Cell Tissue Res (2009) 337:269–280 DOI 10.1007/s00441-009-0801-2
Carnitine reduces testicular damage in rats treated with etoposide in the prepubertal phase Fatima Kazue Okada & Taiza Stumpp & Sandra Maria Miraglia
Received: 16 January 2009 / Accepted: 30 March 2009 / Published online: 15 May 2009 # Springer-Verlag 2009
Abstract Etoposide is a chemotherapeutic agent that induces cell death by blocking topoisomerase II catalytic function. Although etoposide is effective in the treatment of cancer, it also causes the death of normal proliferating cells, including male germ cells. Administration of etoposide during the prepubertal phase causes diturbances in several testicular morphometric parameters and in Sertoli cells. Cytoprotection of the seminiferous epithelium is the only means of preserving potential male reproduction in prepubertal cancer patients. Carnitine, an amino acid naturally present in normal cells, is a promising cryoprotectant as it is concentrated in the epididymis and promotes sperm maturation. We have therefore investigated whether carnitine protects rat testes against etoposide and, thus, improves fertility in adulthood. Our results suggest that carnitine partially protects the testis against damage caused by etoposide, although the mechanism by which it happens remains unknown. Keywords Carnitine . Etoposide . Apoptosis . Spermatogenesis . Testis . Rat (Wistar)
Introduction Etoposide is a chemotherapeutic agent that interacts with topoisomerase II, a nuclear enzyme that is essential for Electronic supplementary material The online version of this article (doi:10.1007/s00441-009-0801-2) contains supplementary material, which is available to authorized users. F. K. Okada (*) : T. Stumpp : S. M. Miraglia Laboratory of Developmental Biology, Federal University of Sao Paulo, 740 Botucatu, Ed. Leitao da Cunha (2nd floor), Sao Paulo, SP, Brazil 04023-900 e-mail: [email protected]
DNA replication, transcription, chromosomal segregation, and recombination. Etoposide induces cell death by blocking topoisomerase II catalytic function leading to high levels of DNA strand breaks (Hande 1998). Although etoposide is effective in the treatment of cancer, it does not act solely on cancer cells; it also causes the death of normal proliferating cells, including the male germ cells. In the testis, etoposide acts on the pre-mitotic synthesis of DNA, causing the death of intermediate and type B spermatogonia. However, it can also act on premeiotic DNA synthesis and kills primary spermatocytes (Hakovirta et al. 1993). Sjöblom et al. (1998) and Stumpp et al. (2004) have shown an increase in the number of apoptotic germ cells in rats treated with etoposide during adulthood and pre-puberty, respectively. Other studies have demonstrated that the administration of etoposide during the prepubertal phase causes the reduction of several testicular morphometric parameters (Freitas et al. 2002; Stumpp et al. 2004) and Sertoli cell alterations (Stumpp et al. 2006, 2008). Much progress in cancer treatment has recently been achieved, resulting in a significant increase of patients restored to health, including children (Gommersall et al. 2005; Kaspers and Creutzig 2005; Oliver 2007). In view of this, oncology protocols have to take into account the life quality of the patients once their anticancer treatment has been completed; fertility preservation is one of the main concerns. Thus, in an attempt to preserve male reproduction, sperm collection and storage are important procedures before anticancer treatment of adult patients of reproductive age. However, this is impracticable in child patients, making the cytoprotection of the seminiferous epithelium a suitable alternative. Carnitine, an amino acid naturally present in normal cells, might be a promising option for such cryoprotection. In the male genital system, carnitine is
Cell Tissue Res (2009) 337:269–280
concentrated in the epididymis and promotes sperm maturation (Jeulin and Lewin 1996). Because of its antioxidant properties, carnitine has been used in the treatment of male infertility (Vicari et al. 2002; Lenzi et al. 2003) and as a cytoprotective substance against the toxicity of drugs such as alcohol and bleomycin (Arafa and Sayed-Ahmed 2003; Demirdag et al. 2004; Sayed-Ahmed et al. 2004). Moreover, Palmero et al. (2000) have demonstrated that carnitine acts directly on the Sertoli cells in vitro, improving their physiology and, consequently, the development of the germ cells. In view of the therapeutic, antioxidant, and cytoprotective properties of carnitine in other tissues, we have investigated whether carnitine protects rat testes against etoposide and, thus, improves fertility in adulthood.
