Protective effect of ascorbic acid on cyclophosphamide-induced testicular gametogenic and androgenic disorders in male rats

Asian J Androl 2002 Sep; 4: 201-207 

Keywords: Cyclophosphamide; androgenesis; gametogenesis; oxidative stress; free radicals; ascorbic acid; catalase; peroxidase; testosterone

Abstract

Aim: To study the detrimental effects of cyclophosphamide on the testicular androgenic and gametogenic activities through endocrine inhibition and/or induction of oxidative stress in male albino rats and to evaluate the protective effect of ascorbic acid. Methods: The testicular D⁵, 3b-hydroxysteroid dehydrogenase (HSD), 17b-HSD, peroxidase and catalase activities along with the levels of malondialdehyde (MDA) and conjugated dienes in testicular tissue were measured for the evaluation of testicular oxidative stress. The plasma testosterone (T) level was measured by immunoassay. Various germ cells at stage VII of spermatogenic cycle were quantified from testicular stained sections. Results: Cyclophosphamide treatment results in a significant inhibition in the testicular D⁵, 3b-HSD and 17b-HSD activities, a decrease in plasma T level and a diminution in the counts of various germ cells. Moreover, this treatment was also associated with a significant inhibition of the peroxidase and catalase activities along with high levels of MDA and conjugated dienes in the testis. All these changes were reversed by ascorbic acid co-administration. Conclusion: Cyclophosphamide treatment at the dosage used caused testicular gametogenic and androgenic disorders as well as induced testicular oxidative stress that can be reversed by ascorbic acid co-administration.

Cyclophosphamide is used widely as an anticancer and immuno suppressive drug [1, 2] and in the treatment of nephrotic syndrome [3, 4]. Although the therapeutic usefulness of this alkylating agent remains indis-putable, a wide range of adverse effects including reproductive toxicity has been demonstrated in the humans and animals. Cyclophosphamide treatment in patients is associated with oligozospermia and azoospermia [3,5, 6] as well as biochemical and histological alterations in the testes and epididymis of rats and humans [3,7]. Moreover, disturbance in gonadotrophin secretion and testicular damage and decreased blood testosterone (T) levels are found in cyclophosphamide-treated male patients [8, 9]. In male rats, chronic cyclophosphamide treatment injures the progeny, decreases the reproductive organ weights and impairs the fertility [10]. Its frequent use is associated with significant gonadal damages in men and women [11]. We reported that the anti-folliculogenic activity of cyclophosphamide in rat could be reversed by co-administration of human chorionic gonadotrophin (hCG) [12] and that cyclophosphamide treatment in rat was associated with inhibition in testicular androgenesis and spermatogenesis, which were protected by hCG co-administration [13]. We also reported that treatment of cyclophosphamide in adult rats caused toxic effect in liver and kidney and inhibited the ovarian steroidogenesis, that was ameliorated by ascorbic acid supplementation [14, 15]. Cyclophosphamide treatment is associated with induction of oxidative stress by the generation of free radicals and reactive oxygen species (ROS) [16]. The testicular steroidogenic and gametogenic activities are affected by free radical and ROS [17, 18], though their correlation is not well established.

The potential role of dietary antioxidants, as ascorbic acid, tocopherol, b-carotene, etc., to reduce the activity of free radical-induced reactions has drawn increasing attention [19]. Ascorbic acid may prevent cellular degenerative diseases associated with lipid peroxida-tion [20, 21], reduce ROS generation [22] and stimulate steroidogenic activity [23].

2 Materials and methods

2.1 Animals

Thirty sexually mature male Wistar rats, 3 months of age and weighing 13010 g, were used. The animals were kept under standard laboratory conditions (12 hours light: 12 hours dark, 26-28) for a period of 10 days prior to experimentation. Body weights were checked weekly. Animal pellets and tap water were provided ad libitum and the principles of Laboratory Animal Care (NIH publication No. 85-93, revised 1985) were followed throughout the experimental period.

Thirty rats were divided at random into 3 groups of 10 animals each. the two experimental groups were gavaged cyclophosphamide at a dose of 5 mg in 5 ml water/kg/day, which is in correspondance to the therapeutic dose [1,4]. the treatment period was 28 days. The controls were given a similar amount of distilled water. one experimental group was also gavaged ascorbic acid for 28 days at a dose of 25 mg/kg/day 4 h after cyclo-phosphamide. The other two groups were similarly ga-vaged distilled water.

