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Poult Sci 2007. 86:2008-2012
© 2007 Poultry Science Association
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PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION

The Isoflavonoid Daidzein Attenuates the Oxidative Damage Induced by Polychlorinated Biphenyls on Cultured Chicken Testicular Cells

Y. L. Mi, C. Q. Zhang1, W. D. Zeng, J. X. Liu and H. Y. Liu

College of Animal Sciences, Zhejiang University, Hangzhou 310029, China

1 Corresponding author: cqzhang{at}zju.edu.cn


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The soy isoflavonoid daidzein (DAI) is one of the most abundant phenolic compounds in the human diet and in animal feedstuffs. Daidzein possesses a wide spectrum of physiological and pharmacological functions related to human health. The aim of this study was to evaluate the effect of DAI on oxidative damage induced by a potential carcinogen, polychlorinated biphenyl. Testicular cells were dispersed from 18-d-old chicken embryos and exposed to DAI alone or in combination with a mixture of polychlorinated biphenyls, Aroclor 1254 (A1254), in culture. Oxidative damage was estimated by measuring the lipid peroxidation and superoxide dismutase activities and glutathione content. Results showed that DAI (10 µg/mL) increased germ cell numbers, which were inhibited by cotreatment with the estrogen receptor antagonist tamoxifen at 0.1 µg/mL. Exposure to A1254 (10 µg/mL) reduced superoxide dismutase activity and glutathione content but increased lipid peroxidation significantly. However, simultaneous supplementation with 10 µg/mL of DAI restored these parameters. The above results indicated that DAI may exert weak estrogenic activity, and more important, that DAI may display an antioxidant effect to prevent oxidative damage induced by the oxidant A1254.

Key Words: daidzein • antioxidant • polychlorinated biphenyl • oxidative damage • testicular cell


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Daidzein (DAI), a phytoestrogen, is one of the main soy isoflavonoids. Daidzein exhibits a wide range of physiological and pharmacological effects relevant to human health (Honoré et al., 1997; MacDonald et al., 2005). These bioactive benefits are, at least in part, attributed to its antioxidant properties and estrogenic activity. Although DAI can bind to estrogen receptors (ER), its occupancy time and affinity for the receptor are greatly reduced compared with endogenous estrogen (Casanova et al., 1999). As a phytoestrogen, DAI also exhibits some detrimental responses. Soy-derived phytoestrogens can exert modest endocrine disruptor-like effects on reproduction (Wilhelms et al., 2006). Although phytoestrogens are believed to have health implications for humans, the beneficial effects are debatable.

In addition to the estrogenic activity of DAI, it is an effective antioxidant in vivo and in vitro. For instance, DAI exhibited high antioxidant potency in the liposome system (Arora et al., 1998). Isoflavones manifested a potent quenching effect on 8-hydroxy-2-deoxyguanosine formation induced by UV light irradiation (Cai et al., 1997). Because humans and domestic animals consume soybeans and soybean-based products, DAI, a bioactive food component, is subsequently assimilated in relatively large quantities.

Oxidative modification to cells and tissues is thought to be relevant to the development of chronic diseases. Polychlorinated biphenyl (PCB), a persistent organic pollutant, has been used extensively as a commercial product and imposes health hazards on humans and animals. Polychlorinated biphenyls can cause lipid peroxidation, can damage the antioxidant enzymes and antioxidant status, and can cause DNA oxidative damage (Srinivasan et al., 2001; Mi and Zhang, 2005). The natural antioxidant DAI is thought to prevent or inhibit free radical-induced oxidative damage. Our previous study showed that DAI can inhibit oxidative modification induced by reactive oxygen species in ovarian cells (Liu et al., 2006). However, the effect of DAI on testicular cell development still remains unknown.

Aroclor 1254 (A1254), a commercial mixture of PCB, was shown to impose oxidative damage in embryonic chickens and adverse effects on spermatogenesis in adult cockerels (Mi and Zhang, 2005; Zhang et al., 2002). The current study was designed to evaluate the relation between the bioactive food component DAI and the oxidative pollutant A1254 to explore potential dietary prevention of oxidative damage induced by PCB on testicular cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of Testicular Cells and Chemical Treatments

