Protective Effect of Quercetin on Aroclor 1254–Induced Oxidative Damag
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《毒物学科学杂志》
Department of Veterinary Medicine, College of Animal Sciences, Zhejiang University, Hangzhou 310029, China
ABSTRACT
Quercetin, a dietary-derived falvonol-type flavonoid, is ubiquitous in fruits and vegetables and plays important roles in human health by virtue of its antioxidant function. The present study was performed to investigate effects of quercetin on oxidative damage that was induced by an environmental endocrine disrupter, Aroclor 1254 (A1254), in cultured spermatogonial cells of embryonic chickens. Spermatogonial cells were dispersed from 18-day-old embryo and exposed to A1254 alone or in combination with quercetin. The oxidative damage was estimated by measuring contents of thiobarbituric acid–reactive substances (TBARS, an indicator of lipid peroxidation), activity of superoxide dismutase (SOD, a scavenger of superoxide), and activity of glutathione (GSH, an intracellular antioxidant). Results showed that quercetin had no deleterious effect on spermatogonial cells at 0.011 μg/ml. Exposure to A1254 (10 μg/ml) induced an increase of spermatogonial cell number, and membrane integrity was damaged by elevation of lactate dehydrogenase (LDH) leakage. Exposure to A1254 also induced an elevation in TBARS but a decrease in SOD activity and GSH content. However, compared with A1254 treatment alone, simultaneous supplementation with quercetin decreased LDH leakage to maintain the cell integrity, decreased the levels of TBARS to quench the free radicals, increased SOD activity and GSH content to restore the endogenous antioxidant defense system. Thus, quercetin displayed protective effects on spermatogonial cells from A1254-induced oxidative damage through increasing intracellular antioxidant levels and decreasing lipid peroxidation. Consequently, the antioxidant, such as quercetin, from food or feed consumed by human and animals may attenuate the negative effects of environmental endocrine disrupters.
Key Words: quercetin; polychlorinated biphenyls; oxidative damage; chicken; spermatogonial cells.
INTRODUCTION
In the past decade, the bioactivities of flavonoids on human health have given rise to much attention, especially the antioxidant activity. Flavonoids are important phytochemicals that cannot be synthesized by humans. Quercetin (3,3',4',5,7-pentahydroxyflavone) is one of the ubiquitous flavonol-type flavonoids predominant in edible vegetables and fruits. As a member of the flavonol family, quercetin exhibited multiple biological effects on human health. It has been shown to prevent cardiovascular disease (Sesso et al., 2003), inhibit platelet aggregation and prevent atherosclerosis and thrombosis (Formica and Regelson, 1995), have anticancer, anti-inflammatory responses, as well as antiulcer, antiallergic, and antiviral activities (Brown, 1980; Middleton and Kandaswami, 1992). Most of the pharmacological effects of quercetin are ascribed in part to its antioxidant activity.
Quercetin was well known for its antioxidant potential, among other biological properties. One mechanism of the antioxidant action of quercetin was involved in scavenging free radicals, such as superoxide radicals generated by xanthine/xanthine oxidase (Dok-Go et al., 2003). Additionally, quercetin exhibited peroxyl radical–scavenging activity, which was determined by measuring the inhibition of hydroperoxidation of methyl linoleate initiated by a radical initiator, 2,2'-azobis(2,4-dimethylvaleronitrile) (Ioku et al., 1995). The hydroxyl free radical scavenging activity of quercetin was determined by a chemiluminescence-based assay (Kefalas et al., 2003). The antioxidant properties of quercetin might also be due to its ability to chelate transition metal ions. Moreover, quercetin inhibited divalent cation-mediated lipid peroxidation, such as Fe2+ and Cu2+ (da Silva et al., 1998; Ferrali et al., 1997). The protection was related to the intracellular chelation. Quercetin could reduce oxidative damage to macromolecules such as lipids and DNA. Negre-Salvayre and Salvayre (1992) demonstrated that quercetin prevented the cytotoxicity of oxidized low density lipoprotein induced by ultraviolet irradiation on lymphoid cell lines. In addition, previous studies have showed that quercetin inhibited the oxidative DNA damage induced by hydrogen peroxide (Musonda and Chipman, 1998). Quercetin also quenched 8-hydroxy-2'-deoxyguanosine formation and suppressed DNA strand scission (Cai et al., 1997; Sestili et al., 1998). Therefore, quercetin exerted the antioxidant activity as chelators of divalent cations, free radical scavengers, as well as DNA damage protectors, and thus may be involved in preventing free radical–mediated cytotoxicity and lipid peroxidation.
