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Melatonin preserves arachidonic and docosapentaenoic acids during ascorbate-Fe2+ peroxidation of rat testis microsomes and mitochondria
The International Journal of Biochemistry & Cell Biology 35 (2003) 359–366
Melatonin preserves arachidonic and docosapentaenoic acids during ascorbate-Fe2+ peroxidation of rat testis microsomes and mitochondria Mariana Gavazza, Angel Catalá∗,1 Facultad de Ciencias Veterinarias, Cátedra de Bioqu´ımica, Universidad Nacional de La Plata CC 296, B1900 AVW La Plata, Argentina Received 4 January 2001; received in revised form 8 July 2002; accepted 20 August 2002
Abstract The pineal hormone melatonin (N-acetyl, 5-methoxytryptamine) was recently accepted to act as an antioxidant under both in vivo and in vitro conditions. In this study, we examined the possible preventive effect of melatonin on ascorbate-Fe2+ lipid peroxidation of rat testis microsomes and mitochondria. Special attention was paid to the changes produced on the highly polyunsaturated fatty acids C20:4 n6 and C22:5 n6. The lipid peroxidation of testis microsomes or mitochondria produced a significant decrease of C20:4 n6 and C22:5 n6. The light emission (chemiluminescence) used as a marker of lipid peroxidation was similar in both kinds of organelles when the control and peroxidized groups were compared. Both long chain polyunsaturated fatty acids were protected when melatonin was incorporated either in microsomes or mitochondria. The melatonin concentration required to inhibit by 100% the lipid peroxidation process was 5.0 and 1.0 mM in rat testis microsomes and mitochondria, respectively. IC 50 values calculated from the inhibition curve of melatonin on the chemiluminescence rates were higher in microsomes (4.98 mM) than in mitochondria (0.67 mM). The protective effect observed by melatonin in rat testis mitochondria was higher than that observed in microsomes which could be explained if we consider that the sum of C20:4 n6 + C22:5 n6 in testis microsomes is two-fold greater than present in mitochondria. © 2002 Published by Elsevier Science Ltd. Keywords: Lipid peroxidation; Melatonin; Testis; Mitochondria; Microsomes
1. Introduction The content of fatty acids in testis is very high with a prevalence of polyunsaturated fatty acids (PUFA) which renders this tissue very susceptible to lipid peroxidation. In this regard, we have demonstrated that supplementation with ␣-tocopherol reduces the sus∗
Corresponding author. Fax: +54-221-4257980. E-mail address: [email protected] (A. Catal´a). 1 Member of Carrera del Investigador Cient´ıfico, Consejo Nacional de Investigaciones Cient´ıficas y T´ecnicas de la Rep´ublica, Argentina.
ceptibility to lipid peroxidation of PUFA present in rat testis microsomes and mitochondria [1]. Damage to testicular long chain fatty acids adversely affects their normal physiology and may lead to disturbances during the process of spermatogenesis [2]. In a very recent report, we have showed that melatonin (N-acetyl-5-methoxytryptamine), the main secretory product of the pineal gland, may act in “vivo” and in “vitro” as antioxidant protecting long chain polyunsaturated fatty acids present in rat liver microsomes from the deleterious effect by a selective mechanism that reduces the loss of docosahexaenoic and arachidonic acids [3]. The role of lipids in the structure and
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function of the male reproductive system continues to be an interesting and important area of investigation. The predominant lipids of whole testis are phospholipids, with smaller amounts of acylglycerols (chiefly triacylglycerols) free and esterified cholesterol and even smaller amounts of gangliosides and sulfolipids [4]. The phospholipids of testis are characterized by extremely high proportions of long chain highly PUFAs with a prevalence of 4,7,10,13,16-eicosapentaenoic acid, C22:5 n6 [5]. In addition to the saturated and unsaturated fatty acids commonly found in mammalian tissues, testicular lipids have been shown to be enriched with 20- and 22-carbon polyenes and to contain 24-carbon polyenes [6]. The efficient synthesis of C22:5 n6 may also partly explain why this is the major 22-carbon fatty acid in rat testis [7]. Many studies related with lipid chemistry and metabolism of testicular tissue have led to the suggestion that polyenoic acids, particularly C22:5 n6 have an important role in the process of spermatogenesis in the rat [6]. Karbownik et al. [8] have demonstrated that melatonin and related indoles at pharmacological concentrations protect against both the autoxidation of lipids as well as induced peroxidation of lipids in testis. In doing so, these agents would be expected to reduce testicular cancer that is initiated by products of lipid peroxidation. The aim of the current study was to evaluate the potential protective effect of melatonin against ascorbate-Fe2+ induced lipid peroxidation in rat testis microsomes and mitochondria. Chemiluminescence and fatty acid composition of both organelles were used as an index of the oxidative destruction of lipids. 2. Materials and methods 2.1. Chemicals Butylated hydroxytoluene (BHT), phenylmethylsulfonyl fluoride (PMSF), and melatonin were from Sigma, standards of fatty acids methyl esters were from Nu Chek Prep Inc. (Elysian, MN, USA). All other reagents and chemicals were of analytical grade from Sigma. 2.2. Animals and membrane preparation Male Wistar rats, two months old, weighting 200– 250 g were used. Rats were maintained on a commer-
cial standard pellet diet and tap water ad libitum. The diet contained 4% of total lipid with a fatty acid composition of 19.14% palmitic acid C16:0, 0.184% palmitoleic acid C16:1, 4.10% stearic acid C18:0, 19.34% oleic acid C18:1 n9, 51.53% linoleic acid C18:2 n6 and 4.83% linolenic acid C18:3 n3. The rats were sacrificed by cervical dislocation and testis rapidly removed, cut into small pieces and washed extensively with 0.15 M NaCl. An homogenate of each tissue was prepared in sol. A (0.25 M sucrose, 10 mM Tris–HCl pH 7.4, PMSF 0.1 mM), 3 ml of solution per gram of tissue, using the potterElvejhem homogeneizer. The homogenate was spun at 3000 × g, pellets were discarded, the supernatant was spun at 20,000 × g for 10 min to obtain testis mitochondria [9]. After the centrifugation, 5 ml of the resultant supernatant was applied to a Sepharose 4B column (1.6 cm × 12 cm) equilibrated and eluted with 10 mM Tris–HCl pH 7.4, 0.01% NaN3 [10]. The microsomal fraction appearing in the void volume (12–20 ml) was used and cytosol (30–40 ml) discarded. All operations were performed at 4 ◦ C. Microsomes and mitochondria were stored at −83 ◦ C and used within a week of its preparation, after one cycle of freezing and thawing. Tissues and membrane preparations were protected from light during the procedures. 2.3. Lipid peroxidation of mitochondria and microsomes Chemiluminescence and lipid peroxidation were initiated by adding ascorbate to microsomal or mitochondrial preparations [11]. Microsomes and/or mitochondria (1 mg of protein) were preincubated previously during 15 min at room temperature with different amounts of melatonin, and then were incubated at 37 ◦ C with 0.01 M phosphate buffer (pH 7.4), 0.4 mM ascorbate, final volume 1 ml. Microsomal and mitochondrial preparations which lacked ascorbate were carried out simultaneously. Membrane light emission was determined over a 180 min period; chemiluminescence was recorded as counts per minute (cpm) every 10 min and the sum of the total chemiluminescence was used to calculate cpm/mg protein. Chemiluminescence was measured as counts per min in a liquid scintillation analyzer Packard 1900 TR.
