Antiandrogenic Activities of Diesel Exhaust Particle Extracts in PC3/AR Human Prostate Carcinoma Cells

Ryoichi Kizu*,{dagger},1, Kazumasa Okamura*, Akira Toriba*, Atsushi Mizokami{ddagger}, Kerry L. Burnstein§, Carolyn M. Klinge and Kazuichi Hayakawa*,{dagger}

* Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa 920-0934, Japan; {dagger} Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Corporation, Kawaguchi 332-0012, Japan; {ddagger} Department of Urology, School of Medicine, Kanazawa University, Kanazawa 920-8641, Japan; § Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, Miami, Florida 33101; and Department of Biochemistry and Molecular Biology, University of Louisville School of Medicine, Louisville, Kentucky 40292

Received August 16, 2003; accepted August 19, 2003


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We collected diesel exhaust particles (DEPs) emitted from three diesel-engine vehicles—a car, a bus, and a truck—in daily use, and prepared DEP extracts (DEPEs), designated as EC, EB, or ET, respectively. The androgenic and antiandrogenic effects of the DEPE samples were examined by a luciferase reporter assay in human prostate carcinoma PC3/AR cells transiently transfected with a prostate specific antigen gene promoter-driven luciferase expression vector pGLPSA5.8. PC3/AR is a subline of human prostate carcinoma PC3 transformed to stably express wild-type human androgen receptor (AR). While DEPE samples did not exhibit any androgenic effect, they exerted antiandrogenic effect, inhibiting dihydrotestosterone (10 pM) -induced luciferase activity by 24 to 52% at an extract concentration of 10 µg/ml. The antiandrogenic effect was greater in the following order: ET > EB > EC. Co-treatment of PC3/AR cells with SKF-525A, a nonselective inhibitor of cytochrome P450 (CYP) enzymes, enhanced the antiandrogenic effect, indicating that the antiandrogenic effect is caused by intact species of DEPE constituents. The antiandrogenic effect of DEPE samples was reversed by {alpha}-naphthoflavone, an aryl hydrocarbon receptor (AhR) antagonist. The antiandrogenic activity of a DEPE sample correlated with its AhR agonist activity assayed in PC3/AR cells transiently transfected with CYP1A1 gene promoter-driven luciferase expression vector pLUC1A1. Equimolar mixtures of ten polycyclic aromatic hydrocarbons (PAHs) having four or more rings, structures found in the DEPEs, showed significant antiandrogenic effects and AhR agonist activity at concentrations equivalent to those found in DEPE samples. Further, DEPE samples elicited only antiandrogenic effects in recombinant yeast cells, which express ß-galactosidase in response to androgen. A competitive AR binding assay showed that AR-binding constituents exist in DEPE samples, indicating that greater part of AR-binding constituents in DEPEs are AR antagonists. All these findings show that DEPE samples exhibit significant antiandrogenic effect in cell-based transcription assay and that this effect is due in part to the constituents with AhR agonist activity including PAHs and to the constituents with AR antagonist activity.

Key Words: diesel exhaust particulate; antiandrogenic effect; androgen receptor; aryl hydrocarbon receptor; polycyclic aromatic hydrocarbon; PC3/AR cell.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In recent years, there has been a growing concern over the effects of exogenous chemicals that exhibit "endocrine disruptor" or "endocrine mimetic" activities (Gray et al., 2002Go; Hester and Harrison, 1999Go; Tyler et al., 1998Go). This class of chemicals may be producing adverse effects in humans and wild life by directly or indirectly disrupting the endocrine system through mimicking or antagonizing natural hormones. Possible effects of endocrine disruptors include reproductive and developmental abnormalities, increases in certain hormone-related cancers such as breast and prostate, immune system deficiencies, and declines in wild-life populations (Kavlock et al., 1996Go; Melnick, 1999Go). While a wide variety of chemicals including environmental pollutants, industrial chemicals, and natural products have been studied for their endocrine disrupting effects, little is known about those of air pollutants.

The main air pollutants in urban areas of Japan and Europe are nitrogen dioxide and suspended particulate matter (SPM). The greater part of SPM is derived from vehicle emission (Ando et al., 1996Go; Lloyd and Cackette, 2001Go; Tamura et al., 1996Go). The number of diesel-powered vehicles has been increasing in Japan and Europe because of the diesel engine’s greater efficiency and lower cost compared with gasoline engines. Diesel-powered vehicles emit some 30–100 times more particles than do gasoline-powered cars, and consequently diesel exhaust particles (DEPs) comprise most of SPM in the urban atmosphere. DEPs contain carbon nuclei, which absorb heavy metals such as iron, copper and nickel and a vast number of chemicals such as polycyclic aromatic hydrocarbons (PAHs), heterocyclics, quinones, aldehydes, and aliphatic hydrocarbons (WHO/IPCS, 1996Go). While the principal concern regarding exposure to DEPs has been the risks for cancers, especially for lung cancer, and chronic respiratory diseases (Nel et al., 2001Go; Schwela, 2000Go), more recently DEPs have also attracted attention in terms of endocrine disrupting effects. For example, DEP extracts (DEPEs) were reported to exhibit the estrogenic and/or antiestrogenic activities in human MCF-7 breast cancer cells and recombinant yeast cells (Meek, 1998Go; Okamura et al., 2002Go; Taneda et al., 2002Go). The masculinization of fetus during pregnancy was caused by inhalation exposure to diesel exhaust (DE) in rats (Watanabe and Kurita, 2001Go).

