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Modulatory role of lipoic acid on adriamycin-induced testicular injury
Chemico-Biological Interactions 160 (2006) 108–114
Modulatory role of lipoic acid on adriamycin-induced testicular injury Chidambaram Prahalathan, Elangovan Selvakumar, Palaninathan Varalakshmi ∗ Department of Medical Biochemistry, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai 600113, India Received 2 September 2005; received in revised form 13 December 2005; accepted 20 December 2005 Available online 24 January 2006
Abstract The present study investigated the protective efficacy of dl-␣-lipoic acid (LA) on adriamycin (ADR)-induced oxidative damage in rat testis. Adult male albino rats of Wistar strain were administered ADR (1 mg/kg body weight, i.v.), once a week for 10 weeks. ADR injected rats showed increased oxidative stress with a concomitant decrease in cellular thiols. The mRNA level for phospholipid hydroperoxide glutathione peroxidase (PHGPx) was also significantly decreased by ADR administration. Transmission electron microscopic (TEM) observations of testicular germ cells revealed abnormal ultrastructural changes in ADR treated rats. Treatment with lipoic acid (35 mg/kg body weight, i.p.) 1 day prior to ADR administration, effectively reverted these abnormal changes towards normalcy. These findings indicate a cytoprotective role of LA in this experimental model of testicular toxicity. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Adriamycin; Lipoic acid; Oxidative stress; Cellular thiols; PHGPx; TEM; Testicular toxicity
1. Introduction Adriamycin (ADR) is an anthracycline antibiotic that exhibits excellent antitumor activity against a variety of solid tumors. The clinical use of ADR is associated with testicular dysfunction characterized by altered sperm development, production, structural integrity and motility rates in association with increased cellular apoptosis [1–4]. Although antitumor action of ADR may be mediated by a wide number of mechanisms, oxidative stress and the generation of toxic reactive oxygen species (ROS) are the main cause of drug toxicity [5]. The oxyradicals cause damage to mitochondrial and other
∗
Corresponding author. Tel.: +91 44 24480767; fax: +91 44 24926709. E-mail address: [email protected] (P. Varalakshmi).
cytoplasmic membrane structures through peroxidation of phospholipids, proteins and nucleotides that can be detrimental to male fertility [6]. Thus, the combination of the drug delivery together with a potent antioxidant may be an appropriate approach to reduce the toxic side effects of ADR. Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenium containing antioxidant that metabolizes phospholipids in membranes and protects them from oxidative damage. The mRNA for PHGPx is highly expressed in testis especially in specific spermatogenic cells during spermatogenesis and the down-regulation in PHGPx expression due to increased oxidative stress could lead to infertility [7]. Hence, it would be worthwhile to know whether PHGPx expression in the testis is influenced by ADR treatment. Lipoic acid (LA), a naturally occurring nutraceutical, functions as an essential cofactor in metabolic
0009-2797/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2005.12.007
C. Prahalathan et al. / Chemico-Biological Interactions 160 (2006) 108–114
reactions involved in energy utilization. LA and its reduced form dihydrolipoic acid are effective against conditions in which oxidative stress has a role [8]. It shows beneficial effects in oxidative stress conditions because of its synergistic action with other antioxidants [9]. LA, which is a universal antioxidant functions both in aqueous and membrane phases [10]. The present study describes the protective effect of LA; a multifunctional antioxidant on ADR-induced testicular toxicity, in correlation with testicular thiol contents and PHGPx gene expression. 2. Materials and methods 2.1. Drugs and chemicals ADR (doxorubicin hydrochloride-Adrim) procured from Dabur Pharmaceuticals; New Delhi, India was used in the present experimental study. dl-␣-lipoic acid, bovine serum albumin and 2 ,7 -dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma Chemicals Co., St. Louis, USA. All other chemicals and solvents were of analytical grade.
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10th week, the animals were killed by decapitation under anesthesia and both testes were excised immediately and used for the following analyses. 2.4. Determination of cellular oxidative stress A 10% homogenate of the tissue was prepared in 0.01 M Tris–HCl buffer (pH 7.4) and protein content was determined by the method of Lowry et al. [11]. Oxidative stress was determined by using DCFH as a probe, according to the method of LeBel et al. [12]. In brief, the assay buffer contained 20 mM Tris–HCl, 130 mM KCl, 5 mM MgCl2 , 20 mM NaH2 PO4 , 30 mM glucose and 5 M DCFH-DA. The assay medium was incubated at 37 ◦ C for 15 min and 1 mol of H2 O2 was added to the mixture at the end of assay. DCF formation was measured at the excitation 488 nm and emission at 525 nm for 30 min by Shimadzu fluorescence spectrometer. 2.5. Determination of cellular thiols Total thiol and non-protein thiols were estimated by the method of Sedlak and Lindsay [13].
