Ketoconazole-induced testicular damage in rats

ARTICLE IN PRESS

Experimental and Toxicologic Pathology 59 (2008) 377–384 www.elsevier.de/etp

Ketoconazole-induced testicular damage in rats reduced by Gentiana extract Amr Amin,1 Biology Department, UAE University, P.O. Box 17551, Al-Ain, UAE Received 4 October 2007; accepted 31 October 2007

Abstract Ketoconazole (KET) is an antifungal drug with a broad spectrum of activity that also induces reproductive toxicity in humans and animals. The protective effect of Gentiana (GEN) extract (Gentiana lutea) against KET-induced testicular damage was evaluated in male Wistar rats. GEN extract was administered orally (1 g/kg b wt/day) for 26 days. Three weeks after extract administration, KET was co-administered intraperitoneally at a dose of 100 mg/kg once a day for 5 days. KET-induced reproductive toxicity was associated with clear reductions of the weights of testes and epididymides, sperm indices and serum testosterone levels. KET also induced severe testicular histopathological lesions such as degeneration of the seminiferous tubules and depletion of germ cells. In addition, marked oxidative damage to testicular lipids and alterations of natural antioxidants (catalase (CAT) and superoxide dismutase (SOD)) were reported in association with KET toxicity. Most of the KET-induced effects were greatly decreased with the concomitant application of GEN extract. This study suggests a protective role of GEN extract that could be attributed to its antioxidant properties. r 2007 Elsevier GmbH. All rights reserved. Keywords: Gentiana; Ketoconazole; Testicular damage; Antioxidant properties

Introduction Ketoconazole (KET) is an antifungal drug that is a member of imidazole drug family (Kinobe et al., 2006). KET is also used as an anticancer agent in the treatment of advanced prostate cancer (Rodriguez and Acosta, 1995). KET has been shown to induce a dose-dependent decrease in serum testosterone levels in patients (Pon, 1987) and in rats (English et al., 1986; Adams et al., 1998). N-deacetyl KET is the major metabolite which Tel.: +971 3 7134381; fax: +971 3 7671291.

E-mail address: [email protected]. Permanent address: Department of Zoology, Faculty of Science, Cairo University, Egypt. 1

0940-2993/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2007.10.008

undergoes further metabolism by the flavin-containing mono-oxygenase to form a potentially toxic reactive metabolite (Rodriguez et al., 1999). It was reported that KET inhibits C17-20 lyase which blocks the conversion of 17 /aS-hydroxyprogesterone to andostenedione (Santen et al., 1983). KET was also found to decrease serum testosterone levels with no significant increase in serum luteinizing hormone (LH) levels (Adams et al., 1998). These results suggest that KET suppresses negative feed back mechanisms in the pituitary by suppressing regulatory changes in LH and follicle stimulating hormone (FSH) secretion. Oligospermia and azoospermia have been reported at the therapeutic doses of KET (Marwaha and Maheshwari, 1999). Our earlier study revealed that KET-induced liver damage

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A. Amin / Experimental and Toxicologic Pathology 59 (2008) 377–384

was associated with oxidative stress damage and antioxidant depletion in rat livers, which suggests that free radicals may be responsible for the hepatotoxicity induced by KET (Amin and Hamza, 2005). The protective effects of phenolic antioxidants extracted from medicinal plants against oxidative stressmediated disorders have been reported (Soobrattee et al., 2005). The pharmacological actions of phenolic antioxidants stem mainly from their free radical scavenging and metal chelating properties as well as their effects on cell signaling pathways and on gene expression (Urquiaga and Leighton, 2000; Soobrattee et al., 2005). GEN roots are used in ayurvedic medicine to treat fevers, jaundice and other types of liver damage (Duke, 1988). Recently, the hepatoprotective effects of other GEN species (Gentiana olivieri) were investigated in a carbon tetrachloride-induced liver injury in an animal model (Orhan et al., 2003). Several photochemical ingredients were isolated from GEN roots such as the alkaloid gentianine (Duke, 1988), gentiopicroside, swertiamarine, sweroside and secoiridoids (Ozturk et al., 2006). In addition to these active substances, isoorientin (C-glycosylflavone) and gentiopicroside (secoiridoid glycoside) were isolated from different species of Gentiana and were shown to have antioxidant properties (Ko et al., 1998; Kumarasamy et al., 2003). The present investigation was set to evaluate the protective effects of GEN extracts against KET-induced acute testicular toxicity in male rats and to study the mechanisms underlying these effects. This study assesses sensitive biomarkers of testis toxicity such as weights of testes and epididymides, histopathological changes, sperm analysis data and serum concentrations of sexual hormones. Oxidative stress and antioxidant potential were also addressed by the determination of reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) enzyme activities and malondialdehyde (MDA).