Androgens have a pivotal role in the development and maintenance of the male reproductive system (Lindzey et al., 1994Go; Quigley et al., 1995Go). An important endocrine disrupting effect of DEPs is their potential adverse impact on the male reproductive-system functions. It was reported that DE inhalation exposure suppressed the daily sperm production in adult mice (Yoshida et al., 1999Go) and rats (Tsukue et al., 2001Go) and in growing rats (Watanabe and Oonuki, 1999Go). Although the experimental conditions of these studies were different in several critical respects including engine type, species and strain of animal, dose of DEP, and period and schedule of exposure, a common adverse outcome, i.e., inhibited spermatogenesis, was observed. Two of these three studies reported that DE inhalation exposure elevated serum testosterone levels (Tsukue et al., 2001Go; Watanabe and Oonuki, 1999Go). Pathological examination of seminiferous tubules showed decreased numbers of step 18 and step 19 spermatids in stage VI, VII, and VIII tubules, increased numbers of degenerated cells intermediate in development between spermatocytes and terminal stage spermatids and in the seminiferous lumen of DE-exposed animals, while no remarkable histopathological changes in Leydig and Sertoli cells were observed (Watanabe and Oonuki, 1999Go). Further, the decrease in spermatogenesis was more pronounced in the total DE exposure group compared to the filtered DE exposure group (Watanabe and Oonuki, 1999Go). These findings suggest that the suppression in sperm production is caused at least in part by antiandrogenic effects of DEPs. It is possible that DEPs contain antiandrogenic constituents.

As described above, DEPs contain numerous chemicals. It seems likely that some DEP constituents can interact with androgen receptor (AR) and/or other nuclear transcription factors. In fact, PAHs having four or more rings have been reported to be constituents of DEPs (WHO/IPCS, 1996Go) and that they antagonize androgen action through activation of aryl hydrocarbon receptor (AhR; Kizu et al., 2003Go; Vinggaard et al., 2000Go). It is thus important to clarify the DEP constituents implicated in the dysfunction of male reproductive system and the molecular mechanisms of their action.

While several environmental chemicals with the androgenic or antiandrogenic properties have been identified (Gray et al., 2001Go), there is no study on androgenic and antiandrogenic activities of DEP constituents. Here we report results of the first study on the androgenic and antiandrogenic effects of extracts of DEPs emitted from diesel-powered vehicles under daily use in order to obtain knowledge on the constituents involved in DE-induced dysfunction of male reproductive systems. In this study, androgenic and antiandrogenic activities of DEPEs were assayed in two cell systems. One is human prostate carcinoma PC3/AR cells transiently transfected with a prostate specific antigen (PSA) gene promoter-driven luciferase expression vector pGLPSA5.8 constructed by Mizokami et al.(2000)Go. PC3/AR cells established by Burnstein et al. (Dai et al., 1996Go) were derived from human prostate carcinoma PC3 cell by transforming to stably express wild-type human AR. The other is recombinant yeast cells which express ß-galactosidase in response to androgens by two-hybrid mechanism (Nishikawa et al., 1999Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
Coronene (99% pure) and perylene (>99.5% pure) were purchased from Aldrich (Milwaukee, WI). Benzo[b]chrysene (>98% pure) was from AccuStandard (New Haven, CT). Benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[ghi]perylene, benzo[a]pyrene, chrysene, indeno[1,2,3-cd]pyrene and pyrene of environmental analysis standard grade (>99.5% pure), dihydrotestosterone (DHT) of biochemical study grade and ethanol of ultra pure grade were purchased from Wako Pure Chemicals (Tokyo, Japan). {alpha}-Naphthoflavone ({alpha}-NF) and SKF-525A (or proadifen) were purchased from Sigma (St. Louis, MO). Bicalutamide (BCT; Casodex) was a gift from AstraZeneca (Cheshire, UK). All other chemicals were of reagent grade or better from commercial sources and were used as received. PAHs, DHT, {alpha}-NF and SKF-525A were dissolved in 50% (v/v) ethanol.

