Effects of Squalene/Squalane on Dopamine Levels, Antioxidant

Effects of squalene/squalane on dopamine levels, antioxidant enzyme activity, and fatty acid composition in the striatum of Parkinson’s disease mouse model Hideaki Kabuto1＊ , Tomoko T. Yamanushi1, Najma Janjua1, Fusako Takayama2 and Mitsumasa Mankura2 1 Department of Health Sciences, Kagawa Prefectural University of Health Sciences, 281-1, Mure-cho, Takamatsu 761-0123, Japan. 2 ‌Faculty of Pharmaceutical Sciences, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1, Tsushima-naka, Kitaku, Okayama 700-8530, Japan.

Abstract: Active oxygen has been implicated in the pathogenesis of Parkinson’s disease (PD); therefore, antioxidants have attracted attention as a potential way to prevent this disease. Squalene, a natural triterpene and an intermediate in the biosynthesis of cholesterol, is known to have active oxygen scavenging activities. Squalane, synthesized by complete hydrogenation of squalene, does not have active oxygen scavenging activities. We examined the effects of oral administration of squalene or squalane on a PD mouse model, which was developed by intracerebroventricular injection of 6-hydroxydopamine (6-OHDA). Squalene administration 7 days before and 7 days after one 6-OHDA injection prevented a reduction in striatal dopamine (DA) levels, while the same administration of squalane enhanced the levels. Neither squalene nor squalane administration for 7 days changed the levels of catalase, glutathione peroxidase, or superoxide dismutase activities in the striatum. Squalane increased thiobarbituric acid reactive substances, a marker of lipid peroxidation, in the striatum. Both squalane and squalene increased the ratio of linoleic acid/linolenic acid in the striatum. These results suggest that the administration of squalene or squalane induces similar changes in the composition of fatty acids and has no effect on the activities of active oxygen scavenging enzymes in the striatum. However, squalane increases oxidative damage in the striatum and exacerbates the toxicity of 6-OHDA, while squalene prevents it. The effects of squalene or squalane treatment in this model suggest their possible uses and risks in the treatment of PD.

Key words: Parkinson’s disease, squalene, squalane, fatty acid composition, oxidative stress

1 INTRODUCTION These active oxygen species are neurotoxic because of Parkinson’s diseas（e PD）is characterized by the progres- their strong oxidizing potential. We have already reported sive degeneration of dopaminergic neurons of the nigros- that the phosphoric diester of alpha-tocopherol and ascor- triatal system and dopamin（e DA）depletion in the striatum. bic acid, which has active oxygen scavenging activity, can While the pathogenesis of PD is not clear, damage to the prevent 6-OHDA-induced neurotoxicity6）. We also reported dopaminergic neurons by oxygen-derived free radicals is that some alkaloids, such as zingerone and eugenol, which considered to be an important contributing mechanism1）. are known antioxidants, provide protection against 6-OH- 6-Hydroxydopamin（e 6-OHDA）is used to produce an excel- DA-induced depletion7, 8）. lent animal model of PD2）, and is considered to be an en- DA is a neurotransmitter. Once released to synaptic dogenous toxin because it is found in the urine of PD pa- cleft, it binds to and activates postsynaptic neuron, and is tients3）. The toxicity of 6-OHDA is thought to be related to inactivated by reuptake into the presynaptic cell, then en- its ability to produce free radicals and to cause oxidative zymatic breakdown by monoamine oxidase into 3,4-dihy- stress4, 5）. 6-OHDA is susceptible to auto-oxidation, result- droxyphenylacetic acid（DOPAC）. Finally, catechol-O- ing in the formation of 6-OHDA quinine and hydrogen per- methyl transferase reduces DOPAC to homovanillic acid oxide, as well as superoxide and hydroxyl radicals4, 5）. （HVA）.

