Lipoic acid ameliorates adriamycin-induced testicular mitochondriopathy

Reproductive Toxicology 20 (2005) 111–116

Lipoic acid ameliorates adriamycin-induced testicular mitochondriopathy Chidambaram Prahalathan, Elangovan Selvakumar, Palaninathan Varalakshmi∗ Department of Medical Biochemistry, Dr. A.L.M. Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai 600 113, India Received 15 October 2004; received in revised form 6 December 2004; accepted 24 December 2004 Available online 21 January 2005

Abstract Adriamycin (ADR), an anthracycline antibiotic, which is widely used as an antineoplastic drug in the treatment of various solid tumors, has been shown to induce reproductive abnormalities in males. In the present study, the effect of lipoic acid (LA), a universal antioxidant was investigated on ADR-induced testicular toxicity in rats. Adult male albino rats of Wistar strain were administered ADR (1 mg/kg body weight, i.v.), once a week for 10 weeks. Mitochondrial fractions of the testis were obtained by differential centrifugation. The activities of mitochondrial antioxidant enzymes such as superoxide dismutase, glutathione peroxidase and glutathione reductase were decreased significantly in the animals treated with ADR. The levels of mitochondrial lipid peroxides and hydrogen peroxide were increased in ADR-treated rats. ADRtreated rats also showed decline in the activities of mitochondrial enzymes such as succinate dehydrogenase (SDH), malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICDH). Treatment with lipoic acid (35 mg/kg body weight, i.p.) 1 day prior to ADR administration, maintained near normal activities of the enzymes, thereby proving to be an effective cytoprotectant. © 2005 Elsevier Inc. All rights reserved. Keywords: Adriamycin; Lipoic acid; Mitochondria; Lipid peroxidation; Antioxidants; TCA cycle enzymes; Testicular toxicity

1. Introduction Adriamycin (ADR), a widely used anthracycline antibiotic in the treatment of various solid tumors, is recognized to alter sperm development, production, structural integrity and motility rates in association with increased cellular apoptosis [1–4]. While the high affinity of anthracyclines towards chromosomal DNA has been held responsible for their antitumor activity, these drugs also target mitochondria thus interfering with major mitochondrial functions [5]. ADR treatment is always associated with severe oxidative damage and is mediated by a complex oxyradical cascade involving superoxide, hydroxyl radical and small amounts of iron [6]. The oxyradicals cause damage to mitochondrial and other cytoplasmic organelle membrane structures through peroxidation of phospholipids, proteins and nucleotides. The ∗

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semiquinone radical produced by ADR participates in the inactivation of mitochondrial enzymes [7]. It was found that ADR treatment causes increased production of lipid peroxides with a decrease in thiol content and subsequent reduction in the enzyme activities in rat heart mitochondria [8]. Biological compounds with antioxidant properties may contribute to the protection of cells and tissues against deleterious effects of reactive oxygen species (ROS) and other free radicals induced by ADR [9,10]. Lipoic acid (LA), a naturally occurring nutraceutical, functions as an essential cofactor for mitochondrial pyruvate dehydrogenase and ␣-ketoglutarate dehydrogenase, leading to the production of cellular energy. LA, a universal antioxidant, functions both in aqueous and membrane phases [11]. LA administration has been shown to be beneficial in various pathologies in which ROS have been implicated [12,13]. The present study is an attempt to evaluate the role of LA in ADR-induced testicular mitochondrial dysfunction in rats.