DEP collection and DEPE preparation.
DEPs were collected as described previously (Hayakawa et al., 2000Go; Murahashi et al., 1999Go; Okamura et al., 2002Go). Briefly, three diesel-engine vehicles in daily use, a car (made in Japan, 2500 cc, direct injection type, 1996 model), a bus (made in Japan, 4160 cc, direct injection type, 1990 model) and a truck (made in Japan, 7410 cc, direct injection type, 1989 model), were used under idling conditions with commercial light oil (JIS No.2). DEPs were collected on glass-fiber filters (Pallflex T60A20, 55 mm i.d.) by a low-volume air sampler. The filters were exchanged every 5 min. Five sheets of filters (approximately 10 mg of DEPs) were ultrasonically extracted with benzene/ethanol (3:1) and the extracts were reconstituted in 0.47 ml, 0.52 ml, and 0.55 ml of EtOH for car-, bus- and truck-DEPE, respectively, to give an extract concentration of 10 mg/ml. The extract samples originated from the car, bus, and truck were designated as EC, EB, and ET, respectively. A filter blank sample designated as FB was prepared similarly from five sheets of new unused filters by reconstituting the extract in 0.47 mL of EtOH, which is the smallest volume of EtOH used for reconstitution of DEPEs.

Culture of PC3/AR cells.
PC3/AR cells were cultured at 37°C in a humidified atmosphere of 5% CO2–95% air. In routine maintenance the cells were grown in phenol red-free RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 100 µg/ml streptomycin, 10 units/ml penicillin, and 50 µg/ml geneticin (GIBCO, Rockville, MD) and passaged with trypsinization every fourth day. In assays, cells were cultured in an assay medium of phenol red-free RPMI-1640 medium supplemented with 5% charcoal/dextran-treated FBS (Hyclone, Logan, UT), 100 µg/ml streptomycin, and 10 units/ml penicillin.

PC3/AR luciferase assay.
PC3/AR cells (5 x 106 cells) were harvested, washed once with cold phosphate-buffered saline. The cells were suspended in 5 ml of transfection medium of FBS-free OPTI-MEM I medium (GIBCO) containing 20 µg of a luciferase reporter vector and 50 µl of LipofectAMINE (GIBCO) and transiently transfected with a luciferase expressing plasmid for 30 min at 37°C. The vector transfected to PC3/AR cells was PSA promoter-driven luciferase expressing plasmid pGLPSA5.8, cytochrome P450 (CYP) 1A1 promoter-driven luciferase expressing plasmid pLUC1A1 kindly provided by Dr. R. H. Tukey (Postlind et al., 1993Go) or pGL3-control vector (Promega, Madison, WI). Then, 25 ml of assay medium was added to the cell suspension solution and the cells were plated on 48-well plates at a cell density of 5 x 104 cells per well (300 µl of diluted cell suspension solution per well). After 24 h, the cells were washed once with fresh assay medium and treated with 0.2% (v/v) ethanol (blank), 10 pM DHT (DHT control), FB or a DEPE sample alone or in combination with 10 pM DHT, BCT, {alpha}-NF, or SKF-525A for 24 h. The DEPE sample and other reagent solutions were added to the medium at volume ratios of 1:1000 (v/v). The final ethanol concentration in the medium was adjusted at 0.2% (v/v). After the treatment, the cells were lysed with 50 µl of PicaGene cell lysis buffer LUC (Toyo Ink, Tokyo, Japan). Luciferase activity in a cell lysate was assayed using a PicaGene luciferase kit (Toyo Ink, Tokyo, Japan) according to the manufacture’s protocol and normalized to protein concentration measured by a protein assay kit (Bio-Rad, Hercules, CA). Luciferase activities obtained on pGLPSA5.8, pLUC1A1 and pGL3-control vectors were designated as Luc_AR, Luc_AhR, and Luc_Control, respectively.

Ethoxyresorufin-O-deethylase (EROD) assay.
EROD activity was measured by a micro-assay method (Donato et al., 1993Go) according to our preceding article (Okamura et al., 2002Go). Fluorescence intensity of resorufin yielded from ethoxyresorufin was measured at excitation 550 nm and emission 585 nm and normalized to protein concentration.

Yeast two-hybrid assay.
A transformant of the yeast Saccharomyces cerevisiae, Y190 strain that was developed for two-hybrid assay of androgenic activity (Nishikawa et al., 1999Go) was kindly provided by Dr. T. Nishihara. This transformant is designated as Y190_AR. Yeast cells (Y190 strain) and yeast two-hybrid system 2 were purchased from Clontech (Palo Alto, CA, USA). Yeast cells were transformed with yeast two-hybrid system control vectors, pGBKT7-53 and pGADT7-T, using a lithium acetate method and grown on SD medium lacking tryptophan and leucine to yield a transformant that constitutively expresses ß-galactosidase. This transformant was designated as Y190_p53-SV40LT. Yeast cells were treated with 1.1% (v/v) ethanol (blank), 0.5 nM DHT (DHT control), FB, or a DEPE sample alone or in combination with 500 pM DHT for 4 h. DEPE sample and DHT solutions were added to medium at volume ratios of 1:100 and 1:1000 (v/v), respectively. The final ethanol concentration in medium was adjusted at 1.1% (v/v). After treatment, a portion of cell suspension solution was withdrawn and measured for absorbance at 620 nm as an indication of cell density. ß-Galactosidase activity was measured as described previously (Hirose et al., 2001Go) and normalized to absorbance at 620 nm (cell density). ß-Galactosidase activities obtained from Y190_AR and Y190_p53-SV40LT cells were designated as ß-Gal_AR and ß-Gal_Control, respectively.

