospital ‘‘Sisters of Charity’’, Zagreb, Croatia b c
A R T I C L E I N F O
Article history: Received 3 November 2014 Received in revised form 28 January 2015 Accepted 13 February 2015 Available online 26 February 2015 Keywords: Simvastatin Paraoxonase 1 Butyrylcholinesterase Malondialdehyde
A B S T R A C T
Background: We investigated the possible non-lipid effects of simvastatin (SIMV) on paraoxonase 1 (PON1) and butyrylcholinesterase (BuChE) activity, as well as on malondialdehyde (MDA) levels in normolipidemic rats. Methods: Two experimental groups of Wistar rats (10 mg/kg/day of SIMV) and two control groups (saline) underwent a 21-day treatment period (TP). On the 22nd day one experimental and one control group of rats were sacrificed. Remaining groups of animals were sacrificied on the 32nd day of the study (10-day after-treatment period (AT)). Blood samples and slices of liver, heart, kidney, and brain tissue were obtained for the measurement of PON1 and BuChE activity and levels of MDA. Data were analyzed by means of t-test for independent samples. p values 0.05 were considered as statistically significant. Results: SIMV caused a significant decrease of serum and liver PON1 activity (18–24%, p 0.05) and MDA concentrations in the plasma, heart, liver, kidney, and brain (9–40%, p 0.05), while plasma and liver BuChE activity increased by 29% (p 0.05) and 18%, respectively. All effects of SIMV were largely diminished following AT. The exception was MDA, which remained significantly decreased in plasma and all tissues analyzed. Conclusion: SIMV significantly decreased PON1 activity and MDA levels and increased BuChE activity. We suggest that the decrease of MDA levels is a beneficial therapeutic effect of SIMV, for example in cardiovascular disorders, while the increase of BuChE activity, especially in brain, may be a potential adverse effect in patients with Alzheimer disease. ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
Introduction Statins represent drugs of first choice for treatment of hypercholesterolemia since they inhibit 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase, a rate-limiting enzyme in mevalonate and cholesterol biosynthesis [1]. Hypercholesterolemia leads to a pronounced increase in reactive oxygen species (ROS) like free oxygen radicals, oxygen ions, and hydrogen
* Corresponding author. E-mail address: [email protected] (V. Bradamante).
peroxides (H2O2) [1]. Large amounts of H2O2 stimulate the peroxidation of LDL-cholesterol, which causes a significant amount of malondialdehyde (MDA) formation. Therefore MDA is very often used as an index of oxidative status [2]. Individuals with high exposure to oxidative stress, such as those with hypertension [3], have an increased formation of oxidized LDL-cholesterol that is accumulated in the arterial walls and causes endothelial dysfunction. In this situation the endogenous antioxidant defensive effects of superoxid dismutase, catalase, and glutathione peroxidase are very important [3]. Experiments in vitro and in vivo have shown that statins contribute to the protection of organism against oxidative stress
http://dx.doi.org/10.1016/j.pharep.2015.02.005 1734-1140/ß 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
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through their anti-inflammatory and anti-thrombotic actions, and through the scavenging of superoxide anions and hydroxyl radicals [1,4–6]. All these effects are well-known cholesterol-independent or ‘‘pleiotropic’’ effects of statins. It is also suggested that antioxidative effects of statins depend on patients’ cholesterol levels. Thus, in hypercholesterolemic patients atorvastatin and pravastatin were capable of protecting LDL-cholesterol from oxidation in vivo only in early treatment phase [5] while fluvastatin treatment did not improve oxidative stress or inflammation in patients with arterial hypertension and normal cholesterol levels [6]. Statins may also modulate the activity of two serum esterases, paraoxonase 1 (PON1) [7] and butyrylcholinesterase (BuChE) [8], with unknown physiological function at present. Both enzymes are synthesized in the liver and hypothetically are involved in metabolism of lipids. Paraoxonase 