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2. Materials and methods 2.1. Drugs and chemicals ADR (doxorubicin hydrochloride-Adrim; Dabur pharmaceuticals, New Delhi, India) has been used in the present experimental study. dl-␣-Lipoic acid, bovine serum albumin and 1,1,3,3-tetraethoxypropane were purchased from Sigma Chemicals Co., St. Louis, USA. All other chemicals and solvents were of analytical grade. 2.2. Animal model Adult male albino rats of Wistar strain weighing 120 ± 10 g (10–12 weeks old) were used throughout the study. The animals were maintained under standard conditions of humidity, temperature (25 ± 2 ◦ C) and light (12h light/12-h dark). They were fed with a standard rat pelleted diet (M/s Pranav Agro Industries Ltd., India) under the trade name Amrut rat/mice feed and had free access to water. The animal experiments were conducted according to the guidelines of Institutional Animal Ethics Committee (IAEC). 2.3. Experimental protocol The animals were randomly divided into four groups of six rats each as follows. Group I (control) received normal saline throughout the course of the study. Group II (ADR) received intravenous injections of ADR (1 mg/kg body weight) through the tail vein once a week for a period of 10 weeks. Group III (LA) received LA (35 mg/kg body weight) dissolved in saline at alkaline pH (7.8), intraperitoneally once a week for a period of 10 weeks. Group IV (ADR + LA) received a single injection of LA (35 mg/kg body weight) intraperitoneally 24 h prior to the administration of intravenous injections of ADR (1 mg/kg body weight) through the tail vein once a week for a period of 10 weeks. At the end of the 10th week, the animals were killed by decapitation under anesthesia and both testes were excised immediately and washed in ice-cold saline. A 20% homogenate of the tissue was prepared in ice-cold 0.25 M sucrose solution and it was centrifuged at 1000 × g for 10 min at 4 ◦ C to obtain the nuclear pellet. Mitochondrial fraction was obtained by centrifuging the post-nuclear supernatant at 10,000 × g for 10 min at 4 ◦ C [14] and it was used for the following biochemical analyses.

the intensity of oxidative stress. MDA reacts with thiobarbituric acid to generate a coloured product, which absorbs at 532 nm. The ferrous sulphate and ascorbate-induced lipid peroxidation system contained 10 mM ferrous sulphate and 0.2 mM ascorbate as inducers [17]. The enzyme superoxide dismutase (SOD) was assayed according to the method of Marklund and Marklund [18]. The unit of enzyme activity is defined as the amount of enzyme required to give 50% inhibition of pyrogallol auto-oxidation. Glutathione peroxidase (GPx) was determined by the method of Rotruck et al. [19] with some modifications, which is based on the reaction between glutathione remaining after the action of GPx and 5,5 -dithio-bis(2-nitrobenzoic acid) to form a complex that absorbs maximally at 412 nm. Glutathione reductase (GR) was assayed by the method of Staal et al. [20], which utilizes NADPH to convert oxidized glutathione (GSSG) to reduced form (GSH). Hydrogen peroxide (H2 O2 ) generation was estimated by the method of Pick and Keisari [21]. Total reduced glutathione (GSH) in the mitochondrial fraction was estimated by the method of Moron et al. [22], where the color developed was read at 412 nm. Vitamin C (Vit C) was estimated by the method of Omaye et al. [23]. Vitamin E (Vit E) was estimated by the method of Desai [24]. Isocitrate dehydrogenase (ICDH) was determined by the method of Bernt and Bergmeyer [25]. The amount of isocitrate oxidized was determined by the increase in extinction due to the formation of NADPH. Succinate dehydrogenase (SDH) was assayed by the modified method of Slater and Bonner [26]. The principle involves the measurement of the rate of reduction of potassium ferricyanide in the presence of sufficient potassium cyanide to inhibit cytochrome oxidase by following the rate of decrease in optical density at 400 nm. The method adopted for the estimation of malate dehydrogenase (MDH) was that of Mehler et al. [27]. Oxaloacetate was used as the substrate and the enzyme activity was determined by measuring the rate of oxidation of NADH in the presence of the enzyme and excess of substrate. 2.5. Data analysis The values are expressed as mean ± standard deviation for six animals. Differences between groups were assessed by one-way analysis of variance (ANOVA), using the SPSS software package for Windows. Post hoc testing was performed for inter-group comparisons by the least significance difference (LSD) test. A P-value < 0.05 was considered significant.