With the rapid development of industry and agriculture, environmental endocrine disruptors have drawn more and more concerns because of their potential health impacts on human and animals. Among those environmental endocrine disruptors, polychlorinated biphenyls (PCBs) are members of the halogenated hydrocarbon class of lipophilic environmental contaminants. Indeed, PCBs are still regarded as a major global environmental problem, although most industrialized countries have strictly prohibited their use. PCBs may be biomagnified along food chains to increase the risk of human exposure because of the ubiquitous, persistent, and lipophilic character of these chemicals. Voluminous studies have revealed that PCBs have the potential to interfere with the reproductive system. Our previous studies suggested that exposure to Aroclor 1254 (A1254), a commercial mixture of PCBs, interfered with gonadal germ cell proliferation and caused reproductive disorders via both toxic and estrogenic actions in embryonic chickens, as well as spermatogenesis in adult cocks (Mi and Zhang, 2005; Zhang et al., 2002). PCBs were considered potential endocrine disruptors because of their ability to act as estrogen, antiestrogen, and antiandrogen (Bonefeld-Jrgensen et al., 2001). A recent study showed that A1254 exposure may generate reactive oxygen species and decrease specific activities of superoxide dismutase (SOD), glutathione (GSH) peroxidase, and reductase, and they may also increase lipid peroxidation in rat Sertoli cells (Kumar et al., 2004). Thus PCB-induced toxicity may be associated with oxidative damage.
In the present study, we chose quercetin, a typical flavonol that is widespread in plant foods, to test its protective effect against oxidative damage induced by A1254 in spermatogonial cells through a germ–somatic cell co-culture model. The cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) leakage. Lipid peroxidation was explored by determinations of malondialdehyde (MDA). The activity of SOD and GSH content were determined to reflect the level of the intracellular antioxidant defense system. These results would help to provide protection against environmental endocrine disruptor-induced oxidative damage and to generate more comprehensive and reliable data for toxicological risk evaluation.
MATERIALS AND METHODS
Isolation and culture of spermatogonial cells.
Fertilized avian chicken eggs were obtained from a commercial hatchery and incubated at 38.5°C and 60% humidity until day 18 in a rotatory egg incubator (Victoria SRL, Italy). Testicular cells were prepared according to a previous study (Mi et al., 2004). The cells were cultured in collagen-treated 96-well culture plates (Costar, Corning Inc., Corning, NY) at a density of 105/well in 200 μl serum-free McCoy's 5A medium (HyClone, Logan, UT) supplemented with 10 μg/ml insulin, 5 μg/ml transferrin, and 3 x 10–8 M selenium (Sigma, St. Louis, MO) as ITS medium. The medium also contained 2 mM glutamine, 1.75 mM HEPES, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Cells were cultured in a water-saturated atmosphere of 95% air and 5% CO2 at 39°C.
Treatments of cultured cells with chemicals.
Stock solutions of quercetin (Sigma, St. Louis, MO) and Aroclor1254 (A1254, lot number 124–191-A, 99.99% pure, Accustandard Inc., New Haven, CT) were prepared in ethanol and diluted in medium at a maximum of 0.1% ethanol. Cells were incubated in medium with A1254 (10 μg/ml), quercetin (0.01, 0.1, 1 μg/ml) alone or in combinations. The control group received vehicle only. At the end of culture the media were quickly collected, frozen, and stored at –20°C. After they were washed three times the cultured cells were resuspended in an appropriate volume of PBS to determine the cellular LDH and SOD activities, as well as the GSH concentrations.
Morphological studies of spermatogonial cells.
Morphological changes of spermatogonial cells were observed under an IX70 phase contrast microscope (Olympus, Japan) after culture for 48 h, and images were captured with a video camera (Pixera Pro 150ES, Pixera, Los Gatos, CA) to a computer. The number of spermatogonial cells was achieved in each image by using Simple PCI Advanced Imaging Software (Compix, Inc., Torrance, CA).
Determination of cell viability by MTT assay.
Cell viability was quantified by measurement of the mitochondrial reduction of MTT to produce a dark-blue formazan product (Lee et al., 2002). Briefly, testicular cells were treated with chemicals at the indicated concentrations for 48 h. MTT (0.25 mg/ml, Sigma) was added to each well. After incubating for 4 h at 39°C, the medium was removed and 100 μl dimethylsulfoxide was added to solubilize the formazan crystals. The color developed was measured at 570 nm by a microplate reader (Multiskan MK3, Thermo Labsystems Co., China). Cell viability was expressed as the proportion of absorbance values to the control group.
Determination of LDH release.
The cytotoxicity was estimated by quantification of LDH activity in the culture medium versus total LDH activity in the samples after treatment for 48 h. Cultured cells were treated with 10% Triton X-100, and LDH activity was assayed by absorbance change at a wavelength of 440 nm with an LDH kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Determination of TBARS.