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2.4. Measurement of fatty acid composition Microsomal or mitochondrial lipids were extracted with chloroform/methanol (2:1 (v/v)) [12]. Fatty acids were transmethylated with 20% F3 B in methanol at 60 ◦ C for 3 h. Fatty acids methyl esters were analyzed with a GC-14A gas chromatograph (Shimadzu, Kyoto, Japan) equipped with a packed column (1.80 mm × 4 mm i.d.) GP 10% DEGS-PS on 80/100 Supelcoport. Nitrogen was used as a carrier gas. The injector and detector temperatures were maintained at 250 ◦ C, the column temperature was held to 200 ◦ C during 60 min. The fatty acid methyl esters were identified by comparing retention times with standard compounds. All compositions were expressed as area percentage of total fatty acids. 2.5. Peroxidizability index (PI) Peroxidizability index (PI) was calculated according to the formula PI = (percent of monoenoic acids × 0.025) + (percent of dienoic acids × 1) + (percent of tetraenoic acids × 4) + (percent of pentaenoic acids × 6). 2.6. Protein determination Proteins were determined by the method of Lowry et al. [13] using BSA as standard. 2.7. Statistical analysis Results were expressed as means ± S.D. of three independent determinations. Data were evaluated statistically by one-way analysis of variance (ANOVA) and Tukey test. Statistical criterion for significance was selected at different P-values and indicated in each case.
3. Results 3.1. Light emission of rat testis microsomes and mitochondria during lipid peroxidation: melatonin effect The incubation of rat testis microsomes or mitochondria in the presence of ascorbate-Fe2+ resulted
Fig. 1. (A) Light emission produced by rat testis mitochondria and microsomes incubated with (䊏) or without (䊐) ascorbic acid. (B) Ascorbate-Fe2+ induced chemiluminescence as a function of time of rat testis microsomes incubated with (䊊—䊊) or without (䊉—䊉) ascorbic acid. (C) Ascorbate-Fe2+ induced chemiluminescence as a function of time of rat testis mitochondria incubated with (䊊—䊊) or without (䊉—䊉) ascorbic acid. Values are mean ± S.D. of three independent experiments.
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in the peroxidation of membranes as evidenced by the emission of light (chemiluminescence). The light emission was similar in both kinds of organelles when the control and peroxidized groups were compared (Fig. 1A). Chemiluminescence as a function of time was measured during 180 min at 37 ◦ C, in rat testis microsomes or mitochondria with and without 0.4 mM ascorbate. Over the time course studies analysis of chemiluminescence demonstrated that the lipid peroxidation in the presence of ascorbic acid reach a maximum at 120 min (Fig. 1B and C). The time course of the chemiluminescence resulting from the addition of different concentrations of melatonin to rat testis
Fig. 3. (A) Inhibition of lipid peroxidation (light emission) of rat testis microsomes by melatonin. (B) Inhibition of lipid peroxidation (light emission) of rat testis mitochondria by melatonin. Values are mean ± S.D. of three independent experiments.
microsomes and mitochondria are shown in Fig. 2A and B, respectively. Inhibition of lipid peroxidation in microsomes by melatonin, was almost linear up to 5 mM, whereas in mitochondria melatonin inhibition showed linearity up to 1 mM Fig. 3A and B, respectively. Fig. 2. (A) Lipid peroxidation of rat testis microsomes without ascorbic acid (䊉—䊉) and with the addition of 0 (䊊—䊊); 1.0 (䊏—䊏); 2.5 (䉫—䉫); 5.0 (䉱—䉱) and 10.0 mM (䊐—䊐) melatonin. (B) Lipid peroxidation of rat testis mitochondria without ascorbic acid (䊉—䊉) and with the addition of 0 (䊊—䊊); 0.125 (䊏—䊏); 0.25 (䉫—䉫); 0.5 (䉱—䉱) and 1.0 mM (䊐—䊐) melatonin.
