Alpha lipoic acid and vitamin E improve atorvastatin-induced mitochondrial dysfunctions in rats

(1)

Contents lists available atScienceDirect

Mitochondrion

journal homepage:www.elsevier.com/locate/mito

Alpha lipoic acid and vitamin E improve atorvastatin-induced mitochondrial

dysfunctions in rats

Hatice Eser Faki

⁎,1

, Bunyamin Tras, Kamil Uney

Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, University of Selcuk, 42031 Konya, Turkey

A R T I C L E I N F O Keywords: Mitochondria ATP Complex I Atorvastatin Alfa lipoic acid Vitamin E

A B S T R A C T

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 im-proved the ATP levels in the liver, heart and kidney, while Vit E imim-proved 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.

1. Introduction

Mitochondria, known as cellular energy centers and found in most eukaryotic organism, produce adenosine triphosphate (ATP) by oxida-tive phosphorylation (OXPHOS) in the respiratory chain (electron transport chain, ETC). All cell types have mitochondria, except for er-ythrocytes. 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 mi-tochondrion consists of four main parts: the outer mitochondrial membrane (OMM), the intermembrane space (IMS), the inner mi-tochondrial membrane (IMM) and the mimi-tochondrial matrix (MM). The ETC, which plays an important role in energy production in aerobic organisms, consists of ﬁve protein complexes in IMM: NADH dehy-drogenase (complex I), succinate dehydehy-drogenase (complex II), ubiqui-none 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 ulti-mately the reduction of the terminal electron acceptor, O2. The in-hibition 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 meta-bolic 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), mitochon-drial content, mitochonmitochon-drial 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, 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;

https://doi.org/10.1016/j.mito.2020.02.011

Received 20 September 2019; Received in revised form 12 December 2019; Accepted 27 February 2020 ⁎_{Corresponding author.}

E-mail address:haticeeser@selcuk.edu.tr(H. Eser Faki). 1_{ORCID: 0000-0002-6124-7168.}

Available online 28 February 2020

(2)

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 mi-tochondrial dysfunctions (decreases in ATP and NADPH (nicotinamide adenine dinucleotide phosphate) levels, complex I, II, III and IV activ-ities) (Berson et al., 1998; Kashimshetty et al., 2009; Lewis and Dalakas, 1995; Lewis et al., 2003; Varga et al., 2015).

Statins, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) re-ductase inhibitors, are commonly used for the prevention cardiovas-cular diseases in lipid metabolism disorders such as hypercholester-olemia. 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 mor-tality of cardiovascular disease but also, cause adverse effects (rhab-domyolysis, myopathy, myoglobinemia, arthralgia, etc.) that require one to quit taking the drug due to long-term use in the indicated in-dication (Jasiñska et al., 2007). In addition, other adverse effects at-tributed to the effects of these drugs on mitochondria have also been reported, such as diabetes, increase in transaminase level, polyneuro-pathy and myoglobinemia-related renal failure (Naci et al., 2013; Ruscica et al., 2014; Sathasivam, 2012). The effects of statins on mi-tochondria 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 (ubi-quinone) and an important component in the mitochondrial respiratory chain, and impair mitochondrial function as they reduce the coenzyme Q10 (Wagner et al., 2008). Statins are responsible for 0.5–1% of myo-pathies associated with coenzyme Q10 level decrease and electron transport block (Dirks and Jones, 2006). Recent studies have high-lighted 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 sup-port healthy mitochondrial biogenesis process by reducing mitochon-drial 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, neuro-pathic 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) tran-scription factor (Fuchs, 1997; Packer et al., 1995).

Vit E has anti-obesity, anti-hypercholesterolemic, anti-diabetic and anti-hypertensive eﬀects, as well as preventive eﬀects on lipid perox-idation 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 mi-tochondrial 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 inTable 1. ALA and Vit E were co-administered with atorvastatin for 20 days. The doses of atorvastatin, ALA and Vit E were determined by evaluating previous studies in thisﬁeld (Savitha et al., 2005; Kuhad and Chopra, 2009; Pal et al., 2015).

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 (gastro-cnemius), 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 (gastro-cnemius muscle), heart, kidney and brain tissues of rats using the ex-traction method reportedChida et al. (2012)and a commercial ATP kit (Luminescent ATP Detection Assay Kit ab113849). All the Table 1

The substances administered and their doses in groups.a

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.

