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Alpha lipoic acid and vitamin E improve atorvastatin-induced mitochondrial dysfunctions in rats
Journal Pre-proofs Alpha lipoic acid and Vitamin E improve atorvastatin-induced mitochondrial dysfunctions in rats Hatice Eser Faki, Bunyamin Tras, Kamil Uney PII: DOI: Reference:
S1567-7249(19)30253-3 https://doi.org/10.1016/j.mito.2020.02.011 MITOCH 1454
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Mitochondrion
Received Date: Revised Date: Accepted Date:
20 September 2019 12 December 2019 27 February 2020
Please cite this article as: Faki, H.E., Tras, B., Uney, K., Alpha lipoic acid and Vitamin E improve atorvastatininduced mitochondrial dysfunctions in rats, Mitochondrion (2020), doi: https://doi.org/10.1016/j.mito. 2020.02.011
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© 2020 Published by Elsevier B.V.
Alpha lipoic acid and Vitamin E improve atorvastatin-induced mitochondrial dysfunctions in rats
Hatice Eser Faki1,*, Bunyamin Tras1, Kamil Uney1
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Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University
of Selcuk, 42031 Konya, Turkey *Corresponding Author Full Name: Hatice ESER FAKI E-mail: [email protected] Address:Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Selcuk, 42031 Konya, Turkey Phone Number: +90332 2232684 Fax Number: +90332 2410063 ORCID: 0000-0002-6124-7168
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Summary To determine the effects of alpha lipoic acid (ALA) and vitamin E (Vit E) on mitochondrial dysfunction caused by statins. A total of 38 Wistar Albino rats were used in this study. The control group received dimethyl sulfoxide. The atorvastatin (A) group received atorvastatin (10 mg/kg). The A+ALA group received atorvastatin (10 mg/kg) and ALA (100 mg/kg). The A+Vit E group was administered atorvastatin (10 mg/kg) and Vit E (100 mg/kg). The A+ALA+Vit E group was administered atorvastatin (10 mg/kg), ALA (100 mg/kg) and Vit E (100 mg/kg). All applications were administered simultaneously by gavage for 20 days. ATP level and complex I activity were measured from liver, muscle, heart, kidney and brain. Atorvastatin significantly decreased the ATP levels in heart and kidney, while a slight decrease was seen in liver, muscle and brain. Atorvastatin caused an insignificant decrease in the complex I activity in all tissues examined. ALA administration significantly improved the ATP levels in the liver, heart and kidney, while Vit E improved the ATP levels in all tissues except the muscle compared to Atorvastatin group. Single administration of both ALA and vit E ameliorated complex I activity in the muscle, heart, kidney and brain. The combination of ALA and Vit E significantly improved the ATP levels in the liver, heart, kidney and brain and also provided significant improvements the complex I activity in all tissues. The undesirable effects of Atorvastatin on mitochondrial functions in this study ameliorated by using ALA and/or Vit E alone and in combination. Key words: Mitochondria, ATP, Complex I, Atorvastatin, Alfa lipoic acid, Vitamin E
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1. Introduction Mitochondria, known as cellular energy centers and found in most eukaryotic organism, produce adenosine triphosphate (ATP) by oxidative phosphorylation (OXPHOS) in the respiratory chain (electron transport chain, ETC). All cell types have mitochondria, except for erythrocytes. The number of mitochondria of the cells is associated with their energy need. Heart, muscle, brain and gastrointestinal cells have a large number of mitochondria, whereas cells using low energy such as skin cells have less mitochondria (Frye and Rossignol, 2011). The mitochondrion consists of four main parts: the outer mitochondrial membrane (OMM), the intermembrane space (IMS), the inner mitochondrial membrane (IMM) and the mitochondrial matrix (MM). The ETC, which plays an important role in energy production in aerobic organisms, consists of five protein complexes in IMM: NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), ubiquinone cytochrome c oxidoreductase (complex III), cytochrome c oxidase (complex IV) and ATP-synthase (complex V) (Paradies et al., 2011). The complex I, which is measured to determine mitochondrial dysfunction in this study, catalyzes the 2 electron oxidation of NADH followed by the reduction of ubiquinone (Q) to form ubiquinol (QH2), and ultimately the reduction of the terminal electron acceptor, O2. The inhibition of complex I causes mitochondrial dysfunction and increases ROS production (Paradies et al., 2002; Papa et al., 2012). Mitochondrial dysfunction is a prominent factor in various metabolic diseases, intoxications, aging process and especially in muscle and nervous system diseases due to its effect in cell metabolism (Cardinali et al., 2013; Jang et al., 2017; Johannsen and Ravussin, 2009). The ATP level, ETC enzyme activities, mitochondrial DNA (mtDNA), mitochondrial content, mitochondrial membrane potential, ROS production, deteriorated apoptosis, lactate level and intracellular calcium content are considered as biomarkers of this organelle dysfunction (Brand and Nicholls, 2011; Delbarba et al., 2016; Montgomery and Turner, 3
2015). The causes of mitochondrial dysfunction are mitochondrial and genomic DNA mutations, supercomplex destabilization, mitochondrial protein accumulations, endoplasmic reticulum stress, aging, exposure to xenobiotics such as statins, and modern lifestyle (Dudkina et al., 2010; Lim et al., 2010; Park and Larsson, 2011; Tuppen et al., 2010; Pellegrino et al., 2013; Vannuvel et al., 2013). Many drugs used in treatment (flutamide, paclitaxel, oxaliplatin, cisplatin, methotrexate, valproic acid, ketamine, fipronil, buspirone, trazodone) cause mitochondrial dysfunctions (decreases in ATP and NADPH (nicotinamide adenine dinucleotide phosphate) levels, complex I, II, III and IV activities) (Berson et al., 1998; Kashimshetty et al., 2009; Lewis ve Dalakas, 1995; Lewis et al., 2003; Varga et al., 2015). Statins, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, are commonly used for the prevention cardiovascular diseases in lipid metabolism disorders such as hypercholesterolemia. These drugs, which have anti-inflammatory, antiproliferative and antithrombotic effects, can also inhibit the growth of tumor cells and increase intracellular calcium mobilization (Arnaud et al., 2005; Bellosta et al., 2000; Futterman and