3.2. Fatty acid composition of rat testis microsomes and mitochondria during lipid peroxidation: melatonin effect In testis microsomes, Table 1, the proportions of linoleic C18:2 n6, arachidonic C20:4 n6 and
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Table 1 Fatty acid composition of rat testis microsomes during lipid peroxidation in the presence of different amounts of melation Fatty acids
Control
Melatonin (mM)
∗
C16:0 C16:1 n7 C18:0 C18:1 n9 C18:2 n6 C20:4 n6 C22:5 n6 Saturated Monounsaturated Polyunsaturated Total unsaturated Saturated/ unsaturated PI
0 29.08 3.80 7.48 12.46 17.46 25.17 26.98 36.57 16.26 69.61 85.88 0.42
± ± ± ± ± ± ± ± ± ± ± ±
1.52 1.08 2.02 3.01 4.66 3.40 3.20 2.50 2.90 10.17 11.45 0.04
280.44 ± 36.10
38.19 1.04 12.74 7.92 16.93 13.37 15.56 50.93 8.96 45.87 54.83 0.92
1.0 ± ± ± ± ± ± ± ± ± ± ± ±
1.50 0.80 3.10 1.00 0.80 1.30 b 3.60 b 4.60 1.50 3.50 b 4.77 0.10
164.03 ± 20.13 a
2.5
38.30 0.95 19.27 12.04 15.55 12.78 14.60 57.57 12.99 42.93 55.93 1.02
± ± ± ± ± ± ± ± ± ± ± ±
4.20 0.20 2.50 1.40 2.10 2.30 b 4.40 b 5.30 1.60 3.20 a 2.90 0.10
154.27 ± 25.00 a
5.0
31.91 0.75 12.57 15.38 9.38 14.64 20.62 44.48 16.02 44.65 60.78 0.72
± ± ± ± ± ± ± ± ± ± ± ±
2.60 0.40 3.20 4.30 0.80 2.40 b 0.90 5.60 4.10 1.60 a 4.10 0.10
191.95 ± 7.90 b
10.0
35.02 0.99 13.99 12.53 8.61 22.45 22.82 49.03 13.52 50.88 64.41 0.75
± ± ± ± ± ± ± ± ± ± ± ±
2.90 0.80 4.80 2.10 0.80 6.50 0.50 7.30 1.60 0.90 b 1.90 0.10
40.44 2.3 9.12 11.65 17.00 24.98 24.86 49.56 13.91 66.85 80.77 0.60
210.50 ± 9.60 c
± ± ± ± ± ± ± ± ± ± ± ±
14.00 0.50 2.50 0.90 1.10 2.80 d 1.70 e 16.00 0.70 0.30 d 0.90 0.10
266.40 ± 1.04 d
(∗ )
Data shown are given in percentages of total fatty acid content and are mean ± S.D. of three separated experiments. Without ascorbic acid. PI was calculated according to the formula PI = (percent of monoenoic acids × 0.025) + (percent of dienoic acids × 1) + (percent of tetraenoic acids × 4) + (percent of pentaenoic acids × 6). Statistically significant differences between control vs. peroxidized, peroxidized + 1.0; 2.5; 5.0 and 10 mM melatonin are indicated by letter ‘a’ P < 0.0005; ‘b’ P < 0.005; ‘c’ P < 0.05. Statistically significant differences between peroxidized vs. peroxidized + 1.0; 2.5; 5.0 and 10 mM melatonin are indicated by ‘d’ P < 0.005; ‘e’ P < 0.05 using analysis of variance (ANOVA).
Table 2 Fatty acid composition of rat testis mitochondria during lipid peroxidation in the presence of different amounts of melatonin Fatty acids
Control
Melatonin (mM)
∗
C16:0 C16:1 n7 C18:0 C18:1 n9 C18:2 n6 C20:4 n6 C22:5 n6 Saturated Monounsaturated Polyunsaturated Total unsaturated Saturated/ unsaturated PI
0 31.36 0.13 8.59 13.00 11.65 15.02 13.71 39.35 13.76 40.39 54.16 0.73
± ± ± ± ± ± ± ± ± ± ± ±
3.30 0.10 1.40 1.62 3.98 2.10 1.86 1.98 2.70 2.10 0.60 0.10
154.37 ± 16.00
42.24 0.21 16.50 19.39 8.20 5.18 2.85 58.74 19.60 16.24 35.84 1.67
0.125 ± ± ± ± ± ± ± ± ± ± ± ±
1.80 0.10 4.37 1.80 2.87 1.90 b 0.80 a 5.90 1.89 5.60 a 5.40 0.40
46.54 ± 15.40 a
41.05 0.54 12.84 20.96 8.89 5.78 4.32 53.89 21.50 18.99 40.50 1.33
0.25 ± ± ± ± ± ± ± ± ± ± ± ±
1.10 0.10 2.65 3.20 0.10 2.00 b 1.20 a 3.60 3.30 3.03 a 2.50 0.10
58.48 ± 14.40 a
46.05 0.33 12.55 19.33 8.15 6.25 5.53 58.61 19.66 19.94 39.60 1.48
0.5 ± ± ± ± ± ± ± ± ± ± ± ±
3.90 0.10 3.13 1.00 0.10 2.91 b 0.50 a 7.10 1.10 3.10 2.20 0.20
66.86 ± 13.00 a
38.90 0.18 12.60 18.32 9.07 7.51 5.73 51.51 18.50 18.65 37.32 1.38
1.0 ± ± ± ± ± ± ± ± ± ± ± ±
1.40 0.10 2.80 1.10 0.10 1.90 c 0.90 a 3.60 1.03 2.80 a 3.10 0.10
70.32 ± 12.30 a
30.49 0.15 10.97 13.59 11.03 15.70 14.55 41.46 13.74 41.29 55.04 0.75
± ± ± ± ± ± ± ± ± ± ± ±
6.10 0.02 2.70 2.10 4.80 2.10 e 1.70 d 6.10 2.10 5.63 4.30 0.10
161.50 ± 18.40 d
Data shown are given in percentages of total fatty acid content and are mean ± S.D. of three separated experiments. (∗ ) Without ascorbic acid. Peroxidizability index (PI) was calculated according to the formula PI = (percent of monoenoic acids × 0.025) + (percent of dienoic acids × 1) + (percent of tetraenoic acids × 4) + (percent of pentaenoic acids × 6). Statistically significant differences between control vs. peroxidized, peroxidized + 0.125; 0.25; 0.5 and 1.0 mM melatonin are indicated by letter ‘a’ P < 0.0005; ‘b’ P < 0.005; ‘c’ P < 0.05. Statistically significant differences between peroxidized vs. peroxidized + 0.125; 0.25; 0.5 and 1.0 mM melatonin are indicated by ‘d’ P < 0.0005; ‘e’ P < 0.005; ‘f’ P < 0.05 using analysis of variance (ANOVA).