Animals were sacrificed 24 h after the last ascorbic acid treatment by light ether anesthesia. Blood was collected from dorsal aorta using a heparinized syringe with a 21-gauge needle for the preparation of plasma, which was then stored at -20 for hormone assay. The testes, prostate, seminal vesicles and epididymis were dissected, weighed and expressed in g per 100 g body weight. One testis of each animal was used for histological study and the other, for the study of steroidogenic activity, scavenger enzymes and the levels of free radicals.

One testis from each animal was fixed in Bouin's fixative and embedded in paraffin wax. Five mm sections were cut from the middle portion of the testis and stained with hematoxylin-eosin.

The stained slides were examined under a light microscope. The quantitative analysis of the seminiferous epithelium was carried out at stage VII according to the method of Leblond and Clermont [24]. The cells present in this stage are spermatogonia-A (ASg), prelep-totine spermatocytes (pLSc), midpachytene spermatocytes (mPSc), step 7 spermatids (7Sd) and step 19 spermatids (19Sd). The different nuclei of the germ cells (except step 19 spermatids, which can not be precisely counted) were counted at 20 round tubular cross-sections in each rat. All the nuclear count (crude counts) of the germ cells were corrected for differences in nuclear diameter by the formula of Abercrombie [25], True count = (Crude countsection thickness) / (section thickness + nuclear diameter of germ cells), and tubular shrinkage by the Sertoli cell correction factor [26]. Stage VII of seminiferous epithelial cycle was selected for the quantitative study of spermatogenesis because all varieties of germ cells are present [27] and this stage is T dependent[23].

The testicular D⁵, 3b-HSD activity was measured as described by Talalay [28]. The testis was homogenized at 4 in 20 % spectroscopic grade glycerol (BDH Chemical Division, Mumbai, India) containing 5 mmol potassium phosphate (Loba Chemical Company, Mumbai, India) and 1 mmol EDTA (Organon, Calcutta, India) at a tissue concentration of 100 mg/mL homogenizing mixture and centrifuged at 10,000 g for 30 min at 4. One ml of the supernatant was mixed with 1 mL of 100 mmol sodium pyrophosphate buffer (pH-8.9, Loba Chemical Company, Mumbai, India), 40 mL of 0.3 mmol dehydroepiendosterone (DHEA) (Organon, Calcutta, India) and 960 mL of 25 mg % BSA, making the incubation mixture 3 mL in toto. The enzyme activity was measured after the addition of 0.5 mmol of NAD to the tissue supernatant in a spectrophotometer cuvette at 340 nm against a blank (without NAD). One unit of enzyme activity is the amount causing a change in absorbance of 0.001/min at 340 nm.

The activity of testicular 17b-HSD was measured according to Jarabak, et al [29]. The same supernatant prepared for the assay of D⁵, 3b-HSD (above) was used. One ml of the supernatant was mixed with 1 mL of 440 mmol sodium pyrophosphate buffer (pH-10.2, Loba Chemical Company, Mumbai, India), 40 mL of 0.3 mmol T (Sigma, MO, USA) and 960 mL of 25 mg % BSA, making the incubation mixture 3 mL in toto. The enzyme activity was measured after the addition of 1.1 mmol of NAD to the supernatant in a spectrophotometer cuvette at 340 nm against a blank. One unit of enzyme activity is equivalent to a change in absorbance of 0.001/min at 340 nm.

The testicular catalase activity was measured as per Beers and Sizer [30]. Testicular tissue was homogenized in an ice cold medium containing 0.05 mol tris HCl buffer at a tissue concentration of 20 mg/mL. The mixture was centrifuged at 10,000 g for 20 min at 4. in a spectrophotometer cuvette, 0.5 mL hydrogen peroxide solution (100 mL of 30 % H₂O₂ + 99 mL double distilled water) and 2.5 mL double distilled water were mixed well and read at 240 nm. Then 40 mL of the supernatant was added, mixed well and six reading were taken at 30-second intervals.

Testicular peroxidase activity was determined by the procedure of Sadasivam and Manickam [31] with a slight modification. Testicular tissue was homogenized in a medium consisting of equal parts of 0.9 % saline and 0.1 mol sodium phosphate buffer (pH 7.4) with a tissue concentration of 100 mg/mL. The homogenate was centrifuged at 10,000 g for 15 min at 5. in a spectrophotometer cuvette, 0.1 mL of the supernatant was mixed with 3 mL of phosphate buffer (pH 7.4) and 0.05 mL of guaiacol solution at 25. The reading was taken causing a change in absorbance 0.05/min at 436 nm. After this initial reading, 0.3 mL of H₂O₂ solution (0.042 %) was immediately added to the cuvette, mixed well and read at the same wave length. As the absorbance reached 0.05, a stopwatch was started and noted the time (Dt in min) required to increase the absorbance by 0.1.