＊Correspondence to: Hideaki Kabuto, Department of Health Sciences, Kagawa Prefectural University of Health Sciences, 281-1 Mure-cho, Takamatsu 761-0123, JAPAN E-mail: [email protected] Accepted August 1, 2012 (recieved for review May 25, 2012) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs

21 H. Kabuto, T. Yamanushi, N. Janjua et al.

Squalen（e SQLE）is an isoprenoid compound structurally acids levels in mouse striatum similar to beta-carotene, coenzyme Q10, vitamin A, vitamin Mice were orally administered with SQLE or SQLA at a E, and vitamin K, which are physiologically active sub- dose of 1 g/kg body weight. The control was given vehicle stances9）. It has six double bonds and is a highly effective （0.1％ Tween 20/water solution）. Thirty minutes after the oxygen scavenging agent10）, and protects human skin sur- final administration of a 7-day course of SQLE, SQLA or faces from lipid peroxidation as a quencher of singlet vehicle（once daily）, mice were sacrificed by deep sodium oxygen11）. Furthermore, it is reported to have antitumor pentobarbital anesthesia. The brain was removed and the properties and immune enhancing activities12, 13）. Squalane striatum was separated immediately on an ice plate, and （SQLA）is found naturally in sebaceous secretions14）. It is a stored at －80℃ until analysis. saturated analogue of SQLE, from which it can also be pro- duced by complete hydrogenation and possessing no active 2.4 DA and its metabolites（DOPAC and HVA）levels oxygen scavenging effects. Like SQLE, SQLA is widely em- Striatal tissues were homogenized with 300 μL of 200 ployed as a skin lubricant, ingredient of suppositories, mM ice-cold perchloric acid containing 10 mM disodium carrier of lipid-soluble drugs and adjuvants9, 15）. EDTA. After centrifugation（12000×g for 10 min at 4℃）, In this study, we examined the effects of SQLE and the supernatant was filtered（0.45 μm pore size）and then SQLA on 6-OHDA-induced DA depletion in the mouse stri- injected directly into an HPLC column（Shimadzu, Kyoto, atum, and on lipid peroxidation-inhibiting activity using an Japan）with an electrochemical detecto（r ECD; Eicom, in vivo model. The purpose of this study is to investigate Kyoto, Japan）. The appendant potential of the ECD how SQLE/SQLA has the preventive effect against Parkin- （carbon electrode vs. Ag/AgCl reference electrode）was set son’s disease pathogenesis and its mechanisms. at 700 mV. The analytical column was TSKgel ODS-80TM （4.6 mm I.D.×250 mm; Tosoh, Tokyo, Japan）, and the mobile phase consisted of 0.1 M citrate-sodium acetate buffe（r pH 3.9）containing methano（l 18％, v/v）, disodium 2 MATERIALS AND METHODS EDTA（4 mg/L）, and sodium octanesulfonat（e 0.8 mM）. 2.1 Animals Male ICR mice（Charles River Japan, Yokohama, Japan） 2.5 SOD activity weighing 30 to 35 g were used in all experiments. They Cytosolic SOD activity was assayed using a SOD assay were provided with water and a standard laboratory diet ki（t SOD Test Wako; Wako Pure Chemical, Osaka, Japan）, containing 24％ protein（MF; Oriental Yeast, Tokyo, Japan） based on the method described by Oberley and Spitz 16）. ad libitum, and housed at 25℃ with a 12-h light, 12-h dark Briefly, tissues were homogenized in 20 volumes of ice-cold cycle（lights on 7 am to 7 pm）. New mice were used for phosphate buffe（r 10 mM, pH 8.0）and centrifuged at 12000 each assay. The experimental protocol was approved by ×g for 10 min at 4℃. Xanthine（0.4 mM）, nitroblue tetra- the Ethics Review Committee for Animal Experimentation zolium（0.24 mM）, and xanthine oxidase（0.049 U/mL）dis- of Kagawa Prefectural College of Health Sciences. solved in phosphate butte（r 0.1 M, pH 8.0）were added to the supernatants, and the mixture was incubated for 20 2.2 Effect of treatment with SQLE/SQLA on 6-OHDA tox- min at 37℃. The reaction was stopped by adding sodium icity dodecyl sulfat（e 69 mM）. Absorbance was measured at 560 SQLE or SQLA was administered orally at a dose of 1.0 nm. SOD from bovine erythrocyte（s Sigma Life Science, St. or 0.1 g/kg body weight for 14 days. After 7-day administra- Louis, MO, USA）was used as the standard. tion（once daily）, 6-OHDA was injected intracerebroven- tricularly（i.c.v.）at a dose of 60 μg in 2 μL of 0.05％ L- 2.6 Catalase activity ascorbic acid/saline 30 min after SQLE or SQLA Catalase activity was assayed according to the method administration. SQLE/SQLA administration was continued described by Aebi17）. In brief, the striatum was homoge- for 7 days after the 6-OHDA injection. The control was nized in 20 volumes of ice-cold RIPA buffe［r 0.1 M phos- given vehicle（0.1％ Tween 20/water solution for SQLE/ phate buffe（r pH 7.4）containing 5 mM EDTA, 0.01％ digito- SQLA administration and 0.05％ L-ascorbic acid/saline for nin, and 0.25％ sodium cholate］and centrifuged at 12000 6-OHDA injection）in the same manner. Twenty-four hours ×g for 30 min at 4℃. Phosphate buffe（r 50 mM, pH 7.0）

