Cadmium-induced damage to primary cultures of rat Leydig cells

Reproductive Toxicology 17 (2003) 553–560

Cadmium-induced damage to primary cultures of rat Leydig cells Jian-Ming Yang a,b,∗ , Marc Arnush b , Qiong-Yu Chen c , Xiang-Dong Wu a , Bing Pang a , Xue-Zhi Jiang a b

a Department of Toxicology, Shanghai Medical University, Shanghai 200032, PR China Department of Immunology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA c Department of Toxicology, Henan Medical University, Zhengzhou 450052, PR China

Received 12 December 2002; received in revised form 3 April 2003; accepted 3 July 2003

Abstract The mechanism of testicular toxicity of cadmium is poorly understood. Previous studies focusing on cadmium-related changes in testicular histopathology have implicated testicular blood vessel damage as the main cause of cadmium toxicity. To further explore the toxic effects of cadmium on testis, we isolated and cultured rat Leydig cells, exposed to 10, 20, and 40 ␮M of cadmium chloride (base doses). After 24 h of exposure, cells and supernatants were harvested to examine cytotoxicity and genotoxicity of cadmium. The results show that both cell viability and concentration of testosterone excretion in primary Leydig cells are significantly lower in cadmium-exposed groups compared to the controls. Changes in testosterone excretion with human chorionic gonadotropin (hCG) stimulation is especially profound. The contents of malondialdehyde (MDA) and the activity of glutathione peroxidase (GSH-Px) in exposed groups are significantly higher than those in the control group, but the activity of superoxide dismutase (SOD) is lower. The number of cells with DNA single strand breaks and the levels of cellular DNA damage in all three exposure groups are significantly higher than in controls. These results indicate that cadmium is directly toxic to primary Leydig cells, and that the decreased percentage of normal cells and the increased level of DNA damage in cadmium-exposed Leydig cells may be responsible for decreased testosterone secretion. © 2003 Published by Elsevier Inc. Keywords: Cadmium; Leydig cell; Viability; Testosterone; hCG; Lipid peroxidation; Comet assay; DNA damage; Rat

1. Introduction Cadmium is widely used in industry, such as in plating, metal smelting, batteries, plastics, and semiconductors [1]. It is an important environmental pollutant and is ranked as the seventh toxic compound in priority by the Agency for Toxic Substances and Disease Registry (ATSDR). Cadmium pollution is increasing along with the development of industry and agriculture [2]. At the beginning of the last century, Alsberg and Schwartze found that testicular necrosis was induced by cadmium [3], but this observation was not taken seriously at that time. In the 1930s, Itai-itai disease in Japan was identified to be associated with cadmium exposure, triggering concerns about the adverse effects of cadmium on human health. Due to a very long biological half-life, cadmium exposure is often associated with significant accumulation in soft tissues [1]. Cadmium is associated with numerous adverse toxic effects, including prostate and lung

∗ Corresponding author. Tel.: +1-858-784-2741; fax: +1-858-784-8805. E-mail address: [email protected] (J.-M. Yang).

0890-6238/$ – see front matter © 2003 Published by Elsevier Inc. doi:10.1016/S0890-6238(03)00100-X

cancer in humans, and damage to the testis, lung, prostate, and hematological system in animals [4,5]. As early as the 1950s, the adverse effect of cadmium on reproduction was reported [6]. Many studies indicate that cadmium induces testicular damage in many species of animals, including mice, hamsters, rabbits, guinea pigs, dogs, and squirrels [7–12]. Bergh indicated that the acute vascular effects of cadmium in the testis do not require the presence of Leydig cells [13]. These findings, based mainly on whole animal experiments and morphological observations, resulted in the theory that this testicular damage was the result of testicular blood vessel toxicity. The exact mechanism, however, remains unclear. Cultured Leydig cells from different mouse strains that have been injected with cadmium have demonstrated changes in androgen levels and in the activities of lactate dehydrogenase, succinic dehydrogenase, 17␤-hydroxysteroid dehydrogenase and NADP-diaphorase. For example, KP mice that are treated with cadmium demonstrate a marked decrease of activity of all studied dehydrogenases and a decrease in the androgen level, whereas the CBA strain which is resistant to the toxic effects of cadmium show no