Malondialdehyde (MDA) is a breakdown product of the oxidative degradation of cell membrane lipids and is generally considered an indicator of lipid peroxidation. In the present study, lipid peroxidation was thus evaluated by measuring MDA concentrations according to the method of Agostinho et al. (1997). The method was based on the spectrophotometric measurement of the color produced during the reaction to thiobarbituric acid (TBA) with MDA. MDA concentrations were calculated by the absorbance of thiobarbituric acid reactive substances (TBARS) at 532 nm and were expressed in nmol/ml. Analyses were performed in duplicates.
Determination of SOD activities.
SOD is a scavenger of superoxide. Total SOD activity was evaluated by the inhibition in the rate of the superoxide radicals–dependent cytochrome C reduction (Flohe and Otting,1984). The assay used the xanthine–xanthine oxidase system as the source of superoxide ions, and the absorbance at 550 nm was determined. The values were expressed as units per milliliter, where one unit of SOD was defined as the amount of SOD inhibiting the rate of reaction by 50% at 25°C.
Estimation of GSH content.
GSH is an important cellular non-enzymatic antioxidant. The total GSH was determined by measuring the rate of reduction of 5,5'-dithiobis-2- nitrobenzoate (DTNB) to 2-nitro-5-thiobenzoate according to the method (Zakowski and Tappel, 1978) with absorption maximum at 412 nm. The GSH level was expressed as milligrams per liter.
Statistical analysis.
The experiment was repeated three times with quadruplications. All data were expressed as the means ± SD. The statistical differences among the groups were determined by analysis of variance (ANOVA) and Duncan's multiple range test using the GLM procedure of SAS 6.12 software. A level of p < 0.05 was considered significantly different.
RESULTS
Effect of Quercetin and A1254 on Spermatogonial Cell Morphology
In the spermatogonial-somatic cell co-culture model, spermatogonial cells were round in shape and anchored on the overslip by the filopodia of somatic cells in the ITS medium after culture for 48 h (Fig. 1A). In the quercetin-treated groups, spermatogonial cells showed morphology similar to cells in the control group (Fig. 1B). Abnormal morphology in spermatogonial cells and somatic cells was observed in 48-h culture after A1254 treatment when compared to the control. Somatic cells decreased expansion at the bottom of the culture plate (Fig. 1C). The integrity of most spermatogonial cells could be kept in combination with quercetin (Fig. 1D).
Changes in Number of Spermatogonial Cells and Cell Viability
Morphological analysis showed that quercetin had no deleterious effect on spermatogonial cells at the indicated concentrations. Spermatogonial cells grew well in groups treated with quercetin alone (Fig. 2A). The MTT assay confirmed no deleterious effect of quercetin (Fig. 2B). Although A1254 exhibited a toxic effect, surviving spermatogonial cell number showed an increment after culture for 48 h (p < 0.05; Fig. 2A). In the combination groups, quercetin (1 μg/ml) significantly increased spermatogonial cell number beyond that observed with A1254 alone (p < 0.05; Fig. 2A). In addition, MTT assay confirmed that quercetin decreased the mortality of spermatogonial cells from damage induced by A1254 (Fig. 2B), but the attenuating effect of quercetin was not observed at the lower doses groups (0.01 and 0.1 μg/ml).
Assessment of Cytotoxicity by LDH Release Assay
Besides the morphological changes and cell viability, testicular cell injuries were also measured by determining LDH activities in the media and cells after 48-h culture (Fig. 3). The LDH estimation was consistent with the results obtained by the morphological observations. No significant differences in LDH activities were observed between quercetin and the control group. However, in the A1254 group, LDH leakage from testicular cells was significantly elevated (p < 0.05; Fig. 3), but this increase was significantly reduced after combined administration of quercetin (p < 0.05; Fig. 3).
Measurement of Oxidative Damage by MDA Formation, SOD Activity, and GSH Content
There were no obvious differences in MDA formation, SOD activity, and GSH level between quercetin and the control group (p < 0.05; Figs. 4, 5, and 6). After exposure to 10 μg/ml A1254 for 48 h, MDA production was increased significantly; but SOD activities and GSH levels were decreased significantly (p < 0.05; Figs. 4, 5, and 6). However, in combinations with quercetin, there was a significant decrease in MDA production and increases in the SOD activity and GSH level (p < 0.05; Figs. 4, 5 and 6).
DISCUSSION
Among so many environmental endocrine disruptors, PCBs continued to be of concern to biologists because of the disrupting effect of these chemicals on reproduction and development. Reproductive abnormalities caused by PCBs were observed in many vertebrates, such as the reductions in count and activity of sperm, average life span of sperm trials in zebra fish, inhibition of spermatogenesis in cod and newly hatched or adult chickens (Njiwa et al., 2004; Sangalang et al., 1981; Zhang et al., 2002). In the present study a germline stem cell, the spermatogonial cell, was used in toxicological assessment of A1254. Stem cell technology provides a new promising and innovative tool for toxicity screening and better understanding of the mechanisms involved in chemically induced adverse reactions. They also have the potential to predict and avoid toxicity in humans and animals. Moreover, the use of stem cell-derived in vitro models will overcome costly and labor-intensive toxicological studies performed in the in vivo animal models (Davila et al., 2004).