2.4. Biochemical analyses Protein content in the mitochondrial fraction was determined by the method of Lowry et al. [15]. Lipid peroxidation (LPO) was determined by the procedure of Hogberg et al. [16]. Malondialdehyde (MDA), formed as an end product of the peroxidation of lipids, served as an index of

3. Results There were no deaths recorded in any of the experimental groups during the study period. Table 1 represents the abnormal elevation (P < 0.05) in LPO in the testicular mitochondria

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Table 1 Effect of ADR and LA on testicular mitochondrial lipid peroxidation Lipid peroxidation

Group I (control)

Group II (ADR)

Group III (LA)

Group IV (ADR + LA)

Basal Ascorbate-induced Ferrous sulphate-induced

3.04 ± 0.32 5.22 ± 0.46 9.00 ± 0.95

4.33 ± 0.60 8.37 ± 0.98 a* 14.91 ± 1.92 a*

2.94 ± 0.28 5.27 ± 0.45 8.55 ± 0.92

3.52 ± 0.35 b* 6.01 ± 0.44 b* 9.75 ± 1.09 a* b*

a*

Unit: lipid peroxidation (nmol of malondialdehyde formed/mg protein). Values represent mean ± S.D. for six rats. Comparisons are made as follows: (a) with group I; (b) with group II. Values are statistically significant at * P < 0.05. Table 2 Effect of ADR and LA on the activities of mitochondrial antioxidant enzymes and levels of H2 O2 of rat testis Parameters (U/mg protein) SOD GPx GR H2 O2

Group I (control) 12.88 15.79 17.37 18.82

± ± ± ±

1.47 1.69 1.53 1.58

Group II (ADR) 8.19 10.20 12.42 37.37

± ± ± ±

a*

0.87 1.09 a* 1.48 a* 3.36 a*

Group III (LA) 12.88 15.33 17.32 18.70

± ± ± ±

1.29 1.19 1.69 2.18

Group IV (ADR + LA) 11.63 14.65 16.87 21.69

± ± ± ±

1.31 b* 1.50 b* 1.58 b* 1.96 a* b*

Units: SOD, superoxide dismutase (units/min), where one unit is equal to the amount of enzyme required to inhibit auto oxidation of pyrogallol by 50%; GPx, glutathione peroxidase (␮g of reduced glutathione utilized/min); GR, glutathione reductase (nmol of NADPH oxidized/min); H2 O2 , hydrogen peroxide (nmol of H2 O2 formed/min). Values represent mean ± S.D. for six rats. Comparisons are made as follows: (a) with group I; (b) with group II. Values are statistically significant at * P < 0.05. Table 3 Effect of ADR and LA on the levels of mitochondrial non-enzymic antioxidants of rat testis Parameters (␮g/mg protein)

Group I (control)

Group II (ADR)

Group III (LA)

Group IV (ADR + LA)

GSH Vitamin C Vitamin E

7.40 ± 0.69 1.23 ± 0.15 1.49 ± 0.14

4.27 ± 0.42 0.85 ± 0.07 a* 0.92 ± 0.09 a*

7.48 ± 0.80 1.21 ± 0.11 1.50 ± 0.16

7.05 ± 0.88 b* 1.07 ± 0.17 b* 1.33 ± 0.19 b*

a*

Values represent mean ± S.D. for six rats. Comparisons are made as follows: (a) with group I; (b) with group II. Values are statistically significant at * P < 0.05.

of ADR-treated rats. Group II animals showed a 42.43% rise in basal LPO in mitochondrial fractions, as well as a 60.34% and 65.66% increase in LPO following addition of exogenous inducers such as ascorbate and ferrous sulphate, respectively, versus group I. LA pretreatment reverted the values to near normalcy (P < 0.05). Table 2 shows the decrease (P < 0.05) in the activities of mitochondrial SOD (37%), GPx (36%) and GR (29%) with a concomitant increase (P < 0.05) in the levels of H2 O2 (98%) in the testis of ADR administered rats (group II), while their activities were nearly normal on LA pretreatment (group IV).