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docosapentaenoic acids 22:5 n-6 were characteristically higher than in testis mitochondria, Table 2. The total proportions of n-6 PUFA were also higher in testis microsomes than in mitochondria. Both polyunsaturated fatty acids were preserved when melatonin was present in the lipid peroxidation system. Thus, in rat testis microsomes and mitochondria the melatonin concentration required to inhibit by 100% the lipid peroxidation process was 5.0, and 1.0 mM, respectively, Tables 1 and 2. In testis microsomes significant differences in the content of arachidonic acid C 20:4 n6 were observed when control versus peroxidized and control versus peroxidized + 1.0; and 2.5 mM melatonin groups were compared. Significant differences in the content of docosapentaenoic acid C 22:5 n6 were observed when control versus peroxidized and control versus peroxidized + 1.0 mM melatonin groups were compared. In testis mitochondria significant differences in the content of arachidonic acid C 20:4 n6 and docosapentaenoic acid C 22:5 n6 were observed when control versus peroxidized and control versus peroxidized + 0.125; +0.25; and +0.5 mM melatonin groups were compared. 3.3. Peroxidizability index and IC50 values of organelles lipid peroxidized and protected by melatonin In testis microsomes the peroxidizability index (PI) which indicates the degree of vulnerability to degradation of a selected membrane showed significant differences between control versus peroxidized and control versus peroxidized + 1.0; +2.5; and +5.0 mM melatonin. In testis mitochondria the PI showed significant differences between control versus peroxidized and control versus peroxidized+0.125; +0.25; and 0.5 mM melatonin. IC 50 values were calculated from the inhibition curve of melatonin on the chemiluminiscence rates Fig. 2A and B, respectively. The data showed that IC 50 was higher in microsomes (4.98 mM) than in mitochondria (0.67 mM). Our results clearly demonstrate that the preservation of the most abundant polyunsaturated fatty acids C20:4 n6 and C22:5 n6 by melatonin during ascorbate-Fe2+ lipid peroxidation is more effective in rat testis microsomes than in mitochondria. Furthermore, the PI was positively correlated with the level of these long chain fatty acids, Fig. 4A and B.
Fig. 4. (A) Fatty acid percent and PI as a function of melatonin concentration in rat testis microsomes. (B) Fatty acid percent and PI as a function of melatonin concentration in rat testis mitochondria. PI (䊏—䊏 ); C 20:4 n6 (䊐—䊐) and C 22:5 n6 (䉱—䉱). Values are mean ± S.D. of three independent experiments.
4. Discussion Approximately 70% of the fatty acids located in rat testis microsomes and mitochondria are polyunsaturated with a prevalence of arachidonic C20:4 n6 and docosapentaenoic C22:5 n6 acids [1]. This high concentration of polyunsaturated fatty acids in these membranes causes susceptibility to lipid peroxidative degradation. This study has examined some of the effects by which melatonin protect lipid peroxidation of rat testis microsomes and mitochondria in vitro. Experimental studies have shown that melatonin directly scavenges the hydroxyl radical, peroxyl radical,
M. Gavazza, A. Catal´a / The International Journal of Biochemistry & Cell Biology 35 (2003) 359–366
peroxynitrite anion, and singlet oxygen. Furthermore, this tryptophan derivative stimulates a number of antioxidative enzymes and stabilizes cell membranes; this latter action helps membranes to resist free radical damage [14]. While the antioxidative actions of most molecules are limited by their specific intracellular distribution, e.g. Vitamin E in lipid-rich membranes [15], melatonin’s antioxidative actions include the protection of lipids in the cell membrane, proteins in the cytosol, and DNA in the nucleus. Our results have shown that the addition of ascorbate to rat testis microsomes or mitochondria increased lipid peroxidation several-fold over organelles not treated with ascorbic acid. Ascorbate-stimulated lipid peroxidation was substantially suppressed in rat testis microsomes or mitochondria previously incubated with melatonin. These results are consistent with our previous results in which melatonin has been shown to prevent polyunsaturated fatty acid degradation by inhibiting lipid peroxidation [3]. The vulnerability to lipid peroxidation of rat testis microsomes and mitochondria assessed by the sum of the most polyunsaturated fatty acids (C20:4 n6 and C22:5 n6, Tables 1 and 2) is coincident with the results obtained from the chemiluminescence intensity (Fig. 1B and C). In this regard, it is important to note that arachidonic acid was protected more efficiently than docosahexaenoic acid at all melatonin concentrations tested when rat testis microsomes or mitochondria were incubated with ascorbic acid (Tables 1 and 2). The damaging effects of lipid peroxidation on membrane structure and function are well documented [16,17]. Many studies have shown that free radical damage and lipid peroxidation increase as a function of the degree of unsaturation of the fatty acids present in the phospholipids of biological membranes. In this regard it has been demonstrated that the number of bis-allylic positions contained in the cellular lipids of intact cells determines their susceptibility, i.e. oxidizability, to free radical-mediated peroxidative events [18]. Tang et al. [19] have demonstrated the ability of melatonin to protect against iron-induced lipid peroxidation in various rat cell membranes from the brain, heart, kidney and liver of the male Sprague–Dawley rat. The concentration of cellular membrane malondialdehyde (MDA) was used as an index of induced oxidative membrane damage. This peroxidative effect was partially suppressed by melatonin (ED50
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0.886 mM) for the liver. Thus, the direct effect of lipid peroxidation on cellular membranes following iron exposure was markedly reduced by melatonin. In conclusion, the present study demonstrates that melatonin suppresses the ascorbate-Fe2+ induced lipid peroxidation process in rat testis microsomes and mitochondria and protects the most abundant polyunsaturated fatty acids, arachidonic C20:4 n6 and docosapentaenoic C22:5 n6 acids, from damage. The protective effect observed by melatonin in rat testis mitochondria was higher than that observed in microsomes which could be explained if we consider that the sum of C20:4 n6+C22 : 5 n6 in testis microsomes is two-fold than that present in mitochondria. Besides its action in direct free radical scavenging (OH• and the peroxyl radical-LOO• ) and in membrane stabilization, melatonin has actions on enzymes, such as superoxide dismutase and gluthathione peroxidase that either generate or metabolise reactive oxygen intermediates. Studies in vivo provides evidence that melatonin activates those enzymes but in studies in vitro similar to our study, the activities of these enzymes were not actually measured [20]. The physiological potential of melatonin in preventing lipid degradation of polyunsaturated fatty acids in vivo in different organelles of rat testis and studies aimed to determine the presence of different enzyme activities that either generate or metabolise reactive oxygen species in those organelles which could interact with melatonin deserves further investigation.