In the present study, treatment with A1254 resulted in decreased cell viabilities, elevation of LDH leakage, and serious damage to the membrane integrity. These results were in accordance with our previous findings of direct toxic effects induced by A1254 on chicken spermatogonial cells. A1254 also induced an increase in spermatogonial cell numbers. This stimulating effect of A1254 could be blocked by the androgen receptor antagonist flutamide or the estrogen receptor antagonist tamoxifen, suggesting that A1254 promoted spermatogonial cell proliferation through hormonal effects (Mi and Zhang, 2005). The excessive production of free radicals by A1254 exposure induced lipid peroxidation that ultimately caused damage to the membrane integrity. In the present model, exposure to A1254 led to reduced antioxidant enzyme (SOD) activity and endogenous antioxidant (GSH) content, which suggests that the intracellular antioxidant defense system was damaged. Kumar et al. (2004) demonstrated that A1254 exposure increased the levels of hydrogen peroxide, hydroxyl radical, and lipid peroxidation but decreased lactate and SOD activity in rat Sertoli cells. In their study the toxic manifestations induced by A1254 were associated with oxidative stress, including lipid peroxidation and production of reactive oxygen species. These observations are in agreement with our present results, suggesting that A1254-mediated cytotoxicity might be induced by the formation of excessive free radicals, which caused lipid peroxidation and membrane impairment.
In recent years, the positive effects of flavonoids on human health have attracted more attention. Especially, quercetin, a flavonol-type flavonoid found in many plants, is widely distributed in fruits and vegetables. Most of the biological actions of quercetin seem to be associated with its potency as an antioxidant. Our present work aimed to investigate the potential protective value of quercetin on cytotoxicity induced by A1254. The results showed that quercetin had no deleterious effects on spermatogonial cells at the indicated concentrations. The simultaneous supplementation with antioxidant quercetin recovered the normal level of cell viability, SOD activity, and GSH content, while it prevented LDH leakage and TBARS production. These results revealed that A1254-induced testicular cell toxicity can be prevented by quercetin. The low toxicity of quercetin has also been demonstrated in various studies (Alía et al., 2005; Caltagirone et al., 2000). Our results confirmed the findings of these studies at the indicated dose range. However, some studies revealed that the harmful effects of quercetin were attributed to its prooxidant activities through mutagenic and DNA-damaging activities (Johnson and Loo, 2000; van Duursen et al., 2004). The conflicting effects of quercetin might relate to the redox state of the cell and the dose used. Additionally, our observations about the protective effects of quercetin are in agreement with previous observations that vitamins E and C, as the effective antioxidants, protected against oxidative stress caused by A1254 (Kumar et al., 2004). In another study, Dunlap et al. (1999) revealed that quercetin reduced the toxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The protective effects of quercetin may be due to inhibition of lipid peroxidation by its antioxidant nature, because it also showed dose-dependent antioxidative activity against metal-induced lipid peroxidation assessed by measurement of MDA levels (Sugihara et al., 1999). Quercetin protected glutathione depletion induced by dehydroascorbic acid in rabbit red blood cells (Fiorani et al., 2001) and restored the decreased levels of antioxidant defense enzymes that was induced by ultraviolet light (Inal et al., 2001). Moreover, Ramadass et al. (2003) described that the oxidative stress induced by PCB could be inhibited by the dietary flavonoids quercetin and epigallocatechin-3-gallate.
The structure of quercetin plays an important role in its antioxidant effect. The o-dihydroxy-structure in the B-ring has been observed to confer higher stability to the radical form and to participate in electronic delocalization (Cao et al., 1997; Rice-Evans et al., 1996). However, Saija et al. (1995) suggested that the antioxidant activities were dictated both by their structural features and by their location in the membrane. Flavonoids are known to anchor on the polar head of the main phospholipids. Hence, quercetin distributed on the surface of the lipid bilayers as well as in the aqueous phase could scavenge free radicals.
In conclusion, quercetin had no deleterious effect on spermatogonial cells at 0.011 μg/ml. Oxidative damage was induced by exposure to A1254 in embryonic chicken spermatogonial cells as increased lipid peroxidation and a reduced intracellular antioxidant system. However, quercetin could inhibit these negative effects through a reduction in MDA and an increase in SOD and GSH to maintain membrane integrity. Thus, the protective effect of quercetin on oxidative damage was attributable to its antioxidant nature. These results therefore indicated that antioxidants from food or feed consumed by human and animals, such as quercetin, can attenuate the negative effects of environmental endocrine disrupters.
ACKNOWLEDGMENTS
This study was supported by the National Natural Science Foundation of China (No. 30471245). We thank Mr. Weidong Zeng for help during the experiment.