Lemberg, 2004; Liao and Laufs, 2005; Stancu and Sima, 2001). Statins reduce the morbidity and mortality of cardiovascular disease but also, cause adverse effects (rhabdomyolysis, myopathy, myoglobinemia, arthralgia, etc.) that require one to quit taking the drug due to long-term use in the indicated indication (Jasiñska et al., 2007). In addition, other adverse effects attributed to the effects of these drugs on mitochondria have also been reported, such as diabetes, increase in transaminase level, polyneuropathy and myoglobinemia-related renal failure (Naci et al., 2013; Ruscica et al., 2014; Sathasivam, 2012). The effects of statins on mitochondria are dose-related (Cafforio et al., 2005; Dirks and Jones, 2006; Parihar et al., 2012; Wagner et al., 2008). These drugs inhibit the synthesis of mevalonate, which is a precursor of coenzyme Q10 (ubiquinone) and an important component in the mitochondrial respiratory chain, and impair mitochondrial function as they reduce the coenzyme Q10 4
(Wagner et al., 2008). Statins are responsible for 0.5-1% of myopathies associated with coenzyme Q10 level decrease and electron transport block (Dirks and Jones, 2006). Recent studies have highlighted that the adverse effects of statins on muscle, liver and brain are related to mitochondria (Abdoli et al., 2013; Galtier et al., 2012; Golomb and Evans, 2008; Kaufmann et al., 2006; Nadanaciva et al., 2007; Sirvent et al., 2012). Coenzyme Q-10, carnitine, alpha lipoic acid (ALA), vitamin E (vit E) and C, glutathione, ginkgo biloba, curcumin, omega-3 polyunsaturated fatty acids, N-acetylcysteine (NAC) and melatonin, which have a healing effect on mitochondrial dysfunction, are recommended against diseases such as diabetes, Alzheimer’s, Parkinson’s, ALS and cancer, which are presumed to be associated with mitochondrial dysfunction (Kontush et al., 2001; Rastogi et al., 2008; Wadsworth et al., 2008). It has been suggested that these substances with antioxidant effects support healthy mitochondrial biogenesis process by reducing mitochondrial dysfunction (de Oliveira et al., 2016; Hickey et al., 2012; Kumar and Singh, 2015; Legido et al., 2018). ALA provides the recycling of cellular antioxidants, including coenzyme Q, vitamin C and E, glutathione, iron and copper chelates (Moini et al., 2002). ALA, which is involved in many multi-enzyme complexes in mitochondria, is required to metabolize carbohydrates, proteins and fats and to convert them to ATP. In addition, ALA has antimutagenic and anticarcinogenic effects owing to its antioxidant effect (Cremer et al., 2006; Miadokova et al., 2000; Novotny et al., 2008). ALA, which can cross the blood brain barrier, is useful in cases such as diabetes, heart failure, cataract, myocardial infarction, neuropathic pain and neurodegeneration (Beckman et al., 2002). In addition, this substance is a redox regulator of proteins such as myoglobin, prolactin, thioredoxin and nuclear factor kappa beta (NF-κB) transcription factor (Fuchs, 1997; Packer et al., 1995).
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Vit E has anti-obesity, anti-hypercholesterolemic, anti-diabetic and anti-hypertensive effects, as well as preventive effects on lipid peroxidation and other radical-induced oxidative events (Budin et al., 2009; Matough et al., 2014; Newaz et al., 2003; Zaiden et al., 2010; Zhao et al., 2015). This vitamin plays an important role in maintaining the integrity of lipid rich organelles such as membranes (Greene et al., 2017). While β-tocopherol is mostly localized in mitochondrial fractions, α-tocopherol takes an important role in maintaining mitochondrial integrity (Li and May, 2003). The aim of this study was to determine whether antioxidant ALA and vit E can improve atorvastatin-induced mitochondrial dysfunctions and to reveal whether these can be used to prevent the adverse effects of statins and other drugs that cause mitochondrial dysfunction. 2. Materials and methods 2.1. Animals and study design Wistar Albino male rats (38 in total, 6-10 weeks, 242±1.92 g) were used in this study, utilizing research procedures approved by the ethics committee (SUDAM, 2017-42). During the experimental period, the rats were housed in standard rat cages in a central facility under controlled conditions (12 h light/dark cycle and room temperature of 20°C ± 2°C) at SUDAM and allowed water and food ad libitum. In this study, the rats were divided into 5 groups with a control group (C) consisting of 6 rats and the remaining 32 rats were divided into 4 equal groups. The substances administered are presented in Table 1. ALA and Vit E were coadministered with atorvastatin for 20 days. The doses of atorvastatin, ALA and Vit E were determined by evaluating previous studies in this field (Savitha et al., 2005; Kuhad and Chopra, 2009; Pal et al., 2015).
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At the end of the experiment, the rats were euthanized by cervical dislocation under anesthesia (ketamine 95 mg/kg, SC+xylazine 5 mg/kg, SC). Immediately after the euthanasia, the liver, muscle (gastrocnemius), heart, kidney and brain tissue samples were received and were snap frozen immediately in liquid nitrogen and stored at −70°C until analysis time. 2.2. Concomitant medications Atorvastatin (Cholvast, 10 mg tablet, Biofarma Pharmaceutical Industry Ltd. Sti., Turkey), Vitamin E (150 mg/ml, IM, Evigen, Aksu Farma Medicinal Products and Pharmaceuticals Co., Turkey), Alfa Lipoic Acid (İlko Pharmaceuticals Industry and Trade Inc.). For animal administrations, atorvastatin (1 mg/ml) and alfa lipoic acid (100 mg/ml) dissolved in water and dimethyl sulfoxide (DMSO) (Çakır, et al., 2015), respectively. Chemicals with analytical purity was used in the study. 2.3. Laboratory tests 2.3.1. Measurement of ATP The ATP levels were determined from the liver, muscle (gastrocnemius muscle), heart, kidney and brain tissues of rats using the extraction method reported Chida et al. (2012) and a commercial ATP kit (Luminescent ATP Detection Assay Kit ab113849). All the measurements for ATP were performed according to the commercial kit procedures. In this study, ATP and luciferin were used to determine total ATP levels, oxyluciferin and firefly luciferase to convert it to light. The light emitted in this reaction is directly proportional to the concentration of the ATP present. This kit neutralizes adenylpyrophosphatase (ATPase) during the lysis step and ensures that the obtained light signal corresponds only to cellular ATP levels.