1 (PON1, aryldialkilphosphatase, EC 3.1.8.1, PON1) is secreted into the plasma where it is associated with HDL-cholesterol and apolipoprotein (apo) AI. PON1 is included in the protection of the organism against oxidative stress [7] and it has been suggested that possible antioxidant actions of PON1 are hydrolysis of oxidized lipids formed on LDL- and HDLcholesterol and protection of HDL-cholesterol from peroxidation [9]. The evidence about the effect of statins on PON1 activity remains equivocal [7,10]. The enzyme BuChE (EC 3.1.1.8, pseudocholinesterase, serum cholinesterase) is secreted in the blood after its synthesis in the liver [11]. A known special target in the organism is cleavage of choline esters like acetylcholine (ACh) and butyrylthiocholine. The hypothesis that LDL-cholesterol is formed from very low-density lipoproteins (VLDL-cholesterol) in the presence of BuChE is supported by the fact that increased BuChE activity is associated with an abnormal lipid metabolism in humans [11]. It has been shown that BuChE activity is increased in patients with hypercholesterolemia, hypertension, obesity, and diabetes, where this increased enzyme activity correlates positively with serum levels of LDL-cholesterol and triacylglycerols, and inversely with HDL-C [11,12]. The presence of BuChE has also been detected in various tissues [11], specially in neurons and glial cells of the human brain [13] where it hydrolyses ACh, but less efficiently than acetylcholinesterase (AChE) [13]. It is known that the most striking neurochemical disturbance in Alzheimer disease (AD) is a deficiency of ACh [14,15]. Since both cholinesterases are involved in cholinergic transmission in the brain, cholinesterase inhibitors are used for treatment of AD [14,15]. According to the results in vitro and in vivo, some statins can cause either the inhibition of both enzymes or have no effect on AChE and BuChE activity [16,17]. Since the results of human and animal studies about the effects of statins on BuChE and PON1 activity and oxidative stress vary, our aims were to investigate the effects of multiple administration of lipophilic simvastatin (SIMV) on BuChE and PON1 activities and MDA level in the serum and different tissues of normolipidemic rats, as well as to observe the levels of enzyme activity and MDA 10 days after discontinuation of the treatment. Material and methods Test substances SIMV (CAS-79902-63-9) (Statex1 20) was obtained from Pliva, Croatia. It was suspended in saline and administered daily (9.00– 10.00 a.m.) into the stomach by oral gavage. The daily dose was 10 mg/kg body weight during 21 day.
Treatment of animals Male Wistar rats (Department of Pharmacology, School of Medicine, University of Zagreb) weighing 250–350 g were used in this study. Animals were maintained under controlled laboratory conditions. Standard diet in pellet form was available ad libitum. Handling and treatment of the animals were conducted according to international guidelines regulating the use of laboratory animals. The experiments had been approved by the local ethics committee. Study design The study was divided into a 21-day treatment period (TP) and a 10-day after-treatment period (AT). Two experimental groups of Wistar rats were on SIMV treatment (10 mg/kg/day) and two control groups on saline. In each group there were 7–8 animals. SIMV and saline were given for a 21-day TP. After an overnight fast of 12 h, one experimental and one control group of rats were sacrificed under diethyl ether anesthesia on the 22nd day of the study. The remaining groups of animals which received no SIMV or saline for the 10-day ATP were sacrificied after a 12-h overnight fast on the 32nd day of the study. Blood samples for measuring serum PON1 and plasma BuChE activity, plasma lipids, and plasma MDA levels in all groups of rats were obtained by cardiac puncture. Plasma lipids were measured 2–4 h after sampling. All other serum and plasma samples were frozen at 20 8C immediately after sampling, until further processing. Heart, liver, kidney, and brain tissues for determining both PON1 and BuChE activity and MDA level were frozen at 70 8C immediately after sacrifice of the animals, until further processing. Before