2.2. Animal model 2.6. RT-PCR analysis Adult male albino rats of Wistar strain weighing 140 ± 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 (12 h 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
To determine the expression of PHGPx mRNA in each group, total RNA was isolated from testes using a total RNA extraction kit (Eppendorf, Germany). The specific primers for PHGPx were used for amplification [14,15]. PCR amplification was carried out with a thermal cycler using one step RT-PCR kit (Eppendorf, Germany) according to a protocol for the initial denaturing step at 95 ◦ C for 10 min; then 30 cycles at 95 ◦ C for 1 min (denaturing), at 55 ◦ C for 1 min (annealing) and 72 ◦ C for 1.5 min (extension); and a further extension at 72 ◦ C for 10 min. The PCR products were run on a 2% agarose gel in Tris-borate-EDTA buffer. Rat -actin was used as an internal standard. 2.7. Electron microscopy studies Representative electron micrographs from testis were obtained as previously [16]. Briefly, thin slices of the testis were fixed in 2.5% glutaraldehyde and post-fixed in 1% osmium tetroxide. Then tissue was dehydrated in graded acetone solutions and finally embedded in epon-araldite. Ultra-thin sections were stained in uranyl acetate and lead citrate. Sections were transmitted in a Philips 201C transmission electron microscope (TEM) and photographed.
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2.8. 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 (L.S.D.) test. A Pvalue < 0.05 was considered significant. 3. Results There were no deaths recorded in any of the experimental groups during the study period. Fig. 1 represents abnormal elevation in testicular oxidative stress in ADR administered rats (Group II), which exhibited a 2.06-fold
increase as compared to those of control animals. LA pretreatment in ADR injected rats significantly (P < 0.05) reduced the level of oxidative stress to near normalcy. The levels of total thiol and non-protein thiols were significantly (P < 0.05) decreased (36.9% and 44.7%, respectively) in the testis of ADR treated rats as compared to those of control animals (Fig. 2). LA supplementation restored the thiol levels considerably in Group IV animals. Fig. 3 presents the mRNA level of PHGPx in various groups. A single transcript (461 bp) was observed in all the groups. RT-PCR analysis of PHGPx mRNA showed a significant (P < 0.05) decrease in ADR administered rats, while the expression pattern of PHGPx mRNA in
Fig. 1. Effect of ADR and LA on testicular oxidative stress indices. Values represent mean ± S.D. for six rats. Comparisons are made as follows: (a) with Group I and (b) with Group II. Values are statistically significant at * P < 0.05.
Fig. 2. Effect of ADR and LA on thiol levels in testis. Values represent mean ± S.D. for six rats. Comparisons are made as follows: (a) with Group I and (b) with Group II. Values are statistically significant at * P < 0.05.
Fig. 3. Effect of ADR and LA on expression pattern of PHGPx mRNA level in testis. (a) The 461 and 375 bp fragments represent PHGPx transcript and -actin as internal standard, respectively; lane 1:100 bp DNA ladder, lane 2: control, lane 3: ADR, lane 4: LA and lane 5: LA + ADR. (b) Relative levels of PHGPx mRNA expression compared to values for -actin mRNA. Values represent mean ± S.D. for six rats. Comparisons are made as follows: (a) with Group I and (b) with Group II. Values are statistically significant at * P < 0.05.
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Fig. 4. Transmission electron microscopic observations of germ cells in rat testis treated with ADR and/LA. Germ cells from control (a) and LA treated (d) show normal morphological features. However, the sections from ADR treated rat testis (b and c) reveals the following abnormalities. The cytoplasm contains abnormal vesicles (arrow head) and has some marginal chromatins (star) along the nuclear membrane. Mitochondrial distension with loss of cristae (#) and electron dense materials (arrow) are also visible. The testicular germ cells of rat treated with LA and ADR (e) shows nearly normal ultrastructural features.