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2.3.2. Measurement of mitochondrial complex I Mitochondria were isolated with mitochondria isolation kit (Mitochondria Isolation Kit Sigma MITOISO1) from the liver, muscle (gastrocnemius muscle), heart, kidney and brain tissues of rats, and isolation was confirmed by Bradford protein analysis. The activity of NAD (Nicotinamide Dinucleotide) dehydrogenase (complex I), one of the mitochondrial complex enzymes, was determined by ELISA reader (MWGt Lambda Scan 200, Bio-Tek Instruments, Winooski, VT, USA) using commercial mitochondrial complex I activity kit (MitoCheck Complex I Activity Assay Kit Cayman 700930). All the measurements for complex I were performed according to the commercial kit procedures. This kit allows for direct inhibitory effects on complex I to be easily observed. Inhibition of complex I activity was performed with potassium cyanide. The NADH oxidation rate is measured by a decrease in absorbance at 340 nm and is proportional to the activity of complex I. 2.4. Statistical analyses All data are presented as mean ± SD. The Mann-Whitney U test was used to assess the group differences in the tissue ATP and the mitochondrial complex I activity. The control and other groups or atorvastatin group and other groups were compared in terms of tissue ATP level and mitochondrial complex I activity. The SPSS 22.0 program (IBM Corp, Armonk, NY) was used for statistical analysis. Statistical significance was assigned at P<0.05. 3. Results Atorvastatin caused a decrease in ATP level in the heart and kidney compared to the control group, but not in the liver, muscle or brain (P<0.05). On the other hand, atorvastatin insignificantly decreased the complex I activity in all tissues.
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ALA administration significantly improved the ATP levels in the liver, heart and kidney, while Vit E improved the ATP levels in all tissues except the muscle compared to Atorvastatin group (P<0.05). Single administration of both ALA and vit E ameliorated complex I activity in the muscle, heart, kidney and brain compared to Atorvastatin group (P<0.05). The combination of ALA and Vit E significantly improved the ATP levels in the liver, heart, kidney and brain and also provided significant improvements the complex I activity
in
all
tissues
compared
to
Atorvastatin
group
(P<0.05).
4. Discussion The long-term medication is used in the treatment of certain diseases such as diabetes and cardiovascular diseases. Chronic drug use increases the incidence of adverse effects of drugs. Strategies are being developed to reduce adverse drug effects in long-term drug use cases. Long-term use of statins to prevent cardiovascular diseases have mitochondrial dysfunction-based side effects on tissues and organs such as muscle, liver and brain. In this study, the effects of ALA, vit E and their combinations on atorvastatin-induced mitochondrial dysfunction were determined by ATP and complex I biomarkers. In the present study, atorvastatin caused statistically significant decrease in ATP levels (P<0.05) in the heart, kidney and brain compared to the control group; but did not cause significant changes in ATP levels in the muscle or liver or complex I enzyme activity in any tissues. ALA, vit E and their combinations improved atorvastatin-induced reductions in ATP levels in all tissues except muscle. We determined that each of these substances recovers the reductions in complex I activity caused by atorvastatin in all tissues except liver; on the other hand, combined use of these substances also improves the complex I activity of liver. Similar to the findings of our study, it was reported that there were no statistically significant changes in mitochondrial function biomarkers (mitochondrial membrane potential, 9
ATP synthesis, cytochrome c oxidase and citrate synthase activity and mitochondrial respiratory functions) on the 5th and 10th days of a 15 day cerivastatin (0.1, 0.5, and 1.0 mg/kg/day) administration to rats at different doses, but there were weak changes that were not statistically significant on the 15th day (Schaefer et al., 2004). In another study conducted by Mohammadi-Bardbori et al. (2015) it was found that simvastatin (30 mg/kg/bw/day) and atorvastatin (30 mg/kg/bw/day) significantly changed some mitochondrial dysfunction biomarkers (complex II activity, ATP level, mitochondrial membrane potential and mitochondrial permeability transition pore) in liver mitochondria in rats, in contrast to our findings. The difference between the findings of the mentioned study and our study may be due to the differences in dosage regimen (dose, administration period) and the sensitivity of the measured parameters to the drugs. In a study evaluating the toxicity of statins in rat skeletal muscle cell lines (L6 cells), cerivastatin, fluvastatin, atorvastatin, simvastatin and pravastatin were administered. As a result, it was reported that cerivastatin, fluvastatin and atorvastatin decreased mitochondrial membrane potential by 49-65%, while the other two did not. In addition, it was determined that lipophilic statins caused mitochondrial swelling, cytochrome c release and DNA fragmentation, and lipophilic statins were more toxic to mitochondrial functions of the skeletal muscle than hydrophilic statins (pravastatin) (Kaufmann et al., 2006). So far, a lot of in vitro and in vivo studies have been carried out on different tissues and organs of different organisms to ameliorate mitochondrial dysfunction caused by drugs and various chemicals and to identify substances that can be used against the predicted diseases caused by mitochondrial dysfunction. The mentioned studies were carried out on different cell lines and organs of different organisms. The efficacy of the substances investigated in these studies was evaluated on mitochondrial dysfunction biomarkers, as in our study. 10