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ABSTRACT
Quercetin, a dietary-derived falvonol-type flavonoid, is ubiquitous in fruits and vegetables and plays important roles in human health by virtue of its antioxidant function. The present study was performed to investigate effects of quercetin on oxidative damage that was induced by an environmental endocrine disrupter, Aroclor 1254 (A1254), in cultured spermatogonial cells of embryonic chickens. Spermatogonial cells were dispersed from 18-day-old embryo and exposed to A1254 alone or in combination with quercetin. The oxidative damage was estimated by measuring contents of thiobarbituric acid–reactive substances (TBARS, an indicator of lipid peroxidation), activity of superoxide dismutase (SOD, a scavenger of superoxide), and activity of glutathione (GSH, an intracellular antioxidant). Results showed that quercetin had no deleterious effect on spermatogonial cells at 0.011 μg/ml. Exposure to A1254 (10 μg/ml) induced an increase of spermatogonial cell number, and membrane integrity was damaged by elevation of lactate dehydrogenase (LDH) leakage. Exposure to A1254 also induced an elevation in TBARS but a decrease in SOD activity and GSH content. However, compared with A1254 treatment alone, simultaneous supplementation with quercetin decreased LDH leakage to maintain the cell integrity, decreased the levels of TBARS to quench the free radicals, increased SOD activity and GSH content to restore the endogenous antioxidant defense system. Thus, quercetin displayed protective effects on spermatogonial cells from A1254-induced oxidative damage through increasing intracellular antioxidant levels and decreasing lipid peroxidation. Consequently, the antioxidant, such as quercetin, from food or feed consumed by human and animals may attenuate the negative effects of environmental endocrine disrupters.
Key Words: quercetin; polychlorinated biphenyls; oxidative damage; chicken; spermatogonial cells.
INTRODUCTION
In the past decade, the bioactivities of flavonoids on human health have given rise to much attention, especially the antioxidant activity. Flavonoids are important phytochemicals that cannot be synthesized by humans. Quercetin (3,3',4',5,7-pentahydroxyflavone) is one of the ubiquitous flavonol-type flavonoids predominant in edible vegetables and fruits. As a member of the flavonol family, quercetin exhibited multiple biological effects on human health. It has been shown to prevent cardiovascular disease (Sesso et al., 2003), inhibit platelet aggregation and prevent atherosclerosis and thrombosis (Formica and Regelson, 1995), have anticancer, anti-inflammatory responses, as well as antiulcer, antiallergic, and antiviral activities (Brown, 1980; Middleton and Kandaswami, 1992). Most of the pharmacological effects of quercetin are ascribed in part to its antioxidant activity.
Quercetin was well known for its antioxidant potential, among other biological properties. One mechanism of the antioxidant action of quercetin was involved in scavenging free radicals, such as superoxide radicals generated by xanthine/xanthine oxidase (Dok-Go et al., 2003). Additionally, quercetin exhibited peroxyl radical–scavenging activity, which was determined by measuring the inhibition of hydroperoxidation of methyl linoleate initiated by a radical initiator, 2,2'-azobis(2,4-dimethylvaleronitrile) (Ioku et al., 1995). The hydroxyl free radical scavenging activity of quercetin was determined by a chemiluminescence-based assay (Kefalas et al., 2003). The antioxidant properties of quercetin might also be due to its ability to chelate transition metal ions. Moreover, quercetin inhibited divalent cation-mediated lipid peroxidation, such as Fe2+ and Cu2+ (da Silva et al., 1998; Ferrali et al., 1997). The protection was related to the intracellular chelation. Quercetin could reduce oxidative damage to macromolecules such as lipids and DNA. Negre-Salvayre and Salvayre (1992) demonstrated that quercetin prevented the cytotoxicity of oxidized low density lipoprotein induced by ultraviolet irradiation on lymphoid cell lines. In addition, previous studies have showed that quercetin inhibited the oxidative DNA damage induced by hydrogen peroxide (Musonda and Chipman, 1998). Quercetin also quenched 8-hydroxy-2'-deoxyguanosine formation and suppressed DNA strand scission (Cai et al., 1997; Sestili et al., 1998). Therefore, quercetin exerted the antioxidant activity as chelators of divalent cations, free radical scavengers, as well as DNA damage protectors, and thus may be involved in preventing free radical–mediated cytotoxicity and lipid peroxidation.