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differences in measured enzyme activity and endocrine function of gonads [14]. This in vivo cadmium exposure may involve a different mechanism compared to in vitro studies. Waalkes and Poirier found that cadmium can be taken up into Leydig cells by a transport system, which supports our hypothesis that Leydig cells may be a direct target of cadmium on male reproduction beyond Leydig cell tumors [15]. There was once considerable disagreement about whether the testis contains metallothioneins (MTs), a group of cysteine-rich heavy metal binding proteins, which play an important role in detoxifying heavy metals. Japanese researchers successfully isolated and identified cadmiumbinding proteins with sequences identical to those previously described for MT-1 and MT-2 in rat testes [16]. Rat Leydig cells are not only able to synthesize MT under basal conditions, but they also respond to exogenous cadmium by increased synthesis of MT (35 S-cysteine incorporation) and an increased MT-mRNA content [17]. McKenna et al. reported that low-dose cadmium exposure resulted in the increase of MT mRNA but not MT synthesis [18]. Thus, the testes of rats indeed contain MTs. Zn-induced synthesis of MT was once thought to account for tolerance to cadmium in most tissues, but Waalkes and Perantoni indicated that toxicokinetic alterations leading to reduced cadmium accumulation may play an important role in Zn induction of tolerance to cadmium carcinogenesis in the testis [19]. Intracellular MT is protective for cadmium exposure, whereas extracellular cadmium containing MT might be toxic [20]. Liu et al. recently indicated that the sensitivity of cadmium-induced testicular injury in mice is dependent on genetic strain, not MT genotype [21]. Cadmium is a strong inducer of lipid peroxidation and is potentially genotoxic. Koizumi and Li reported that carcinogenic dose of CdCl2 (i.e. 30 ␮mol/kg) induced oxidative stress that may play an important role in the initiation of carcinogenesis within the target cell population [22]. Chang et al. observed that increased lipid peroxidation was associated with chromosomal aberrations in animals exposed to cadmium [23]. However, Wang et al. reported no observed changes in lipid peroxidation in testicular tissue after mice were injected with cadmium at a dosage of 0.23 mg/kg body weight [24]. The action of lipid peroxidation is implicated in the pathogenesis of many diseases, and antioxidative action plays an important role in maintaining normal body function [25]. Since studies of the lipid peroxidation and genotoxicity of cadmium are controversial [26], further studies are warranted to understand the relationship between cadmium and lipid peroxidation as well as its potential genotoxicity. Therefore, we evaluated antioxidative action, the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and lipid peroxidation with the content of malondialdehyde (MDA) in Leydig cells. The integrity of Leydig cell DNA is vital for spermatogenesis. The regulation of spermatogenesis is dependent upon a complex hormonal system, but directly requires testosterone. Leydig cells are the testicular cell type primarily responsible

for androgen synthesis and secretion. Cadmium reduced circulating testosterone [27] and also damaged testicular blood vessels [28], suggesting a possible role of testicular blood vessel toxicity as the mediator of this impaired testosterone secretion. However, this does not exclude the possibility of direct toxicity of cadmium to Leydig cells. Testosterone secretion by Leydig cells is controlled by luteinizing hormone (LH) from the pituitary, with the latter receiving stimulation from gonadotropin-releasing hormone (GnRH) from the hypothalamus. Secretion of testosterone therefore involves complex regulation by the hypothalamic–pituitary–testicular axis. Laskey et al. observed that decreased testosterone levels appeared before morphological changes in testis from rats injected subcutaneously with 0.1 mg/kg cadmium chloride [29]. The decease in testosterone occurred before increases of low molecular weight protein and alkaline phosphorylase in urine, indicating that changes in testosterone levels occur earlier than kidney damage [30]. Isolation and culture of Leydig cells in vitro is very useful to the investigation of chemicals that may affect or interfere with regulatory mechanisms of synthesis and secretion of testosterone and spermatogenesis. The present study investigated the effects of cadmium on cultured primary rat Leydig cells, and its possible mechanism of testicular toxicity. 2. Materials and methods 2.1. Animals and materials Male Sprague–Dawley rats weighing 200 ± 20 g (9 weeks old) were purchased from Sino-British SIPPR/BK laboratory animal Co., China. M199 medium, bovine serum albumin, soybean trypsin inhibitor, RPMI 1640 medium, nitroblue tetrazolium (NBT), etiocholan-3␣-ol-17-one, dimethyl sulfoxide (DMSO) and ␤-nicotinamide adenine dinucleotide (␤-NAD) were obtained from the Sigma Co. (St. Louis, MO). Hank’s balanced salt solution (HBSS), Dubecco’s phosphate-buffered saline (DPBS), and Agarose were purchased from GIBCO-BRL (Life Technologies). Percoll was from Pharmacia Co., Sweden. Polybatum-80 was from Da Zhong Pharmaceutical Co., China. Cadmium chloride, ethiodium bromide, and sodium sarcosinate were from Shanghai Chemical Reagent Co., China. Human chorionic gonadotropin (hCG), trypsin, Trypan blue dye, trypsin, and collagenase were obtained from Sino-American Biotech Co., China. Sodium hydroxide, sodium chloride, Tris(hydroxymethyl) aminomethane, and Na2 EDTA were from FisherBiotech (Fisher Scientific). 2.2. Leydig cell isolation 2.2.1. Preparation of testicular cells Testes were sterilely obtained from Sprague–Dawley adult male rats. The arteries of the testis were perfused with flushing collagenase solution (containing M199 medium,