2.9 Testicular conjugated dienes

Testicular conjugated dienes were measured according to the method of Slater [32]. Testicular tissue was homogenized in 0.1 mol phosphate buffer (pH 7.4) containing 0.1 mol Na₂HPO₄ and 0.1 mol anhydrous NaH₂PO₄ at a tissue concentration of 50 mg/mL. Lipids of the homogenizing mixture (0.5 mL) were extracted with chloroform-methanol (2:1) mixture. It was then centrifuged at 1000 g for 5 min at room temperature. Chloroform was evaporated under a stream of nitrogen and the lipid residue was dissolved in 1.5 mL cyclohexane (Marck, Calcutta, India). The absorbance of this dissolved conjugated diene was read spectrophotometrically at 233 nm.

2.10 Testicular malondialdehyde ( MDA )

MDA of the testis was measured by the method of Ohkawa et al [33]. Testicular tissue was homogenized in the same way as in 2.9. The homogenizing mixture (0.5 ml) was mixed with 0.5 mL 0.9 % saline and 2 ml of TBA-TCA mixture (0.392 gm thiobarbituric acid in 75 mL of 0.25 N HCl with 15 gm trichloroacetic acid, volume up to 100 mL by 95 % ethanol) and boiled for 10 min. Cooled to room temperature and centrifuged at 4000 rpm for 10 min. The supernatant was placed in a spectrophotometer cuvette and read at 535 nm.

2.11 Plasma T

Plasma T was measured with the immunoenzymatic method according to the protocol described by Srivastava [34]. Here we followed the commercially available competitive solid phase enzyme immunoassay. After incuba-tion, bound/free enzyme labeled antigen separation was performed by simple solid-phase washing. The substrate of enzyme, H₂O₂ and the chromogen (TMB) were added and after the scheduled time the enzyme reaction was stopped by the stop solution supplied by IBL (USA). The T concentration in the sample was calculated based on the five standards supplied by IBL. The absorbance of the standards and sample was monitored against the blank at 450 nm. The cross-reaction of the T antibody to dihydrotestosterone is 10 % and the intra-run precision had a coefficient of variation of 6.2 %.

2.12 Statistical Analysis

Statistical analysis was performed with the analysis of variance followed by a multiple two-tailed t-test [35]. One-way ANOVA is used to investigate the effects of single independent variable on the dependent variable. Differences were considered significant when P<0.05.

3 Results

As can be seen from Table 1, there is no significant difference in body weight increase among the three groups, while the relative weights of the testis and accessory sex organs are significantly lower in the cyclophosphamide-treated animals compared with the controls; co-administration with ascorbic acid in cyclophosphamide-treated animals restores the weights of the testis and accessory organs to the control levels.

Table 1. Effect of ascorbic acid co-administration on body weight and testicular, prostatic, seminal vesicular and epididymal weights in rats treated with cyclophosphamide for 28 days, meanSD, ^bP<0.05, compared with controls; ^eP<0.05, compared with cyclophosphamide-treated (ANOVA followed by multiple t-test).

The testicular D⁵, 3b-HSD and 17b-HSD activities and the plasma T levels were significantly lowered in cyclophosphamide-treated animals compared to the controls (Figure 1, 2). Co-administration of ascorbic acid in cyclophosphamide-treated rats restored the enzyme activities to the control levels and significantly increased the T level (Figure 1, 2).

Figure 1. Effect of ascorbic acid co-administration on testicular D⁵, 3b-HSD and 17b-HSD activities in cyclophosphamide-treated albino rats. Each bar represents meanSD (n = 10), ^bP<0.05, compared with controls; ^eP<0.05, compared with cyclophosphamide-treated ( ANOVA followed by multiple t-test).

Figure 2. Effect of ascorbic acid co-administration on plasma level of testosterone in cyclophosphamide-treated albino rats. Each bar represents meanSD (n = 10), ^bP<0.05, compared with controls; ^eP<0.05, compared with cyclophosphamide-treated ( ANOVA followed by multiple t-test).