collecting, the liver had been washed out of blood with saline in situ via the vena cava superior. The brain was also rinsed with saline. Measurement of PON1 activity in serum and liver Serum PON1 activity was measured using synthetic diethyl-pnitrophenyl phosphate (paraoxon, o,o-diethyl-p-nitrophenylphosphate; Sigma Chemical Co., London, UK) and CaCl2 (1 mM in 0.1 M TRIS buffer pH 7.4) as moderator. The activity toward paraoxon was determined by measuring the initial rate of substrate hydrolysis to p-nitrophenol. Slices of liver tissue (0.2 g) were homogenized in four volumes of saline and centrifuged at 3500 g for 15 min. In brief, the reaction for the hydrolysis of paraoxon contained 200 mL of 0.1 M TRIS buffer (pH 7.4)-CaCl2 and 800 mL of 1 mM paraoxon solution, to which 100 mL undiluted serum or 100 mL of supernatant (for measurement of liver PON1 activity) was added to start the reaction. The increase in absorbance at 405 nm was monitored for 3 min [18]. Measurement of BuChE activity in plasma and liver Plasma and liver BuChE activity was measured by spectrophotometric method of Ellman et al. [19], using butyrylthiocholine (0.9 mM) (Sigma ChemCo, St. Louis, USA) as the substrate. Slices of liver tissue (0.2 g) were homogenized in four volumes of saline and centrifuged at 3500 g for 15 min. The reaction for the hydrolysis of butyrythiocholine in plasma and liver contained 1 mL of mixture of 3 mL 0.1 M phosphate buffer and 100 mL 0.38 mM 5.5-dithio-bis(2-nitrobenzoic acid) (DTNB), 100 mL of butyrylthiocholine and 50 mL of plasma or 50 mL of supernatant. The reaction for the measurement of liver BuChE activity was repeated after the addition of 50 mL of etopropazine hydrochloride, which is a specific BuChE inhibitor. The increase in absorbance at 412 nm and a temperature of 25 8C was monitored for 3 min.
M. Macan et al. / Pharmacological Reports 67 (2015) 907–913
During the measurement of PON1 and BuChE activity a blank sample containing the incubation mixture without serum/plasma/ supernatant was run simultaneously to correct the spontaneous substrate breakdown. The activity of serum PON1 and plasma BuChE was expressed as mmol of substrate hydrolyzed per min per ml of serum or plasma. The activity of PON1 and BuChE in the liver was expressed as mmol of substrate hydrolyzed per min per g of tissue. Measurement of MDA-TBA MDA in plasma MDA levels were determined by the MDA-TBA test. The TBA assay was performed according to a previously published method [20,21]. In the first step, an aliquot of plasma (250 mL) was added to 25 mL 0.2% BHT (2.6 di-ter-butyl-4-methylphenol, 99.0%, GC; Sigma–Aldrich Inc. St. Louis, USA), in ethanol (Merck, Darmstadt, Germany) and 1 mL 15% aqueous TCA (trichloracetic acid, 98%; Sigma–Aldrich Inc.). The mixture was then centrifuged (Hettich Universal 32 R Tuttingen Germany) at 4000 g for 15 min at 4 8C. The deproteinized supernatant (stock) was stored at 70 8C. An aliquot of 500 mL of the stoch solution was added to 1 mL TBA (0.375% in 0.25 M) HCl (37%; puriss. p.a., Sigma–Aldrich Inc.). The mixture was then heated at 100 8C for 15 min. After cooling, the solution was analyzed by spectrophotometry [20,21]. MDA in tissue samples The assay was adopted from Angulo et al. [20] and Botsoglou et al. [21] and adjusted for our purposes by Lovric´ et al. [22]. 0.2 g of tissues (liver, heart, kidney, and brain) were used to obtain 10% homogenates in 0.15 M KCl that were further treated with 25 mL 0.2% BHT in 98% ethanol as antioxidant. The homogenates were centrifuged at 18,890 g for 20 min. The separated supernatant was mixed with 5% aqueous TCA in proportion 1:4, and recentrifuged (Hettich Universal 32 R) at 1780 g for 15 min. The deproteinized supernatant was separated and aliquots of 500 mL were mixed with 500 mL TBA 0.375% in 0.25 M HCl. The mixture was heated at 100 8C for 15 min, followed by cooling of the samples to room temperature and measuring the MDA level by UV–vis spectrophotometer (HPV-220, Iskra, Slovenia) using a 1-cm absorption cell. The concentration of MDA was calculated by the absorbance at 532 nm, using a molar extinction coefficient of e = 1.56 105 M/cm [23]. During the whole procedure, until heating, the samples were kept on ice.