With the rapid development of industry and agriculture, environmental endocrine disruptors have drawn more and more concerns because of their potential health impacts on human and animals. Among those environmental endocrine disruptors, polychlorinated biphenyls (PCBs) are members of the halogenated hydrocarbon class of lipophilic environmental contaminants. Indeed, PCBs are still regarded as a major global environmental problem, although most industrialized countries have strictly prohibited their use. PCBs may be biomagnified along food chains to increase the risk of human exposure because of the ubiquitous, persistent, and lipophilic character of these chemicals. Voluminous studies have revealed that PCBs have the potential to interfere with the reproductive system. Our previous studies suggested that exposure to Aroclor 1254 (A1254), a commercial mixture of PCBs, interfered with gonadal germ cell proliferation and caused reproductive disorders via both toxic and estrogenic actions in embryonic chickens, as well as spermatogenesis in adult cocks (Mi and Zhang, 2005; Zhang et al., 2002). PCBs were considered potential endocrine disruptors because of their ability to act as estrogen, antiestrogen, and antiandrogen (Bonefeld-Jrgensen et al., 2001). A recent study showed that A1254 exposure may generate reactive oxygen species and decrease specific activities of superoxide dismutase (SOD), glutathione (GSH) peroxidase, and reductase, and they may also increase lipid peroxidation in rat Sertoli cells (Kumar et al., 2004). Thus PCB-induced toxicity may be associated with oxidative damage.
In the present study, we chose quercetin, a typical flavonol that is widespread in plant foods, to test its protective effect against oxidative damage induced by A1254 in spermatogonial cells through a germ–somatic cell co-culture model. The cytotoxicity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) leakage. Lipid peroxidation was explored by determinations of malondialdehyde (MDA). The activity of SOD and GSH content were determined to reflect the level of the intracellular antioxidant defense system. These results would help to provide protection against environmental endocrine disruptor-induced oxidative damage and to generate more comprehensive and reliable data for toxicological risk evaluation.
MATERIALS AND METHODS
Isolation and culture of spermatogonial cells.
Fertilized avian chicken eggs were obtained from a commercial hatchery and incubated at 38.5°C and 60% humidity until day 18 in a rotatory egg incubator (Victoria SRL, Italy). Testicular cells were prepared according to a previous study (Mi et al., 2004). The cells were cultured in collagen-treated 96-well culture plates (Costar, Corning Inc., Corning, NY) at a density of 105/well in 200 μl serum-free McCoy's 5A medium (HyClone, Logan, UT) supplemented with 10 μg/ml insulin, 5 μg/ml transferrin, and 3 x 10–8 M selenium (Sigma, St. Louis, MO) as ITS medium. The medium also contained 2 mM glutamine, 1.75 mM HEPES, 100 IU/ml penicillin, and 100 μg/ml streptomycin. Cells were cultured in a water-saturated atmosphere of 95% air and 5% CO2 at 39°C.
Treatments of cultured cells with chemicals.
Stock solutions of quercetin (Sigma, St. Louis, MO) and Aroclor1254 (A1254, lot number 124–191-A, 99.99% pure, Accustandard Inc., New Haven, CT) were prepared in ethanol and diluted in medium at a maximum of 0.1% ethanol. Cells were incubated in medium with A1254 (10 μg/ml), quercetin (0.01, 0.1, 1 μg/ml) alone or in combinations. The control group received vehicle only. At the end of culture the media were quickly collected, frozen, and stored at –20°C. After they were washed three times the cultured cells were resuspended in an appropriate volume of PBS to determine the cellular LDH and SOD activities, as well as the GSH concentrations.
Morphological studies of spermatogonial cells.
Morphological changes of spermatogonial cells were observed under an IX70 phase contrast microscope (Olympus, Japan) after culture for 48 h, and images were captured with a video camera (Pixera Pro 150ES, Pixera, Los Gatos, CA) to a computer. The number of spermatogonial cells was achieved in each image by using Simple PCI Advanced Imaging Software (Compix, Inc., Torrance, CA).
Determination of cell viability by MTT assay.
Cell viability was quantified by measurement of the mitochondrial reduction of MTT to produce a dark-blue formazan product (Lee et al., 2002). Briefly, testicular cells were treated with chemicals at the indicated concentrations for 48 h. MTT (0.25 mg/ml, Sigma) was added to each well. After incubating for 4 h at 39°C, the medium was removed and 100 μl dimethylsulfoxide was added to solubilize the formazan crystals. The color developed was measured at 570 nm by a microplate reader (Multiskan MK3, Thermo Labsystems Co., China). Cell viability was expressed as the proportion of absorbance values to the control group.
Determination of LDH release.
The cytotoxicity was estimated by quantification of LDH activity in the culture medium versus total LDH activity in the samples after treatment for 48 h. Cultured cells were treated with 10% Triton X-100, and LDH activity was assayed by absorbance change at a wavelength of 440 nm with an LDH kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).
Determination of TBARS.
Malondialdehyde (MDA) is a breakdown product of the oxidative degradation of cell membrane lipids and is generally considered an indicator of lipid peroxidation. In the present study, lipid peroxidation was thus evaluated by measuring MDA concentrations according to the method of Agostinho et al. (1997). The method was based on the spectrophotometric measurement of the color produced during the reaction to thiobarbituric acid (TBA) with MDA. MDA concentrations were calculated by the absorbance of thiobarbituric acid reactive substances (TBARS) at 532 nm and were expressed in nmol/ml. Analyses were performed in duplicates.