after the final administration, mice were sacrificed under containing EDTA（5 mM）and H2O（2 10 mM）pre-incubated sodium pentobarbital anesthesia and the striatum was for 10 min at 37℃ was added to the supernatant, and the

removed on an ice plate and stored at －80℃ until analysis. decomposition of H2O2 was assayed directly by measuring the decrease in absorbance at 240 nm for 3 min. Catalase 2.3 Effect of treatment with SQLE/SQLA on activity of from bovine live（r Wako Pure Chemical, Osaka, Japan）was striatal active oxygen scavenging enzymes, thiobar- used as a standard. bituric acid reactive substances（TBARS）, and fatty

22 J. Oleo Sci. 62, (1) 21-28 (2013) Neuroprotective effects of squalene and squalane

2.7 GPx activity ratio of linoleic acid/linolenic acid was examined for statis- GPx activity was assayed using a Bioxytech GPx-340 kit tical significance using Mann-Whitney U test. A p-value of （OXIS International., Portland, OR, USA）. The striatum less than 0.05 was considered to indicate statistical signifi- was homogenized with 10 volumes of ice-cold 50 mM Tris- cance. HCl buffe（r pH 7.5）containing 5 mM EDTA and 1 mM 2-mercaptoethanol, and centrifuged at 12000×g for 10 min at 4℃. Supernatant was used for the analysis. GPx from bovine erythrocyte（s Sigma Life Science, St. Louis, MO, 3 RESULTS USA）was used as a standard. 3.1 Effect of SQLE/SQLA administration for 7 days before and 7 days following one 6-OHDA i.c.v. injection on 2.8 Thiobarbituric acid reactive substance（TBARS）levels DA, DOPAC, and HVA levels in mouse striatum The tissue concentration of TBARS was measured ac- The mean tissue concentrations of DA and its metabo- cording to the method described by Ohishi18）. In brief, lite（s DOPAC and HVA）in the mouse striatum are shown in tissues were homogenized with ten volumes of ice-cold Figs. 1 and 2. DA and DOPAC levels were significantly de- saline containing EDTA（20 mM）. Two hundred μL of 8.1％ creased by 6-OHDA injection compared with those in the sodium dodecyl sulfate, 1.5 mL of 20％ sodium acetate striatum of the contro（l Fig. 1）. SQLE treatment blunted buffe（r pH.3.5）, 1.5 mL of 0.8％ sodium thiobarbiturate, the 6-OHDA-induced DA and DOPAC reduction. DA recov- and 600 μL of distilled water were added to 200 μL of the ered but not to a significant level compared with the homogenate and the mixture was heated at 100℃ for 60 control when 1.0 mg/kg of SQLE was administered（Fig. 1）. min. TBARS were extracted with a mixture of n- SQLA treatment enhanced the 6-OHDA-induced DA and butanol:pyridine（15:1, V/V）, and the fluorescence was DOPAC reduction（Fig. 2）. SQLE or SQLA alone did not measured（Ex. 515 nm, Em. 532 nm）. Malonaldehyde significantly change the levels of striatal DA or its metabo- （Tokyo Kasei Kogyo, Tokyo, Japan）was used as a standard. lites at this time poin（t Figs.1 and 2）.