(3)

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 andﬁreﬂy luciferase to convert it to light. The light emitted in this reaction is directly proportional to the con-centration of the ATP present. This kit neutralizes adenylpyropho-sphatase (ATPase) during the lysis step and ensures that the obtained light signal corresponds only to cellular ATP levels.

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 proce-dures. 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 mi-tochondrial complex I activity. The control and other groups or ator-vastatin 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.

ALA administration significantly improved the ATP levels in the liver, heart and kidney, while Vit E improved the ATP levels in all tis-sues 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) (Tables 2 and 3).

4. Discussion

The long-term medication is used in the treatment of certain dis-eases such as diabetes and cardiovascular disdis-eases. 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 mi-tochondrial 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-in-duced reductions in ATP levels in all tissues except muscle. We de-termined 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 thefindings of our study, it was reported that there were no statistically significant changes in mitochondrial function bio-markers (mitochondrial membrane potential, ATP synthesis, cyto-chrome c oxidase and citrate synthase activity and mitochondrial re-spiratory 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 ac-tivity, ATP level, mitochondrial membrane potential and mitochondrial permeability transition pore) in liver mitochondria in rats, in contrast to ourfindings. 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, ator-vastatin, 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 mi-tochondrial 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 mi-tochondrial dysfunction caused by drugs and various chemicals and to identify substances that can be used against the predicted diseases caused by mitochondrial dysfunction.

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 ± 0.3846}a _{2.7270 ± 0.5829}b _{0.0206 ± 0.0096}c _{0.7047 ± 0.1714}c

A (n = 8) 0.0172 ± 0.0178d _{4.7252 ± 0.3846}a _{2.4238 ± 0.3683}c _{0.0085 ± 0.0096}d _{0.6493 ± 0.0366}c

A + ALA (n = 8) 0.3228 ± 0.1240a _{5.3519 ± 0.9384}a _{4.5044 ± 0.4847}a _{0.2500 ± 0.1268}b _{0.8438 ± 0.2947}bc

A + Vit E (n = 8) 0.1001 ± 0.0374c _{5.4006 ± 0.3469}a _{3.2661 ± 1.0230}b _{0.4128 ± 0.0606}a _{1.1019 ± 0.1909}ab

A + ALA + Vit E (n = 8) 0.2002 ± 0.0968b _{5.0549 ± 1.0800}a _{0.3243 ± 0.1633}b _{0.3243 ± 0.1633}ab _{1.7431 ± 0.8190}a

*The values are presented as nM.

a,b,c_{: Di}_{ﬀerent letters in the same line are statistically diﬀerent (P < 0.05).}

(4)

The mentioned studies were carried out on different cell lines and organs of different organisms. The efficacy of the substances in-vestigated in these studies was evaluated on mitochondrial dysfunction biomarkers, as in our study.

In the present study, the decrease in atorvastatin-induced liver mi-tochondria ATP level was improved by the administration of Vit E alone and in combination with ALA, whereas the reduction in complex I ac-tivity in the liver mitochondri was improved only by combined ad-ministration of these substances. Mohammadi-Bardbori et al. (2015) reported that oral co-administration 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, mi-tochondrial 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 im-prove 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 cyto-chrome c reductase, cytocyto-chrome 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-hydro-xydopamine-induced Parkinson’s animal model in rats. Also, the ad-ministration of vit E together with endosulfan and gossypol to experi-mental 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.

Declaration of Competing Interest

The authors declare that they have no known competingﬁnancial interests or personal relationships that could have appeared to in ﬂu-ence the work reported in this paper.

Acknowledgments

This research was summarized from the PhD thesis of theﬁrst au-thor. 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“.

Authors' contributions

Hatice Eser Faki contributed the conception, experimental admin-istration, analysis, drafted to manuscript, critically revised the manu-script and agreed to be accountable for all aspects of 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.

References

Abdoli, N., Heidari, R., Azarmi, Y., Eghbal, M.A., 2013. Mechanisms of the statins cyto-toxicity in freshly isolated rat hepatocytes. J. Biochem. Mol. Toxicol. 27, 287–294.

Arivazhagan, P., Ramanathan, K., Panneerselvam, C., 2001. Eﬀect ofDL-α-lipoic acid on

mitochondrial enzymes in aged rats. Chem. Biol. Interact. 138, 189–198.