University, UAE. They were maintained on standard pellet diet and tap water ad libitum and were kept in polycarbonate cages with wood chip bedding under a 12 h light/dark cycle and room temperature of 22–24 1C. Rats were acclimatized to the environment for 1 week prior to experimental use. Animals were treated following the UAEU Animal Research Ethics Committee guidelines.

Extraction To increase the yield of extraction in a shorter time and at a lower temperature, the liquid-phase microwaveassisted process was used for extraction of GEN according to the method described by Pan et al. (2001). These microwave-assisted extraction applications are based upon the selective heating of the matrix that contains the target extract when the matrix is immersed in a solvent such as ethanol and water, which is transparent to the microwaves. This solvent allows for selective heating of particular components within the materials being treated, without using excessive energy. About 100 g of GEN dried roots were mixed in 1000 ml of 70% ethanol. Every 10 g of ground herb was mixed with 100 ml of 70% ethanol in a 250-ml conical flask. The mixtures were then irradiated in a 300 W microwave for 2 min. The suspension was irradiated for 25 s with power on to give the desired temperature of about 80 1C, for an additional 5 s after the desired temperature was achieved, with power on for heating and then cooled in the microwave for 10 s. The extract was finally filtered through gauze and evaporated under vacuum at 40 1C using a rotary evaporator. The dried extract was dissolved in 2.5% dimethyl sulfoxide (DMSO) before animal administration.

Treatment regime

Materials and methods Chemicals KET (Nizoral) was purchased from Janssen-Cilag pharmaceutical company (Beerse, Belgium). The dry roots of GEN were purchased from local markets. Thiobarbituric acid, Folin’s reagent, epinephrine, SOD enzyme, H2O2, and bovine albumin were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were purchased from local commercial suppliers.

Animals Adult male albino rats (150–200 g) of the Wistar strain were obtained from the Animal House, UAE

The solution of KET was protected from light in saline solution and was administered to animals at a volume of 1 ml/100 g b wt. The control animals and the protected animal group received an equivalent volume of saline based on body weight. The GEN extract was given orally by gavage at a concentration 1 g/kg of body weight. Rats were randomly divided into four groups (n ¼ 6 males) and were subjected to one of the following treatments: animals of the KET-treated group were given daily 2.5% DMSO for 26 days and after 3 weeks of DMSO treatments, the rats were injected with daily intraperitoneal dose of KET (100 mg/kg b wt) for 5 days. This toxic dose of KET has been used previously to induce oxidative stress in the livers of male Wistar rats (Amin and Hamza, 2005). Animals of the protected group were treated daily for 26 days with GEN extract and after 3 weeks of extract administration, were

ARTICLE IN PRESS A. Amin / Experimental and Toxicologic Pathology 59 (2008) 377–384

injected daily with the same dose of KET for 5 days. Rats from the control group were treated daily with an equivalent volume of DMSO for 26 days and were injected with saline after 21 days of DMSO administration for 5 days. Animals from the GEN group were treated daily for 26 days with GEN extract and after 3 weeks of extract administration, were injected daily with the same volume of saline solution (1 ml/100 g) for 5 days. Twenty-four hours after their last treatment with KET, herbal extract or vehicle solution, blood and organ samples were collected from animals in each group.

Sample preparation Following diethyl ether anesthesia, blood was collected from the retro-orbital plexus. Following the sacrifice, testes and epididymides were removed and weighed. The percentage of organ weight/body weight was calculated and expressed as a relative organ weight. For histopathological examinations, the left testis was immediately fixed in 10% buffered formalin. The right testis was homogenized in ice-cold KCl (150 mM) for further biochemical analyses. The ratio of tissue weight to homogenization buffer was 1:10. Then further dilutions of testis homogenates were made in suitable buffers to determine levels of GSH, MDA and total proteins and to assess the activity of SOD. Serum was prepared by collecting blood in centrifuge tubes and spinning them in a refrigerated centrifuge (4 1C) at 3000 rpm for 20 min. Serum samples were then used for the determination of testosterone, LH and FSH concentrations.