2.9 Measurement of fatty acids concentrations in the 3.2 Effect of SQLE/SQLA administration for 7 days on the striatum levels of TBARS in the mouse striatum The tissue was homogenized with 10 volumes of chloro- The mean tissue concentrations of TBARS in the mouse form（67％）/ methano（l 33％）solution containing 1 μg hep- striatum are shown in Fig. 3. SQLE treatment did not tadecanoic acid as an internal standard. After centrifuga- change the level of TBARS. However, SQLA treatment in- tion, the supernatant was added to 2 mL of distilled water, creased the level of TBARS significantly compared with mixed, and centrifuged again. The chloroform layer was that in the control striatum. isolated, evaporated, and dissolved in 0.2 mL of n-hexane. The extracted samples were methylated using a fatty acid 3.3 Effect of SQLE/SQLA administration for 7 days on the methylation ki（t Nacalai Tesque, Kyoto, Japan）. Samples activities of SOD, catalase and GPx in mouse stria- were injected into a gas chromatography-mass spectrome- tum te（r GC/MS, Shimazu GCMS-QP5050A, Shimazu, Kyoto, The activities of SOD, catalase, and GPx in the mouse Japan）, using a DB-5 column（J and W Scientific, Folsom, striatum after the administration of SQLE or SQLA for 7 CA, USA）and helium as the carrier gas. days are shown in Table 1. No significant changes in SOD, catalase, or GPx activity were found. 2.10 Protein levels The protein assay was performed by the method of 3.4 Effect of SQLE/SQLA administration for 7 days on the Lowry et al.19） using bovine serum albumin as the standard. levels of fatty acids in the mouse brain The mean tissue concentrations of DHA, EPA, arachi- 2.11 Chemicals donic acid, linolenic acid, linoleic acid, and the ratio of lin- All chemicals and reagents were of the highest quality oleic acid/linolenic acid in the mouse brain are shown in available and obtained either from Wako Pure Chemical Table 2. SQLE or SQLA treatment did not change the （Osaka, Japan）or Sigma Life Scienc（e St. Louis, MO, USA）. levels of these fatty acids; however, there was a significant decrease in the ratio of linoleic acid/linolenic acid. 2.12 Statistical analysis All data are expressed as means±SEM. Differences between each groups were examined for statistical significance using two-way analysis of variance（DA, DOPAC, 4 DISCUSSION HVA）, and one-way analysis of varianc（e TBARS, SOD, cat- Previous studies reported the presence of increased oxi- alase, GPx, fatty acids）followed by Scheffe’s F-test. The dative stress, low levels of endogenous antioxidants, and

23 J. Oleo Sci. 62, (1) 21-28 (2013) H. Kabuto, T. Yamanushi, N. Janjua et al.

Fig. 2 Effects ‌ of oral administration of SQLA for the Fig. 1 Effects ‌ of oral administration of SQLE for the 7 days before and 7 days after i.c.v. injection of 7 days before and 7 days after i.c.v. injection of 6-OHDA on DA, DOPAC, and HVA levels in 6-OHDA on DA, DOPAC, and HVA levels in the mouse striatum. Symbols are as described the mouse striatum. Data are expressed as means for Fig. 1. n=6-8. ± SEM of 6-8 mice in each group. p <0.05, compared with each other using two-way ANOVA. ubiquitously in tissues26）. SQLE has been reported to possess antioxidant properties due to double bonds in the low activity of free radical-scavenging enzymes in the structure, and in vitro experimental evidence indicates that brains of patients with PD20－23）. These ﬁndings suggest that it is a highly effective oxygen-scavenging agent10, 11）. Fur- free radicals and oxidative stress are important contribu- thermore, SQLE is a unique antioxidant molecule with high tors to the pathogenesis of PD24）. scavenging activity toward singlet oxygen11）. The high SQLE is easily absorbed when it is orally administrat- singlet oxygen quenching ability is due to its chemical ed25）. It is transported in serum, generally in association structure consisting of a six 2-methyl-2-pentene unit11）. with very low density lipoproteins, until it is distributed In the present study, we first examined the effects of