Arnaud, C., Burger, F., Steffens, S., Veillard, N.R., Nguyen, T.H., Trono, D., Mach, F., 2005. Statins reduce interleukin-6–induced C-reactive protein in human hepatocytes: new evidence for direct antiinflammatory effects of statins. Arterioscler. Thromb. Vasc. Biol. 25, 1231–1236.

Beckman, J.A., Creager, M.A., Libby, P., 2002. Diabetes and atherosclerosis: epide-miology, pathophysiology, and management. JAMA 287, 2570–2581.

Bellosta, S., Bernini, F., Paoletti, R., Corsini, A., 2000. Non-lipid-related eﬀects of statins. Ann. Med. 32, 164–176.

Berson, A., De Beco, V., Lettéron, P., Robin, M.A., Moreau, C., El Kahwaji, J., Verthier, N., Feldmann, G., Fromenty, B., Pessayre, D., 1998. Steatohepatitis-inducing drugs cause mitochondrial dysfunction and lipid peroxidation in rat hepatocytes.

Gastroenterology 114, 764–774.

Brand, M.D., Nicholls, D.G., 2011. Assessing mitochondrial dysfunction in cells. Biochem. J. 435, 297–312.

Budin, S.B., Othman, F., Louis, S.R., Bakar, M.A., Das, S., Mohamed, J., 2009. The eﬀects of palm oil tocotrienol-rich fraction supplementation on biochemical parameters, oxidative stress and the vascular wall of streptozotocin-induced diabetic rats. Clinics 64, 235–244.

Caﬀorio, P., Dammacco, F., Gernone, A., Silvestris, F., 2005. Statins activate the mi-tochondrial pathway of apoptosis in human lymphoblasts and myeloma cells. Carcinogenesis 26, 883–891.

Cardinali, D.P., Pagano, E.S., Bernasconi, P.A.S., Reynoso, R., Scacchi, P., 2013. 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 Heart Kidney Brain

C (n = 6) 100 ± 34b _{99 ± 14}b _{99 ± 55}ab _{100 ± 22}bc _{100 ± 21}b

A (n = 8) 89 ± 13bc _{93 ± 6}b _{91 ± 10}b _{94 ± 13}c _{94 ± 19}b

A + ALA (n = 8) 124 ± 11ab _{136 ± 20}a _{136 ± 15}a _{123 ± 21}ab _{138 ± 13}a

A + Vit E (n = 8) 127 ± 12ab _{138 ± 13}a _{130 ± 16}a _{125 ± 7}a _{144 ± 16}a

A + ALA + Vit E (n = 8) 131 ± 9a _{134 ± 11}a _{128 ± 6}a _{136 ± 23}ab _{152 ± 23}a

*The values are presented as % activity.

a,b,c_{: Di}_{ﬀerent letters in the same line are statistically diﬀerent (P < 0.05).}

(5)

Melatonin and mitochondrial dysfunction in the central nervous system. Horm. Behav. 63, 322–330.

Cardoso, S.M., Santos, S., Swerdlow, R.H., Oliveira, C.R., 2001. Functional mitochondria are required for amyloidβ-mediated neurotoxicity. FASEB J. 15, 1439–1441.

Chida, J., Yamane, K., Takei, T., Kido, H., 2012. An eﬃcient extraction method for quantitation of adenosine triphosphate in mammalian tissues and cells. Anal. Chim. Acta 727, 8–12.

Cremer, D., Rabeler, R., Roberts, A., Lynch, B., 2006. Safety evaluation ofα-lipoic acid (ALA). Regul. Toxicol. Pharm. 46, 29–41.

Çakır, T., Baştürk, A., Polat, C., Aslaner, A., Durgut, H., Şehirli, A.Ö., Gül, M., Öğünç, A.V., Gül, S., Sabuncuoğlu, M.Z., 2015. Does alfa lipoic acid prevent liver from methotrexate induced oxidative injury in rats? Acta Cir. Bras. 30, 247–252.

Delbarba, A., Abate, G., Prandelli, C., Marziano, M., Buizza, L., Arce Varas, N., Novelli, A., Cuetos, F., Martinez, C., Lanni, C., Memo, M., Uberti, D., 2016. Mitochondrial al-terations in peripheral mononuclear blood cells from Alzheimer’s disease and mild cognitive impairment patients. Oxid. Med. Cell. Longevity.

de Oliveira, M.R., Jardim, F.R., Setzer, W.N., Nabavi, S.M., Nabavi, S.F., 2016. Curcumin, mitochondrial biogenesis, and mitophagy: exploring recent data and indicating future needs. Biotechnol. Adv. 34, 813–826.