J.-M. Yang et al. / Reproductive Toxicology 17 (2003) 553–560

0.1% bovine serum albumin (1 g/l), 25 mg/l soybean trypsin inhibitor, and 1.0 mg/ml collagenase) to remove blood. Testes were decapsulated and transferred to 50 ml centrifuge tubes with 5 ml of M199 media in a 34 ◦ C water bath for 15 min. Five milliliters of collagenase solution (containing M199 medium, 0.1% bovine serum albumin, 25 mg/l soybean trypsin inhibitor, and 0.5 mg/ml collagenase) was added to each tube and shaken at 90 cycles per minute at 34 ◦ C for 15 min and then filtered through medium size Nitex filters. The cellular suspension was diluted twofold to threefold with M199 media and centrifuged at 250 × g at 4 ◦ C for 10 min; the supernatant was discarded and the cells were resuspended in M199 media. 2.2.2. Isolation of Leydig cells Ninety percent stock Percoll containing 90 ml of Percoll and 10 ml of 10 × HBSS was mixed with different volumes of M199 media to make 5, 30, 58, and 70% Percoll gradients. The cellular suspension was loaded onto the preformed Percoll discontinuous gradient carefully and centrifuged for 30 min at 800×g at 4 ◦ C. Four cell bands were visible: three distinct cell bands and a fourth opaque band which contained the Leydig cells. All cell bands were carefully removed and resuspended in 5 ml RPMI 1640 culture media. 2.3. Staining for β-hydroxysteroid dehydrogenase (β-HSD) The purity of isolated Leydig cells was ascertained by staining for ␤-HSD activity as previously described [31]. 2.4. Leydig cell culture and treatment Leydig cells were plated in 24-well culture dishes, 2 × 105 cells per well, and placed in a 5% CO2 incubator (Hirasawa Works, Japan) at 34 ◦ C for 2 h. After incubation, most of the Leydig cells were attached to the substrate of the culture dishes. The media were changed and cadmium was added at a final concentration of 10, 20, or 40 ␮M, respectively. The doses designed were referring to the LC50 of Leydig cells, 19.6 ␮M CdCl2 [32]. At the same time, 1 U of hCG was also added to half of the wells. After 24 h, media were collected for analysis of indices of lipid peroxidation, and the levels of testosterone. Leydig cells were harvested to assay cell viability and DNA single strand damage. Each treated group and the control group contained six parallel wells and all experiments were repeated three times. 2.5. Lipid peroxidation assay The activity of SOD was determined using The Institute of Nanjing Jiancheng Biological Product, China kit according to the instructions supplied with the kit. The content of MDA was measured by a thiobarbituric acid reactive substance assay [33]. The determination of GSH-Px activity was by the method of Curi et al. [34].