AR competitive binding assay.
The ability of DEPE samples to bind AR was assayed by competitive ligand binding assay using a Ligand Screening System: Androgen Receptor Kit (Toyobo, Osaka, Japan). FB and DEPE samples were diluted 100, 333, or 1000 times with dimethyl sulfoxide (DMSO) and then subjected to the assay according to the manufacturer’s instructions. An androgen standard and a reference competitor employed in the kit were testosterone and mibolerone, respectively. DMSO and 250 nM milbolerone solutions were used as a blank (inhibition 0%) and a positive control (inhibition 100%), respectively.

Analysis of PAHs in DEPE samples.
PAHs in DEPE samples were analyzed with high-performance liquid chromatography coupled with fluorescence detection (Hayakawa et al., 2000Go). Concentrations of PAHs found in samples were determined by the peak height method.

Statistical analysis.
Statistical analyses were performed using unpaired Student’s t-test with StatView-J 5.0 for Macintosh computer (Nankodo; Tokyo, Japan). A value of p < 0.05 was considered to be significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Androgenic and Antiandrogenic Effects of DEPE Samples
PSA is a well-known androgen-regulated gene expressed in the prostate, an accessory sex organ. In this study, androgenic and antiandrogenic effects of DEPE samples were evaluated on the basis of PSA promoter-induced luciferase expression in PC3/AR wild-type human AR-expressing prostate carcinoma cells (Dai et al., 1996Go). First, we examined the responsiveness of this assay system to DHT and an AR antagonist BCT. Figure 1AGo shows that the expression of Luc_AR increased with DHT concentration with a maximal response (approximately 28-fold) at 1 to 10 nM DHT. On the other hand, Luc_AR activity induced by 1 nM DHT was completely inhibited to blank level by 10 µM BCT (Fig. 1BGo). Therefore, the Luc_AR is a robust system to detect androgenic activity.



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FIG. 1. Responsiveness of PC3/AR cells transiently transfected with pGLPSA5.8 vector to DHT (A) and BCT (B). (A) The cells were treated with 0.2% ethanol (blank) or DHT at indicated concentrations. (B) The cells were treated with 0.2% ethanol (blank) or 1 nM DHT in combination with BCT at indicated concentration. After treatment for 24 h, the cells were processed for measurement of luciferase activity as described in the Materials and Methods. Each column and vertical bar represent the mean and SD, respectively, from five separate cultures.

 
Since FB did not exhibit any significant effects on Luc_AR and Luc_Control activities in the absence and presence of DHT (Figs. 2AGo, 2BGo, and 2CGo), the androgenic and antiandrogenic effects of DEPE samples were then examined. No androgen agonist activity was detected in PC3/AR cells treated with each DEPE sample at 0.1, 1, or 10 µg/ml alone (Fig. 2AGo). Next, the cells were treated with each DEPE sample in the presence of 10 pM DHT, which gives approximately 60% of the maximum response, to examine the antiandrogenic activities of DEPE samples. The DEPE samples significantly reduced the DHT-induced Luc_AR activity in concentration-dependent manner, suggesting that DEPE samples have antiandrogenic activity in PC3/AR cells (Fig. 2BGo). EC, EB, and ET inhibited DHT-induced Luc_AR activity by approximately 26, 45, and 52%, respectively, at an extract concentration of 10 µg/ml. To address whether DEPEs lowered general gene transcription of PC3/AR cells, we treated PC3/AR cells transiently transfected with pGL3-control vector with each DEPE sample and measured Luc_Control activity. None of the DEPEs affected Luc_Control activity (Fig. 2CGo). This finding confirms that the observed reduction in DHT-induced Luc_AR activity in DEPE-treated cells was due to antiandrogenic effects of DEPEs mediated through the PSA promoter and that the antiandrogenic effect was greater in the following order: ET > EB > EC.



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FIG. 2. PC3/AR luciferase assay for androgenic (A), antiandrogenic (B), and cytotoxic (C) effects of DEPE samples. The cells transiently transfected with pGLPSA5.8 or pGL3-control vector were treated with 0.2% ethanol (blank), 10 pM DHT (DHT control), FB or each DEPE sample alone or in combination with 10 pM DHT for 24 h and then processed for measurement of luciferase activity. Each column and vertical bar represents the mean and SD, respectively, from five separate cultures. *,**Significantly different from DHT control (p < 0.05 and p < 0.01, respectively).