Acknowledgements We are grateful to Cesar Arcemis for performing GLC analysis. This work was carried out with a grant from Secretaria de Ciencia y Técnica Universidad Nacional de La Plata.
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Melatonin preserves arachidonic and docosapentaenoic acids during ascorbate-Fe2+ peroxidation of rat testis microsomes and mitochondria Mariana Gavazza, Angel Catalá∗,1 Facultad de Ciencias Veterinarias, Cátedra de Bioqu´ımica, Universidad Nacional de La Plata CC 296, B1900 AVW La Plata, Argentina Received 4 January 2001; received in revised form 8 July 2002; accepted 20 August 2002
Abstract The pineal hormone melatonin (N-acetyl, 5-methoxytryptamine) was recently accepted to act as an antioxidant under both in vivo and in vitro conditions. In this study, we examined the possible preventive effect of melatonin on ascorbate-Fe2+ lipid peroxidation of rat testis microsomes and mitochondria. Special attention was paid to the changes produced on the highly polyunsaturated fatty acids C20:4 n6 and C22:5 n6. The lipid peroxidation of testis microsomes or mitochondria produced a significant decrease of C20:4 n6 and C22:5 n6. The light emission (chemiluminescence) used as a marker of lipid peroxidation was similar in both kinds of organelles when the control and peroxidized groups were compared. Both long chain polyunsaturated fatty acids were protected when melatonin was incorporated either in microsomes or mitochondria. The melatonin concentration required to inhibit by 100% the lipid peroxidation process was 5.0 and 1.0 mM in rat testis microsomes and mitochondria, respectively. IC 50 values calculated from the inhibition curve of melatonin on the chemiluminescence rates were higher in microsomes (4.98 mM) than in mitochondria (0.67 mM). The protective effect observed by melatonin in rat testis mitochondria was higher than that observed in microsomes which could be explained if we consider that the sum of C20:4 n6 + C22:5 n6 in testis microsomes is two-fold greater than present in mitochondria. © 2002 Published by Elsevier Science Ltd. Keywords: Lipid peroxidation; Melatonin; Testis; Mitochondria; Microsomes
1. Introduction The content of fatty acids in testis is very high with a prevalence of polyunsaturated fatty acids (PUFA) which renders this tissue very susceptible to lipid peroxidation. In this regard, we have demonstrated that supplementation with ␣-tocopherol reduces the sus∗
Corresponding author. Fax: +54-221-4257980. E-mail address: [email protected] (A. Catal´a). 1 Member of Carrera del Investigador Cient´ıfico, Consejo Nacional de Investigaciones Cient´ıficas y T´ecnicas de la Rep´ublica, Argentina.
ceptibility to lipid peroxidation of PUFA present in rat testis microsomes and mitochondria [1]. Damage to testicular long chain fatty acids adversely affects their normal physiology and may lead to disturbances during the process of spermatogenesis [2]. In a very recent report, we have showed that melatonin (N-acetyl-5-methoxytryptamine), the main secretory product of the pineal gland, may act in “vivo” and in “vitro” as antioxidant protecting long chain polyunsaturated fatty acids present in rat liver microsomes from the deleterious effect by a selective mechanism that reduces the loss of docosahexaenoic and arachidonic acids [3]. The role of lipids in the structure and
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function of the male reproductive system continues to be an interesting and important area of investigation. The predominant lipids of whole testis are phospholipids, with smaller amounts of acylglycerols (chiefly triacylglycerols) free and esterified cholesterol and even smaller amounts of gangliosides and sulfolipids [4]. The phospholipids of testis are characterized by extremely high proportions of long chain highly PUFAs with a prevalence of 4,7,10,13,16-eicosapentaenoic acid, C22:5 n6 [5]. In addition to the saturated and unsaturated fatty acids commonly found in mammalian tissues, testicular lipids have been shown to be enriched with 20- and 22-carbon polyenes and to contain 24-carbon polyenes [6]. The efficient synthesis of C22:5 n6 may also partly explain why this is the major 22-carbon fatty acid in rat testis [7]. Many studies related with lipid chemistry and metabolism of testicular tissue have led to the suggestion that polyenoic acids, particularly C22:5 n6 have an important role in the process of spermatogenesis in the rat [6]. Karbownik et al. [8] have demonstrated that melatonin and related indoles at pharmacological concentrations protect against both the autoxidation of lipids as well as induced peroxidation of lipids in testis. In doing so, these agents would be expected to reduce testicular cancer that is initiated by products of lipid peroxidation. The aim of the current study was to evaluate the potential protective effect of melatonin against ascorbate-Fe2+ induced lipid peroxidation in rat testis microsomes and mitochondria. Chemiluminescence and fatty acid composition of both organelles were used as an index of the oxidative destruction of lipids. 2. Materials and methods 2.1. Chemicals Butylated hydroxytoluene (BHT), phenylmethylsulfonyl fluoride (PMSF), and melatonin were from Sigma, standards of fatty acids methyl esters were from Nu Chek Prep Inc. (Elysian, MN, USA). All other reagents and chemicals were of analytical grade from Sigma. 2.2. Animals and membrane preparation Male Wistar rats, two months old, weighting 200– 250 g were used. Rats were maintained on a commer-
cial standard pellet diet and tap water ad libitum. The diet contained 4% of total lipid with a fatty acid composition of 19.14% palmitic acid C16:0, 0.184% palmitoleic acid C16:1, 4.10% stearic acid C18:0, 19.34% oleic acid C18:1 n9, 51.53% linoleic acid C18:2 n6 and 4.83% linolenic acid C18:3 n3. The rats were sacrificed by cervical dislocation and testis rapidly removed, cut into small pieces and washed extensively with 0.15 M NaCl. An homogenate of each tissue was prepared in sol. A (0.25 M sucrose, 10 mM Tris–HCl pH 7.4, PMSF 0.1 mM), 3 ml of solution per gram of tissue, using the potterElvejhem homogeneizer. The homogenate was spun at 3000 × g, pellets were discarded, the supernatant was spun at 20,000 × g for 10 min to obtain testis mitochondria [9]. After the centrifugation, 5 ml of the resultant supernatant was applied to a Sepharose 4B column (1.6 cm × 12 cm) equilibrated and eluted with 10 mM Tris–HCl pH 7.4, 0.01% NaN3 [10]. The microsomal fraction appearing in the void volume (12–20 ml) was used and cytosol (30–40 ml) discarded. All operations were performed at 4 ◦ C. Microsomes and mitochondria were stored at −83 ◦ C and used within a week of its preparation, after one cycle of freezing and thawing. Tissues and membrane preparations were protected from light during the procedures. 2.3. Lipid peroxidation of mitochondria and microsomes Chemiluminescence and lipid peroxidation were initiated by adding ascorbate to microsomal or mitochondrial preparations [11]. Microsomes and/or mitochondria (1 mg of protein) were preincubated previously during 15 min at room temperature with different amounts of melatonin, and then were incubated at 37 ◦ C with 0.01 M phosphate buffer (pH 7.4), 0.4 mM ascorbate, final volume 1 ml. Microsomal and mitochondrial preparations which lacked ascorbate were carried out simultaneously. Membrane light emission was determined over a 180 min period; chemiluminescence was recorded as counts per minute (cpm) every 10 min and the sum of the total chemiluminescence was used to calculate cpm/mg protein. Chemiluminescence was measured as counts per min in a liquid scintillation analyzer Packard 1900 TR.