Fertilized Arbor Acres broiler chicken eggs were incubated at 38.5°C and 60% humidity until d 18. Testicular cells were prepared according to the method in a previous study (Mi et al., 2004). Stock solutions of A1254 (lot number 124-191-A, Accustandard Inc., New Haven, CT), tamoxifen (TMX), and DAI (Sigma Co., St. Louis, MO) were prepared in ethanol and dimethylsulfoxide, respectively. Cells were incubated in medium with DAI (0.1 to 10 µg/mL) alone or in combination with A1254 (10 µg/mL) and TMX (0.1 µg/mL). The final concentration of dimethylsulfoxide or ethanol in the medium was ≤0.1%. The control treatment received the vehicle only. Cells were cultured in collagen-treated 24-well culture plates (Costar, Corning Inc., Corning, NY) at a density of 2 x 105/well in a water-saturated atmosphere of 95% air and 5% CO2 at 39°C.

Morphological Studies of Testicular Cells

Morphological changes in testicular cells were observed under an IX70 phase-contrast microscope (Olympus, Tokyo, Japan). The image was transferred with a video camera (Pixera Pro 150ES, Los Gatos, CA) to a computer. The number of germ cells was counted in each image.

Biochemical Determinations

After a 48-h culture, the media and cells were used for biochemical analysis. Lipid peroxidation was evaluated by malondialdehyde (MDA) concentrations with the spectrophotometric measurement of the color produced during the reaction to TBA with MDA (Agostinho et al., 1997). Total superoxide dismutase (SOD) activity was determined by inhibition of the rate of superoxide radical-dependent cytochrome C reduction (Flohé and Otting, 1984). Total glutathione (GSH) was determined by measuring the rate of reduction of 5,5'-dithiobis-2-nitrobenzoate to 2-nitro-5-thiobenzoate (Zakowski and Tappel, 1978).

Statistical Analysis

The experiment was repeated 3 times. All data were expressed as means ± SD and analyzed by ANOVA and Duncan’s multiple range test with the GLM procedure of SAS (version 6.12 software, SAS Inst. Inc., Cary, NC). A P value of <0.05 was considered significantly different.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Estrogenic Effect of DAI

Germ cells were round in shape and somatic cells spread filopodia in the control group after a 48-h culture (Figure 1AGo). Analysis showed that germ cell numbers in the DAI (10 µg/mL) group were significantly increased above those observed with the control group (Figures 1BGo and 2Go; P < 0.05). However, in combination with 0.1 µg/mL of TMX, a distinct pattern of germ cells did not appear and the number elevated by DAI was significantly decreased (Figure 2Go; P < 0.05).


Figure 1
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  		Figure 1. Morphological changes in embryonic chicken testicular cells after daidzein (DAI) and Aroclor 1254 (A1254) treatments for 48 h. Panels indicate testicular cells of the A) control, B) DAI (10 µg/mL), C) A1254 (10 µg/mL), D) DAI + A1254, E) tamoxifen (TMX, 0.1 µg/mL), and F) DAI + A1254 + TMX groups, respectively. Note the somatic cells (arrowheads) and germ cells (arrows). Scale bar: 10 µm.

 

Figure 2
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  		Figure 2. Effects of daidzein (DAI) and Aroclor 1254 (A1254) on germ cell numbers after a 48-h treatment. Values represent the means ± SD (n = 4). Bars with different letters were different (P < 0.05).

 
Antioxidant Effects of DAI

The expansion of somatic cells at the bottom of the culture plate decreased after treatment with 10 µg/mL A1254 (Figure 1CGo). In combination with DAI, the integrity of most cells could be maintained and more germ cells survived (Figure 1DGo). The germ cell numbers increased compared with those of the A1254 group. In the TMX treatment alone, the germ cells grew well and remained alive (Figure 1EGo). After exposure to A1254 + TMX, the germ cell numbers decreased significantly compared with those in the A1254 group (Figure 2Go; P < 0.05). In the DAI + A1254 + TMX group, the integrity of most germ cells was retained and the number was significantly increased (Figures 1FGo and 2Go; P < 0.05).

Measurement of Oxidative Damage

No significant differences in MDA production, SOD activities, and GSH levels were observed in the DAI (10 µg/mL), TMX (0.1 µg/mL), or DAI + TMX groups compared with the control group. After incubation with 10 µg/mL of A1254 for 48 h, MDA production was increased, whereas SOD activities and GSH levels were significantly decreased. Especially in combination with TMX, A1254 significantly decreased the SOD activity and GSH level and increased the production of MDA (Figures 3Go and 4Go; P < 0.05). However, after the combined treatment with DAI there was a significant decrease in MDA production and an increase in SOD activities and GSH levels in these 2 groups.


