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Environmental polycyclic aromatic hydrocarbons affect androgen receptor activation in vitro
Toxicology 145 (2000) 173 – 183
www.elsevier.com/locate/toxicol
Environmental polycyclic aromatic hydrocarbons affect androgen receptor activation in vitro Anne Marie Vinggaard *, Christina Hnida, John Christian Larsen Institute of Food Safety and Toxicology, Di6ision of Biochemical and Molecular Toxicology, Danish Veterinary and Food Administration, Morkhoj Bygade 19, 2860 Soborg, Denmark Received 19 August 1999; accepted 30 December 1999
Abstract Nine structurally different polycyclic aromatic hydrocarbons (PAHs) were tested for their ability to either agonize or antagonize the human androgen receptor (hAR) in a sensitive reporter gene assay based on CHO cells transiently cotransfected with a hAR vector and an MMTV-LUC vector. Benz[a]anthracene (B[a]A), benzo[a]pyrene (B[a]P), fluoranthene, chrysene and 7,12-dimethylbenz[a]anthracene (DMBA) were acting as antiandrogens in vitro, resulting in IC50 values of 3.2, 3.9, 4.6, 10.3 and 10.4 mM, respectively. Only at the highest concentration tested (10 mM), a slight inhibitory effect by pyrene, phenanthrene, and anthracene was observed. In contrast, dibenzo[a,h]anthracene (DB[a,h]A) gave rise to an agonistic effect, which was added upon the effect of the androgen receptor agonist R1881 (0.1 nM). The antiandrogenic responses by PAHs (10 mM) were found to be fully reversible, determined in the presence of increasing concentrations of R1881. No cytotoxic effects of the tested compounds were observed as determined either by metabolic reduction using AlamarBlue (up to 20 mM) or determined in cells transfected with a constitutively active hAR (up to 10 mM). The well-known ability of certain PAHs to activate the Ah receptor was assessed in H4IIE liver cancer cells, stably transfected with a luciferase reporter gene system. The positive control 2,3,7,8-tetrachlorodibenzodioxin (TCDD) caused a 13 – 14-fold induction of luciferase activity reaching maximum activity at 0.1 nM. DB[a,h]A, B[a]P, Chrysene, B[a]A and DMBA gave rise to a 4.5-fold induction of luciferase activity at 0.03, 0.4, 0.89, 3.06, and 9.27 mM, respectively, whereas fluoranthene, pyrene, phenanthrene and anthracene were without effect. In conclusion, no clear correlation between the antiandrogenic effects and the Ah receptor activation in vitro was seen. However, the Ah receptor agonists containing four or five aromatic rings (i.e. B [a] A, B [a] P, chrysene, DMBA) appeared to be the most potent antiandrogens (with the exception of DB [a, h] A), whereas those not able to activate the Ah receptor containing three or four aromatic rings (i.e. pyrene, phenanthrene, anthracene) displayed either very weak or no antiandrogenic effect at concentrations up to 10 mM (with the exception of fluoranthene which blocked the hAR at lower concentrations, but did not activate the Ah receptor). © 2000
Abbre6iations: B[a]A, benz[a]anthracene; B[a]P, benzo[a]pyrene; DB[a,h]A, dibenzo[a,h]anthracene; DES, diethylstilbestrol; DMBA, 7,12-dimethylbenz[a]anthracene; hAR, human androgen receptor; PAH, polycyclic aromatic hydrocarbon; p,p%-DDE, 1,1%(2,2-dichloroethylidene)bis(4-chlorobenzene); p,p%-DDT, 1,1%(2,2,2-trichloroethylidene)bis(4-chlorobenzene); R1881, methyltrienolone; TCDD, 2,3,7,8-tetrachlorodibenzodioxin. * Corresponding author. Tel.: + 45-33-956000; fax: +45-33-956696. E-mail address: [email protected] (A.M. Vinggaard) 0300-483X/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 0 0 ) 0 0 1 4 3 - 8
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Elsevier Science Ireland Ltd. All rights reserved. Keywords: Androgen receptor; Polycyclic aromatic hydrocarbons; Ah receptor; Endocrine disruptors; Antiandrogen; Reporter gene assay
1. Introduction Environmental chemicals may disrupt reproductive development in wildlife and humans by either mimicking or inhibiting the action of the gonadal steroid hormones, estradiol and testosterone. Several pieces of evidence indicate that environmental chemicals, which are able to bind to the androgen receptor (AR) may have an important impact on abnormalities associated with the developing male reproductive system (Kelce and Wilson, 1997; Gray et al., 1999). An inhibition of the action of the AR during the embryonic stage very often leads to alterations in the development of the male external genitalia such as cryptorchidism and hypospadia (Foster, 1997). Thus, environmental antiandrogens may have contributed to the increasing incidence of reproductive abnormalities observed in the human male population and potentially