Quantitative study on spermatogenesis revealed that the detrimental effect of cyclophosphamide was significantly less in animals co-treated with ascorbic acid. The numbers of ASg, pLSc, mPSc and 7Sd were significantly lowered in the cyclophosphamide-treated rats compared to the controls; with co-administration of ascorbic acid, the numbers of ASg, pLSc, mPSc, 7Sd returned to the control levels (Table 2).

Table 2. Quantitative study on spermatogenesis at stage VII in rats, meanSD, ^bP<0.05, compared with controls; ^eP<0.05, compared with cyclophosphamide-treated (ANOVA followed by multiple t-test).

Testicular peroxidase and catalase activities were significantly decreased in the cyclophosphamide-treated group in comparison to the controls; co-administration of ascorbic acid restored the activities of both enzymes to the control levels (Table 3).

Table 3. Effect of ascorbic acid co-administration on testicular antioxidant enzyme activities and lipid peroxidation of testis in cyclophosphamide-treated rats, meanSD, ^bP<0.05, compared with controls; ^eP<0.05, compared with cyclophosphamide-treated (ANOVA followed by multiple t-test).

The levels of MDA and conjugated dienes were significantly increased after cyclophosphamide treatment and co-administration of ascorbic acid resulted in the restoration to the control values (Table 3).

The study demonstrates the adverse effect of cyclophosphamide on testicular gametogenic and androgenic activities and its protection by ascorbic acid co-administration. Moreover, attempts have been made to study the cyclophosphamide-induced testicular oxidative stress and its correction by ascorbic acid. The decrease in D⁵, 3b-HSD and 17b-HSD activities in cyclophosphamide-treated rat may be the result of a decrease in gonadotrophins or an elevation in testicular conjugated dienes and MDA, as the enzyme activities are reduced in the presence of these free radical products [36]. The elevation in testicular free radicals in cyclophosphamide-treated rats was further supported by the diminution in testicular peroxidase and catalase, important scavenger enzymes against free radicals [37]. The decreased plasma T level in cyclophosphamide-treated rats corresponds to the observation that cyclophosphamide inhibits testicular steroidogenesis [13]. The spermatogenic inhibition in cyclophosphamide-treated rats indicated in the present study may be the result of lowered plasma T level. Besides the hormonal alteration, the spermatogenic inhibition may also be due to the formation of free radical products in the testicular tissue as they exert a detrimental effect on spermatogenesis [13, 17]. Diminution in the accessory gland weights in cyclophosphamide-treated rats also supports the inhibition in testicular androgenesis and pituitary gonadotrophin secretion [10]. As the body growth was not significantly altered in cyclophosphamide-treated rats, the effect of cyclophosphamide on the testis may be due to its specific toxic effect on the target organ and not the result of its general toxicity.

Ascorbic acid co-administration in cyclophosphamide-treated rats resulted in a significant elevation in the activities of testicular D⁵, 3b-HSD and 17b-HSD, which may be due to the direct stimulatory effect of this vitamin on the enzymes [13, 23]. The protection in gametogenic activity after ascorbic acid co-administration in cyclophosphamide-treated rat may be the result of restoration of testicular androgenesis, as androgen is a prime regulator of gametogenesis [38]. It may also be due to the antioxidant effect of ascorbic acid [21, 39, 40] against oxidative stress induced by cyclophosphamide. The latter possibility is supported by the facts that ascorbic acid reversed the testicular MDA and conjugated diene levels and restored the testicular peroxidase and catalase activities.

From the results of this experiment it may be concluded that ascorbic acid co-administration in cyclophosphamide-treated rat has a protective effect on cyclophosphamide-induced testicular androgenic and gametogenic dysfunction. Moreover, ascorbic acid also alleviates the cyclophosphamide-induced oxidative stress. Ascorbic acid may execute its role by modulating testicular free radical production and/or stimulating testicular andro-genesis. As to which one is more important, further investigation is needed. The protective effect of ascorbic acid may have some clinical implication in patients treated with cyclophosphamide in diminishing some of its adverse effects.

The authors gratefully acknowledge the financial assistance from the Major Research Project (Project No. F-3 / 50 / 99 dated 31-3-99) provided by the University Grants Commission (UGC), New Delhi, India.

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Correspondence to: Dr. Debidas Ghosh, Reproductive Endocrinology and Family Welfare Research Unit, department of Human Physiology with Community Health, Vidyasagar University, Midnapore-721 102, West Bengal, India.
Fax: +91-32-226 2329
E-mail: debidasghosh@yahoo.com
Received 2002-04-10      Accepted 2002-08-01