24 J. Oleo Sci. 62, (1) 21-28 (2013) Neuroprotective effects of squalene and squalane

enhanced 6-OHDA-induced neurotoxicity, inducing a further reduction in striatal tissue DA and DOPAC. The lipid peroxidation levels, and the antioxidative con- dition of the striatum at the time 6-OHDA was injected were important because 6-OHDA is susceptible to autooxi- dation, resulting in the formation of 6-OHDA quinine and hydrogen peroxide, as well as superoxide and hydroxyl radicals4, 5）. Administration of 1.0 g/kg/day of SQLE/SQLA was effective to the 6-OHDA induced DA depression in the striatum. Therefore, we measured the effects of SQLE/ SQLA（1.0 mg/kg/day administration for 7 days）on lipid peroxidation levels and the activities of active oxygen scav- Fig. 3 Effects ‌ of oral administration of SQLE and enging enzymes, such as SOD, catalase, and GPx in the SQLA for 7 days on TBARS levels in the mouse striatum. Treatment with SQLA for 7 days increased the striatum. Data are expressed as means ± SEM of striatal TBARS level but SQLE treatment had no effect. 6-8 mice in each group. p <0.05, compared with 6-OHDA is taken in the DA neuron specifically and formed each other using one-way ANOVA. 6-OHDA quinine and hydrogen peroxide. Therefore, oxidative damage by 6-OHDA is induced in the DA neuron spe- Table 1 Activities of SOD, catalase, and GPx in cifically. However, SQLA may be ingested into all cells in mouse striatum after one week administra- the brain. SQLA may change lipid composition and make tion of squalene or squalane. the cells sensitive to oxidative stress, and increased TBARS levels. This increase in oxidative damage sensitivity control SQLE SQLA induced by SQLA administration, combined with the toxic- SOD 473.6±38.9 465.5±22.6 464.4±12.7 ity of 6-OHDA, may exacerbate the level of DA depression catalase 549.9±22.9 549.4±16.1 605.0±29.7 in the striatum. Santhilkumar et al. reported that the oral GPx 2.09±0.20 2.20±0.08 2.18±0.16 administration of SQLE did not change the active oxygen Data (U/mg protein) are expressed as the mean ± SEM. scavenging activities of SOD, catalase, and GPx in heart 27） n=5-8. Significance was determined at p<0.05 level usinga tissue and erythrocyte hemolysate in rats . Likewise, we one-way ANOVA followed by Sheffe's F test. No significant did not observe significant differences in the activities of difference was observed. these enzymes in the mouse striatum after oral administration of SQLE for 7 days. SQLE/SQLA on 6-OHDA neurotoxicity by measuring We measured the effect of SQLE/SQLA on the composi- changes in DA, DOPAC, and HVA as markers of dopaminer- tion of fatty acids in the striatum. SQLE or SQLA adminis- gic neuronal injury. Treatment with SQLE（for 7 days tration, carried out daily for one week, did not change the before and 7 days after 6-OHDA injection）inhibited 6-OH- levels of these fatty acids in the striatum in a statistical DA-induced neurotoxicity, lessening the reductions in stri- sense. We compared the ratio of linolenic acid and linoleic atal tissue DA and DOPAC. However, treatment with SQLA acid. That of the SQLE and SQLA groups was significantly

Table 2 Concentrations of fatty acids in mouse striatum after one week administration of squalene or squalane. control SQLE SQLA Stearic Acid (C18) 703.7±78.2 820.9±127.2 656.7±87.1 Linolenic Acid (C18:2n-3) 64.3±7.6 78.0±9.7 61.9±4.6 EPA (C20:5n-3) 12.6±1.5 19.5±2.7 15.8±2.2 DHA (C22:6n-3) 1517.1±184.8 1933.4±305.8 1517.1±183.0 Linoleic Acid (C18:3n-6) 55.3±5.5 50.7±6.6 42.7±2.3 Arachidonic Acid (C20:4n-6) 1256.3±139.2 1542.1±227.9 1106.6±140.2 Linoleic Acid/Linolenic Acid 0.944±0.069 0.651±0.026a 0.697±0.027a Data (ng/mg wet weight and the ratio) are expressed as the mean±SEM. n=5-8. Differences were examined using ANOVA followed by Sheffe's F test (fatty acids) and MannMann- Whitney’s U test (Linoleic Acid/Linolenic Acid). a:p<0.05

25 J. Oleo Sci. 62, (1) 21-28 (2013) H. Kabuto, T. Yamanushi, N. Janjua et al.

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