Sperm motility and count Epididymides were dissected out, weighed, immediately minced in 5 ml of physiological saline and then incubated at 37 1C for 30 min to allow spermatozoa to leave the epididymal tubules. The percentage of motile sperm was recorded using a phase contrast microscope at a magnification of 400 . Total sperm number was determined by using a Neubauer hemocytometer by a method of Yokoi et al. (2003). The total number of sperm per gram of epididymides was then calculated. Since epididymal weight varies among rats according to their body weight and age, the epididymal sperm numbers were expressed per gram of epididymides.

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the thiol group of GSH at pH 8.0 to produce 5-thiol-2nitrobenzoate, which is yellow. MDA is the most abundant individual aldehyde resulting from the process of lipid peroxidation (LP) in biological systems and is used as an indirect index of LP (Packer and Cadenas, 2002). Determination of MDA in biological samples is based on its reaction with thiobarbituric acid to form a pink complex with absorption maximum at 535 nm (Uchiyama and Mihara, 1978). CAT activity was determined by measuring the exponential disappearance of H2O2 at 240 nm and expressed as units/mg of protein as described by Aebi (1984). The activity of SOD enzyme in testis homogenate was determined according to the method described by Sun and Zigman (1978). This method is based on the ability of SOD to inhibit the auto-oxidation of epinephrine to adrenochrome and other derivatives at alkaline pH. These derivatives can easily be monitored in the nearUV region of the absorption spectrum. The serum concentrations of testosterone, LH and FSH were determined using enzyme-linked immunosorbent assay (ELISA) kits (Diagnostic Systems Laboratories, Inc. Corporate Headquarters, 445 Medical Center Boulevard. Webster, TX, USA), according to the manufacturer’s instruction. The total protein content of testes was determined by Lowry’s method as modified by Peterson (1977). Absorbance was recorded using a Shimadzu recording spectrophotometer (UV-160) in all measurements.

Histology For the histological examinations, small pieces of testis were fixed in 10% neutral phosphate-buffered (pH 7.4) formalin, dehydrated, embedded in paraffin and then sectioned (5 mm in thickness). Sections were then stained with hematoxylin and eosin and examined under a Leica DMRB/E light microscope.

Statistical analysis SPSS (version 10) statistical program (SPSS Inc., Chicago, IL, USA) was used to carry out a one-way analysis of variance (ANOVA) on our data. When significant differences by ANOVA were detected, analyses of differences between the means of the treated and control groups were performed by using Dunnett’s t-test.

Biochemical analysis

Results

GSH content of testis homogenate was determined using the method described by Van Dooran et al. (1978). This method is based on the reaction of Ellman’s reagent 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) with

Sperm indices The relative weight of testes is the percentage of organ weight to body weight. Treatment with KET



significantly (Po0.001) decreased absolute and relative weights of both testes and epididymides (Table 1). GEN extract pre-treatment followed by GEN/KET co-treatment abolished the decrease in absolute and relative weights of testes and epididymides. The number of spermatozoa per gram of epididymides was significantly (Po0.001) reduced post-treatment with KET (Table 1). GEN pretreatment followed by GEN/KET co-treatment had no effect on this parameter. Sperm motility was reduced (Po0.001) in the KET-treated group. GEN pretreatment followed by GEN/KET co-treatment retained normal sperm motility in the protected group. Rats receiving GEN alone did not show any significant change in the absolute and relative weights of both testes and epididymides as well as in the quality of sperm indices.

Effects on reproductive hormones Serum sex hormones (testosterone, FSH and LH) were monitored in male rats (Table 2). The level of testosterone dramatically dropped in KET-treated rats compared with the control group. In the GEN-only group and the GEN+KET (protected) group, the levels of testosterone did not significantly change from the control value. With respect to FSH and LH levels, there

were no significant changes in their main values in all experimental groups.

Effects on testicular MDA In KET-treated rats, testicular MDA levels (Fig. 1a) were significantly (Po0.001) increased, compared with the control group. In the GEN alone and the GEN+ KET groups, the levels of MDA did not significantly differ from those of the control group.

Effects on antioxidant substances Testes from rats in the KET-treated group showed a significant depletion (Po0.05) in SOD activity (Fig. 2b) and elevation (Po0.05) in CAT activity (Fig. 2a). The level of these markers of oxidative stress did not significantly differ from control level after GEN/KET co-treatment. With respect to GSH content, there was no significant change in the main values from all experimental groups (Fig. 1b).