Determination of SOD activities.
SOD is a scavenger of superoxide. Total SOD activity was evaluated by the inhibition in the rate of the superoxide radicals–dependent cytochrome C reduction (Flohe and Otting,1984). The assay used the xanthine–xanthine oxidase system as the source of superoxide ions, and the absorbance at 550 nm was determined. The values were expressed as units per milliliter, where one unit of SOD was defined as the amount of SOD inhibiting the rate of reaction by 50% at 25°C.
Estimation of GSH content.
GSH is an important cellular non-enzymatic antioxidant. The total GSH was determined by measuring the rate of reduction of 5,5'-dithiobis-2- nitrobenzoate (DTNB) to 2-nitro-5-thiobenzoate according to the method (Zakowski and Tappel, 1978) with absorption maximum at 412 nm. The GSH level was expressed as milligrams per liter.
Statistical analysis.
The experiment was repeated three times with quadruplications. All data were expressed as the means ± SD. The statistical differences among the groups were determined by analysis of variance (ANOVA) and Duncan's multiple range test using the GLM procedure of SAS 6.12 software. A level of p < 0.05 was considered significantly different.
RESULTS
Effect of Quercetin and A1254 on Spermatogonial Cell Morphology
In the spermatogonial-somatic cell co-culture model, spermatogonial cells were round in shape and anchored on the overslip by the filopodia of somatic cells in the ITS medium after culture for 48 h (Fig. 1A). In the quercetin-treated groups, spermatogonial cells showed morphology similar to cells in the control group (Fig. 1B). Abnormal morphology in spermatogonial cells and somatic cells was observed in 48-h culture after A1254 treatment when compared to the control. Somatic cells decreased expansion at the bottom of the culture plate (Fig. 1C). The integrity of most spermatogonial cells could be kept in combination with quercetin (Fig. 1D).
Changes in Number of Spermatogonial Cells and Cell Viability
Morphological analysis showed that quercetin had no deleterious effect on spermatogonial cells at the indicated concentrations. Spermatogonial cells grew well in groups treated with quercetin alone (Fig. 2A). The MTT assay confirmed no deleterious effect of quercetin (Fig. 2B). Although A1254 exhibited a toxic effect, surviving spermatogonial cell number showed an increment after culture for 48 h (p < 0.05; Fig. 2A). In the combination groups, quercetin (1 μg/ml) significantly increased spermatogonial cell number beyond that observed with A1254 alone (p < 0.05; Fig. 2A). In addition, MTT assay confirmed that quercetin decreased the mortality of spermatogonial cells from damage induced by A1254 (Fig. 2B), but the attenuating effect of quercetin was not observed at the lower doses groups (0.01 and 0.1 μg/ml).
Assessment of Cytotoxicity by LDH Release Assay
Besides the morphological changes and cell viability, testicular cell injuries were also measured by determining LDH activities in the media and cells after 48-h culture (Fig. 3). The LDH estimation was consistent with the results obtained by the morphological observations. No significant differences in LDH activities were observed between quercetin and the control group. However, in the A1254 group, LDH leakage from testicular cells was significantly elevated (p < 0.05; Fig. 3), but this increase was significantly reduced after combined administration of quercetin (p < 0.05; Fig. 3).
Measurement of Oxidative Damage by MDA Formation, SOD Activity, and GSH Content
There were no obvious differences in MDA formation, SOD activity, and GSH level between quercetin and the control group (p < 0.05; Figs. 4, 5, and 6). After exposure to 10 μg/ml A1254 for 48 h, MDA production was increased significantly; but SOD activities and GSH levels were decreased significantly (p < 0.05; Figs. 4, 5, and 6). However, in combinations with quercetin, there was a significant decrease in MDA production and increases in the SOD activity and GSH level (p < 0.05; Figs. 4, 5 and 6).
DISCUSSION
Among so many environmental endocrine disruptors, PCBs continued to be of concern to biologists because of the disrupting effect of these chemicals on reproduction and development. Reproductive abnormalities caused by PCBs were observed in many vertebrates, such as the reductions in count and activity of sperm, average life span of sperm trials in zebra fish, inhibition of spermatogenesis in cod and newly hatched or adult chickens (Njiwa et al., 2004; Sangalang et al., 1981; Zhang et al., 2002). In the present study a germline stem cell, the spermatogonial cell, was used in toxicological assessment of A1254. Stem cell technology provides a new promising and innovative tool for toxicity screening and better understanding of the mechanisms involved in chemically induced adverse reactions. They also have the potential to predict and avoid toxicity in humans and animals. Moreover, the use of stem cell-derived in vitro models will overcome costly and labor-intensive toxicological studies performed in the in vivo animal models (Davila et al., 2004).