Dirks, A.J., Jones, K.M., 2006. Statin-induced apoptosis and skeletal myopathy. Am. J. Physiol. Cell Physiol. 291, 1208–1212.

Dudkina, N.V., Kouril, R., Peters, K., Braun, H.P., Boekema, E.J., 2010. Structure and function of mitochondrial supercomplexes. Biochim. Biophys. Acta 1797, 664–670.

El-Sayed, N.K., Gaafar, K.M., El-Ansary, A.K., Osman, A.I., 2002. The protective potency of vitamins E and C in methanol-induced oxidative stress and retinotoxicity. J. Toxicol.: Cutan Ocul. Toxicol. 21, 307–327.

Fuchs, J., 1997. In: Lipoic Acid in Health and Disease. CRC Press, pp. 504.

Futterman, L.G., Lemberg, L., 2004. Statin pleiotropy: fact orﬁction? Am. J. Crit. Care 13, 244–249.

Frye, R.E., Rossignol, D.A., 2011. Mitochondrial dysfunction can connect the diverse medical symptoms associated with autism spectrum disorders. Water Res. 69, 41–47.

Galtier, F., Mura, T., de Mauverger, E.R., Chevassus, H., Farret, A., Gagnol, J.P., Costa, F., Dupuy, A., Petit, P., Cristol, J., 2012. Eﬀect of a high dose of simvastatin on muscle mitochondrial metabolism and calcium signaling in healthy volunteers. Toxicol. Appl. Pharmacol. 263, 281–286.

Golomb, B.A., Evans, M.A., 2008. Statin adverse eﬀects. Am. J. Cardiovasc. Drugs 8, 373–418.

Greene, L.E., Lincoln, R., Cosa, G., 2017. Rate of lipid peroxyl radical production during cellular homeostasis unraveled viaﬂuorescence imaging. J. Am. Chem. Soc. 139, 15801–15811.

Hagen, T.M., Ingersoll, R.T., Lykkesfeldt, J., Liu, J., Wehr, C.M., Vinarsky, V., Bartholomew, J.C., Ames, N.B., 1999. (R)-α-lipoic acid-supplemented old rats have improved mitochondrial function, decreased oxidative damage, and increased me-tabolic rate. FASEB J. 13, 411–418.

Hickey, M.A., Zhu, C., Medvedeva, V., Franich, N.R., Levine, M.S., Chesselet, M.F., 2012. Evidence for behavioral beneﬁts of early dietary supplementation with CoEnzyme Q10 in a slowly progressing mouse model of Huntington's disease. Mol. Cell. Neurosci. 49, 149–157.

Hiller, S., DeKroon, R., Xu, L., Robinette, J., Winnik, W., Alzate, O., Simington, S., Maeda, N., Yi, X., 2014.α-Lipoic acid protects mitochondrial enzymes and attenuates lipo-polysaccharide-induced hypothermia in mice. Free Radic. Biol. Med. 71, 362–367.

Hiller, S., DeKroon, R., Hamlett, E.D., Xu, L., Osorio, C., Robinette, J., Winnik, W., Simington, S., Maeda, N., Alzate, O., 2016. Alpha-lipoic acid supplementation pro-tects enzymes from damage by nitrosative and oxidative stress. Biochim. Biophys. Acta 1860, 36–45.

Huang, T., Fang, F., Chen, L., Zhu, Y., Zhang, J., Chen, X., Shidu, Y.S., 2012. Ginsenoside Rg1 attenuates oligomeric Aβ1-42-induced mitochondrial dysfunction. Curr. Alzheimer Res. 9, 388–395.

Jang, D.H., Greenwood, J.C., Spyres, M.B., Eckmann, D.M., 2017. Measurement of mi-tochondrial respiration and motility in acute care: sepsis, trauma, and poisoning. J. Intensive Care Med. 32, 86–94.

Jasiñska, M., Owczarek, J., Orszulak-Michalak, D., 2007. Statins: a new insight into their mechanisms of action and consequent pleiotropic eﬀects. Pharmacol. Rep. 59, 483–499.