Materials and methods Animals and groups Male Wistar rats (Rattus norvegicus albinus; n=120; age: 25 days) were distributed into four groups: a sham-control group (S) treated with 0.9% saline solution; a carnitine group (C) treated with carnitine (250 mg/kg); an etoposide group (E) treated with etoposide (40 mg/kg Oncosideo, Darrow-Brazil); and a carnitine+etoposide group (CE) treated with carnitine and etoposide (250 mg/kg Lcarnitine plus 40 mg/kg Oncosideo). The animals were housed under controlled conditions (12/12 h light/dark cycles at 23°C to 25°C); water and food were provided ad libitum. All groups were divided into three subgroups formed according to the age at which they were killed: 32, 64, and 127 days (Stumpp et al. 2004; de Oliva and Miraglia 2008; Table 1). Each subgroup contained ten rats.
Etoposide and carnitine administration The animals of the E and CE groups received intraperitoneal cumulative doses of 5 mg/kg etoposide (Oncosideo, Darrow-Brazil) per day for 8 consecutive days, completing the total dose of 40 mg/Kg. Treatment started when the rats were 25 days old. Just before administration, the etoposide was diluted in 0.9% sterile saline solution. The animals of the C and CE groups received, intraperitoneally, 250 mg/kg of L-carnitine (Sigma) for the same 8 consecutive days of etoposide administration. The dose was established based in the literature (Sayed-Ahmed et al. 2001, 2004). In the animals of the CE group, carnitine was administered 1 h before the etoposide injection. Carnitine was diluted in twice-distilled water, according to the manufacturer’s directions. The sham-control rats received 0.9% sterile physiological saline solution (Table 1). The gain of body weight during the treatment was recorded. This study was performed according to the rules of the Ethical Committee for Animal Research of the Federal University of São Paulo (UNIFESP). Morphometric and histological analyses Immediately before being killed, the rats were weighed and anesthetized with an intraperitoneal injection of thiopental (Thiopentax, Cristália, SP, Brazil). Subsequently, the testes were carefully removed from the scrota and weighed, and their axes (the major and minor axes) and volume were measured (Scherle 1970). The testes were fixed in Bouin’s liquid and embedded in paraffin. Two non-consecutive cross sections (2.5 µm thick) per animal were stained by the periodic-acid-Schiff method and counterstained with Harris’ hematoxylin (PAS+H) for histopathological and histomorphometric analyses; the latter involved the measurement of the diameter of the seminiferous tubule and the height of the
Table 1 Groups and subgroups according to applied treatment Groups
Age when killed(days)
Type of treatment
S32 S64 S127 C32 C64
32 64 127 32 64
12 32 95 12 32
h days days h days
C127 E32 E64 E127 CE32 CE64 CE127
127 32 64 127 32 64 127
95 12 32 95 12 32 95
days h days days h days days
L-carnitine, 250 mg/kg
Oncosideo (Darrow-Brazil), 40 mg/kg
L-carnitine 250 mg/kg+Oncosideo 40 mg/kg
Cell Tissue Res (2009) 337:269–280
seminiferous epithelium via a binocular microscope coupled to a Leica Q550IW (Cambridge, England) image analysis system. Fifty random seminiferous tubule cross sections per testicular section were examined (see also Electronic Supplementary Material).
according to Stumpp et al. (2004) and Lirdi et al. (2008; see also Electronic Supplementary Material).
Apoptotic germ cell numerical density
Samples of epididymal fluid were obtained from the caudal region of the epididymis of 127-day-old rats for the analyses of the concentration and morphology of spermatozoa (see Electronic Supplementary Material).