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2.6. Cell viability determination Cell viability was determined using the Trypan blue dye exclusion test. The cells were removed from the plate with 0.2% trypsin, and 50 ␮l of the diluted cell preparation was combined with 50 ␮l of the 4% Trypan blue dye solution. The cells were counted using a hemocytometer under an Olympus microscope (Olympus 1M, Japan). 2.7. Testosterone radioimmunoassay Testosterone in the media was measured by specific radioimmunoassay as described in the manual of the World Health Organization collaborating center for research and reference services in the immunoassay of hormones in human reproduction [35]. 2.8. Assessment of DNA damage The single cell gel electrophoresis assay (SCGE) or comet assay was used for the detection of single strand DNA breaks in individual cells. The method has been modified from that reported by Gutierrez et al. [36] as follows. 2.8.1. Preparation of microgels Slides were cleaned with acid wash, ethanol treated, and coated with 75 ␮l of 1% agarose, a cover glass (22 mm × 22 mm) added and were allowed to stand for about 5 min at 4 ◦ C. Ten microliters cell suspension (about 1 × 104 viable cells that detached with 0.2% trypsin) and 70 ␮l 0.5% low-melting agarose were mixed and added to the first gel layer after removing the cover glass. A third layer of 1% agarose gel was then added. Slides were immersed in cold lysis buffer (2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Tris, pH 10, 1% sodium sarcosinate, 1% Triton X-100, and 10% DMSO) for at least 1 h at 4 ◦ C. 2.8.2. Single cell gel electrophoresis Slides were arranged on the electrophoresis chamber and fresh electrophoresis buffer (1 mM Na2 EDTA and 300 mM NaOH, pH 13.5) was added. After standing for 20 min at 4 ◦ C, electrophoresis was performed for 20 min at 24 V at 4 ◦ C. All procedures were performed in the absence of strong light. Neutralization and washing steps in 0.4 M Tris–HCl buffer, pH 7.5, were performed three times for 5 min each. 2.8.3. Dye DNA and photograph Forty micrograms per milliliter ethidium bromide was applied to dye DNA and 50 randomly selected cells were counted per slide. Using a fluorescence microscope (Olympus, Japan) with wavelengths of 535 and 590 nm, a photograph of a representative image of each slide was taken. The standard of classifying DNA single strand breaks was based on the percentage of DNA in the comet assay tail by visual estimation. Observations to the treatment groups and the controls were blinded. Zero indicated “no DNA damage” if

J.-M. Yang et al. / Reproductive Toxicology 17 (2003) 553–560

2.9. Statistical analyses Mean±standard errors were calculated from at least three replicate determinations of the endpoints examined, including cell number, cell viability, MDA, SOD, GSH-Px, and testosterone. All data were analyzed using one-way ANOVA among the groups and Student’s t-test was applied between each exposed group and the control group. The analysis of Mantel–Haenszel chi-square was carried out to compare the rate of comet in Leydig cells among various groups. Differences were regarded as significant when the P value was less than 0.05. 3. Results 3.1. Cell purity The Leydig cell-rich fraction obtained in the Percoll gradients contained more than 95% Leydig cells, while the other three bands contained less than 5% (Fig. 1A). The testosterone concentrations in the Leydig cell bands, with or without hCG, were significantly higher than those in other bands. This was especially obvious with hCG stimulation of Leydig cells (Fig. 1B).

120

Cell number (%)

the percentage of DNA in the tail was less than 5% (Fig. 5, Panel A); 1 was “very low DNA damage” if the percentage of DNA in the tail was 5–20% (Fig. 5, Panels B and C); 2 was “light DNA damage” if the percentage of DNA in the tail was 21–40% (Fig. 5, Panel D); 3 was “medium DNA damage” if the percentage of DNA amount in the tail was 41–95% (Fig. 5, Panel E); 4 was “heavy DNA damage” if the percentage of DNA in the tail was more than 95% (Fig. 5, Panel F). The rate of comet (percentage of DNA damage cells in 300 viable cells) also was used to assess the effects of cadmium chloride on DNA damage of Leydig cell.

80 60 40 20 0

Crude Band 1Band 2Band 3 Band 4 40 35

hCG+T

30

T

**

25 20 15 10

*

5 0

Crude Band 1Band 2Band 3Band 4

(B)

Fig. 1. (A) ␤-HSD staining in Leydig cells. Crude cells contained 11.9% Leydig cells, Bands 1, 2, 3 contained 3.5, 0.4, and 0.6% Leydig cells, respectively, and Band 4 contained more than 95% Leydig cells. (B) The concentrations of testosterone in the Leydig cell bands with and without hCG were 30.3 and 6.6 ng/ml, respectively, which were significantly higher than those in Bands 1, 2, 3, and in crude cells. T represents Leydig cell-rich fraction.

activities of SOD in all three exposed groups were significantly lower than in the control (Fig. 3B). Correlation analysis showed a negative correlation between SOD and MDA (r = −0.2015, P > 0.05), a significant negative correlation between SOD and GSH-Px (r = −0.4339, P < 0.05), and a

Cell viability (%)

3.2. Cell viability Leydig cell viability was assessed by means of Trypan blue dye elusion test at 24 h after exposure to cadmium. The cell viabilities in the 10, 20, and 40 ␮M groups were 78.1, 52.7, and 32.6%, respectively, which were significantly lower than in the control group (96.5%) (P < 0.01). The cell viabilities in the 40 and 20 ␮M groups were markedly lower than those in the 20 and 10 ␮M groups, respectively (P < 0.001). There was an obvious dose–response relationship between cell viability and cadmium dosage (Fig. 2).