Figure 3
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  		Figure 3. Determination of lipid peroxidation by malondialdehyde (MDA) formation for a 48-h culture. Values represent the means ± SD (n = 4). Bars with different letters were different (P < 0.05).

 

Figure 4
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  		Figure 4. Determination of oxidative damage by A) superoxide dis-mutase (SOD) activity and B) glutathione (GSH) level for a 48-h culture. Values represent the means ± SD (n = 4). Bars with different letters were different (P < 0.05).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Estrogenic isoflavonoids, such as DAI and genistein, have attracted increasing attention because of their implications for human health. Daidzein is derived from plants and is consumed by human beings and domestic animals in relatively large quantities. In the present study, DAI increased germ cell numbers, and this effect was blocked by TMX. Therefore, DAI exerted a weak estrogenic activity on the germ cells. This result was consistent with other studies. For example, Ju et al. (2006) found that DAI significantly stimulated the growth of MCF-7 cells. Daidzein and genistein are structurally similar to endogenous estrogens and can bind to ER to exert estrogenic activity (Casanova et al., 1999). Therefore, the effect of DAI was mediated partly through weak estrogenic action without marked cytotoxicity.

Moreover, our previous study showed that A1254 exhibited both a hormonal and toxic effect on testicular cells. This study showed that DAI in combination with A1254 increased germ cell numbers compared with DAI or A1254 alone. This elevated effect was blocked by TMX, although the number of germ cells was still higher than in the treatment with DAI or A1254 + TMX. Thus, in addition to the estrogenic effect, DAI exhibited another mechanism that inhibited the cytotoxicity of A1254. Furthermore, phytoestrogen is known as a plant endocrine-disrupting chemical that disrupts the reproductive system (Woclawek-Potocka et al., 2005). Payne et al. (2001) found that at very high concentrations in vivo, soy isoflavones depressed the growth rate and the gain:feed rate in commercial broilers. Even if no significant changes of the conventional reproductive-toxicological markers appeared, isoflavones disrupted mRNA levels of ER{alpha} and androgen receptors (Shibayama et al., 2001). The present study showed that at levels up to 10 µg/mL, DAI imposed no significant negative effect on testicular cells, despite its weak estrogenic activity. Furthermore, it was able to inhibit the cytotoxicity of oxidative agents such A1254 by maintaining normal cell morphology.

The antioxidant capacity of DAI was also revealed in the present study. The formation of MDA was significantly elevated, but SOD activity and the GSH content decreased in testicular cells after exposure to A1254. However, these parameters were restored by the combined treatment with DAI. This result clearly demonstrates the antioxidant activity of DAI and is in accordance with previous observations that A1254 caused oxidative stress but was protected by the well-known antioxidants vitamins E and C (Kumar et al., 2004). Cai and Wei (1996) found that soybean isoflavones significantly increased SOD activity. Depletion of GSH led to oxidative damage at toxic levels and ultimately induced cell death. In this study, DAI may have restored the decrease in GSH content caused by the A1254 treatment. Such an effect of DAI is probably due to its chemical structure, which can inhibit the diffusion of free radicals and thereby hinder the attack of free radicals (Arora et al., 2000). Daidzein is mainly derived from soybeans, a feedstuff ingredient consumed by poultry in relatively large quantities. Aroclor 1254 can be also ingested by poultry in the contaminated feedstuff. In line with the above results, DAI manifested much weaker estrogenic activity than endogenous 17ß-estradiol and imposed a highly beneficial preventive effect on A1254-induced oxidative damage.

In conclusion, the present study revealed the dual effect of DAI on testicular cells, including weak estrogenic and antioxidant effects. Although DAI is regarded as an endocrine-disrupting chemical, its estrogenic activity was quite low compared with endogenous estrogen. As an effective antioxidant, DAI protected testicular cells against A1254-induced toxicity by restoring the intracellular antioxidant system. Therefore, supplementation of diets with the dietary antioxidant isoflavonoid DAI may prevent oxidative damage, which could contribute to protection against testicular toxicity and reduce the risk of cancer in animals.


   ACKNOWLEDGMENTS
 
This study was supported by the Natural Science Foundation of China (30471245 and 30371051) and the Chinese Ministry of Education (NCET-05-0514).

Received for publication February 13, 2007. Accepted for publication May 18, 2007.


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