Johannsen, D.L., Ravussin, E., 2009. The role of mitochondria in health and disease. Curr. Opin. Pharmacol. 9, 780–786.

Kashimshetty, R., Desai, V.G., Kale, V.M., Lee, T., Moland, C.L., Branham, W.S., New, L.S., Chan, E.C., Younis, H., Boelsterli, U.A., 2009. Underlying mitochondrial dysfunction triggersﬂutamide-induced oxidative liver injury in a mouse model of idiosyncratic drug toxicity. Toxicol. Appl. Pharmacol. 238, 150–159.

Kaufmann, P., Török, M., Zahno, A., Waldhauser, K., Brecht, K., Krähenbühl, S., 2006. Toxicity of statins on rat skeletal muscle mitochondria. Cell. Mol. Life Sci. 63, 2415–2425.

Kheir-Eldin, A.A., Motawi, T.K., Gad, M.Z., Abd-ElGawad, H.M., 2001. Protective eﬀect of vitamin E,β-carotene and N-acetylcysteine from the brain oxidative stress induced in rats by lipopolysaccharide. Int. J. Biochem. Cell Biol. 33, 475–482.

Kontush, A., Mann, U., Arlt, S., Ujeyl, A., Lührs, C., Müller-Thomsen, T., Beisiegel, U., 2001. Inﬂuence of vitamin E and C supplementation on lipoprotein oxidation in patients with Alzheimer’s disease. Free Radic. Biol. Med. 31, 345–354.

Kuhad, A., Chopra, K., 2009. Attenuation of diabetic nephropathy by tocotrienol: in-volvement of NFkB signaling pathway. Life Sci. 84, 296–301.

Kumar, A., Singh, A., 2015. A review on mitochondrial restorative mechanism of anti-oxidants in Alzheimer's disease and other neurological conditions. Front. Pharmacol. 6, 206.

Legido, A., Goldenthal, M., Garvin, B., Damle, S., Corrigan, K., Connell, J., Thao, D., Valencia, I., Melvin, J., Khurana, D., 2018. Eﬀect of a Combination of Carnitine,

Coenzyme Q10 and Alpha-lipoic acid (MitoCocktail) on Mitochondrial Function and Neurobehavioral Performance in Children with Autism Spectrum Disorder (P3. 313). AAN Enterprises.

Lei, L., Zhu, Y., Gao, W., Du, X., Zhang, M., Peng, Z., Fu, S., Li, X., Zhe, W., Li, X., 2016. Alpha-lipoic acid attenuates endoplasmic reticulum stress-induced insulin resistance by improving mitochondrial function in HepG2 cells. Cell. Signal. 28, 1441–1450.

Lewis, W., Dalakas, M.C., 1995. Mitochondrial toxicity of antiviral drugs. Nat. Med. 5, 417–422.

Lewis, W., Day, B.J., Copeland, W.C., 2003. Mitochondrial toxicity of NRTI antiviral drugs: an integrated cellular perspective. Nat. Rev. Drug Discov. 10, 812–822.

Li, X., May, J.M., 2003. Location and recycling of mitochondrialα-tocopherol. Mitochondrion 3, 29–38.

Liao, J.K., Laufs, U., 2005. Pleiotropic eﬀects of statins. Annu. Rev. Pharmacol. Toxicol. 45, 89–118.

Lim, S., Cho, Y.M., Park, K.S., Lee, H.K., 2010. Persistent organic pollutants, mitochon-drial dysfunction, and metabolic syndrome. Ann. NY Acad. Sci. 1201, 166–176.

Liu, Z., Guo, J., Sun, H., Huang, Y., Zhao, R., Yang, X., 2015.α-Lipoic acid attenuates LPS-induced liver injury by improving mitochondrial function in association with GR mitochondrial DNA occupancy. Biochimie 116, 52–60.

Matough, F.A., Budin, S.B., Hamid, Z.A., Abdul-Rahman, M., Al-Wahaibi, N., Mohammed, J., 2014. Tocotrienol-rich fraction from palm oil prevents oxidative damage in dia-betic rats. Sultan Qaboos Univ. Med. J. 14, 95–103.

Miadokova, E., Vlckova, V., Duhova, V., 2000. Antimutagenic eﬀect of alpha-lipoic acid on three model test systems. Pharmazie 55, 862–863.