Changes in the levels of mitochondrial non-enzymic antioxidants GSH, Vit C and Vit E are given in Table 3. ADRtreated animals (group II) demonstrated a decrease in the levels of GSH and antioxidant vitamins in the testicular mitochondria (P < 0.05). Replenishing activity was found to be pronounced in group IV animals, which might be attributed to the antioxidative nature of LA. Fig. 1 highlights the effect of ADR and LA administrations on the activities of tricarboxyclic acid (TCA) cycle enzymes of testis. ADR-treated animals (group II) demonstrated a 1.95-, 1.61- and 1.55-fold decrease (P < 0.05) in the

Fig. 1. Effect of ADR and LA on the activities of TCA cycle enzymes of rat testis. Units: ICDH, isocitrate dehydrogenase (nmol of NADPH formed/min); MDH, malate dehydrogenase (nmol of NADH oxidized/min); SDH, succinate dehydrogenase (nmol of succinate formed/min). Values represent mean ± S.D. for 6 rats. Comparisons are made as follows: a–with group I; b–with group II. Values are statistically significant at * P < 0.05.

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activities of ICDH, MDH and SDH, respectively. LA pretreatment (group IV) resulted in near normal activities of TCA cycle enzymes in ADR administered rats.

4. Discussion Testicular dysfunction is the most common side effect of cytotoxic chemotherapy. ADR is one of the widely used cytotoxic agents for the treatment of various cancers. ADR-treated animals have shown shrunken seminiferous tubules with decreased germ cells [3]. Mitochondrial dysfunction is an early indicator of ADR-induced apoptosis and cytotoxicity [28]. ADR treatment causes alterations in mitochondrial DNA and defects in respiratory chain functions [29]. Mitochondrial respiration is the main biological source of superoxide anion radicals under normal physiological conditions. The elevated levels of lipid peroxides resulting from increased formation of free radicals are recognized as one of the possible biochemical mechanisms for ADR associated side effects [6]. In the present study, administration of ADR decreased the activities of SOD, GPx and GR with a concomitant increase in the levels of H2 O2 and LPO in the mitochondrial fractions of rat testis. These changes may adversely affect the testicular mitochondria in ADR-treated rats. It was found that ADR interacts with mitochondrial NADH–dehydrogenase to facilitate the production of the ADR semiquinone free radical intermediate, which in the presence of oxygen redox cycle generates superoxide anion, H2 O2 and hydroxyl radical. This ROS greatly enhances NADH-dependent mitochondrial membrane peroxidation of phospholipids in spite of the presence of endogenous biological defenses against oxyradicals [30]. The increased LPO due to ADR administration is a consequence of impaired antioxidant enzyme activities and depletion in GSH levels. Our results also suggest that ADR-treated rats may be more susceptible to LPO in the presence of promoters like ascorbate and ferrous sulphate. The enhanced LPO in mitochondria may decrease mitochondrial membrane fluidity, increase the negative surface charge distribution and alter membrane ionic permeability including proton permeability, which uncouples oxidative phosphorylation [31]. It is therefore obvious that the enhanced lipid peroxidation due to ADR administration can be extremely damaging to both mitochondrial structure and function. SOD acts as the first line of defense against deleterious effects of oxyradicals in cells by catalyzing the dismutation of superoxide radicals to H2 O2 and molecular oxygen. The decreased activities of SOD in ADR-treated rats may lead to the continuous production of superoxide anions. In the presence of inadequate GPx activity in group II animals to degrade H2 O2 , more H2 O2 would be converted to toxic hydroxyl radicals that may contribute to severe oxidative damage to mitochondrial membranes. SOD plays an important role in rat testicular maturation and spermatogenesis [32]. Any alteration in the activity of testicular SOD may lead to