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Group IV animals was not much altered when compared to control animals. Transmission electron microscopic studies of the testis revealed the following changes. The germ cells of control animals showed normal morphological features (Fig. 4a and d). ADR-induced dramatic ultrastructural changes in germ cell morphology (Fig. 4b and c). ADR treated rats showed numerous vesicles and electron dense materials in their germ cell cytoplasm. Some marginated chromatins along the nuclear membrane and mitochondrial distension were frequently recognized within the germ cell cytoplasm of ADR treated rats. Ultrastructurally, LA pretreatment in ADR administered group showed significant improvement in their germ cell morphology (Fig. 4e). 4. Discussion “Oxidative stress” is a condition associated with an increased rate of cellular damage induced by oxygen and oxygen-derived oxidants commonly known as ROS [6]. Oxidative damage induced by ROS is implicated as an important contributing factor in male infertility. An excessive production of ROS may result in an impaired steroidogenesis [17] and spermatogenesis [18]. These ROS include oxygen free radicals such as superoxide, hydroxyl, peroxyl, alkoxyl and hydroperoxyl radicals which cause tissue damage by a variety of mechanisms including DNA damage, lipid peroxidation, protein oxidation and depletion of thiols. It is widely accepted that the cytotoxic effect of ADR on normal differentiated cells is due to production of ROS at high levels [5]. We have already reported that ADR administration resulted in excessive lipid peroxidation with concomitant decrease in reduced glutathione (GSH) [19], increased protein oxidation and DNA damage [20] in rat testes. It has been reported that ADR interacts with mitochondrial NADH-dehydrogenase to facilitate the production of the ADR semiquinone free radical intermediate, which in a subsequent reaction with molecular oxygen forms ROS [21]. This might be the possible reason for the increased cellular oxidative stress in ADR administered rats in the present study. Thiols are the major components of cellular antioxidant system, which play an important role in detoxification of xenobiotic compounds and in the antioxidation of ROS and free radicals [22]. The interaction of ADR with protein thiols is one of the vital mechanisms involved in its toxicity [23]. It has been shown that ADR significantly lowers GSH levels in various tissues [19,24,25]. GSH is the predominant intracellular non-protein sulphydryl and its depletion results in an increased vulnerability of
the cell to oxidative stress [22]. Olson et al. reported that ADR toxicity could be potentiated with GSH depletion [26]. The observed reduction in the levels of total and non-protein thiols in ADR treated rats in the present study implies an exhausted antioxidant capacity of testis and thus a disturbance in redox reactions and thioldisulfide exchanges. The observed protective action of LA against ADR-induced oxidative damage is possibly due to its antioxidant property. LA is a thiol containing nucleophile that reacts with endogenous electrophiles including free radicals and reactive drug metabolites. LA is also reported to regenerate the glutathione pool by reduction of oxidized glutathione [8]. PHGPx is another constituent of glutathione redox cycle and its activity is particularly high in testis. It is responsible for the protection of membranes against oxidative damage since it can metabolize phospholipid hydroperoxide in membranes. PHGPx-defective spermatozoa showed extreme variation and abnormality in their mitochondrial ultrastructure compared to that of normal spermatozoa [7]. PHGPx present in the nuclei of sperm plays an important role in the stabilization of condensed chromatin and protection of sperm DNA from oxidation and thus helps in sperm maturation [27,28]. Hence, an imbalance in PHGPx activity leads to the production of abnormal spermatozoa that are compromised in terms of the integrity of their DNA and their potential for fertilization. According to our RT-PCR analysis, PHGPx mRNA levels in the testis were significantly decreased by ADR administration. Kang et al. have already shown that ADR administration resulted in decreased testicular PHGPx mRNA level [29] and our findings also corroborate with their results. Oxidative stress resulting from increased ROS production is the most important factor in down-regulating the expression of various genes [30]. Moreover, it has been shown that ADR interferes with the specific stages in spermatogenic process by inducing apoptosis and causing depletion of germ cells [31]. We have already shown that LA pretreatment helps in restoring ADR-induced arrest of spermatogenesis at specific stages [20]. In the present study, LA pretreatment significantly increased PHGPx mRNA level in ADR administered rats. These findings suggest that ADR-induced oxidative stress and depletion of germ cells might be the possible reasons for reduction in PHGPx transcript in the testis and LA may enhance transcription of PHGPx in the spermatogenic cells by decreasing oxidative stress and germ cell apoptosis. The testicular toxicity induced by ADR is further confirmed by the abnormal ultrastructural changes in germ cells of ADR exposed animals. The ADR treated rats