With the aim of investigating whether carnitine protected germ cells against apoptosis caused by etoposide, we determined the numerical density of apoptotic cells in the seminiferous epithelium (Stumpp et al. 2004; Lirdi et al. 2008). To detect apoptosis, the TUNEL method (ApopTag Plus Peroxidase kit, Intergen Company, N.Y.) and the analysis of nuclear morphology were performed (Stumpp et al. 2004; Lirdi et al. 2008; see Electronic Supplementary Material). Because the TUNEL method detects late apoptotic cells (Barroso et al. 2000; Huerta et al. 2007), it does not label the final stages of apoptosis (Stumpp et al. 2004). Cells in the final stages of apoptosis, i.e., with condensed and peripheral chromatin, were scored in the same sections submitted to the TUNEL reaction (Stumpp et al. 2004; Lirdi et al. 2008). These cells were referred as TUNELnegative cells with abnormal nuclear morphology (ANM; Lirdi et al. 2008). The numerical densities of TUNEL-positive (Nvt1) and TUNEL-negative cells with ANM (Nvt2) were obtained. Then, the total numerical density of apoptotic cells per cubic centimeter, including the TUNEL-positive cells and TUNEL-negative cells with ANM (Nvt3), was calculated
Concentration and morphological analyses of spermatozoa in epididymal fluid
Statistical analysis Morphometric and stereological data were submitted to parametric methods by using Jandel Statistical SigmaStat software 2.0. The parametric analysis of variance test was performed to compare data that passed the normality test. The non-parametric Kruskall-Wallis test was used to compare data that failed the normality test. When the differences were statistically significant (P≤0.05), analysis was completed with the multiple comparison test (StudentNewman-Keuls method).
Results Body weight and testicular morphometry The rats of the E and CE groups showed a lower gain of body weight during the treatment than the rats of the S and C groups (Table 2).
Table 2 Final body weight (mean±SD), gain of body weight (body weight at 25 days subtracted from the body weight at 32 days; median and interquartile range [Q1–Q3]), and testicular morphometric data (mean±SD) of the variously treated groups Groups
Final body weight (g)
Testicular weight (g)
Gain of body weight (g)
S32 S64 S127 C32 C64 C127 E32 E64 E127 CE32 CE64 CE127
109±15.27 319±29.36 429±31.93 93±6.73*c 286±22.77*c 424±35.74 58±7.55*a,b 234±20.83*b 376±38.29*b 70±10.63*a,b 238±25.52*b 394±35.47
0.47±0.09 1.64±0.13 1.85±0.11 0.40±0.05 1.55±0.16 1.96±0.19 0.26±0.04*b 0.46±0.11*b 1.21±0.26*b 0.28±0.03*b 0.51±0.11*b 1.31±0.30*b
ES and CE
(1.4–2.1) (2.8–5.3)*e (6.4–13.3)*c,d (1.5–3.0)*c
Cell Tissue Res (2009) 337:269–280
further studies are necessary to verify the way that carnitine promotes these effects. Carnitine has been shown to protect the heart from the damage caused by chemotherapeutic drugs such as cisplatin and doxorubicin (Sayed-Ahmed et al. 2001, 2004; Al-Majed et al. 2006) and can also improve male reproduction (Vicari et al. 2002; Lenzi et al. 2003). The results obtained in the present study reinforce the hypothesis that etoposide harms stem spermatogonia. The mutagenicity of etoposide has been shown by Sjöblom et al. (1998) and Martin et al. (1999). Although we suggest that the protective effect of carnitine occurs through its action on stem spermatogonia, the mechanism of action of this drug is not clear. On the other hand, the effects of etoposide and carnitine on the Sertoli cells should also be considered. As previously mentioned, carnitine is known to improve Sertoli cell metabolism, and the carnitine/organic cation transporter (OCTN) is present in the Sertoli cell membrane. When these cells are injured, spermatogenesis is impaired. According to Stumpp et al. (2006, 2008), the testicular damage caused by etoposide might be mediated, to some extent, by the Sertoli cells. Thus, the protective effect of carnitine might also be mediated by the Sertoli cells. Moreover, carnitine is known to protect the cell membrane against damage induced by free oxygen radicals (Lenzi et al. 2003), whereas etoposide has been shown to cause free radical production (Oh et al. 2007). Thus, this might be another way in which carnitine improves testicular parameters and fertility. In conclusion, our results suggest that, under the schedule used in this study, carnitine partially protects the testis against damage caused by etoposide, although the mechanism by which this happens is unknown. Carnitine seems to be a promising drug in the fertility field, but further studies are necessary to confirm the protective effect of carnitine and to comprehend its action in the testis, especially during the prepubertal phase. These studies are particularly important because they might contribute to avoiding fertility problems in patients subjected to chemotherapeutic treatment during childhood or adolescence. Finally, the increase in the number of cancer patients restored to health in the past few years (Gommersall et al. 2005; Kaspers and Creutzig 2005; Oliver 2007) emphasizes the importance of preserving abilities that, although not vital, represent an important part of life, such as fertility.