Mohammadi-Bardbori, A., Najibi, A., Amirzadegan, N., Gharibi, R., Dashti, A., Omidi, M., Saeedi, A., Ghafarian-Bahreman, A., Niknahad, H., 2015. Coenzyme Q10 remarkably improves the bio-energetic function of rat liver mitochondria treated with statins. Eur. J. Pharmacol. 762, 270–274.

Moini, H., Packer, L., Saris, N.-E.L., 2002. Antioxidant and prooxidant activities of α-lipoic acid and dihydroα-lipoic acid. Toxicol. Appl. Pharmacol. 182, 84–90.

Montgomery, M.K., Turner, N., 2015. Mitochondrial dysfunction and insulin resistance: an update. Endocr. Connect. 4, 1–15.

Naci, H., Brugts, J., Ades, T., 2013. Comparative tolerability and harms of individual statins: a study-level network meta-analysis of 246 955 participants from 135 ran-domized, controlled trials. Circ. Cardiovasc. Qual. Outcomes 6, 390–399.

Nadanaciva, S., Dykens, J.A., Bernal, A., Capaldi, R.A., Will, Y., 2007. Mitochondrial impairment by PPAR agonists and statins identiﬁed via immunocaptured OXPHOS complex activities and respiration. Toxicol. Appl. Pharmacol. 223, 277–287.

Newaz, M., Youseﬁpour, Z., Nawal, N., Adeeb, N., 2003. Nitric oxide synthase activity in blood vessels of spontaneously hypertensive rats: antioxidant protection by gamma-tocotrienol. J. Physiol. Pharmacol. 54, 319–327.

Novotny, L., Rauko, P., Cojocel, C., 2008. Alpha-lipoic acid-the potential for use in cancer therapy minireview. Neoplasma 55, 81–86.

Packer, L., Witt, E.H., Tritschler, H.J., 1995. Alpha-lipoic acid as a biological antioxidant. Free Radic. Biol. Med. 19, 227–250.

Padma, P., Setty, O., 1997. Protective eﬀect of vitamin E against ethionine toxicity. IUBMB Life 41, 785–795.

Pal, S., Ghosh, M., Ghosh, S., Bhattacharyya, S., Sil, P.C., 2015. Atorvastatin induced hepatic oxidative stress and apoptotic damage via MAPKs, mitochondria, calpain and caspase 12 dependent pathways. Food Chem. Toxicol. 83, 36–47.

Palaniappan, A., Dai, A., 2007. Mitochondrial ageing and the beneﬁcial role of α-lipoic acid. Neurochem. Res. 32, 1552–1558.

Papa, S., Martino, P.L., Capitanio, G., Gaballo, A., De Rasmo, D., Signorile, A., Petruzzella, V., 2012. The oxidative phosphorylation system in mammalian mitochondria. In: Advances in Mitochondrial Medicine. Springer, pp. 3–37.

Paradies, G., Petrosillo, G., Pistolese, M., Ruggiero, F.M., 2002. Reactive oxygen species aﬀect mitochondrial electron transport complex I activity through oxidative cardio-lipin damage. Gene 286, 135–141.

Paradies, G., Petrosillo, G., Paradies, V., Ruggiero, F.M., 2011. Mitochondrial dysfunction in brain aging: role of oxidative stress and cardiolipin. Neurochem. Int. 58, 447–457.

Parihar, A., Parihar, M., Zenebe, W., Ghafourifar, P., 2012. Statins lower calcium-induced oxidative stress in isolated mitochondria. Hum. Exp. Toxicol. 31, 355–363.

Park, C.B., Larsson, N.G., 2011. Mitochondrial DNA mutations in disease and aging. J. Cell Biol. 193, 809–818.

Pellegrino, M.W., Nargund, A.M., Haynes, C.M., 2013. Signaling the mitochondrial un-folded protein response. Biochim. Biophys. Acta 1833, 410–416.

Rastogi, M., Ojha, R.P., Rajamanickam, G., Agrawal, A., Aggarwal, A., Dubey, G., 2008. Curcuminoids modulates oxidative damage and mitochondrial dysfunction in dia-betic rat brain. Free Radic. Res. 42, 999–1005.

Ruscica, M., Macchi, C., Morlotti, B., Sirtori, C.R., Magni, P., 2014. Statin therapy and related risk of new-onset type 2 diabetes mellitus. Eur. J. Intern. Med. 25, 401–406.