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showed numerous electron dense materials and vesicles in their germ cell cytoplasm with some marginal chromatins along the nuclear membrane. The toxic manifestations were also revealed by mitochondrial distension. The decline in PHGPx mRNA level in ADR administration may be one of the possible reasons for the abnormal ultrastructural changes in Group II animals. In our study, LA treated group showed significant improvement, thereby proving its cytoprotective role in ameliorating the testicular injury caused by ADR. In summary, the findings of our study indicate that administration of ADR induces significant oxidative stress in the testis of rats which is associated with decreased PHGPx mRNA level and abnormal ultrastructural findings in germ cells. LA supplementation restored the PHGPx mRNA level as well as the levels of thiols to near normalcy. The transmission electron microscopic studies confirmed the cytoprotection rendered by LA. Although the exact mechanisms remain to be clarified, LA could be an effective regimen to enhance therapeutic efficacy and reduce toxic side effects of ADR in clinical chemotherapy. Acknowledgement The first and second author gratefully acknowledge the financial assistance in the form of Senior Research Fellowship awarded by the Indian Council of Medical Research (ICMR), New Delhi, India. References [1] R.C. Lui, M.C. Laregina, D.R. Herbold, F.E. Johnson, Testicular cytotoxicity of intravenous doxorubicin in rats, J. Urol. 136 (1986) 940–943. [2] M. Kato, S. Makino, H. Kimura, T. Ota, T. Furuhashi, Y. Nagamura, Sperm motion analysis in rats treated with adriamycin and its applicability to male reproductive toxicity studies, J. Toxicol. Sci. 26 (2001) 51–59. [3] C. Prahalathan, E. Selvakumar, P. Varalakshmi, Protective effect of lipoic acid on adriamycin-induced testicular toxicity, Clin. Chim. Acta 360 (2005) 160–166. [4] T. Sjoblom, A. West, J. Landetie, Apoptotic response of spermatogenic cells to germ cell mutagens etopside, adriamycin and diepoxybutane, Environ. Mol. Mutagen. 31 (1998) 133–148. [5] K. Kiyomiya, S. Matsuo, M. Kurebe, Differences in intracellular sites of action of adriamycin in neoplastic and normal differentiated cells, Cancer Chemother. Pharmacol. 47 (2001) 51–56. [6] S.C. Sikka, Oxidative stress and role of antioxidants in normal and abnormal sperm function, Front. Biosci. 1 (1996) e78–e86. [7] H. Imai, Y. Nakagawa, Biological significance of phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) in mammalian cells, Free Radic. Biol. Med. 34 (2003) 145–169. [8] L. Packer, E.H. Witt, H.J. Tritschler, Alpha-lipoic as biological antioxidant. A review, Free Radic. Biol. Med. 19 (1995) 227–250.
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[9] Y.J. Suzuki, M. Tsuchiya, L. Packer, Antioxidant activities of dihydrolipoic acid and its structural homologues, Free Radic. Res. Commun. 18 (1993) 115–122. [10] V.E. Kagan, A. Shvedova, E. Serbinova, S. Khan, C. Swanson, R. Powell, L. Packer, Dihydrolipoic acid—a universal antioxidant both in the membrane and in the aqueous phase, Biochem. Pharmacol. 44 (1992) 1637–1649. [11] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275. [12] C.P. LeBel, H. Ischiropoulos, S.C. Bondy, Evaluation of the probe 2 ,7 -dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress, Chem. Res. Toxicol. 5 (1992) 227–231. [13] J. Sedlak, R.H. Lindsay, Estimation of total, protein-bound and non-protein sulfhydryl groups in tissue with Ellman’s reagent, Anal. Biochem. 25 (1968) 192–205. [14] S.Y. Nam, N. Nakamuta, M. Kurohmaru, Y. Hayashi, Cloning and sequencing of the mouse cDNA encoding phospholipid hydroperoxide glutathione peroxidase, Gene 198 (1997) 245–249. [15] S.Y. Nam, I.J. Baek, B.J. Lee, C.H. In, E.Y. Jung, J.M. Yon, B. Ahn, J.K. Kang, W.J. Yu, Y.W. Yun, Effects of 17-estradiol and tamoxifen on the selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA expression in male reproductive organs of rats, J. Reprod. Dev. 49 (2003) 389– 396. [16] V.F. Agnes, M.A. Akbarsha, Spermatotoxic effect of aflatoxin B1 in the albino mouse, Food Chem. Toxicol. 