 
Cytochrome P450-dependent enzymes are involved in the synthesis and/or degradation of many exogenous as well as endogenous compounds. Although the details of CYP enzyme expression in PC3/AR cells is unknown, it is possible that metabolites of DEPE constituents contributed to the observed antiandrogenic effects. In fact, EROD activity, which mainly reflects CYP1 family enzyme activity (Rendic and Di Carlo, 1997Go), was significantly induced by DEPE samples in PC3/AR cells (Fig. 3AGo). Next, we examined the effect of SKF-525A, a well-known nonspecific CYP inhibitor, on the antiandrogenic effects of DEPE samples in the Luc_AR assay (Fig. 3BGo). In parallel, the CYP-inhibiting effect of SKF-525A was evaluated based on EROD activity (Fig. 3AGo). SKF-525A inhibited DEPE-induced EROD activities by approximately 70% at a SKF-525A concentration of 1 µM (Fig. 3AGo). SKF-525A had no effect on Luc_AR activities of blank and DHT control (Fig. 3BGo), indicating that SKF-525A does not cause alteration in cellular DHT concentration leading to the decrease or increase in Luc_AR activity in the absence and presence of DHT. On the other hand, SKF-525A further inhibited the DEPE-reduced Luc_AR activities by approximately 25% at 1 µM (Fig. 3BGo). Thus, blockade of CYP enzyme activity enhances the antiandrogenic effects of DEPEs, indicating that the intact species of DEPE constituents are in part responsible for the antiandrogenic effects observed in the Luc_AR assay rather than their metabolites.



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FIG. 3. Effect of SKF-525A on DEPE-induced EROD activity (A) and antiandrogenic effect (B) in PC3/AR cells. The cells transiently transfected with pGLPSA5.8 vector were treated with 0.2% ethanol (blank), 10 pM DHT (DHT control), 10 pM DHT—FB or 10 pM DHT—10 µg/ml DEPE sample without and with 1 µM SKF-525A for 24 h and then processed for measurement of EROD or luciferase activity. Each column and vertical bar represents the mean and SD, respectively, from five separate cultures. *,**Significantly different (p < 0.05 and p < 0.01, respectively).

 
Contribution of the Constituents with AhR Agonist Activity
Next, we examined the contribution of constituents with AhR agonist activity to the antiandrogenic effects of DEPE samples. In this experiment, we used {alpha}-NF to antagonize AhR (Merchant et al., 1993Go). Figure 4Go shows the Luc_AhR and Luc_AR activities in PC3/AR cells treated with each DEPE sample alone or in combination with DHT and/or {alpha}-NF. Whereas FB did not cause significant alterations in Luc_AhR activity, DEPE samples significantly increased Luc_AhR activity (approximate 2-fold by EC and 3-fold by EB and ET compared with DHT control; Fig. 4AGo) and these effects were concentration-dependent (data not shown). The DEPE-induced Luc_AhR activities were inhibited by approximately 40% at an {alpha}-NF concentration of 1 µM (Fig. 4AGo). It was found that the DEPE samples have AhR agonist activity and the activity was significantly antagonized by {alpha}-NF under the present experimental conditions. In Fig. 4BGo, Luc_AR activities of blank and DHT control shows that {alpha}-NF itself did not impact Luc_AR activity and had no effect on DHT-stimulated Luc_AR activity. These results indicate that {alpha}-NF also does not cause alteration in cellular DHT concentration leading to the decrease or increase in Luc_AR activity in the absence and presence of DHT. However, {alpha}-NF restored approximately 76, 56, and 45% of EC-, EB-, and ET-reduced Luc_AR activities, respectively, that is, the antiandrogenic effects of DEPE samples were attenuated by 1 µM {alpha}-NF which effectively antagonized AhR agonist activities of the DEPEs. The restored Luc_AR activity Further, Luc_AR activity of the cells treated with DHT, BaP, and {alpha}-NF was completely blocked by 10 µM bicalutamide, confirming that the Luc_AR activities observed in this study is mediated by AR. Together these findings indicate that the antiandrogenic effects of the DEPE samples are due in part to the constituents acting as AhR agonists.



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FIG. 4. Effect of {alpha}-NF on AhR agonist activities (A) and antiandrogenic effects (B) of DEPE samples in PC3/AR cells. The cells transiently transfected with pLUC1A1 or pGLPSA5.8 vector were treated with 0.2% ethanol (blank), 10 pM DHT (DHT control), 10 pM DHT—FB or 10 pM DHT—10 µg/ml DEPE sample without and with 1 µM {alpha}-NF for 24 h and then processed for measurement of luciferase activity. Each column and vertical bar represents the mean and SD, respectively, from five separate cultures. *,**Significantly different from DHT control (p < 0.05 and p < 0.01, respectively).

 
Contribution of PAHs
DEPs contain PAHs (WHO/IPCS, 1996Go) and some PAHs have been found to exert antiandrogenic effect (Kizu et al., 2003Go, Vinggaard et al., 2000Go). We determined the concentration of PAH in the DEPE samples by HPLC. A number of peaks corresponding to PAHs were observed in DEPE samples but not in FB (data not shown). Table 1Go lists the concentrations of PAHs having four or more rings that were identified in DEPE samples, i.e., pyrene, benz[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, perylene, benzo[b]chrysene, benzo[ghi]perylene, indeno[1,2,3-cd]pyrene, and coronene.