**

100

(A)

Testosterone (nM)

556

100 90 80 70 60 50 40 30 20 10 0

**

**

**

C

10µM

20µM

40µM

3.3. Lipid peroxidation The MDA content and the activity of GSH-Px was elevated in response to increased cadmium dose (Fig. 3A and C), and the MDA content in the 40 ␮M group was significantly higher than in the control group (Fig. 3A). The

Fig. 2. Assessment of cell viability with Trypan blue dye exclusion testing at 24 h after exposure to cadmium. The cell viabilities in the groups exposed to higher levels of cadmium were significantly lower than in the groups exposed to less cadmium. The decreased cell viabilities were cadmium dose dependent. (∗∗ ) presents P < 0.01 (compared to the control group).

J.-M. Yang et al. / Reproductive Toxicology 17 (2003) 553–560 25

3.5 *

Testosterone (nM)

MDA (µmol/ml)

3 2.5 2 1.5 1

20 **

15 10

aa **

5

**

0.5 0 (A)

**

bb bb aa aa ** **

0

C

10µM

20µM

**

**

40µM

500 450 400

SOD (NU/ml)

557

**

350 300 250 200 150

C

10µM

20µM

40µM

Fig. 4. Comparison of the effects of different cadmium doses on the secretion of testosterone by Leydig cells in vitro. This figure shows the testosterone concentrations following exposure to cadmium for 24 h compared to the control group. The light bars represent treatment without hCG and the black bars represent treatment with hCG. (∗∗ ), aa, and bb represent control, 10, and 20 ␮M cadmium groups, respectively (P < 0.01). There was a dose–response relationship between the secretion of testosterone and the dose of cadmium.

100 50 0

(B)

C

10µM

20µM

40µM

**

*

**

35

GSH-Px (µmol/min)

30 25 20 15 10

munoassay. Cells were grown in RPMI 1640 culture media with and without hCG stimulation. The concentrations of testosterone in three cadmium-exposed groups, with and without hCG, were significantly lower than in the control group (P < 0.01). The concentrations of secreted testosterone in the 40 ␮M group without hCG and in the 20 and 40 ␮M groups with hCG were significantly lower than in the 10 ␮M group; and the concentrations of testosterone in the 40 ␮M group with and without hCG were markedly lower than in the 20 ␮M group. The effects of cadmium on testosterone secretion ability were dose dependent (Fig. 4). 3.5. DNA damage

5 0

(C)

C

10µM

20µM

40µM

Fig. 3. Comparison of MDA content, the activity of GSH-Px, and the activity of SOD between the cadmium-exposed groups and the control group. The units of MDA, SOD, and GSH-Px were ␮mol/ml, NU/ml, and ␮mol/min, respectively. (A) The content of MDA in the 40 ␮M group was significantly higher than that in the control group (P < 0.05). The contents of MDA in the 10 and 20 ␮M groups were higher than in the control but without statistical significance. (B) The activities of SOD in all three exposed groups were significantly lower than in the control (P < 0.01). (C) The activities of GSH-Px in the 10, 20, and 40 ␮M groups were significantly higher than in the control group, and the increased activity of GSH-Px was dose dependent.

significant positive correlation between MDA and GSH-Px (r = 0.4329, P < 0.05). 3.4. Testosterone secretion To explore the mechanism of cadmium’s toxicity in Leydig cells, the androgen secreting ability of Leydig cells 24 h after cadmium exposure was determined using radioim-