In the present study, the decrease in atorvastatin-induced liver mitochondria ATP level was improved by the administration of Vit E alone and in combination with ALA, whereas the reduction in complex I activity in the liver mitochondri was improved only by combined administration of these substances. Mohammadi-Bardbori et al. (2015) reported that oral coadministration of CoQ10 with simvastatin (30 mg/kg/bw/day) and atorvastatin (30 mg/kg/bw/day) to rats for 30 days recovered the changes (complex II activity, ATP level, mitochondrial permeability transition pore) in liver mitochondria caused by the indicated drugs. Some studies investigating the effect of ALA on liver, kidney and brain cell mitochondrial functions in elderly rats showed ALA to improve mitochondrial membrane potential, ATP, mitochondrial enzyme and ETC complex activities (Arivazhagan et al., 2001; Hagen et al., 1999; Palaniappan and Dai; 2007). In addition, it was stated that ALA improved LPS-mediated change in ATP, complex I, IV and V, NADH, mtDNA expression and enzymes in rat liver mitochondria (Hiller et al., 2014; Liu et al., 2015). Kheir-Eldin et al. (2001) reported that decrease in ATP/ADP ratio and increases in mitochondria/cytosolic hexokinase ratio caused by LPS in rat brain mitochondria were ameliorated by vit E, beta carotene, Se and NAC treatments, and Vit E+Se was foundthe most effective. The damage caused by methanol and ethionine in rat liver mitochondria (decrease in NADH dehydrogenase, succinate cytochrome c reductase, cytochrome oxidase activity and ATP level) was improved by 20-28 period oral administration of vit E (Padma and Setty, 1997; El-Sayed et al., 2002). We found that vit E improved atorvastatin-mediated ATP reduction in the liver mitochondria, did not improve the reduction in complex I activity, but its combination with ALA improved the reduction in complex I activity. Consistent with the results of our study, Zhou et al. (2018) reported that alpha lipoamide (neutral amide derivative of lipoic acid) increased brain ATP levels and improved mitochondrial morphology in nerve cells in 6-hydroxydopamine-induced 11
Parkinson’s animal model in rats. Also, the administration of vit E together with endosulfan and gossypol to experimental animals (mice, rat) improved the reduction in testicular mitochondrial ATP levels caused by these substances (Santana et al., 2015; Wang et al., 2012). In vitro studies performed with various cell lines revealed that ALA, vit E, melatonin and ginsenoside corrected mitochondrial dysfunction (impairment in mitochondrial membrane potential and decrease in ATP level and cytochrome c oxidase activity) caused by amyloid beta, S-nitrosylation, acrylamide and tunicamycin (Cardoso et al., 2001; Huang et al., 2012; Hiller et al., 2016; Lei et al., 2016; Song et al., 2017). In conclusion, it can be indicated that i) the effects of statins on mitochondria vary by organs, ii) the ATP level is a more sensitive biomarker for mitochondrial dysfunction than complex I activity, and iii) ALA alone and in combination with vit E can be used concurrently with drugs such as statins, which are used in the long-term and cause mitochondrial dysfunction, to reduce their adverse effects during therapy. ACKNOWLEDGMENTS This research was summarized from the PhD thesis of the first author. Research was supported by OYP (2015-OYP-042). The abstract of this article was presented as an oral presentation at the congress “2nd Internatıonal Eurasıan Conference on Biological and Chemical Sciences, June, 28-29, 2019". CONFLICT OF INTEREST The authors declare that they have no conflict of interest. AUTHORS' CONTRIBUTIONS Hatice Eser Faki contributed the conception, experimental administration, analysis, drafted to manuscript, critically revised the manuscript and agreed to be accountable for all aspects of 12
work ensuring integrity and accuracy. Bunyamin Tras contributed the critically revised the manuscript and agreed to be accountable for all aspects of work ensuring integrity and accuracy. Kamil Uney contributed the analysis, critically revised the manuscript and agreed to be accountable for all aspects of work ensuring integrity and accuracy.
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Table 1 The substances administered and their doses in groupsa Group
Atorvastatin
Alfa Lipoic Acid
Vitamin E
Cb (n=6)
-
-
-
A (n=8)
10 mg/kg
-
-
A+ALA (n=8)
10 mg/kg
100 mg/kg
-
A+Vit E (n=8)
10 mg/kg
-
100 mg/kg
A+ALA+Vit E (n=8)
10 mg/kg
100 mg/kg
100 mg/kg
C: control, A: atorvastatin, A+ALA: atorvastatin + alpha lipoic acid, A+Vit E: atorvastatin + vitamin E, A+ALA+Vit E: atorvastatin + alpha lipoic acid +vitamin E. a
All substances were administered orally. For animal administrations, atorvastatin (1 mg/ml) and alfa lipoic acid (100 mg/ml) dissolved in water and dimethyl sulfoxide,
respectively. b
Dimethyl sulfoxide (100µl/kg) was applied to the control group.
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Table 2 The effects of alpha lipoic acid (100 mg/kg) and vitamin E (100 mg/kg) on the ATP in various tissues of atorvastatin (10 mg/kg) treated rats (mean ± SD)* Group
Liver
Muscle
Heart
Kidney
Brain
C (n=6)
0.0293±0.0372d
4.9224±0.3846a
2.7270±0.5829b
0.0206±0.0096c
0.7047±0.1714c
A (n=8)
0.0172±0.0178d
4.7252±0.3846a
2.4238±0.3683c
0.0085±0.0096d
0.6493±0.0366c
A+ALA (n=8)
0.3228±0.1240a
5.3519±0.9384a
4.5044±0.4847a
0.2500±0.1268b
0.8438±0.2947bc
A+Vit E (n=8)
0.1001±0.0374c
5.4006±0.3469a
3.2661±1.0230b
0.4128±0.0606a
1.1019±0.1909ab
A+ALA+Vit E (n=8)
0.2002±0.0968b
5.0549±1.0800a
0.3243±0.1633b
0.3243±0.1633ab
1.7431±0.8190a
* The values are presented as nM. a,b,c
: Different letters in the same line are statistically different (P˂0.05).
C: control, A: atorvastatin, A+ALA: atorvastatin + alpha lipoic acid, A+Vit E: atorvastatin + vitamin E, A+ALA+Vit E: atorvastatin + alpha lipoic acid + vitamin E.
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Table 3 The effects of alpha lipoic acid (100 mg/kg) and vitamin E (100 mg/kg) on the mitochondrial complex I in various tissues of atorvastatin (10 mg/kg) treated rats (mean ± SD)* Group
Liver
Muscle
C (n=6)
100±34b
99±14b
99±55ab 100±22bc 100±21b
A (n=8)
89±13bc
93±6b
91±10b
A+ALA (n=8)
124±11ab 136±20a 136±15a 123±21ab 138±13a
A+Vit E (n=8)
127±12ab 138±13a 130±16a
A+ALA+Vit E (n=8)
131±9a
134±11a
Heart
128±6a
Kidney
94±13c
125±7a
Brain
94±19b
144±16a
136±23ab 152±23a
* The values are presented as % activity. a,b,c
: Different letters in the same line are statistically different (P˂0.05).
C: control, A: atorvastatin, A+ALA: atorvastatin + alpha lipoic acid, A+Vit E: atorvastatin + vitamin E, A+ALA+Vit E: atorvastatin + alpha lipoic acid + vitamin E.
20
The effects of Atorvastatin on mitochondria vary by organs.