References Al-Majed AA, Sayed-Ahmed MM, Al-Yahya AA, Aleisa AM, Al-Rejaie SS, Al-Shabanah OA (2006) Propionyl-L-carnitine prevents the progression of cisplatin-induced cardiomyopathy in a carnitinedepleted rat model. Pharmacol Res 53:278–286
279 Arafa HMM, Sayed-Ahmed MM (2003) Protective role of carnitine esters against alcohol-induced gastric lesions in rat. Pharmacol Res 48:285–290 Barroso G, Morshedi M, Oehninger S (2000) Analysis of DNA fragmentation, plasma membrane, translocation of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod 15:1338–1344 Billig H, Furuta I, Rivier C, Tapanainen J, Parvinen M, Hsueh AJ (1995) Apoptosis in testis germ cells: developmental changes in gonadotropin dependence and localization to selective tubule stages. Endocrinology 36:5–12 Demirdag K, Bahcecioglu IH, Ozercan IH, Ozden M, Yilmaz S, Kalkan A (2004) Role of L-carnitine in the prevention of acute liver damage induced by carbon tetrachloride in rats. J Gastroenterol Hepatol 19:333–338 Freitas FEL, Cordeiro-Mori F, Sasso-Cerri E, Lucas SRA, Miraglia SM (2002) Alterations of spermatogenesis in etoposide-treated rats: a stereological study. Interciencia 27:227–235 Gommersall LM, Arya M, Mushtaq I, Duffy P (2005) Current challenges in Wilms’ tumor management. Nat Clin Pract Oncol 2:298–304 Hakovirta H, Parvinen M, Lähdetie J (1993) Effects of etoposide on stage-specific DNA synthesis during rat spermatogenesis. Mutat Res 301:189–193 Hande KR (1998) Etoposide: four decades of development of a topoisomerase II inhibitor. Eur J Cancer 34:1514–1521 Huckins C (1965) The duration of spermatogenesis in the pre postpuberal Wistar rats. Anat Rec 151:365 Huerta S, Goulet EJ, Huerta-Yepez S, Livingston EH (2007) Screening and detection of apoptosis. J Surg Res 139:143–156 Jeulin C, Lewin LM (1996) Role of free L-carnitine and acetyl-Lcarnitine in post-gonadal maturation of mammalian spermatozoa. Hum Reprod Update 2:87–102 Kadota T, Chikazawa H, Takahashi N (1989) Toxicological study of etoposide (VP-16) in rats with special emphasis on testicular alteration. Toxicol Lett 45:185–194 Kaspers GJL, Creutzig U (2005) Pediatric acute myeloid leukemia: international progress and future directions. Leukemia 19:2025– 2029 Korst AE, Sterre ML van der, Gall HE, Fichtinger-Schepman AM, Vermorken JB, Vijgh WJ van der (1998) Influence of amifostine on the pharmacokinetics of cisplatin in cancer patients. Clin Cancer Res 4:331–336 Lenzi A, Lombardo F, Sgrò P, Salacone P, Caponecchia L, Dondero F, Gandini L (2003) Use of carnitine therapy in selected cases of male factor infertility: a doble-blind crossover trial. Fertil Steril 79:292–300 Lirdi LC, Stumpp T, Sasso-Cerri E, Miraglia SM (2008) Amifostine protective effect on cisplatin-treated rat testis. Anat Rec 291:797– 808 Marchetti F, Pearson FS, Bishop JB, Wyrobek AJ (2006) Etoposide induces chromosomal abnormalities in mouse spermatocytes and stem cell spermatogonia. Hum Reprod 21:888–895 Martin RH, Ernst S, Rademaker A, Barclay L, Ko E, Summers N (1999) Analysis of sperm chromosome complements before, during and after chemotherapy. Cancer Genet Cytogenet 108:808–812 Oh SY, Sohn YW, Park JW, Park HJ, Jeon HM, Kim TK, et al (2007) Selective cell death of oncogenic Akt-transduced brain cancer cells by etoposide through reactive oxygen species mediated damage. Mol Cancer Ther 6:2178–2187 Oliva SU de, Miraglia SM (2008) Carbamazepine damage to rat spermatogenesis in different sexual developmental phases. Int J Androl doi:10.1111/j.1365-2605.2008.00898 Oliver T (2007) Conservative management of testicular germ-cell tumors. Nat Clin Pract Urol 4:550–560
280 Palmero S, Bottazzi C, Costa M, Leone M, Fugassa E (2000) Metabolic effects of L-carnitine on prepubertal rat Sertoli cells. Horm Metab Res 32:87–90 Rooij DG de, Russell LD (2000) All you wanted to know about spermatogonia but were afraid to ask. J Androl 21:776–798 Russell LD, Ettlin RA, Hikim APS, Clegg ED (1990) Histophatology of testis. In: Russell LD, Ettlin RA, Hikim APS, Clegg ED (eds) Histological and histopathological evaluation of the testis, 1st edn. Cache River, Florida, pp 210–264 Russell LB, Hunsicker PR, Johnson DK, Shelby MD (1998) Unlike other chemicals, etoposide (a topoisomerase-II inhibitor) produces peak mutagenicity in primary spermatocytes of the mouse. Mutat Res 400:279–286 Russell LB, Hunsicker PR, Kerley M, Pyle A, Saxton AM (2004) Etoposide exposure during male mouse pachytene has complex effects on crossing-over and causes nondisjunction. Mutat Res 565:61–77 Sayed-Ahmed MM, Salman TM, Gaballah HE, Abou El-Naga SA, Nicolai R, Calvani M (2001) Propionyl-L-carnitine as protector against adriamycin-induced cardiomyopathy. Pharmacol Res 43:513–520 Sayed-Ahmed MM, Mansour HH, Gharib OA, Hafez HF (2004) Acetyl-L-carnitine modulates bleomycin-induced oxidative stress
Cell Tissue Res (2009) 337:269–280 and energy depletion in lung tissues. J Egypt Natl Canc Inst 16:237–243 Scherle W (1970) A simple method for volumetry of organs in quantitative stereology. Mikroskopie 26:57–60 Sjöblom T, West A, Lähdetie J (1998) Apoptotic response of spermatogenic cells to the germ cell mutagens etoposide, adriamycin, and diepoxybutane. Environ Mol Mutagen 31:133– 148 Stumpp T, Sasso-Cerri E, Freymüller E, Miraglia SM (2004) Apoptosis and testicular alterations in albino rats treated with etoposide during the prepubertal phase. Anat Rec [A] 279:611– 622 Stumpp T, Freymüller E, Miraglia SM (2006) Sertoli cell function in albino rats treated with etoposide during prepubertal phase. Histochem Cell Biol 126:353–361 Stumpp T, Freymüller E, Miraglia SM (2008) Sertoli cell morphological alterations in albino rats treated with etoposide during prepuberal phase. Microsc Microanal 14:225–235 Vicari E, Vignera SL, Calogero AE (2002) Antioxidant treatment with carnitines is effective in infertile patients with prostatovesiculoepididymitis and elevated seminal leukocyte concentrations after treatment with nonsteroidal anti-inflammatory compounds. Fertil Steril 78:1203–1208