Santana, A.T., Guelﬁ, M., Medeiros, H.C., Tavares, M.A., Bizerra, P.F., Mingatto, F.E., 2015. Mechanisms involved in reproductive damage caused by gossypol in rats and protective eﬀects of vitamin E. Biol. Res. 48, 43.

Sathasivam, S., 2012. Statin induced myotoxicity. Eur. J. Intern. Med. 23, 317–324.

Savitha, S., Sivarajan, K., Haripriya, D., Kokilavani, V., Panneerselvam, C., 2005. Eﬃcacy of levo carnitine and alpha lipoic acid in ameliorating the decline in mitochondrial enzymes during aging. Clin. Nutr. 24, 794–800.

Schaefer, W.H., Lawrence, J.W., Loughlin, A.F., Stoﬀregen, D.A., Mixson, L.A., Dean, D.C., Raab, C.E., Nathan, X.Y., Lankas, G.R., Frederick, C.B., 2004. Evaluation of ubiqui-none concentration and mitochondrial function relative to cerivastatin-induced ske-letal myopathy in rats. Toxicol. Appl. Pharmacol. 194, 10–23.

Sirvent, P., Fabre, O., Bordenave, S., Hillaire-Buys, D., De Mauverger, E.R., Lacampagne, A., Mercier, J., 2012. Muscle mitochondrial metabolism and calcium signaling im-pairment in patients treated with statins. Toxicol. Appl. Pharmacol. 259, 263–268.

(6)

eﬀects of lipoic acid against acrylamide-induced neurotoxicity: involvement of mi-tochondrial energy metabolism and autophagy. Food Funct. 8, 4657–4667.

Stancu, C., Sima, A., 2001. Statins: mechanism of action and eﬀects. J. Cell Mol. Med. 5, 378–387.

Tuppen, H.A., Blakely, E.L., Turnbull, D.M., Taylor, R.W., 2010. Mitochondrial DNA mutations and human disease. Biochim. Biophys. Acta 1797, 113–128.

Wadsworth, T.L., Bishop, J.A., Pappu, A.S., Woltjer, R.L., Quinn, J.F., 2008. Evaluation of coenzyme Q as an antioxidant strategy for Alzheimer's disease. J. Alzheimers Dis. 14, 225–234.

Wagner, B.K., Kitami, T., Gilbert, T.J., Peck, D., Ramanathan, A., Schreiber, S.L., Golub, T.R., Mootha, V.K., 2008. Large-scale chemical dissection of mitochondrial function. Nat. Biotechnol. 26, 343–351.

Wang, N., Qian, H.Y., Zhou, X.Q., Li, Y.B., Sun, Z.W., 2012. Mitochondrial energy me-tabolism dysfunction involved in reproductive toxicity of mice caused by endosulfan and protective eﬀects of vitamin E. Ecotoxicol. Environ. Saf. 82, 96–103.

Vannuvel, K., Renard, P., Raes, M., Arnould, T., 2013. Functional and morphological impact of ER stress on mitochondria. J. Cell. Physiol. 228, 1802–1818.

Varga, Z.V., Ferdinandy, P., Liaudet, L., Pacher, P., 2015. Drug-induced mitochondrial dysfunction and cardiotoxicity. Am. J. Physiol. Heart Circ. Physiol. 309, 1453–1467.

Zaiden, N., Yap, W.N., Ong, S., Xu, C.H., Teo, V.H., Chang, C.P., Zhang, X.W., Nesaretnam, K., Shiba, S., Yap, Y.L., 2010. Gamma delta tocotrienols reduce hepatic triglyceride synthesis and VLDL secretion. J. Atheroscler. Thromb. 17, 1019–1032.

Zhao, L., Kang, I., Fang, X., Wang, W., Lee, M., Hollins, R., Marshall, M., Chung, S., 2015. Gamma-tocotrienol attenuates high-fat diet-induced obesity and insulin resistance by inhibiting adipose inﬂammation and M1 macrophage recruitment. Int. J. Obes. 39, 438–446.

Zhou, B., Wen, M., Lin, X., Chen, Y.H., Gou, Y., Li, Y., Zhang, Y., Li, H.W., Tang, L., 2018. Alpha lipoamide ameliorates motor deﬁcits and mitochondrial dynamics in the Parkinson’s disease model induced by 6-hydroxydopamine. Neurotoxicol. Res. 33, 759–767.