The ATP level is a more sensitive biomarker than complex I activity.
Effects of Atorvastatin on mitochondrial functions in this study ameliorated by using ALA and/or Vit E alone and in combination.
21
S1567-7249(19)30253-3 https://doi.org/10.1016/j.mito.2020.02.011 MITOCH 1454
To appear in:
Mitochondrion
Received Date: Revised Date: Accepted Date:
20 September 2019 12 December 2019 27 February 2020
Please cite this article as: Faki, H.E., Tras, B., Uney, K., Alpha lipoic acid and Vitamin E improve atorvastatininduced mitochondrial dysfunctions in rats, Mitochondrion (2020), doi: https://doi.org/10.1016/j.mito. 2020.02.011
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© 2020 Published by Elsevier B.V.
Alpha lipoic acid and Vitamin E improve atorvastatin-induced mitochondrial dysfunctions in rats
Hatice Eser Faki1,*, Bunyamin Tras1, Kamil Uney1
1
Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University
of Selcuk, 42031 Konya, Turkey *Corresponding Author Full Name: Hatice ESER FAKI E-mail: [email protected] Address:Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Selcuk, 42031 Konya, Turkey Phone Number: +90332 2232684 Fax Number: +90332 2410063 ORCID: 0000-0002-6124-7168
1
Summary To determine the effects of alpha lipoic acid (ALA) and vitamin E (Vit E) on mitochondrial dysfunction caused by statins. A total of 38 Wistar Albino rats were used in this study. The control group received dimethyl sulfoxide. The atorvastatin (A) group received atorvastatin (10 mg/kg). The A+ALA group received atorvastatin (10 mg/kg) and ALA (100 mg/kg). The A+Vit E group was administered atorvastatin (10 mg/kg) and Vit E (100 mg/kg). The A+ALA+Vit E group was administered atorvastatin (10 mg/kg), ALA (100 mg/kg) and Vit E (100 mg/kg). All applications were administered simultaneously by gavage for 20 days. ATP level and complex I activity were measured from liver, muscle, heart, kidney and brain. Atorvastatin significantly decreased the ATP levels in heart and kidney, while a slight decrease was seen in liver, muscle and brain. Atorvastatin caused an insignificant decrease in the complex I activity in all tissues examined. ALA administration significantly improved the ATP levels in the liver, heart and kidney, while Vit E improved the ATP levels in all tissues except the muscle compared to Atorvastatin group. Single administration of both ALA and vit E ameliorated complex I activity in the muscle, heart, kidney and brain. The combination of ALA and Vit E significantly improved the ATP levels in the liver, heart, kidney and brain and also provided significant improvements the complex I activity in all tissues. The undesirable effects of Atorvastatin on mitochondrial functions in this study ameliorated by using ALA and/or Vit E alone and in combination. Key words: Mitochondria, ATP, Complex I, Atorvastatin, Alfa lipoic acid, Vitamin E
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1. Introduction Mitochondria, known as cellular energy centers and found in most eukaryotic organism, produce adenosine triphosphate (ATP) by oxidative phosphorylation (OXPHOS) in the respiratory chain (electron transport chain, ETC). All cell types have mitochondria, except for erythrocytes. The number of mitochondria of the cells is associated with their energy need. Heart, muscle, brain and gastrointestinal cells have a large number of mitochondria, whereas cells using low energy such as skin cells have less mitochondria (Frye and Rossignol, 2011). The mitochondrion consists of four main parts: the outer mitochondrial membrane (OMM), the intermembrane space (IMS), the inner mitochondrial membrane (IMM) and the mitochondrial matrix (MM). The ETC, which plays an important role in energy production in aerobic organisms, consists of five protein complexes in IMM: NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), ubiquinone cytochrome c oxidoreductase (complex III), cytochrome c oxidase (complex IV) and ATP-synthase (complex V) (Paradies et al., 2011). The complex I, which is measured to determine mitochondrial dysfunction in this study, catalyzes the 2 electron oxidation of NADH followed by the reduction of ubiquinone (Q) to form ubiquinol (QH2), and ultimately the reduction of the terminal electron acceptor, O2. The inhibition of complex I causes mitochondrial dysfunction and increases ROS production (Paradies et al., 2002; Papa et al., 2012). Mitochondrial dysfunction is a prominent factor in various metabolic diseases, intoxications, aging process and especially in muscle and nervous system diseases due to its effect in cell metabolism (Cardinali et al., 2013; Jang et al., 2017; Johannsen and Ravussin, 2009). The ATP level, ETC enzyme activities, mitochondrial DNA (mtDNA), mitochondrial content, mitochondrial membrane potential, ROS production, deteriorated apoptosis, lactate level and intracellular calcium content are considered as biomarkers of this organelle dysfunction (Brand and Nicholls, 2011; Delbarba et al., 2016; Montgomery and Turner, 3
2015). The causes of mitochondrial dysfunction are mitochondrial and genomic DNA mutations, supercomplex destabilization, mitochondrial protein accumulations, endoplasmic reticulum stress, aging, exposure to xenobiotics such as statins, and modern lifestyle (Dudkina et al., 2010; Lim et al., 2010; Park and Larsson, 2011; Tuppen et al., 2010; Pellegrino et al., 2013; Vannuvel et al., 2013). Many drugs used in treatment (flutamide, paclitaxel, oxaliplatin, cisplatin, methotrexate, valproic acid, ketamine, fipronil, buspirone, trazodone) cause mitochondrial dysfunctions (decreases in ATP and NADPH (nicotinamide adenine dinucleotide phosphate) levels, complex I, II, III and IV activities) (Berson et al., 1998; Kashimshetty et al., 2009; Lewis ve Dalakas, 1995; Lewis et al., 2003; Varga et al., 2015). Statins, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, are commonly used for the prevention cardiovascular diseases in lipid metabolism disorders such as hypercholesterolemia. These drugs, which have anti-inflammatory, antiproliferative and antithrombotic effects, can also inhibit the growth of tumor cells and increase intracellular calcium mobilization (Arnaud et al., 2005; Bellosta et al., 2000; Futterman and Lemberg, 2004; Liao and Laufs, 2005; Stancu and Sima, 2001). Statins reduce the morbidity and mortality of cardiovascular disease but also, cause adverse effects (rhabdomyolysis, myopathy, myoglobinemia, arthralgia, etc.) that require one to quit taking the drug due to long-term use in the indicated indication (Jasiñska et al., 2007). In addition, other adverse effects attributed to the effects of these drugs on mitochondria have also been reported, such as diabetes, increase in transaminase level, polyneuropathy and myoglobinemia-related renal failure (Naci et al., 2013; Ruscica et al., 2014; Sathasivam, 2012). The effects of statins on mitochondria are dose-related (Cafforio et al., 2005; Dirks and Jones, 2006; Parihar et al., 2012; Wagner et al., 2008). These drugs inhibit the synthesis of mevalonate, which is a precursor of coenzyme Q10 (ubiquinone) and an important component in the mitochondrial respiratory chain, and impair mitochondrial function as they reduce the coenzyme Q10 4