41 (2003) 119–130. [17] T. Diemer, J.A. Allen, K.H. Hales, D.B. Hales, Reactive oxygen disturbs mitochondria in MA-10 tumor Leydig cells and inhibits steroidogenic acute regulatory (StAR) protein and steroidogenesis, Endocrinology 144 (2003) 2882–2891. [18] R. Sen Gupta, J. Kim, C. Gomes, S. Oh, J. Park, W.B. Im, J.Y. Seong, R.S. Ahn, H.B. Kwon, J. Soh, Effect of ascorbic acid supplementation on testicular steroidogenesis and germ cell death in cadmium-treated male rats, Mol. Cell. Endocrinol. 221 (2004) 57–66. [19] C. Prahalathan, E. Selvakumar, P. Varalakshmi, Remedial effect of dl-␣-lipoic acid against adriamycin induced testicular lipid peroxidation, Mol. Cell Biochem. 267 (2004) 209–214. [20] C. Prahalathan, E. Selvakumar, P. Varalakshmi, Lipoic acid modulates adriamycin induced testicular toxicity, Reprod. Toxicol. 21 (2006) 54–59. [21] E.G. Mimnaugh, M.A. Trush, M. Bhatnagar, T.E. Gram, Enhancement of reactive oxygen-dependent mitochondrial membrane lipid peroxidation by the anticancer drug adriamycin, Biochem. Pharmacol. 34 (1985) 847–856. [22] G. Atmaca, Antioxidant effects of sulfur-containing aminoacids, Yonsei Med. J. 45 (2004) 776–788. [23] I. Fabregat, J. Satrustegui, A. Machado, Interaction of protein SH groups could be involved in adriamycin cardiotoxicity, Biochem. Med. 32 (1984) 289–295. [24] A.V. Balachandar, K.P. Malarkodi, P. Varalakshmi, Protective role of dl-␣-lipoic acid against adriamycin-induced cardiac lipid peroxidation, Hum. Exp. Toxicol. 22 (2003) 249–254. [25] K.P. Malarkodi, A.V. Balachandar, P. Varalakshmi, Protective effect of lipoic acid on adriamycin induced lipid peroxidation in rat kidney, Mol. Cell. Biochem. 247 (2003) 9–13. [26] R.D. Olson, J.S. MacDonold, C.J. vanBoxtel, R.C. Boerth, A.E. Slonim, R.W. Freeman, J.A. Oates, Regulatory role of glutathione and soluble sulfhydryl groups in the toxicity of adriamycin, J. Pharmacol. Exp. Ther. 215 (1980) 450–454.
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[27] H. Pfeifer, M. Conrad, D. Roethlein, A. Kyriakopoulos, M. Brielmeier, G.W. Bornkamm, D. Behne, Identification of a specific sperm nuclei selenoenzyme necessary for protamine thiol cross-linking during sperm maturation, FASEB J. 15 (2001) 1236–1238. [28] M. Maiorino, P.L. Mauri, A. Roveri, L. Benazzi, S. Toppo, V. Bosello, F. Ursini, Primary structure of the nuclear forms of phospholipid hydroperoxide glutathione peroxidase (PHGPx) in rat spermatozoa, FEBS Lett. 579 (2005) 667– 670.
[29] J.K. Kang, Y.J. Lee, K.O. No, E.Y. Jung, J.H. Sung, Y.B. Kim, S.Y. Nam, Ginseng intestinal metabolite-I (GIM-I) reduces doxorubicin toxicity in the mouse testis, Reprod. Toxicol. 16 (2002) 291–298. [30] Y. Morel, R. Barouki, Repression of gene expression by oxidative stress, Biochem. J. 342 (1999) 481–496. [31] K. Shinoda, K. Mitsumori, K. Yasuhara, C. Uneyama, H. Onodera, M. Hirose, M. Uehara, Genotoxicity: doxorubicin induces male germ cell apoptosis in rats, Arch. Toxicol. 73 (1999) 274–281.
Modulatory role of lipoic acid on adriamycin-induced testicular injury Chidambaram Prahalathan, Elangovan Selvakumar, Palaninathan Varalakshmi ∗ Department of Medical Biochemistry, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai 600113, India Received 2 September 2005; received in revised form 13 December 2005; accepted 20 December 2005 Available online 24 January 2006
Abstract The present study investigated the protective efficacy of dl-␣-lipoic acid (LA) on adriamycin (ADR)-induced oxidative damage in rat testis. Adult male albino rats of Wistar strain were administered ADR (1 mg/kg body weight, i.v.), once a week for 10 weeks. ADR injected rats showed increased oxidative stress with a concomitant decrease in cellular thiols. The mRNA level for phospholipid hydroperoxide glutathione peroxidase (PHGPx) was also significantly decreased by ADR administration. Transmission electron microscopic (TEM) observations of testicular germ cells revealed abnormal ultrastructural changes in ADR treated rats. Treatment with lipoic acid (35 mg/kg body weight, i.p.) 1 day prior to ADR administration, effectively reverted these abnormal changes towards normalcy. These findings indicate a cytoprotective role of LA in this experimental model of testicular toxicity. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Adriamycin; Lipoic acid; Oxidative stress; Cellular thiols; PHGPx; TEM; Testicular toxicity
1. Introduction Adriamycin (ADR) is an anthracycline antibiotic that exhibits excellent antitumor activity against a variety of solid tumors. The clinical use of ADR is associated with testicular dysfunction characterized by altered sperm development, production, structural integrity and motility rates in association with increased cellular apoptosis [1–4]. Although antitumor action of ADR may be mediated by a wide number of mechanisms, oxidative stress and the generation of toxic reactive oxygen species (ROS) are the main cause of drug toxicity [5]. The oxyradicals cause damage to mitochondrial and other
∗
Corresponding author. Tel.: +91 44 24480767; fax: +91 44 24926709. E-mail address: [email protected] (P. Varalakshmi).