In the present study, treatment with A1254 resulted in decreased cell viabilities, elevation of LDH leakage, and serious damage to the membrane integrity. These results were in accordance with our previous findings of direct toxic effects induced by A1254 on chicken spermatogonial cells. A1254 also induced an increase in spermatogonial cell numbers. This stimulating effect of A1254 could be blocked by the androgen receptor antagonist flutamide or the estrogen receptor antagonist tamoxifen, suggesting that A1254 promoted spermatogonial cell proliferation through hormonal effects (Mi and Zhang, 2005). The excessive production of free radicals by A1254 exposure induced lipid peroxidation that ultimately caused damage to the membrane integrity. In the present model, exposure to A1254 led to reduced antioxidant enzyme (SOD) activity and endogenous antioxidant (GSH) content, which suggests that the intracellular antioxidant defense system was damaged. Kumar et al. (2004) demonstrated that A1254 exposure increased the levels of hydrogen peroxide, hydroxyl radical, and lipid peroxidation but decreased lactate and SOD activity in rat Sertoli cells. In their study the toxic manifestations induced by A1254 were associated with oxidative stress, including lipid peroxidation and production of reactive oxygen species. These observations are in agreement with our present results, suggesting that A1254-mediated cytotoxicity might be induced by the formation of excessive free radicals, which caused lipid peroxidation and membrane impairment.
In recent years, the positive effects of flavonoids on human health have attracted more attention. Especially, quercetin, a flavonol-type flavonoid found in many plants, is widely distributed in fruits and vegetables. Most of the biological actions of quercetin seem to be associated with its potency as an antioxidant. Our present work aimed to investigate the potential protective value of quercetin on cytotoxicity induced by A1254. The results showed that quercetin had no deleterious effects on spermatogonial cells at the indicated concentrations. The simultaneous supplementation with antioxidant quercetin recovered the normal level of cell viability, SOD activity, and GSH content, while it prevented LDH leakage and TBARS production. These results revealed that A1254-induced testicular cell toxicity can be prevented by quercetin. The low toxicity of quercetin has also been demonstrated in various studies (Alía et al., 2005; Caltagirone et al., 2000). Our results confirmed the findings of these studies at the indicated dose range. However, some studies revealed that the harmful effects of quercetin were attributed to its prooxidant activities through mutagenic and DNA-damaging activities (Johnson and Loo, 2000; van Duursen et al., 2004). The conflicting effects of quercetin might relate to the redox state of the cell and the dose used. Additionally, our observations about the protective effects of quercetin are in agreement with previous observations that vitamins E and C, as the effective antioxidants, protected against oxidative stress caused by A1254 (Kumar et al., 2004). In another study, Dunlap et al. (1999) revealed that quercetin reduced the toxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The protective effects of quercetin may be due to inhibition of lipid peroxidation by its antioxidant nature, because it also showed dose-dependent antioxidative activity against metal-induced lipid peroxidation assessed by measurement of MDA levels (Sugihara et al., 1999). Quercetin protected glutathione depletion induced by dehydroascorbic acid in rabbit red blood cells (Fiorani et al., 2001) and restored the decreased levels of antioxidant defense enzymes that was induced by ultraviolet light (Inal et al., 2001). Moreover, Ramadass et al. (2003) described that the oxidative stress induced by PCB could be inhibited by the dietary flavonoids quercetin and epigallocatechin-3-gallate.
The structure of quercetin plays an important role in its antioxidant effect. The o-dihydroxy-structure in the B-ring has been observed to confer higher stability to the radical form and to participate in electronic delocalization (Cao et al., 1997; Rice-Evans et al., 1996). However, Saija et al. (1995) suggested that the antioxidant activities were dictated both by their structural features and by their location in the membrane. Flavonoids are known to anchor on the polar head of the main phospholipids. Hence, quercetin distributed on the surface of the lipid bilayers as well as in the aqueous phase could scavenge free radicals.
In conclusion, quercetin had no deleterious effect on spermatogonial cells at 0.011 μg/ml. Oxidative damage was induced by exposure to A1254 in embryonic chicken spermatogonial cells as increased lipid peroxidation and a reduced intracellular antioxidant system. However, quercetin could inhibit these negative effects through a reduction in MDA and an increase in SOD and GSH to maintain membrane integrity. Thus, the protective effect of quercetin on oxidative damage was attributable to its antioxidant nature. These results therefore indicated that antioxidants from food or feed consumed by human and animals, such as quercetin, can attenuate the negative effects of environmental endocrine disrupters.
ACKNOWLEDGMENTS
This study was supported by the National Natural Science Foundation of China (No. 30471245). We thank Mr. Weidong Zeng for help during the experiment.
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