(Wagner et al., 2008). Statins are responsible for 0.5-1% of myopathies associated with coenzyme Q10 level decrease and electron transport block (Dirks and Jones, 2006). Recent studies have highlighted that the adverse effects of statins on muscle, liver and brain are related to mitochondria (Abdoli et al., 2013; Galtier et al., 2012; Golomb and Evans, 2008; Kaufmann et al., 2006; Nadanaciva et al., 2007; Sirvent et al., 2012). Coenzyme Q-10, carnitine, alpha lipoic acid (ALA), vitamin E (vit E) and C, glutathione, ginkgo biloba, curcumin, omega-3 polyunsaturated fatty acids, N-acetylcysteine (NAC) and melatonin, which have a healing effect on mitochondrial dysfunction, are recommended against diseases such as diabetes, Alzheimer’s, Parkinson’s, ALS and cancer, which are presumed to be associated with mitochondrial dysfunction (Kontush et al., 2001; Rastogi et al., 2008; Wadsworth et al., 2008). It has been suggested that these substances with antioxidant effects support healthy mitochondrial biogenesis process by reducing mitochondrial dysfunction (de Oliveira et al., 2016; Hickey et al., 2012; Kumar and Singh, 2015; Legido et al., 2018). ALA provides the recycling of cellular antioxidants, including coenzyme Q, vitamin C and E, glutathione, iron and copper chelates (Moini et al., 2002). ALA, which is involved in many multi-enzyme complexes in mitochondria, is required to metabolize carbohydrates, proteins and fats and to convert them to ATP. In addition, ALA has antimutagenic and anticarcinogenic effects owing to its antioxidant effect (Cremer et al., 2006; Miadokova et al., 2000; Novotny et al., 2008). ALA, which can cross the blood brain barrier, is useful in cases such as diabetes, heart failure, cataract, myocardial infarction, neuropathic pain and neurodegeneration (Beckman et al., 2002). In addition, this substance is a redox regulator of proteins such as myoglobin, prolactin, thioredoxin and nuclear factor kappa beta (NF-κB) transcription factor (Fuchs, 1997; Packer et al., 1995).
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Vit E has anti-obesity, anti-hypercholesterolemic, anti-diabetic and anti-hypertensive effects, as well as preventive effects on lipid peroxidation and other radical-induced oxidative events (Budin et al., 2009; Matough et al., 2014; Newaz et al., 2003; Zaiden et al., 2010; Zhao et al., 2015). This vitamin plays an important role in maintaining the integrity of lipid rich organelles such as membranes (Greene et al., 2017). While β-tocopherol is mostly localized in mitochondrial fractions, α-tocopherol takes an important role in maintaining mitochondrial integrity (Li and May, 2003). The aim of this study was to determine whether antioxidant ALA and vit E can improve atorvastatin-induced mitochondrial dysfunctions and to reveal whether these can be used to prevent the adverse effects of statins and other drugs that cause mitochondrial dysfunction. 2. Materials and methods 2.1. Animals and study design Wistar Albino male rats (38 in total, 6-10 weeks, 242±1.92 g) were used in this study, utilizing research procedures approved by the ethics committee (SUDAM, 2017-42). During the experimental period, the rats were housed in standard rat cages in a central facility under controlled conditions (12 h light/dark cycle and room temperature of 20°C ± 2°C) at SUDAM and allowed water and food ad libitum. In this study, the rats were divided into 5 groups with a control group (C) consisting of 6 rats and the remaining 32 rats were divided into 4 equal groups. The substances administered are presented in Table 1. ALA and Vit E were coadministered with atorvastatin for 20 days. The doses of atorvastatin, ALA and Vit E were determined by evaluating previous studies in this field (Savitha et al., 2005; Kuhad and Chopra, 2009; Pal et al., 2015).
6
At the end of the experiment, the rats were euthanized by cervical dislocation under anesthesia (ketamine 95 mg/kg, SC+xylazine 5 mg/kg, SC). Immediately after the euthanasia, the liver, muscle (gastrocnemius), heart, kidney and brain tissue samples were received and were snap frozen immediately in liquid nitrogen and stored at −70°C until analysis time. 2.2. Concomitant medications Atorvastatin (Cholvast, 10 mg tablet, Biofarma Pharmaceutical Industry Ltd. Sti., Turkey), Vitamin E (150 mg/ml, IM, Evigen, Aksu Farma Medicinal Products and Pharmaceuticals Co., Turkey), Alfa Lipoic Acid (İlko Pharmaceuticals Industry and Trade Inc.). For animal administrations, atorvastatin (1 mg/ml) and alfa lipoic acid (100 mg/ml) dissolved in water and dimethyl sulfoxide (DMSO) (Çakır, et al., 2015), respectively. Chemicals with analytical purity was used in the study. 2.3. Laboratory tests 2.3.1. Measurement of ATP The ATP levels were determined from the liver, muscle (gastrocnemius muscle), heart, kidney and brain tissues of rats using the extraction method reported Chida et al. (2012) and a commercial ATP kit (Luminescent ATP Detection Assay Kit ab113849). All the measurements for ATP were performed according to the commercial kit procedures. In this study, ATP and luciferin were used to determine total ATP levels, oxyluciferin and firefly luciferase to convert it to light. The light emitted in this reaction is directly proportional to the concentration of the ATP present. This kit neutralizes adenylpyrophosphatase (ATPase) during the lysis step and ensures that the obtained light signal corresponds only to cellular ATP levels.