The mean cell numbers showing no DNA damage by comet assay in all three cadmium-exposed groups were significantly lower than in the control group, and the mean cell numbers with no damage in the 40 ␮M group were markedly lower than in the 10 ␮M group. The mean cell numbers of low DNA damage in the 10 and 20 ␮M groups were significantly higher than in the control group, and the mean cell numbers of low DNA damage in the 40 ␮M group were significantly lower than in the 10 and 20 ␮M groups. The cell numbers of light DNA damage in all three cadmium groups were significantly higher than in the control group. The mean cell numbers of medium DNA damage in all three cadmium groups were significantly higher than in the control group, and were markedly higher in the 40 ␮M group compared to the 10 and 20 ␮M groups. No heavy DNA damage cells were found in the control group, and the mean cell numbers in the 40 ␮M group were significantly higher than in the 10 and 20 ␮M groups (Fig. 6). Fig. 5 shows different levels of single strain DNA damage. There was an obvious dose–response relationship between the cadmium dose and the level and number of cells with DNA damage in Leydig cells (Fig. 6).

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Fig. 5. This figure shows representative images in the classification of DNA damage. The standard of classifying DNA damage is based on the percentage of DNA in tail. Panel A shows no DNA damage and Panels B through F show different levels of single strand DNA breaks. Panel A shows no DNA damage (0), Panels B and C demonstrate very low DNA damage (1), Panel D is light DNA damage (2), Panel E is medium DNA damage (3), and Panel F is heavy DNA damage (4).

Mean cell number

50 45 40 35 30 25 20 15 10 5 0

bb aa **

** * **

* aa **

0

b a

1

**

** **

**

2

b aa **

**

**

C 10µ m 20µ m 40µ m

*

3

The comet rates in three cadmium-exposed groups were significantly higher than in the control group (P < 0.0001), and were markedly higher in the 40 and 20 ␮M groups compared to the 20 and 10 ␮M groups, respectively (P < 0.0001). The relative risk (RR) was more than one in the 40 ␮M group compared to the 20 ␮M group, 20 ␮M group compared to the 10 ␮M group, and 10 ␮M group compared to the control. The increased comet rate was dose dependent (Table 1).

4

4. Discussion

DNA damage levels Fig. 6. Comparison of Leydig cell DNA damage levels among various cadmium-exposed groups and the control group. (∗ ), (∗∗ ) are compared to the control group (P < 0.05, P < 0.01); a, aa are compared to the 10 ␮M group (P < 0.05, P < 0.01); b, bb are compared to the 20 ␮M group (P < 0.05, P < 0.01); 0, 1, 2, 3, and 4 represent no, low, light, medium, and heavy DNA damage, respectively. There was an obvious dose–response relationship between the cadmium dose and the level and number of cells with DNA damage in Leydig cells.

Cadmium is a well-recognized environmental pollutant with numerous adverse health effects. Testis is known to serve as one of the important targets of cadmium. Several mechanisms of cadmium-induced testicular toxicity have been proposed. Lafuente et al. reported increased cadmium accumulation in the hypothalamus, pituitary, and testis, and decreased plasma levels of follicle stimulating hormone

Table 1 Comparison of comet rate of different groups (%) Cd concentration (␮M)

Cell numbera

Comet rate

RR

95% CI

2 χM -H

P

0 10 20 40

300 300 300 300

19.56 57.80 86.07 88.11

2.96 1.25 1.22

2.32–3.76 1.12–1.14 1.13–1.32

101.09 15.80 25.71

0.00000b 0.00007c 0.00000d

a

Represents viable cells. This was one representative observation of three experiments. Comparing to the 0 ␮M group (control group). c Comparing to the 10 ␮M group. d Comparing to the 20 ␮M group. b