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Lipids and lipoproteins Lipids were determined on the Olympus automatic analyser AU 2700 with the use of original reagents produced by Olympus Diagnostics GmbH (Irish Branch), Lismeehan, Ireland. Plasma concentrations of total cholesterol and triacylglycerols were determined by enzymatic colorimetric methods [24,25]. The HDL-cholesterol level was estimated using a compound method of immuno-inhibition with b-lipoprotein antibody in the first step, and enzymatic colorimetric measuring of accessible cholesterol in the second step [26]. All plasma lipid concentrations were expressed as mmol/L. Data analysis Data were analyzed using an independent two-sample t-test. All applied tests were two-tailed, and p values 0.05 were considered as statistically significant. The data in table are presented as means and standard deviations. In the graphs, all individual data are presented as points, and the horizontal line denotes the mean. All calculations and data plotting were done using GraphPad Prism for Windows, version 5 [27,28]. Results Effects of SIMV on serum and liver PON1 activity SIMV caused a significant decrease of serum and liver PON1 activity, of 18% and 24%, respectively (Table 1). After withdrawal of SIMV treatment and the AT, PON1 activity in the serum was lower by 12% vs. control. On the contrary, PON1 activity in liver was higher by 35% than the activity measured in the control group (Table 1). Effects of SIMV on plasma and liver BuChE activity Both plasma and liver BuChE activity increased by 29% and 18%, respectively, compared to the values in the control group (Table 1). Following the ATP, the activity of BuChE remained higher in the plasma and liver (17% and 15%, respectively) in comparison with the control group (Table 1). Effects of SIMV on MDA levels in plasma, heart, liver, kidney, and brain Application of SIMV resulted in a significant decrease of MDA levels in the plasma (24%), heart (11%), liver (9%), kidney (13%), and brain (40%) (Fig. 1).
Table 1 Effect of simvastatin (10 mg/kg/day for 3 weeks) on paraoxonase 1 and butyrylcholinesterase activity and level of plasma lipids in Wistar rats. Data are presented as means and standard deviations. Variable
Treatment period Controla
Paraoxonase serum (mM/min/mL) Paraoxonase liver (mM/min/g) BuChE plasma (mM/min/mL) BuChE liver (mM/min/g) Total cholesterol (mM) HDL cholesterol (mM) Triacylglyceroles (mM)
0.33 0.07 0.25 0.08 0.07 0.01 0.28 0.06 1.54 0.22 1.08 0.12 0.93 0.22
Bold p values are significant at the 0.05 level. a The values are mean SD. b p value of independent two-sample t-test.
(n = 7) (n = 7) (n = 7) (n = 7) (n = 7) (n = 7) (n = 7)
After treatment Simvastatina (10 mg/kg/day)
pb Value
Controla
0.27 0.03 0.19 0.07 0.09 0.02 0.33 0.08 1.29 0.17 0.93 0.11 0.71 0.18
0.048 0.036 0.033 0.200 0.036 0.035 0.057
0.25 0.03 0.20 0.08 0.06 0.01 0.27 0.06 1.69 0.21 1.18 0.15 0.93 0.17
(n = 8) (n = 7) (n = 8) (n = 8) (n = 7) (n = 7) (n = 7)
(n = 8) (n = 7) (n = 7) (n = 7) (n = 7) (n = 7) (n = 7)
Simvastatina (10 mg/kg/day)
pb Value
0.22 0.04 0.27 0.08 0.07 0.02 0.31 0.08 1.64 0.23 1.13 0.18 1.12 0.36
0.129 0.107 0.260 0.311 0.697 0.607 0.224
(n = 7) (n = 7) (n = 7) (n = 7) (n = 7) (n = 7) (n = 7)
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Fig. 1. MDA levels in plasma and rat tissue (heart, brain, liver, kidney) after 21 days of treatment with simvastatin as well as 10 days after simvastatin discontinuation. Results are expressed as mean 95% confidence intervals. p 0.05 compared to the control group (TP – treatment period, AT – after treatment).