7
2.3.2. Measurement of mitochondrial complex I Mitochondria were isolated with mitochondria isolation kit (Mitochondria Isolation Kit Sigma MITOISO1) from the liver, muscle (gastrocnemius muscle), heart, kidney and brain tissues of rats, and isolation was confirmed by Bradford protein analysis. The activity of NAD (Nicotinamide Dinucleotide) dehydrogenase (complex I), one of the mitochondrial complex enzymes, was determined by ELISA reader (MWGt Lambda Scan 200, Bio-Tek Instruments, Winooski, VT, USA) using commercial mitochondrial complex I activity kit (MitoCheck Complex I Activity Assay Kit Cayman 700930). All the measurements for complex I were performed according to the commercial kit procedures. This kit allows for direct inhibitory effects on complex I to be easily observed. Inhibition of complex I activity was performed with potassium cyanide. The NADH oxidation rate is measured by a decrease in absorbance at 340 nm and is proportional to the activity of complex I. 2.4. Statistical analyses All data are presented as mean ± SD. The Mann-Whitney U test was used to assess the group differences in the tissue ATP and the mitochondrial complex I activity. The control and other groups or atorvastatin group and other groups were compared in terms of tissue ATP level and mitochondrial complex I activity. The SPSS 22.0 program (IBM Corp, Armonk, NY) was used for statistical analysis. Statistical significance was assigned at P<0.05. 3. Results Atorvastatin caused a decrease in ATP level in the heart and kidney compared to the control group, but not in the liver, muscle or brain (P<0.05). On the other hand, atorvastatin insignificantly decreased the complex I activity in all tissues.
8
ALA administration significantly improved the ATP levels in the liver, heart and kidney, while Vit E improved the ATP levels in all tissues except the muscle compared to Atorvastatin group (P<0.05). Single administration of both ALA and vit E ameliorated complex I activity in the muscle, heart, kidney and brain compared to Atorvastatin group (P<0.05). The combination of ALA and Vit E significantly improved the ATP levels in the liver, heart, kidney and brain and also provided significant improvements the complex I activity
in
all
tissues
compared
to
Atorvastatin
group
(P<0.05).
4. Discussion The long-term medication is used in the treatment of certain diseases such as diabetes and cardiovascular diseases. Chronic drug use increases the incidence of adverse effects of drugs. Strategies are being developed to reduce adverse drug effects in long-term drug use cases. Long-term use of statins to prevent cardiovascular diseases have mitochondrial dysfunction-based side effects on tissues and organs such as muscle, liver and brain. In this study, the effects of ALA, vit E and their combinations on atorvastatin-induced mitochondrial dysfunction were determined by ATP and complex I biomarkers. In the present study, atorvastatin caused statistically significant decrease in ATP levels (P<0.05) in the heart, kidney and brain compared to the control group; but did not cause significant changes in ATP levels in the muscle or liver or complex I enzyme activity in any tissues. ALA, vit E and their combinations improved atorvastatin-induced reductions in ATP levels in all tissues except muscle. We determined that each of these substances recovers the reductions in complex I activity caused by atorvastatin in all tissues except liver; on the other hand, combined use of these substances also improves the complex I activity of liver. Similar to the findings of our study, it was reported that there were no statistically significant changes in mitochondrial function biomarkers (mitochondrial membrane potential, 9
ATP synthesis, cytochrome c oxidase and citrate synthase activity and mitochondrial respiratory functions) on the 5th and 10th days of a 15 day cerivastatin (0.1, 0.5, and 1.0 mg/kg/day) administration to rats at different doses, but there were weak changes that were not statistically significant on the 15th day (Schaefer et al., 2004). In another study conducted by Mohammadi-Bardbori et al. (2015) it was found that simvastatin (30 mg/kg/bw/day) and atorvastatin (30 mg/kg/bw/day) significantly changed some mitochondrial dysfunction biomarkers (complex II activity, ATP level, mitochondrial membrane potential and mitochondrial permeability transition pore) in liver mitochondria in rats, in contrast to our findings. The difference between the findings of the mentioned study and our study may be due to the differences in dosage regimen (dose, administration period) and the sensitivity of the measured parameters to the drugs. In a study evaluating the toxicity of statins in rat skeletal muscle cell lines (L6 cells), cerivastatin, fluvastatin, atorvastatin, simvastatin and pravastatin were administered. As a result, it was reported that cerivastatin, fluvastatin and atorvastatin decreased mitochondrial membrane potential by 49-65%, while the other two did not. In addition, it was determined that lipophilic statins caused mitochondrial swelling, cytochrome c release and DNA fragmentation, and lipophilic statins were more toxic to mitochondrial functions of the skeletal muscle than hydrophilic statins (pravastatin) (Kaufmann et al., 2006). So far, a lot of in vitro and in vivo studies have been carried out on different tissues and organs of different organisms to ameliorate mitochondrial dysfunction caused by drugs and various chemicals and to identify substances that can be used against the predicted diseases caused by mitochondrial dysfunction. The mentioned studies were carried out on different cell lines and organs of different organisms. The efficacy of the substances investigated in these studies was evaluated on mitochondrial dysfunction biomarkers, as in our study. 10