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TABLE 1 Concentrations of Polycyclic Aromatic Hydrocarbons Having Four or More Rings in the DEPE Samples
 
We evaluated the contribution of each of the identified PAHs to the antiandrogenic effects of DEPE samples. Since DEPE samples were added to the medium at a volume ratio of 1:1000, the antiandrogenic effect of each PAH was examined in a concentration range of 0.1 to 1 nM, based on their concentrations in the DEPE samples. None of the individual PAHs listed in Table 1Go showed any significant antiandrogenic effect in that concentration range. The sum concentration of PAHs in each DEPE sample was calculated from ten PAHs excluding pyrene because the ten PAHs exhibited the antiandrogenic effects at concentrations greater than 1 nM, but pyrene did not in the present assay system (data not shown). They were 1.79 µM in EC, 4.75 µM in EB, and 5.26 µM in ET. We then prepared an equimolecular mixture of the ten PAHs (10-PAHs-Mix, excluding pyrene) and examined the AhR agonist and antiandrogenic activities of this PAH mixture at concentrations of 1, 5, and 10 nM in PC3/AR cells transfected with reporter plasmid (Figs. 5AGo and 5BGo). The 10-PAHs-Mix exhibited significant AhR agonist activity at all concentrations tested (Fig. 5AGo). The 10-PAHs-Mix showed significant antiandrogenic activity at 5 nM, reducing DHT-induced Luc_AR activity by approximately 13% (Fig. 5BGo). The concentration of 5 nM is equivalent to the sum PAH concentration in EB or ET (Table 1Go). These data indicate that PAHs having four or more rings contributed to the observed AhR-mediated antiandrogenic effects of DEPE samples.



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FIG. 5. PC3/AR luciferase assay for AhR agonist activity (A) and antiandrogenic effect (B) of 10-PAHs-mix. The cells transiently transfected with pLUC1A1 or pGLPSA5.8 vector were treated with 0.2% ethanol (blank), 10 pM DHT (DHT control), or 10 pM DHT—10-PAHs-mix at indicated concentrations for 24 h and then processed for measurement of luciferase activity. Each column and vertical bar represents the mean and SD, respectively, from five separate cultures. *,**Significantly different (p < 0.05 and p < 0.01, respectively).

 
Contribution of the Constituents Capable of Binding to AR
Since DEPs contain a mixture of chemicals in addition to PAHs, it seems likely that the constituents capable of binding to AR may also be included in the DEPE samples. To test this idea, the androgenic and antiandrogenic effects of DEPE samples were evaluated based on a ß-galactosidase expression in yeast Y190_AR cells (description provided in Materials and Methods). First, we examined the responsiveness of Y190_AR cells to DHT concentrations from 10 pM to 100 nM (Fig. 6Go). DHT induced a concentration-dependent increase in ß-Gal_AR activity with a maximal response (approximately 13-fold) at 10 to 100 nM.



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FIG. 6. Responsiveness of Y190_AR cells to DHT. The cells were treated with 1.1% ethanol (blank) or DHT at indicated concentrations for 4 h and then processed for measurement ß-galactosidase activity as described in the Materials and Methods. Each column and vertical bar represents the mean and SD, respectively, from five separate cultures.

 
The androgenic and antiandrogenic effects of DEPE samples were evaluated by treating Y190_AR cells with each DEPE sample alone or in combination with 0.5 nM DHT which affords approximately 60% of the maximal ß-galactosidase response (Fig. 7Go). While DEPE samples did not increase the ß-Gal_AR activity in an extract concentration range of 1 to 10 ng/ml in the absence of DHT (Fig. 7AGo), they decreased the DHT-induced ß-Gal_AR activity in a concentration-dependent manner in the presence of DHT (Fig. 7BGo). EC, EB, and ET inhibited DHT-induced ß-Gal_AR activity by approximately 24, 38, and 48%, respectively, at an extract concentration of 10 ng/ml. On the other hand, no significant decrease in ß-Gal_Control was observed in Y190_p53-SV40LT cells treated similarly (Fig. 7CGo). FB did not exhibit any effects on ß-Gal_AR activity in Y190_AR and Y190_p53-SV40LT cells (Figs. 7AGo, 7BGo, and 7CGo). These data indicate that the suppression of DHT-induced ß-Gal_AR activity was not due to DEPE-induced cytotoxicities, but is caused by the antiandrogenic effects of DEPE samples. The antiandrogenic effect was greater in the following order: ET > EB > EC.



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FIG. 7. Yeast two-hybrid assay for androgenic (A), antiandrogenic (B), and cytotoxic (C) effects of DEPE samples. The Y190_AR or Y190_p53-SV40LT cells were treated with 1.1% ethanol (blank), 0.5 nM DHT (DHT control), FB or each DEPE sample alone or in combination with 0.5 nM DHT for 4 h and then processed for measurement of ß-galactosidase activity as described in the Materials and Methods. Each column and vertical bar represents the mean and SD, respectively, from five separate cultures. *,**Significantly different from DHT control (p < 0.05 and p < 0.01, respectively).