After the completion of the AT, the concentrations of MDA remained significantly lower in plasma (22%), heart (17%), liver (9%), kidney (13%), and brain (29%), as compared with their control values (Fig. 1).
concentration of triacylglycerols increased by 20% vs. control (Table 1).
Effects of SIMV on plasma lipids
The findings of the present study show that SIMV caused a low but significant decrease of PON1 activity in the serum (18%) and liver (24%) (Table 1). At the same time, SIMV induced a parallel significant lowering of HDL and total cholesterol concentration by 14% and 16% respectively, whilst the level of triacylglycerols showed the tendency to decrease by 24% in the plasma of normolipidemic rats (Table 1).
The plasma levels of total and HDL-cholesterol were significantly lower, by 16% and 14% respectively, and the triacyglycerol level decreased by 24% after SIMV administration (Table 1). After the end of AT, the levels of both total and HDL-cholesterol were similar to the values in the control group, while the plasma
Discussion
M. Macan et al. / Pharmacological Reports 67 (2015) 907–913
Several mechanisms might be responsible for the decrease of PON1 activity caused by SIMV. The first potential mechanism is the enzyme’s close structural bond mostly with HDL-cholesterol, but to a lesser extent also with VLDL-cholesterol and chylomicron particles, which stimulate the secretion of the enzyme from liver and ensure the optimal environment for its catalytic activity [29]. Therefore, it seems logical to assume that every change of the structure and/or concentration of these plasma lipoprotein particles, caused by statins, might lead to an alteration of PON1 activity. Because SIMV in our study significantly decreased total and HDL-cholesterol and also showed a tendency to decrease plasma triacylglycerols, these changes of plasma lipid concentration might partially explain the significant decrease of PON1 activity in the serum of rats, although they were normolipidemic [29]. In our study the decrease of PON1 activity in serum and liver was similar. Because of fact that PON1 is synthesized in the liver, it may be supposed that the second potential mechanism responsible for the reduction of PON1 activity is the inhibition of enzyme synthesis caused by SIMV. Our hypothesis is supported by results of Goue´dard et al. [30], who showed that in HuH7 human hepatoma cells pravastatin, fluvastatin, and SIMV caused a significant decrease of PON1 activity and its mRNA level of 25%, 30%, and 40%, respectively. A significant decrease of PON1 gene promoter activity of 40–60% was also obtained [30]. Since liver PON1 activity in our study showed the tendency to increase by 35% 10 days after cessation of SIMV administration (Table 1), we can conclude that decrease of PON1 activity in liver was possibly mediated by its reversible repressive action on the PON1 gene promoter. The third possible mechanism of significant decrease of PON1 activity might be the activation of peroxisome proliferatoractivated receptor a (PPARa) caused by SIMV. According to Beltowski et al. [10] and Berthou et al. [31], in rodents, as opposed to humans, PPARa agonists inhibit the synthesis of apoA-I in the liver. Since apoA-I is the dominant component of HDL-cholesterol that stimulates the secretion of PON1 from the liver into the blood, SIMV as a possible PPARa agonist could cause the inhibition of apoA-I synthesis in the liver and a decrease of PON1 activity. It is important to mention that HDL is the main cholesterol-transporting fraction of plasma lipoproteins in rat. Fourthly, since SIMV is a compound that contains a lactone ring, and in regard of the fact that PON1 hydrolyzes lactone ringcontaining substances [32], the significant decrease of the liver and serum PON1 activity observed in our experiment may simply reflect the consumption of this enzyme in the process of SIMV breakdown. Our results obtained from the observation of PON1 activity and levels of plasma total cholesterol and triacylglycerols are chiefly supported by the results of other authors. The treatment of normolipidemic rats with fluvastatin (2 or 20 mg/kg/day for 3 