growth arrest and impaired function of the testis. The ability of LA or its reduced form to scavenge hydroxyl radicals and chelate transition metal ions restricts the production of toxic lipid peroxides and so reduces cellular need for enzymic antioxidants in group IV animals. Adriamycin-treated animals show decreased activities of glutathione metabolizing enzymes namely GPx and GR. GSH plays a critical role in several important biological processes including the maintenance of essential sulphydryl groups on membrane proteins, drug detoxification reactions involving glutathione-S-transferase and the breakdown of intracellular peroxides or free radicals. Increased oxidative stress increases the formation and efflux of GSSG [33]. GR mediates the reduction of GSSG to GSH. This reduction reaction requires NADPH, which is supplied by the enzyme ICDH, an intramitochondrial NADPH generator [34]. ICDH is lowered in ADR injected group, which reduces the availability of NADPH, which in turn causes reduction in the activity of GR. All these events eventually lead to imbalance in GSSG/GSH redox couple. The reduced activity of GPx observed in ADR-treated group in the present study may partly be due to lack of the substrate (GSH) and also because of alteration of the protein structure. Vit E is the main lipid soluble antioxidant vitamin, which plays an important role in maintaining the integrity of the intracellular organelles by preventing membrane peroxidation [35]. Vit C is a first line antioxidative defense in watersoluble compartment. The marked decrease of the vitamins in mitochondria of ADR-treated group may be due to over production of ROS in both membrane and aqueous phases. A similar decrease in the activities of enzymic and non-enzymic antioxidants with rise in LPO was observed in testis [36] and renal tissues [37] of ADR administered rats. To the best of our knowledge, the present report is the first one to highlight the testicular mitochondrial antioxidant changes in ADR administered rats. The present findings show decreased activities of TCA cycle enzymes in ADR-treated rats. A constant supply of energy is considered to be an essential requirement for the proper functioning of testis and reduction in the activities of TCA cycle enzymes proves the defect in aerobic oxidation of pyruvate, which might lead to low production of ATP molecules. The enhanced lipid peroxidation in mitochondria of ADR-treated rats may result in the disintegration of the mitochondrial membrane ultrastructure, which in turn affects the membrane bound enzyme functions. It is possible that the reactive aldehydes derived from peroxidized phospholipids might inactivate the mitochondrial enzymes by alkylation. ADR has been shown to inhibit –SH groups containing enzymes such as dehydrogenases [38]. This might be the possible reason for the decrease in the activities of TCA cycle enzymes in ADR-treated animals. The results obtained by us indicate that pretreatment with LA in ADR administered rats may influence TCA cycle enzymes to an appreciable extent. LA is highly effective in reducing free radicals including lipid peroxides in cellular membranes. LA pretreatment protects

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the mitochondrial membrane from peroxidation and subsequent inactivation of enzymes induced by ADR. This might be the possible reason for near normal activities of TCA cycle enzymes in group IV animals. This observation is supported by a similar work in our laboratory on ADR toxicity where the lowered activities of TCA cycle enzymes in kidney was replenished by LA [38]. LA pretreatment showed a significant increase in mitochondrial GSH levels as well as enhanced activities of all enzymic and non-enzymic antioxidants with normal values of LPO in ADR-treated groups. The protection rendered by LA is due to its free radical scavenging ability and antioxidant activity [39]. Thiols are thought to play a vital role in protecting cells against LPO [40]. LA, a dithiol compound, shows beneficial effects in oxidative stress conditions because of its synergistic action with other antioxidants [41]. It has been proved that ␣-lipoic acid minimizes LPO and protects the cells against various toxic conditions [42,43]. To conclude, the present study highlights the biochemical aberrations in the testicular mitochondria caused by ADR administration and the role of LA in combating the abnormalities. By the reversal of biochemical and oxidative markers towards normalcy, the cytoprotective role of LA is illuminated in testicular toxicity. Further studies are in progress to elucidate the mechanisms involved in ADR-induced testicular injury and the protection afforded by LA.

Acknowledgement The first and second authors gratefully acknowledge the financial assistance in the form of Junior Research Fellowship by the Indian Council of Medical Research (ICMR), New Delhi, India.

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