Histopathology Untreated rats showed mostly normal testicular architecture with an orderly arrangement of germinal cells and Sertoli cells (Fig. 3a). KET treatment induced

Table 1. Effect of GEN extract on absolute and relative weights of testes and epididymides and epididymal spermatozoa concentration and motility in control and KET-treated rats Parameters

Control

GEN

KET

GEN+KET

Testes Absolute weights (g) Relative weights (g/100 g)

2.6670.13 1.2970.01

2.5770.05 1.2770.05

2.0770.07*** 0.9670.05***

2.3970.05 1.2270.02

Epididymides Absolute weights (g) Relative weights (g/100 g)

1.4970.07 0.4970.01

1.4870.04 0.4870.02

0.9770.05*** 0.3270.01***

1.4270.04 0.4670.02

486.6757.5 82.4071.57

460.8730.4 83.2071.36

180.20716.09*** 57.0071.22***

433.20757.89 76.0073.35

Epididymal spermatozoa Concentration (106/g epididymides) Sperm motility (%)

Each column represents the mean7S.E.M. for six rats in each group. ***Po0.001 vs. control.

Table 2.

Effect of GEN extract on serum levels of testosterone, FSH and LH in control and KET-treated rats

Parameters

Control

GEN

KET

GEN+KET

Serum testosterone level (ng/ml) Serum FSH level (mIU/ml) Serum LH level (mIU/ml)

4.3670.33 3.2470.04 0.7470.08

4.2270.22 3.2870.07 0.7470.05

1.5070.66*** 3.0870.02 0.8070.09

4.3870.37 3.1970.03 0.7470.08

Each column represents the mean7S.E.M. for six rats in each group. ***Po0.001.


Testicular MDA(nmol/mg protein)

1.0

***

(a)

Testicular CAT activity (µ/mg protein)

1.1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0

Control

KET

GEN+KET

Testicular SOD activity (µ/mg protein)

Testicular GSH (nmol/mg protein)

200

GEN

(b)

180 160 140 120 100 80 60 40 20 0

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

(a)

2.5

(b)

GEN

KET

GEN+KET

*

GEN

KET

GEN+KET

*

2.0

1.5

1.0

0.5

0.0 Control

Control

381

Control

GEN

KET

GEN+KET

Fig. 1. Effect of GEN extract on testicular (a) malondialdehyde level (MDA) and (b) reduced glutathione content (GSH) in control and KET-treated rats. Each column represents the mean7S.E.M. for six rats in each group. ***Po0.001 vs. control.

Fig. 2. Effect of GEN extract on testicular (a) catalase (CAT) and (b) superoxide dismutase (SOD) activities in control and KET-treated rats. Each column represents the mean7S.E.M. for six rats in each group. *Po0.05 vs. control.

testicular atrophy accompanied by the degeneration of germ cells within the seminiferous tubules (Fig. 3c). The tubules were shrunken and greatly depleted of germ cells. Sertoli cells with few germ cells were observed in the lumen. It is to be noted here that this atrophy was seen in many tubules of treated rats and only in very few of controls. Animals pretreated with GEN showed less degeneration of seminiferous epithelium with shedding of germ cells in few tubules.

count and motility). KET-induced testicular toxicity was further confirmed by histological alteration in the testis. The present histological observations correlate well with results from other reports, where short- and long-time KET treatment have been shown to cause adverse effects on testicular function in the rat (English et al., 1986; Adams et al., 1998) and in humans (Pon, 1987; Marwaha and Maheshwari, 1999). GEN pre-treatment followed by GEN/KET co-treatment significantly maintains normal testicular function and the sperm characteristics. The GEN-treated rats also retained normal testicular and epididymal weights, even when treated with KET. Taken together, the present results demonstrate a remarkable protective effect of GEN extract against KET-induced testicular damage. Reactive oxygen species (ROS) are involved in a variety of pathophysiological conditions of testes (Diemer et al., 2003; Agarwal et al., 2006). Oxidation products of lipids, proteins, and DNA are reliable biomarkers of oxidative stress (Dalle-Donne et al.,

Discussion Reproductive toxicology has been receiving increasing interest and concern in recent years (Mangelsdorf et al., 2003). In this study, the testicular damage induced by KET was shown to be associated with reduction in both testicular and epididymal weights as well as with depletion in the quality of epididymal sperm (sperm



Fig. 3. Photomicrograph of the seminiferous tubules of control (a) and GEN-treated rats (b) showing the normal arrangement of germinal cells and Sertoli cells. Testis of KET-treated rats (c) showing tubular atrophy (asterisks) with extensive degeneration of the germinal epithelium. The atrophic tubules contained degenerated Sertoli cells with few germ cells. However, the GEN pretreated group (d) exhibits less degeneration in some tubules and irregular arrangement of germ cells with shedding of cellular materials from the seminiferous epithelium in some tubules (H&E, X200).