weeks) reduced PON1 activity in plasma, liver, heart, and kidney, whereas pravastatin (4 or 40 mg/kg/day for 3 weeks) caused a significant decrease of PON1 only in the kidney [10,18]. These statins had no effect on either total or HDL-cholesterol, but significantly reduced plasma triacylglycerols [10]. PON1 activity also showed the tendency to decrease by 16.1% and 11.6% after treatment with cerivastatin (0.03 or 0.3 mg/kg for 3 weeks) [33]. In contrast to these findings, in the study of Bolayirli et al., 0.3 mg/kg/ day atrovastatin for 4 weeks resulted with a significant increase of PON1 activity and HDL-cholesterol, whilst the level of LDLcholesterol decreased significantly in the plasma of hypercholesterolemic rabbits [34]. It is important to emphasize that studies on the effect of SIMV on the activity of PON1 in humans have yielded discordant results as well [7].
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The significant decrease of HDL-cholesterol (Table 1) obtained in our results is an exception in comparison with the results mentioned above. These decrease of HDL-cholesterol could be the consequence of the inhibition of apoA-I synthesis in the rat liver caused by SIMV due to its agonistic action on PPARa receptors [10]. The lowering of plasma triacylglycerols in our experiments is also partially mediated by activation of PPARa receptors [35], causing in rodents the induction of genes that decrease the availability of triacylglycerols for hepatic VLDL secretion and induction of genes that promote lipoprotein lipase-mediated lipolysis of triacylglycerol-rich plasma lipoproteins [31,35]. According to our results, SIMV caused the significant increase of plasma BuChE activity of 29% (Table 1), while liver BuChE activity showed a tendency to be increased by 18% (Table 1). Following the AT, BuChE activity remained higher in the plasma and liver (17% and 15%, respectively) vs. control, which implies that the effect of SIMV does not cease 10 days after discontinuation (Table 1). Our results show a negative correlation between plasma and liver BuChE activity and the levels of total and HDLcholesterol and triacylglycerols in the plasma of normolipidemic rats (Table 1) which is in the contrast with results of Rustemeijer et al. [12]. This difference can partially be explained by the fact that our results were obtained in a model of normolipidemic rats, whereas in clinical studies changes in BuChE activity and lipid profiles were observed under pathological conditions. It can be assumed that in our experiment the increase in BuChE activity represents a direct consequence of SIMV on the PPARa receptors as well [10]. We suggest that in rodents statins cause induction of beta-oxidation of fatty acids that is followed by an increased production of butyrylcholine as its metabolite [11]. Therefore, the increased BuChE activity in our experiments might actually result from the increased demand for butyrylcholine degradation. If not metabolized, butyrylcholine could cause unwanted toxic effects [11]. Our recent results obtained in hyperlipidemic rats show an increase of BuChE activity in rat brain after SIMV treatment [36]. We suggest that as an adverse effect, especially when the role of cholinesterase in the development of AD is being considered. Our results are completely in contrast with the results of other investigators [16,17], who found that statins cause either the inhibition of both enzymes or have no effect on AChE and BuChE activity. Although it is claimed that statins with high lipid solubility, such as SIMV, suppress AChE activity [37] our results obtained in normolipidemic and hyperlipidemic rats abolish their protective effect against AD, due to the inhibition of cholinesterase activity. In our investigations SIMV treatment resulted in a significant decrease of MDA levels in the plasma (24%), heart (11%), liver (9%), kidney (13%), and brain (40%) (Fig. 1). Our results obtained from the observation of MDA plasma levels are chiefly supported by the results of Beltowski et al. [10,33], who proved a statistically significant decrease in MDA (46.6%) and oxidized lipids (59.3%) in rat plasma