cytoplasmic membrane structures through peroxidation of phospholipids, proteins and nucleotides that can be detrimental to male fertility [6]. Thus, the combination of the drug delivery together with a potent antioxidant may be an appropriate approach to reduce the toxic side effects of ADR. Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenium containing antioxidant that metabolizes phospholipids in membranes and protects them from oxidative damage. The mRNA for PHGPx is highly expressed in testis especially in specific spermatogenic cells during spermatogenesis and the down-regulation in PHGPx expression due to increased oxidative stress could lead to infertility [7]. Hence, it would be worthwhile to know whether PHGPx expression in the testis is influenced by ADR treatment. Lipoic acid (LA), a naturally occurring nutraceutical, functions as an essential cofactor in metabolic
0009-2797/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2005.12.007
C. Prahalathan et al. / Chemico-Biological Interactions 160 (2006) 108–114
reactions involved in energy utilization. LA and its reduced form dihydrolipoic acid are effective against conditions in which oxidative stress has a role [8]. It shows beneficial effects in oxidative stress conditions because of its synergistic action with other antioxidants [9]. LA, which is a universal antioxidant functions both in aqueous and membrane phases [10]. The present study describes the protective effect of LA; a multifunctional antioxidant on ADR-induced testicular toxicity, in correlation with testicular thiol contents and PHGPx gene expression. 2. Materials and methods 2.1. Drugs and chemicals ADR (doxorubicin hydrochloride-Adrim) procured from Dabur Pharmaceuticals; New Delhi, India was used in the present experimental study. dl-␣-lipoic acid, bovine serum albumin and 2 ,7 -dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma Chemicals Co., St. Louis, USA. All other chemicals and solvents were of analytical grade.
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10th week, the animals were killed by decapitation under anesthesia and both testes were excised immediately and used for the following analyses. 2.4. Determination of cellular oxidative stress A 10% homogenate of the tissue was prepared in 0.01 M Tris–HCl buffer (pH 7.4) and protein content was determined by the method of Lowry et al. [11]. Oxidative stress was determined by using DCFH as a probe, according to the method of LeBel et al. [12]. In brief, the assay buffer contained 20 mM Tris–HCl, 130 mM KCl, 5 mM MgCl2 , 20 mM NaH2 PO4 , 30 mM glucose and 5 M DCFH-DA. The assay medium was incubated at 37 ◦ C for 15 min and 1 mol of H2 O2 was added to the mixture at the end of assay. DCF formation was measured at the excitation 488 nm and emission at 525 nm for 30 min by Shimadzu fluorescence spectrometer. 2.5. Determination of cellular thiols Total thiol and non-protein thiols were estimated by the method of Sedlak and Lindsay [13].
2.2. Animal model 2.6. RT-PCR analysis Adult male albino rats of Wistar strain weighing 140 ± 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 (12 h 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
To determine the expression of PHGPx mRNA in each group, total RNA was isolated from testes using a total RNA extraction kit (Eppendorf, Germany). The specific primers for PHGPx were used for amplification [14,15]. PCR amplification was carried out with a thermal cycler using one step RT-PCR kit (Eppendorf, Germany) according to a protocol for the initial denaturing step at 95 ◦ C for 10 min; then 30 cycles at 95 ◦ C for 1 min (denaturing), at 55 ◦ C for 1 min (annealing) and 72 ◦ C for 1.5 min (extension); and a further extension at 72 ◦ C for 10 min. The PCR products were run on a 2% agarose gel in Tris-borate-EDTA buffer. Rat -actin was used as an internal standard. 2.7. Electron microscopy studies Representative electron micrographs from testis were obtained as previously [16]. Briefly, thin slices of the testis were fixed in 2.5% glutaraldehyde and post-fixed in 1% osmium tetroxide. Then tissue was dehydrated in graded acetone solutions and finally embedded in epon-araldite. Ultra-thin sections were stained in uranyl acetate and lead citrate. Sections were transmitted in a Philips 201C transmission electron microscope (TEM) and photographed.