J.-M. Yang et al. / Reproductive Toxicology 17 (2003) 553–560

in rats, suggesting a possible effect of cadmium on the hypothalamic–pituitary–testicular axis [37]. Studies using light and electron microscopy revealed cadmium-induced damage in testicular blood vessels, followed by the degeneration of spermatopoietic epithelia [10,38]. These studies led to the theory that cadmium-induced testicular toxicity is secondary to another process, such as testicular blood vessel toxicity. Our results, however, demonstrate that cadmium is directly toxic to primary cultured Leydig cells at a 10 ␮M concentrations. Previous studies using Leydig cell lines questioned the direct testicular toxicity of cadmium. Low doses of cadmium (25–50 ␮M) are not cytotoxic to R2C cells, a rat testicular Leydig cell line [39]. One hundred micromolar cadmium exerted an adverse effect on the viability of isolated rat Leydig cells and testosterone production [40]. Our results demonstrate that the viability of primary Leydig cells exposed to lower doses of cadmium (10–40 ␮M) is significantly decreased, and that the response is dose related. Compared to R2C cells [39], primary Leydig cells appear more sensitive to the toxicity of cadmium. Cadmium is a strong inducer of lipid peroxidation, and also increases the number of chromosomal aberrations through direct or indirect effects, indicating a possible association between lipid peroxidation and genotoxicity [23]. Shaikh et al. found that chronic cadmium administration in rats caused increased lipid peroxidation in liver and kidney tissue [41]. To our knowledge, there is very little literature about the effects of cadmium on oxidative stress and lipid peroxidation in primary Leydig cells. Our results show that there is a significant increase in the content of MDA and activity of GSH-Px. The latter is consistent with the results of in vivo cadmium-exposed animals [42], as well as the reported decrease in the activity of SOD in response to cadmium in primary Leydig cells. There is a clear dose–response relationship between cadmium exposure and both lipid peroxidation intensity (MDA) and the activity of antioxidants (SOD). These results suggest that cadmium induces an increase in lipid peroxidation and a decrease in antioxidant status. The increased activity of GSH-Px may indicate a stress response of Leydig cells to cadmium toxicity. The single cell gel electrophoresis (Comet) assay was originally developed for mammalian cells by Ostling and Johanson [43], and is a simple, effective, and sensitive method for detection of DNA single strand damage. Our results reveal that cadmium induces DNA single strand breaks in Leydig cells in vitro. The level of DNA damage in Leydig cells is correlated with the level of cadmium exposure. These results confirm that 10 ␮M cadmium is directly toxic to primary cultured Leydig cells, and that this toxicity includes DNA damage. These findings different from other previous reports that theorized that carcinogenesis of Cd in the rat testis is triggered by active oxygen species, which are generated by the metal exposure, rather than by a direct genotoxicity of Cd [44]. Zinc pre-treatment can prevent

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cadmium-induced testicular tumors [45]. This may be due to its ability to reduce the cytotoxicity of cadmium in interstitial cells by enhancing efflux of cadmium and decreasing accumulation of cadmium in nuclei of this target cell population. This suggests that cadmium may be directly toxic to Leydig cells. Testosterone is mainly secreted by Leydig cells and spermatogenesis is dependent on the level of testosterone. Our results indicate that cadmium impairs testosterone secretion directly in primary Leydig cells in vitro. This phenomenon is especially obvious after hCG stimulation. The results were similar to the discovery of Laskey and Phelps [46]. It is likely that the decreased testosterone in cadmium-exposed groups results from decreased cells due to cell death, however the decreased numbers of living cells in cadmium exposure groups were far lower than the observed decreases in testosterone levels. Timbrell recently suggested urinary creatine as a biomarker for cadmium-induced testicular damage [47]. However, two reports have previously shown that the changes in testosterone secretion in response to cadmium occur earlier than the changes in testis morphology and kidney damage [29,30], suggesting that testosterone might be a more sensitive index for assaying testicular toxicity of cadmium. Our results also suggest that measuring testosterone levels may be useful in assaying such damage. In summary, this paper report that cadmium is directly toxic to primary cultured Leydig cell in vitro. This toxicity includes reduced cell viability and testosterone secretion, increased lipid peroxidation, decreased antioxidative ability, and DNA damage. Although previous reports suggested that testicular toxicity is secondary to vascular damage, we revealed a significant effect of cadmium directly on the essential testosterone secreting cell type in the testis. This may play a role in the understanding of the male reproductive toxicology of cadmium.

Acknowledgments We thank Drs. Ding Xuncheng, Wu Xiaoyun, and Gu Dunyu (National Evaluation Center for the Toxicology of Fertility Regulating Drugs, Shanghai, China) for their help in cell culture. We thank Dr. K. Michael Pollard (The Scripps Research Institute, La Jolla, CA 92037) for his suggestions and help in the discussion.

References [1] Friberg L, Kjellstrom T, Nordberg GF. Cadmium. In: Friberg L, Nordberg GF, Vouk V, editors. Handbook on the toxicology of metals. Amsterdam: Elsevier; 1986. p. 130–84. [2] ICPS, editor. Environmental health criteria 134. Cadmium. Geneva: World Health Organization; 1992. [3] Alsberg CL, Schwartze EW. Pharmacological action of Cd. Pharmacology 1919;13:504–10.

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