after administration of cerivastatin (0.3 mg/kg/day for 3 weeks) and fluvastatin (19.8% and 30.9%, respectively, at doses of 2 and 20 mg/kg/day for 3 weeks), and also a tendency for MDA decrease after pravastatin (25.9%, at a dose of 40 mg/kg/day for 3 weeks). Similar results were confirmed by experiments of Bolayirli et al., who showed the antioxidant activity of 0.3 mg/kg/ day atrovastatin for 4 weeks in rabbits with hypercholesterolemia [34]. Investigations of the effects of statins on the MDA level in dyslipidemic type 2 diabetic patients gave the same results [38]. We have shown very clearly that the significant MDAlowering effect of SIMV established in the plasma, heart, kidney, liver, and brain at the end of the treatment was maintained even 10 days after the treatment period (Fig. 1). These results are a
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strong proof for the positive ‘‘pleiotropic’’ effect of SIMV and for the justified statin therapy of some acute cardiovascular diseases. In addition, the antioxidative effect of SIMV on MDA levels in the brain proven in our experiments could also explain its positive effects in patients with AD. The antioxidant effects of statins may be attributed to their antiinflammatory and lipid-lowering action, i.e., that in animals and humans with hyperlipidemia these effects might be the consequence of their lipid-lowering action and thus reduced availability of plasma lipids for the process of peroxidation [32]. Our results show that antioxidative effects of SIMV are also present in normolipidemic rats and that they are completely independent of its other lipid and non-lipid effects. In conclusion, our results have clearly demonstrated that SIMV in normolipidemic rats caused the decrease of PON1 activity, while BuChE activity was increased. Our results also gave proof for the antioxidant action of SIMV, because the MDA level was significantly lower in the plasma, heart, kidney, and brain after SIMV treatment, and was maintained even 10 days after the treatment period. Therefore, we suggest that the positive effects of SIMV in patients with cardiovascular disorders and Alzheimer disease are the consequence of its antioxidative action. The effect of SIMV on BuChE activity represents an adverse event in the context of its action on the brain and possible therapy of Alzheimer disease. Authors’ contribution Marija Macan, Antonija Vuksˇic´, Suzana Zˇunec, Jasna Lovric´, Marta Kelava, Nikola Sˇtambuk, and Vlasta Bradamante participated in the research and the article preparation. Pasˇko Konjevoda participated in the article preparation and conduction of statistical analysis. Funding This study was supported by the Ministry of Science, Education, and Sports of the Republic of Croatia, Grant No. 108-00000000013. Conflict of interest All authors declare no conflict of interest. Acknowledgments We gratefully acknowledge the expert technical assistance of Mrs Jelka Roca. We also wish to thank Ms Aleksandra Zˇmegacˇ Horvat for language editing the text. References [1] Parizadeh SM, Azarpazhooh MR, Moohebati M, Nematy M, Ghayour-Mobarhan M, Tavallaie S, et al. Simvastatin therapy reduces prooxidant-antioxidant balance: results of a placebo-controlled cross-over trial. Lipids 2011;46: 333–40. [2] Janero DR. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radic Biol Med 1990;9:515–40. [3] Giugliano D, Ceriello A, Paolisso G. Diabetes mellitus, hypertension, and cardiovascular disease: which role for oxidative stress? Metabolism 1995; 44:363–8. [4] Endres M. Statins: potential new indications in inflammatory conditions. Atheroscler Suppl 2006;7:31–5. [5] Thallinger C, Urbauer E, Lackner E, Graselli U, Kostner K, Wolzt M, et al. The ability of statins to protect low density lipoprotein from oxidation in hypercholesterolemic patients. Int J Clin Pharmacol Ther 2005;43:551–7. [6] Schneider MP, Schmidt BM, John S, Schmieder RE. Effects of statin treatment on endothelial function, oxidative stress and inflammation in patients with arterial hypertension and normal cholesterol levels. J Hypertens 2011;29: 1757–64.
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