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2.8. 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 (L.S.D.) test. A Pvalue < 0.05 was considered significant. 3. Results There were no deaths recorded in any of the experimental groups during the study period. Fig. 1 represents abnormal elevation in testicular oxidative stress in ADR administered rats (Group II), which exhibited a 2.06-fold
increase as compared to those of control animals. LA pretreatment in ADR injected rats significantly (P < 0.05) reduced the level of oxidative stress to near normalcy. The levels of total thiol and non-protein thiols were significantly (P < 0.05) decreased (36.9% and 44.7%, respectively) in the testis of ADR treated rats as compared to those of control animals (Fig. 2). LA supplementation restored the thiol levels considerably in Group IV animals. Fig. 3 presents the mRNA level of PHGPx in various groups. A single transcript (461 bp) was observed in all the groups. RT-PCR analysis of PHGPx mRNA showed a significant (P < 0.05) decrease in ADR administered rats, while the expression pattern of PHGPx mRNA in
Fig. 1. Effect of ADR and LA on testicular oxidative stress indices. Values represent mean ± S.D. for six rats. Comparisons are made as follows: (a) with Group I and (b) with Group II. Values are statistically significant at * P < 0.05.
Fig. 2. Effect of ADR and LA on thiol levels in testis. Values represent mean ± S.D. for six rats. Comparisons are made as follows: (a) with Group I and (b) with Group II. Values are statistically significant at * P < 0.05.
Fig. 3. Effect of ADR and LA on expression pattern of PHGPx mRNA level in testis. (a) The 461 and 375 bp fragments represent PHGPx transcript and -actin as internal standard, respectively; lane 1:100 bp DNA ladder, lane 2: control, lane 3: ADR, lane 4: LA and lane 5: LA + ADR. (b) Relative levels of PHGPx mRNA expression compared to values for -actin mRNA. Values represent mean ± S.D. for six rats. Comparisons are made as follows: (a) with Group I and (b) with Group II. Values are statistically significant at * P < 0.05.
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Fig. 4. Transmission electron microscopic observations of germ cells in rat testis treated with ADR and/LA. Germ cells from control (a) and LA treated (d) show normal morphological features. However, the sections from ADR treated rat testis (b and c) reveals the following abnormalities. The cytoplasm contains abnormal vesicles (arrow head) and has some marginal chromatins (star) along the nuclear membrane. Mitochondrial distension with loss of cristae (#) and electron dense materials (arrow) are also visible. The testicular germ cells of rat treated with LA and ADR (e) shows nearly normal ultrastructural features.
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Group IV animals was not much altered when compared to control animals. Transmission electron microscopic studies of the testis revealed the following changes. The germ cells of control animals showed normal morphological features (Fig. 4a and d). ADR-induced dramatic ultrastructural changes in germ cell morphology (Fig. 4b and c). ADR treated rats showed numerous vesicles and electron dense materials in their germ cell cytoplasm. Some marginated chromatins along the nuclear membrane and mitochondrial distension were frequently recognized within the germ cell cytoplasm of ADR treated rats. Ultrastructurally, LA pretreatment in ADR administered group showed significant improvement in their germ cell morphology (Fig. 4e). 4. Discussion “Oxidative stress” is a condition associated with an increased rate of cellular damage induced by oxygen and oxygen-derived oxidants commonly known as ROS [6]. Oxidative damage induced by ROS is implicated as an important contributing factor in male infertility. An excessive production of ROS may result in an impaired steroidogenesis [17] and spermatogenesis [18]. These ROS include oxygen free radicals such as superoxide, hydroxyl, peroxyl, alkoxyl and hydroperoxyl radicals which cause tissue damage by a variety of mechanisms including DNA damage, lipid peroxidation, protein oxidation and depletion of thiols. It is widely accepted that the cytotoxic effect of ADR on normal differentiated cells is due to production of ROS at high levels [5]. We have already reported that ADR administration resulted in excessive lipid peroxidation with concomitant decrease in reduced glutathione (GSH) [19], increased protein oxidation and DNA damage [20] in rat testes. It has been reported that ADR interacts with mitochondrial NADH-dehydrogenase to facilitate the production of the ADR semiquinone free radical intermediate, which in a subsequent reaction with molecular oxygen forms ROS [21]. This might be the possible reason for the increased cellular oxidative stress in ADR administered rats in the present study. Thiols are the major components of cellular antioxidant system, which play an important role in detoxification of xenobiotic compounds and in the antioxidation of ROS and free radicals [22]. The interaction of ADR with protein thiols is one of the vital mechanisms involved in its toxicity [23]. It has been shown that ADR significantly lowers GSH levels in various tissues [19,24,25]. GSH is the predominant intracellular non-protein sulphydryl and its depletion results in an increased vulnerability of