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2.4. Measurement of fatty acid composition Microsomal or mitochondrial lipids were extracted with chloroform/methanol (2:1 (v/v)) [12]. Fatty acids were transmethylated with 20% F3 B in methanol at 60 ◦ C for 3 h. Fatty acids methyl esters were analyzed with a GC-14A gas chromatograph (Shimadzu, Kyoto, Japan) equipped with a packed column (1.80 mm × 4 mm i.d.) GP 10% DEGS-PS on 80/100 Supelcoport. Nitrogen was used as a carrier gas. The injector and detector temperatures were maintained at 250 ◦ C, the column temperature was held to 200 ◦ C during 60 min. The fatty acid methyl esters were identified by comparing retention times with standard compounds. All compositions were expressed as area percentage of total fatty acids. 2.5. Peroxidizability index (PI) Peroxidizability index (PI) was calculated according to the formula PI = (percent of monoenoic acids × 0.025) + (percent of dienoic acids × 1) + (percent of tetraenoic acids × 4) + (percent of pentaenoic acids × 6). 2.6. Protein determination Proteins were determined by the method of Lowry et al. [13] using BSA as standard. 2.7. Statistical analysis Results were expressed as means ± S.D. of three independent determinations. Data were evaluated statistically by one-way analysis of variance (ANOVA) and Tukey test. Statistical criterion for significance was selected at different P-values and indicated in each case.
3. Results 3.1. Light emission of rat testis microsomes and mitochondria during lipid peroxidation: melatonin effect The incubation of rat testis microsomes or mitochondria in the presence of ascorbate-Fe2+ resulted
Fig. 1. (A) Light emission produced by rat testis mitochondria and microsomes incubated with (䊏) or without (䊐) ascorbic acid. (B) Ascorbate-Fe2+ induced chemiluminescence as a function of time of rat testis microsomes incubated with (䊊—䊊) or without (䊉—䊉) ascorbic acid. (C) Ascorbate-Fe2+ induced chemiluminescence as a function of time of rat testis mitochondria incubated with (䊊—䊊) or without (䊉—䊉) ascorbic acid. Values are mean ± S.D. of three independent experiments.
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in the peroxidation of membranes as evidenced by the emission of light (chemiluminescence). The light emission was similar in both kinds of organelles when the control and peroxidized groups were compared (Fig. 1A). Chemiluminescence as a function of time was measured during 180 min at 37 ◦ C, in rat testis microsomes or mitochondria with and without 0.4 mM ascorbate. Over the time course studies analysis of chemiluminescence demonstrated that the lipid peroxidation in the presence of ascorbic acid reach a maximum at 120 min (Fig. 1B and C). The time course of the chemiluminescence resulting from the addition of different concentrations of melatonin to rat testis
Fig. 3. (A) Inhibition of lipid peroxidation (light emission) of rat testis microsomes by melatonin. (B) Inhibition of lipid peroxidation (light emission) of rat testis mitochondria by melatonin. Values are mean ± S.D. of three independent experiments.
microsomes and mitochondria are shown in Fig. 2A and B, respectively. Inhibition of lipid peroxidation in microsomes by melatonin, was almost linear up to 5 mM, whereas in mitochondria melatonin inhibition showed linearity up to 1 mM Fig. 3A and B, respectively. Fig. 2. (A) Lipid peroxidation of rat testis microsomes without ascorbic acid (䊉—䊉) and with the addition of 0 (䊊—䊊); 1.0 (䊏—䊏); 2.5 (䉫—䉫); 5.0 (䉱—䉱) and 10.0 mM (䊐—䊐) melatonin. (B) Lipid peroxidation of rat testis mitochondria without ascorbic acid (䊉—䊉) and with the addition of 0 (䊊—䊊); 0.125 (䊏—䊏); 0.25 (䉫—䉫); 0.5 (䉱—䉱) and 1.0 mM (䊐—䊐) melatonin.