 
To address the hypothesis that constituents in the DEPE samples bind directly to the AR, a ligand binding competition assay was performed. Binding of testosterone to AR was inhibited by DEPE samples in a concentration-dependent manner but not by FB (Fig. 8Go). The inhibition was approximately 54% by EC, 74% by EB, and 77% by ET at an extract concentration of 100 µg/ml and thus the inhibitory effect was strongest with EB and ET and least with EC; the same order as the antiandrogenic effects of these DEPE samples in the yeast cell assay (Fig. 7BGo). These results indicate that AR-binding constituents are present in DEPE samples and they are mostly AR-antagonists.



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FIG. 8. Competitive AR binding assay for AR-binding constituents in DEPE samples. FB diluted 100 times and DEPE samples diluted with 100, 333, 1000 times with DMSO were subjected to the assay. Inhibition percentage was calculated by using DMSO and 250 nM milbolerone solutions as a blank (inhibition 0%) and a positive control (inhibition 100%), respectively, as indicated in the instruction of the kit. Each column and vertical bar represents the mean and SD, respectively, of quadruplicate assays.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The aim of present study was to determine whether antiandrogenic constituents are present in DEPs and to evaluate on their molecular mechanisms in cell-based assays. Here we used two assay methods, a luciferase reporter gene assay in PC3/AR human prostate cancer cells and a yeast two-hybrid assay to test the activities of AhR- and AR-binding constituents of three DEPE samples, EC, EB, and ET prepared from DEPs of diesel-powered vehicles, a car, a bus, and a truck. While the DEPE samples did not induce any significant reporter gene expression in the absence of DHT (Figs. 2AGo and 7AGo), they suppressed the DHT-induced gene expression in both assay methods (Figs. 2BGo and 7BGo). Since Luc_Control and ß-Gal_Control activities were not reduced by DEPE samples in PC3/AR or Y190_p53-SV40LT cells (Figs. 2CGo and 7CGo), the observed suppression of DHT-induced reporter gene expression was caused by the antiandrogenic effects of DEPE samples.

The present study demonstrated that the antiandrogenic effects were caused by two types of constituents: one is AhR agonist and the other is AR antagonist. As shown in Fig. 4BGo, {alpha}-NF, an AhR antagonist, decreased the antiandrogenic effects of DEPE samples. {alpha}-NF not only antagonizes AhR but also inactivates CYP 1 family enzymes. Since inhibition of CYP enzymes was shown to enhance the antiandrogenic effects of DEPEs (Fig. 3BGo), results of {alpha}-NF and SKF-525A experiments clearly indicate that activated AhR mediates the antiandrogenic effects of DEPE samples. As shown in Fig. 4BGo, {alpha}-NF restored Luc-AR by approximately half the DEPE-inhibited activity for each DEPE sample, EC, EB, and ET. Taking into consideration that {alpha}-NF did not completely antagonize AhR under the present experimental conditions (Fig. 4AGo), we conclude from our experiments that the major part of the antiandrogenic effects of DEPE samples are caused by constituents with AhR agonist activity.

PAHs are a typical class of chemicals found in DEPs (WHO/IPCS, 1996Go). The 10-PAHs-Mix, an equimolar mixture of ten PAHs having four or more rings identified in DB and DT, inhibited AR reporter activity at 5 nM, a concentration that is almost equivalent to sum concentrations of the ten PAHs found in EB or ET at the concentration of 10 µg/ml used in the PC3/AR assay (Table 1Go, Fig. 2Go). While EB and ET suppressed the DHT-induced Luc_AR by approximately 50% at an extract concentration of 10 µg/mL, 10-PAHs-Mix suppressed DHT-induced Luc_AR by approximately 13% at 5 nM (compare Figs. 2BGo and 6BGo). Thus, the 10-PAHs-Mix did not fully recapitulate the activity of the DEPE samples. One possible explanation for this difference is that several unidentified peaks were observed in chromatograms of the DEPE samples. Since analyses were made by fluorescence detection, unknown peaks should originate from PAHs having four or more ring. In fact, several PAHs other than ones identified in this study have been detected in DEPs: those include benzo[a]fluorene, cyclopenta[cd]pyrene, indeno[1,2,3-cd]fluoranthene, benzo[ghi]fluoranthene, and dibenzopyrene isomers (Sauvain et al., 2001Go; Westerholm et al., 2001Go; WHO/IPCS, 1996Go). Together these findings suggest that PAHs having four or more rings contribute to the observed AhR-mediated antiandrogenic effects of the DEPE samples.

The importance of AhR agonist activity in DEP effects on male reproductive-system functions has been suggested by in vivo studies in mice and rats. Lee et al.(1980)Go found that aryl hydrocarbon hydroxylase (a CYP1 family member) was induced in prostate, liver, and lung of adult male SD rats who had been exposed to DE by inhalation. We (Hatanaka et al., 2001Go) also reported that CYP 1A2 and 1B1 were induced in liver of adult male Fischer 344 rats who received inhalation exposure to DE. It is noteworthy that inhalation exposure to DE activated AhR in organs other than the lung, i.e., prostate and liver. On the other hand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a potent AhR agonist, has been shown to impair male reproductive functions in animal models (Gray et al., 2001Go). While many of the TCDD-induced alterations in male reproductive function in rats have been thought to be consequent to the reduced serum androgen concentration (Moore et al., 1985Go), this idea was contradicted by recent studies reporting that TCDD exerted its adverse effects without affecting circulatory androgen levels and steroidogenic enzyme activities in rats (Cooke et al., 1998Go; Gray et al., 1995Go; Roman et al., 1995Go), suggesting that activated AhR is implicated in the TCDD-induced dysfunction of male reproductive system. These findings strongly support the hypothesis that constituents of DEPs with AhR agonist activity play an important role in inhibiting spermatogenesis in rats and mice exposed to DE.