the cell to oxidative stress [22]. Olson et al. reported that ADR toxicity could be potentiated with GSH depletion [26]. The observed reduction in the levels of total and non-protein thiols in ADR treated rats in the present study implies an exhausted antioxidant capacity of testis and thus a disturbance in redox reactions and thioldisulfide exchanges. The observed protective action of LA against ADR-induced oxidative damage is possibly due to its antioxidant property. LA is a thiol containing nucleophile that reacts with endogenous electrophiles including free radicals and reactive drug metabolites. LA is also reported to regenerate the glutathione pool by reduction of oxidized glutathione [8]. PHGPx is another constituent of glutathione redox cycle and its activity is particularly high in testis. It is responsible for the protection of membranes against oxidative damage since it can metabolize phospholipid hydroperoxide in membranes. PHGPx-defective spermatozoa showed extreme variation and abnormality in their mitochondrial ultrastructure compared to that of normal spermatozoa [7]. PHGPx present in the nuclei of sperm plays an important role in the stabilization of condensed chromatin and protection of sperm DNA from oxidation and thus helps in sperm maturation [27,28]. Hence, an imbalance in PHGPx activity leads to the production of abnormal spermatozoa that are compromised in terms of the integrity of their DNA and their potential for fertilization. According to our RT-PCR analysis, PHGPx mRNA levels in the testis were significantly decreased by ADR administration. Kang et al. have already shown that ADR administration resulted in decreased testicular PHGPx mRNA level [29] and our findings also corroborate with their results. Oxidative stress resulting from increased ROS production is the most important factor in down-regulating the expression of various genes [30]. Moreover, it has been shown that ADR interferes with the specific stages in spermatogenic process by inducing apoptosis and causing depletion of germ cells [31]. We have already shown that LA pretreatment helps in restoring ADR-induced arrest of spermatogenesis at specific stages [20]. In the present study, LA pretreatment significantly increased PHGPx mRNA level in ADR administered rats. These findings suggest that ADR-induced oxidative stress and depletion of germ cells might be the possible reasons for reduction in PHGPx transcript in the testis and LA may enhance transcription of PHGPx in the spermatogenic cells by decreasing oxidative stress and germ cell apoptosis. The testicular toxicity induced by ADR is further confirmed by the abnormal ultrastructural changes in germ cells of ADR exposed animals. The ADR treated rats
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showed numerous electron dense materials and vesicles in their germ cell cytoplasm with some marginal chromatins along the nuclear membrane. The toxic manifestations were also revealed by mitochondrial distension. The decline in PHGPx mRNA level in ADR administration may be one of the possible reasons for the abnormal ultrastructural changes in Group II animals. In our study, LA treated group showed significant improvement, thereby proving its cytoprotective role in ameliorating the testicular injury caused by ADR. In summary, the findings of our study indicate that administration of ADR induces significant oxidative stress in the testis of rats which is associated with decreased PHGPx mRNA level and abnormal ultrastructural findings in germ cells. LA supplementation restored the PHGPx mRNA level as well as the levels of thiols to near normalcy. The transmission electron microscopic studies confirmed the cytoprotection rendered by LA. Although the exact mechanisms remain to be clarified, LA could be an effective regimen to enhance therapeutic efficacy and reduce toxic side effects of ADR in clinical chemotherapy. Acknowledgement The first and second author gratefully acknowledge the financial assistance in the form of Senior Research Fellowship awarded by the Indian Council of Medical Research (ICMR), New Delhi, India. References [1] R.C. Lui, M.C. Laregina, D.R. Herbold, F.E. Johnson, Testicular cytotoxicity of intravenous doxorubicin in rats, J. Urol. 136 (1986) 940–943. [2] M. Kato, S. Makino, H. Kimura, T. Ota, T. Furuhashi, Y. Nagamura, Sperm motion analysis in rats treated with adriamycin and its applicability to male reproductive toxicity studies, J. Toxicol. Sci. 26 (2001) 51–59. [3] C. Prahalathan, E. Selvakumar, P. Varalakshmi, Protective effect of lipoic acid on adriamycin-induced testicular toxicity, Clin. Chim. Acta 360 (2005) 160–166. [4] T. Sjoblom, A. West, J. Landetie, Apoptotic response of spermatogenic cells to germ cell mutagens etopside, adriamycin and diepoxybutane, Environ. Mol. Mutagen. 31 (1998) 133–148. [5] K. Kiyomiya, S. Matsuo, M. Kurebe, Differences in intracellular sites of action of adriamycin in neoplastic and normal differentiated cells, Cancer Chemother. Pharmacol. 47 (2001) 51–56. [6] S.C. Sikka, Oxidative stress and role of antioxidants in normal and abnormal sperm function, Front. Biosci. 1 (1996) e78–e86. [7] H. Imai, Y. Nakagawa, Biological significance of phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) in mammalian cells, Free Radic. Biol. Med. 34 (2003) 145–169. [8] L. Packer, E.H. Witt, H.J. Tritschler, Alpha-lipoic as biological antioxidant. A review, Free Radic. Biol. Med. 19 (1995) 227–250.
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