3.2. Fatty acid composition of rat testis microsomes and mitochondria during lipid peroxidation: melatonin effect In testis microsomes, Table 1, the proportions of linoleic C18:2 n6, arachidonic C20:4 n6 and
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Table 1 Fatty acid composition of rat testis microsomes during lipid peroxidation in the presence of different amounts of melation Fatty acids
Control
Melatonin (mM)
∗
C16:0 C16:1 n7 C18:0 C18:1 n9 C18:2 n6 C20:4 n6 C22:5 n6 Saturated Monounsaturated Polyunsaturated Total unsaturated Saturated/ unsaturated PI
0 29.08 3.80 7.48 12.46 17.46 25.17 26.98 36.57 16.26 69.61 85.88 0.42
± ± ± ± ± ± ± ± ± ± ± ±
1.52 1.08 2.02 3.01 4.66 3.40 3.20 2.50 2.90 10.17 11.45 0.04
280.44 ± 36.10
38.19 1.04 12.74 7.92 16.93 13.37 15.56 50.93 8.96 45.87 54.83 0.92
1.0 ± ± ± ± ± ± ± ± ± ± ± ±
1.50 0.80 3.10 1.00 0.80 1.30 b 3.60 b 4.60 1.50 3.50 b 4.77 0.10
164.03 ± 20.13 a
2.5
38.30 0.95 19.27 12.04 15.55 12.78 14.60 57.57 12.99 42.93 55.93 1.02
± ± ± ± ± ± ± ± ± ± ± ±
4.20 0.20 2.50 1.40 2.10 2.30 b 4.40 b 5.30 1.60 3.20 a 2.90 0.10
154.27 ± 25.00 a
5.0
31.91 0.75 12.57 15.38 9.38 14.64 20.62 44.48 16.02 44.65 60.78 0.72
± ± ± ± ± ± ± ± ± ± ± ±
2.60 0.40 3.20 4.30 0.80 2.40 b 0.90 5.60 4.10 1.60 a 4.10 0.10
191.95 ± 7.90 b
10.0
35.02 0.99 13.99 12.53 8.61 22.45 22.82 49.03 13.52 50.88 64.41 0.75
± ± ± ± ± ± ± ± ± ± ± ±
2.90 0.80 4.80 2.10 0.80 6.50 0.50 7.30 1.60 0.90 b 1.90 0.10
40.44 2.3 9.12 11.65 17.00 24.98 24.86 49.56 13.91 66.85 80.77 0.60
210.50 ± 9.60 c
± ± ± ± ± ± ± ± ± ± ± ±
14.00 0.50 2.50 0.90 1.10 2.80 d 1.70 e 16.00 0.70 0.30 d 0.90 0.10
266.40 ± 1.04 d
(∗ )
Data shown are given in percentages of total fatty acid content and are mean ± S.D. of three separated experiments. Without ascorbic acid. PI was calculated according to the formula PI = (percent of monoenoic acids × 0.025) + (percent of dienoic acids × 1) + (percent of tetraenoic acids × 4) + (percent of pentaenoic acids × 6). Statistically significant differences between control vs. peroxidized, peroxidized + 1.0; 2.5; 5.0 and 10 mM melatonin are indicated by letter ‘a’ P < 0.0005; ‘b’ P < 0.005; ‘c’ P < 0.05. Statistically significant differences between peroxidized vs. peroxidized + 1.0; 2.5; 5.0 and 10 mM melatonin are indicated by ‘d’ P < 0.005; ‘e’ P < 0.05 using analysis of variance (ANOVA).
Table 2 Fatty acid composition of rat testis mitochondria during lipid peroxidation in the presence of different amounts of melatonin Fatty acids
Control
Melatonin (mM)
∗
C16:0 C16:1 n7 C18:0 C18:1 n9 C18:2 n6 C20:4 n6 C22:5 n6 Saturated Monounsaturated Polyunsaturated Total unsaturated Saturated/ unsaturated PI
0 31.36 0.13 8.59 13.00 11.65 15.02 13.71 39.35 13.76 40.39 54.16 0.73
± ± ± ± ± ± ± ± ± ± ± ±
3.30 0.10 1.40 1.62 3.98 2.10 1.86 1.98 2.70 2.10 0.60 0.10
154.37 ± 16.00
42.24 0.21 16.50 19.39 8.20 5.18 2.85 58.74 19.60 16.24 35.84 1.67
0.125 ± ± ± ± ± ± ± ± ± ± ± ±
1.80 0.10 4.37 1.80 2.87 1.90 b 0.80 a 5.90 1.89 5.60 a 5.40 0.40
46.54 ± 15.40 a
41.05 0.54 12.84 20.96 8.89 5.78 4.32 53.89 21.50 18.99 40.50 1.33
0.25 ± ± ± ± ± ± ± ± ± ± ± ±
1.10 0.10 2.65 3.20 0.10 2.00 b 1.20 a 3.60 3.30 3.03 a 2.50 0.10
58.48 ± 14.40 a
46.05 0.33 12.55 19.33 8.15 6.25 5.53 58.61 19.66 19.94 39.60 1.48
0.5 ± ± ± ± ± ± ± ± ± ± ± ±
3.90 0.10 3.13 1.00 0.10 2.91 b 0.50 a 7.10 1.10 3.10 2.20 0.20
66.86 ± 13.00 a
38.90 0.18 12.60 18.32 9.07 7.51 5.73 51.51 18.50 18.65 37.32 1.38
1.0 ± ± ± ± ± ± ± ± ± ± ± ±
1.40 0.10 2.80 1.10 0.10 1.90 c 0.90 a 3.60 1.03 2.80 a 3.10 0.10
70.32 ± 12.30 a
30.49 0.15 10.97 13.59 11.03 15.70 14.55 41.46 13.74 41.29 55.04 0.75
± ± ± ± ± ± ± ± ± ± ± ±
6.10 0.02 2.70 2.10 4.80 2.10 e 1.70 d 6.10 2.10 5.63 4.30 0.10
161.50 ± 18.40 d
Data shown are given in percentages of total fatty acid content and are mean ± S.D. of three separated experiments. (∗ ) Without ascorbic acid. Peroxidizability index (PI) was calculated according to the formula PI = (percent of monoenoic acids × 0.025) + (percent of dienoic acids × 1) + (percent of tetraenoic acids × 4) + (percent of pentaenoic acids × 6). Statistically significant differences between control vs. peroxidized, peroxidized + 0.125; 0.25; 0.5 and 1.0 mM melatonin are indicated by letter ‘a’ P < 0.0005; ‘b’ P < 0.005; ‘c’ P < 0.05. Statistically significant differences between peroxidized vs. peroxidized + 0.125; 0.25; 0.5 and 1.0 mM melatonin are indicated by ‘d’ P < 0.0005; ‘e’ P < 0.005; ‘f’ P < 0.05 using analysis of variance (ANOVA).