2003). The KET-induced testicular damage in this study was accompanied by elevation in MDA, which is the product of lipid peroxidation. It is possible then that ROS generation and the oxidative damage of lipid membranes mediate KET-induced testicular toxicity. SOD and glutathione peroxidase are major enzymes that scavenge harmful ROS in male reproductive organs (Fujii et al., 2003). In the present investigation, treatment with KET has led to the depletion of SOD levels and the elevation of MDA in testes. The resultant overproduction of ROS could thus lead to oxidative stress. Upregulation of CAT enzyme activity, which catalyzes the removal of hydrogen peroxide (Packer and Cadenas, 2002), could be an adaptive response to the massive production of hydrogen peroxide in the testicular tissues. This oxidative stress damage is consistent with our previous study in which the same acute dose of KET induced massive DNA fragmentation, an increase in lipid peroxidation and GSH and SOD depletion in rat livers (Amin and Hamza, 2005). Currently, it is well accepted that the pro-oxidantinduced oxidative damage can effectively be attenuated by the use of various dietary antioxidants (Soobrattee et al., 2005). Owing to the presence of antioxidant ingredients in Gentiana lutea (Ko et al., 1998), it was interesting to examine whether dietary GEN would offer any significant protection against KET-induced oxidative damage. Indeed, GEN crude extract was able to

prevent the increase in testicular MDA and CAT as well as the depletion in testicular SOD activity induced by KET. These results showed that the antioxidant properties of GEN may contribute to the prevention of KETinduced testicular damage. The treatment with GEN alone showed no effect on the levels of testicular antioxidants, which indicates that GEN does not function through the induction of these antioxidants. GEN might reduce the free radical formation, decompose and quench free radicals. This antioxidant activity of our crude extract was confirmed in this study by ferric reducing antioxidant power (FRAP) assays, which indicate the ability of the GEN extract to exhibit significant reducing power. Several photochemical ingredients were isolated from roots of GEN such gentiopicroside, swertiamarine, sweroside and secoiridoids (Ozturk et al., 2006). Isoorientin (C-glycosylflavone) and gentiopicroside (secoiridoid glycoside) were also isolated from different species of Gentiana and were shown to have antioxidant properties (Ko et al., 1998; Kumarasamy et al., 2003). Treatment with KET significantly decreases serum testosterone without any effect on LH and FSH levels. In male rats, acute KET treatment (10–300 mg/kg) was previously shown to decrease serum testosterone within 24 h without any effect on endogenous LH level (Adams et al., 1998). Normal spermatogenesis depends on the testosterone secretion and gonadotropic hormonal


action of LH and FSH. While LH stimulates testosterone production and secretion by Leydig cells, FSH acts directly on the seminiferous tubules (MacLachlan et al., 2002; Spaliviero et al., 2004). The reported depletion of testosterone may have resulted from the direct effect of KET on Leydig cells. KET may inhibit testosterone secretion in rats, in part, due to (Kinobe et al., 2006) a decreased production of hormone in Leydig cells (Rodriguez and Acosta, 1995), a decreased response of testosterone to gonadotropin, and (Pon, 1987) a reduction of the LH response to GnRH. In the present study, KET-induced reduction of serum testosterone level did not upregulate the level of LH. It is possible that KET reduces hypothalamic–pituitary sensitivity to the feedback control of testosterone on LH secretion. Previous results of Adams et al. (1998) suggest that KET suppresses feedback mechanisms in the pituitary, suppressing regulatory changes in LH secretion. This suppressive effect of KET on testosterone production may be attributed to the direct inhibitory effects of ROS on testicular steroidogenic enzymes. Oxidative stress is known to inhibit testicular steroidogenesis (Diemer et al., 2003; Fujii et al., 2003). GEN administration maintains the control value of testosterone in KETtreated rats. The protective effect of GEN against KETinduced testosterone depletion and oxidative stress damage supports the hypothesis that part of the inhibition of testosterone production is attributed to the overproduction of free radicals. In conclusion, a GEN crude extract has the potential to prevent both the reproductive toxicity and oxidative damage induced in rat testes in response to a high acute dose of KET. These data may provide a potential application for crude ethanolic extract of GEN to protect humans from testicular damage induced by KET. This may eventually lead to a possible development of a combination therapeutic strategy.

Acknowledgment The author is grateful to Ms. Karima Al-Mansouri (Biology Department, UAEU) for her valuable assistance in formatting the manuscript.

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