Previous works proposed possible mechanisms for inhibitory effect of activated AhR on androgen-induced AR transcriptional activity. While activated AhR induces expression of c-fos and c-jun genes and consequently an increase in transcription factor activator protein-1 (AP-1; Hoffer et al., 1996Go; Puga et al., 1992Go), AP-1 was documented to suppress the transcriptional activity of AR by two pathways. One pathway is that AP-1 inhibits the formation of AR-DNA complex by binding to DNA-binding domain of AR (Sato et al., 1997Go). Another pathway is that AP-1 competes with AR for limiting amount of CREB (cAMP response element binding protein) -binding protein (CBP), which functions as a coactivator for AR (Frønsdal et al., 1998Go). These mechanisms could be available to the antiandrogenic effect of DEPE samples observed in the present study.

Another type of constituent responsible for the antiandrogenic effects of DEPE samples is compounds with AR antagonist activity. Since we could not exclude the direct interaction of DEPE constituents with AhR in the PC3/AR cell line, i.e., because it expresses functional AhR, we used an yeast two-hybrid assay (Nishikawa et al., 1999Go) and a competitive AR ligand binding assay to examine the AR-mediated androgenic or antiandrogenic effects of DEPE samples. These experiments revealed that constituents capable of binding to AR were present in DEPE samples (Fig. 8Go) and that greater portion of them acted as AR antagonists (Fig. 7BGo). Environmental pollutants identified as AR antagonists are few: those include p,p’-DDT, p,p’-DDE, vinclozolin, and linuron (Gray et al., 2001Go). We are currently conducting chromatographic separation of DEPE constituents and assaying of their antiandrogenic activities to identify the compounds with direct AR antagonist activity.

Various adverse trends in male reproductive functions have been observed during the last decades (Sandblom and Varenhorse, 2001Go; Toppari et al., 1996Go). These trends include the increasing incidence rates of testicular and prostate cancers, declining semen quality, increasing frequencies of undescended testis, and hypospadias. It has been pointed out that environmental chemicals with antiandrogen activity may contribute to those adverse trends (Kelce and Wilson, 1997Go; Skakkebæk et al., 2001Go). DEPs are a possible environmental factor affecting male reproductive functions.

The present study showed that PAHs acting as AhR agonist played a role in the antiandrogenic effect of DEPE samples. PAHs exist ubiquitously in environment. While humans are exposed to PAHs from various sources including air and foods, cigarette smoke is an important source (Harvey, 1997Go). Epidemiological studies have shown that cigarette smoking impacts male fertility (Zinaman et al., 2000Go) and is associated with reduced semen quality (Kunzle et al., 2003Go; Trummer et al., 2002Go). It was reported that serum testosterone level was significantly higher in smokers than nonsmokers (Field et al., 1994Go; Trummer et al., 2002Go), suggesting that action of androgens is inhibited by cigarette smoking. It is possible that PAHs contribute to the effects of cigarette smoking on male fertility and reduced semen quality.

In sum, this is the first report showing that DEPE samples exhibit significant antiandrogenic effect in PC3/AR human prostate carcinoma cells and that these effects are due in part to the constituents with AhR agonist activity and to the constituents with AR antagonist activity. Further, PAHs having four or more rings were shown to contribute to a considerable extent to the AhR-mediated antiandrogenic effects of the DEPE samples. Further study is necessary to identify other DEP constituents with antiandrogenic activity and clarify their mechanisms of action.


    ACKNOWLEDGMENTS
 
The authors would like to thank Dr. J. Nishikawa and Dr. T. Nishihara for providing yeast cells for two-hybrid assay of androgenic activity, and Dr. R. H. Tukey for pLUC1A1 plasmid. This work was supported by Grant-in-Aid for Scientific Research on Priority Areas "The Environmental Risk of Endocrine Disruptor" (No.14042218) and Grant-in-Aid for Scientific Research (C) (No.13672342) to R.K. and Grant-in-Aid for Scientific Research (B) and Grant-in-Aid for the Kanazawa University 21st-Century COE Program to K.H. from the Ministry of Education, Culture, Sports and Science and Technology of Japan.


    NOTES
 
1 To whom correspondence should be addressed at Graduate School of Natural Science and Technology, Kanazawa University, 13-1, Takara-machi, Kanazawa 920-0934, Japan. Fax: +81-76-234-4456. E-mail: kizu{at}p.kanazawa-u.ac.jp. Back


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