In the present study, the decrease in atorvastatin-induced liver mitochondria ATP level was improved by the administration of Vit E alone and in combination with ALA, whereas the reduction in complex I activity in the liver mitochondri was improved only by combined administration of these substances. Mohammadi-Bardbori et al. (2015) reported that oral coadministration of CoQ10 with simvastatin (30 mg/kg/bw/day) and atorvastatin (30 mg/kg/bw/day) to rats for 30 days recovered the changes (complex II activity, ATP level, mitochondrial permeability transition pore) in liver mitochondria caused by the indicated drugs. Some studies investigating the effect of ALA on liver, kidney and brain cell mitochondrial functions in elderly rats showed ALA to improve mitochondrial membrane potential, ATP, mitochondrial enzyme and ETC complex activities (Arivazhagan et al., 2001; Hagen et al., 1999; Palaniappan and Dai; 2007). In addition, it was stated that ALA improved LPS-mediated change in ATP, complex I, IV and V, NADH, mtDNA expression and enzymes in rat liver mitochondria (Hiller et al., 2014; Liu et al., 2015). Kheir-Eldin et al. (2001) reported that decrease in ATP/ADP ratio and increases in mitochondria/cytosolic hexokinase ratio caused by LPS in rat brain mitochondria were ameliorated by vit E, beta carotene, Se and NAC treatments, and Vit E+Se was foundthe most effective. The damage caused by methanol and ethionine in rat liver mitochondria (decrease in NADH dehydrogenase, succinate cytochrome c reductase, cytochrome oxidase activity and ATP level) was improved by 20-28 period oral administration of vit E (Padma and Setty, 1997; El-Sayed et al., 2002). We found that vit E improved atorvastatin-mediated ATP reduction in the liver mitochondria, did not improve the reduction in complex I activity, but its combination with ALA improved the reduction in complex I activity. Consistent with the results of our study, Zhou et al. (2018) reported that alpha lipoamide (neutral amide derivative of lipoic acid) increased brain ATP levels and improved mitochondrial morphology in nerve cells in 6-hydroxydopamine-induced 11
Parkinson’s animal model in rats. Also, the administration of vit E together with endosulfan and gossypol to experimental animals (mice, rat) improved the reduction in testicular mitochondrial ATP levels caused by these substances (Santana et al., 2015; Wang et al., 2012). In vitro studies performed with various cell lines revealed that ALA, vit E, melatonin and ginsenoside corrected mitochondrial dysfunction (impairment in mitochondrial membrane potential and decrease in ATP level and cytochrome c oxidase activity) caused by amyloid beta, S-nitrosylation, acrylamide and tunicamycin (Cardoso et al., 2001; Huang et al., 2012; Hiller et al., 2016; Lei et al., 2016; Song et al., 2017). In conclusion, it can be indicated that i) the effects of statins on mitochondria vary by organs, ii) the ATP level is a more sensitive biomarker for mitochondrial dysfunction than complex I activity, and iii) ALA alone and in combination with vit E can be used concurrently with drugs such as statins, which are used in the long-term and cause mitochondrial dysfunction, to reduce their adverse effects during therapy. ACKNOWLEDGMENTS This research was summarized from the PhD thesis of the first author. Research was supported by OYP (2015-OYP-042). The abstract of this article was presented as an oral presentation at the congress “2nd Internatıonal Eurasıan Conference on Biological and Chemical Sciences, June, 28-29, 2019". CONFLICT OF INTEREST The authors declare that they have no conflict of interest. AUTHORS' CONTRIBUTIONS Hatice Eser Faki contributed the conception, experimental administration, analysis, drafted to manuscript, critically revised the manuscript and agreed to be accountable for all aspects of 12
work ensuring integrity and accuracy. Bunyamin Tras contributed the critically revised the manuscript and agreed to be accountable for all aspects of work ensuring integrity and accuracy. Kamil Uney contributed the analysis, critically revised the manuscript and agreed to be accountable for all aspects of work ensuring integrity and accuracy.
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17
Table 1 The substances administered and their doses in groupsa Group
Atorvastatin
Alfa Lipoic Acid
Vitamin E
Cb (n=6)
-
-
-
A (n=8)
10 mg/kg
-
-
A+ALA (n=8)
10 mg/kg
100 mg/kg
-
A+Vit E (n=8)
10 mg/kg
-
100 mg/kg
A+ALA+Vit E (n=8)
10 mg/kg
100 mg/kg
100 mg/kg
C: control, A: atorvastatin, A+ALA: atorvastatin + alpha lipoic acid, A+Vit E: atorvastatin + vitamin E, A+ALA+Vit E: atorvastatin + alpha lipoic acid +vitamin E. a
All substances were administered orally. For animal administrations, atorvastatin (1 mg/ml) and alfa lipoic acid (100 mg/ml) dissolved in water and dimethyl sulfoxide,
respectively. b
Dimethyl sulfoxide (100µl/kg) was applied to the control group.
18
Table 2 The effects of alpha lipoic acid (100 mg/kg) and vitamin E (100 mg/kg) on the ATP in various tissues of atorvastatin (10 mg/kg) treated rats (mean ± SD)* Group
Liver
Muscle
Heart
Kidney
Brain
C (n=6)
0.0293±0.0372d
4.9224±0.3846a
2.7270±0.5829b
0.0206±0.0096c
0.7047±0.1714c
A (n=8)
0.0172±0.0178d
4.7252±0.3846a
2.4238±0.3683c
0.0085±0.0096d
0.6493±0.0366c
A+ALA (n=8)
0.3228±0.1240a
5.3519±0.9384a
4.5044±0.4847a
0.2500±0.1268b
0.8438±0.2947bc
A+Vit E (n=8)
0.1001±0.0374c
5.4006±0.3469a
3.2661±1.0230b
0.4128±0.0606a
1.1019±0.1909ab
A+ALA+Vit E (n=8)
0.2002±0.0968b
5.0549±1.0800a
0.3243±0.1633b
0.3243±0.1633ab
1.7431±0.8190a
* The values are presented as nM. a,b,c
: Different letters in the same line are statistically different (P˂0.05).
C: control, A: atorvastatin, A+ALA: atorvastatin + alpha lipoic acid, A+Vit E: atorvastatin + vitamin E, A+ALA+Vit E: atorvastatin + alpha lipoic acid + vitamin E.
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Table 3 The effects of alpha lipoic acid (100 mg/kg) and vitamin E (100 mg/kg) on the mitochondrial complex I in various tissues of atorvastatin (10 mg/kg) treated rats (mean ± SD)* Group
Liver
Muscle
C (n=6)
100±34b
99±14b
99±55ab 100±22bc 100±21b
A (n=8)
89±13bc
93±6b
91±10b
A+ALA (n=8)
124±11ab 136±20a 136±15a 123±21ab 138±13a
A+Vit E (n=8)
127±12ab 138±13a 130±16a
A+ALA+Vit E (n=8)
131±9a
134±11a
Heart
128±6a
Kidney
94±13c
125±7a
Brain
94±19b
144±16a
136±23ab 152±23a
* The values are presented as % activity. a,b,c
: Different letters in the same line are statistically different (P˂0.05).
C: control, A: atorvastatin, A+ALA: atorvastatin + alpha lipoic acid, A+Vit E: atorvastatin + vitamin E, A+ALA+Vit E: atorvastatin + alpha lipoic acid + vitamin E.
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The effects of Atorvastatin on mitochondria vary by organs.
The ATP level is a more sensitive biomarker than complex I activity.
Effects of Atorvastatin on mitochondrial functions in this study ameliorated by using ALA and/or Vit E alone and in combination.
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