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docosapentaenoic acids 22:5 n-6 were characteristically higher than in testis mitochondria, Table 2. The total proportions of n-6 PUFA were also higher in testis microsomes than in mitochondria. Both polyunsaturated fatty acids were preserved when melatonin was present in the lipid peroxidation system. Thus, in rat testis microsomes and mitochondria the melatonin concentration required to inhibit by 100% the lipid peroxidation process was 5.0, and 1.0 mM, respectively, Tables 1 and 2. In testis microsomes significant differences in the content of arachidonic acid C 20:4 n6 were observed when control versus peroxidized and control versus peroxidized + 1.0; and 2.5 mM melatonin groups were compared. Significant differences in the content of docosapentaenoic acid C 22:5 n6 were observed when control versus peroxidized and control versus peroxidized + 1.0 mM melatonin groups were compared. In testis mitochondria significant differences in the content of arachidonic acid C 20:4 n6 and docosapentaenoic acid C 22:5 n6 were observed when control versus peroxidized and control versus peroxidized + 0.125; +0.25; and +0.5 mM melatonin groups were compared. 3.3. Peroxidizability index and IC50 values of organelles lipid peroxidized and protected by melatonin In testis microsomes the peroxidizability index (PI) which indicates the degree of vulnerability to degradation of a selected membrane showed significant differences between control versus peroxidized and control versus peroxidized + 1.0; +2.5; and +5.0 mM melatonin. In testis mitochondria the PI showed significant differences between control versus peroxidized and control versus peroxidized+0.125; +0.25; and 0.5 mM melatonin. IC 50 values were calculated from the inhibition curve of melatonin on the chemiluminiscence rates Fig. 2A and B, respectively. The data showed that IC 50 was higher in microsomes (4.98 mM) than in mitochondria (0.67 mM). Our results clearly demonstrate that the preservation of the most abundant polyunsaturated fatty acids C20:4 n6 and C22:5 n6 by melatonin during ascorbate-Fe2+ lipid peroxidation is more effective in rat testis microsomes than in mitochondria. Furthermore, the PI was positively correlated with the level of these long chain fatty acids, Fig. 4A and B.
Fig. 4. (A) Fatty acid percent and PI as a function of melatonin concentration in rat testis microsomes. (B) Fatty acid percent and PI as a function of melatonin concentration in rat testis mitochondria. PI (䊏—䊏 ); C 20:4 n6 (䊐—䊐) and C 22:5 n6 (䉱—䉱). Values are mean ± S.D. of three independent experiments.
4. Discussion Approximately 70% of the fatty acids located in rat testis microsomes and mitochondria are polyunsaturated with a prevalence of arachidonic C20:4 n6 and docosapentaenoic C22:5 n6 acids [1]. This high concentration of polyunsaturated fatty acids in these membranes causes susceptibility to lipid peroxidative degradation. This study has examined some of the effects by which melatonin protect lipid peroxidation of rat testis microsomes and mitochondria in vitro. Experimental studies have shown that melatonin directly scavenges the hydroxyl radical, peroxyl radical,
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peroxynitrite anion, and singlet oxygen. Furthermore, this tryptophan derivative stimulates a number of antioxidative enzymes and stabilizes cell membranes; this latter action helps membranes to resist free radical damage [14]. While the antioxidative actions of most molecules are limited by their specific intracellular distribution, e.g. Vitamin E in lipid-rich membranes [15], melatonin’s antioxidative actions include the protection of lipids in the cell membrane, proteins in the cytosol, and DNA in the nucleus. Our results have shown that the addition of ascorbate to rat testis microsomes or mitochondria increased lipid peroxidation several-fold over organelles not treated with ascorbic acid. Ascorbate-stimulated lipid peroxidation was substantially suppressed in rat testis microsomes or mitochondria previously incubated with melatonin. These results are consistent with our previous results in which melatonin has been shown to prevent polyunsaturated fatty acid degradation by inhibiting lipid peroxidation [3]. The vulnerability to lipid peroxidation of rat testis microsomes and mitochondria assessed by the sum of the most polyunsaturated fatty acids (C20:4 n6 and C22:5 n6, Tables 1 and 2) is coincident with the results obtained from the chemiluminescence intensity (Fig. 1B and C). In this regard, it is important to note that arachidonic acid was protected more efficiently than docosahexaenoic acid at all melatonin concentrations tested when rat testis microsomes or mitochondria were incubated with ascorbic acid (Tables 1 and 2). The damaging effects of lipid peroxidation on membrane structure and function are well documented [16,17]. Many studies have shown that free radical damage and lipid peroxidation increase as a function of the degree of unsaturation of the fatty acids present in the phospholipids of biological membranes. In this regard it has been demonstrated that the number of bis-allylic positions contained in the cellular lipids of intact cells determines their susceptibility, i.e. oxidizability, to free radical-mediated peroxidative events [18]. Tang et al. [19] have demonstrated the ability of melatonin to protect against iron-induced lipid peroxidation in various rat cell membranes from the brain, heart, kidney and liver of the male Sprague–Dawley rat. The concentration of cellular membrane malondialdehyde (MDA) was used as an index of induced oxidative membrane damage. This peroxidative effect was partially suppressed by melatonin (ED50
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0.886 mM) for the liver. Thus, the direct effect of lipid peroxidation on cellular membranes following iron exposure was markedly reduced by melatonin. In conclusion, the present study demonstrates that melatonin suppresses the ascorbate-Fe2+ induced lipid peroxidation process in rat testis microsomes and mitochondria and protects the most abundant polyunsaturated fatty acids, arachidonic C20:4 n6 and docosapentaenoic C22:5 n6 acids, from damage. The protective effect observed by melatonin in rat testis mitochondria was higher than that observed in microsomes which could be explained if we consider that the sum of C20:4 n6+C22 : 5 n6 in testis microsomes is two-fold than that present in mitochondria. Besides its action in direct free radical scavenging (OH• and the peroxyl radical-LOO• ) and in membrane stabilization, melatonin has actions on enzymes, such as superoxide dismutase and gluthathione peroxidase that either generate or metabolise reactive oxygen intermediates. Studies in vivo provides evidence that melatonin activates those enzymes but in studies in vitro similar to our study, the activities of these enzymes were not actually measured [20]. The physiological potential of melatonin in preventing lipid degradation of polyunsaturated fatty acids in vivo in different organelles of rat testis and studies aimed to determine the presence of different enzyme activities that either generate or metabolise reactive oxygen species in those organelles which could interact with melatonin deserves further investigation.
Acknowledgements We are grateful to Cesar Arcemis for performing GLC analysis. This work was carried out with a grant from Secretaria de Ciencia y Técnica Universidad Nacional de La Plata.
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