cause demasculinization of males (Kelce and Wilson, 1997). The number of environmental chemicals identified with antiandrogenic properties is steadily growing (Gray et al., 1999). For these chemicals, receptor mediated as well as non-receptor mediated toxicity has been reported as potential mechanisms of actions. Many small molecules that were hitherto considered to be biologically inert have been found to interact with the AR specifically and affect hormonal activities in vivo. Metabolites of the fungicide vinclozolin (Wong et al., 1995), of the insecticide p,p%-DDT (i.e. p,p%DDE) (Kelce et al., 1995), and the fungicide procymidone (Gray et al., 1993) have been found as potent antagonists of the binding and transactivation of the hAR in vitro and in vivo. In contrast, dibutylphthalate has been found to act as an antiandrogen via a non-receptor mediated mechanism (Mylchreest et al., 1999). The compound caused cryptorchidism and degeneration of testicular tissues, but in contrast to the well-known AR antagonist, flutamide, caused only a low incidence
of hypospadias and prostate agenesis. Subsequent in vitro analysis revealed that the compound did not act directly via the receptor. PAHs are a class of toxic organic chemicals comprising hundreds of congeners that are ubiquitous in the environment and in foodstuffs. They are produced and released into the environment by incomplete combustion of fossil fuel, oil spills, and industrial processes. A number of PAHs have shown carcinogenicity in experimental animals following oral, pulmonary, dermal or subcutaneous administration. Tumor formation has been induced in a number of tissues, such as lung, skin, forestomach, liver, the haematopoeitic system, and the mammary gland (IARC, 1983, 1987; WHO/IPCS, 1998). Most of the carcinogenic PAHs are metabolically activated to DNA-binding, reactive diol-epoxides, which are thought to act as the initiators of the carcinogenicity. In addition the more potent carcinogenic PAHs are able to activate the Ah receptor, which might play a role in promoting carcinogenicity. A growing body of literature identifies PAHs as potential environmental endocrine disruptors (Santodonato, 1997). First of all, PAHs may act as antiestrogens by binding to the Ah receptor (Chaloupka et al., 1992, 1993; Clemons et al. 1998; Arcaro et al. 1999) leading to induction of Ah responsive genes that result in a broad spectrum of antiestrogenic responses. Secondly, PAHs may act as antiestrogens by blocking activation of the estrogen receptor (ER) in transfected yeast cells (Tran et al., 1996), whereas agonistic activities were observed in transfected MCF7 cells (Clemons et al., 1998). Thirdly, simple binding to ER of PAHs or their hydroxylated metabolites has been reported to occur in some cases (Schneider et al., 1976; Ebright et al., 1986; Shen et al., 1993; Arcaro et al., 1999) but not in others (Heizmann and Wyss, 1972; Toft and Spelsberg, 1972; Keightley and Okey, 1973). Binding studies in vitro employing PAHs must, however, be
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viewed with caution in light of the large number of mono- and dihydroxy metabolites that are produced in intact cells or animals, which may serve as ligands with higher ER binding affinity than the parent compound. Furthermore, the presence of different ER subtypes within different tissues may explain some of the conflicting findings of PAH binding. Potent aryl hydrocarbon carcinogens bear a remarkable similarity in their molecular architecture to certain steroid hormones (Santodonato, 1997). Especially, the superimposibility of estradiol with certain hydroxy derivatives of carcinogenic PAH is clear, leading to the suggestion that the apparent steric similarities to steroids is important for the capability for interaction and binding with specific cytoplasmic receptor proteins to cause enhanced gene transcription. Natural and synthetic estrogens (like diethylstilbestrol and 17b-estradiol) and very often so called environmental estrogens are able to activate or block the AR (Kelce et al., 1995; Sohoni and Sumpter, 1998) underlining the relevance of testing interaction of environmental chemicals with both receptor types. In this study, eight structurally different and commonly found PAHs, as well as the experimental carcinogen DMBA, were tested for their ability to either agonize or antagonize the human AR in a sensitive reporter gene assay. These data were related to the ability of the compounds to activate the Ah receptor in a stably transfected liver cancer cell line.
2. Materials and methods
2.1. Androgen receptor assay Chinese Hamster Ovary cells (CHO K1) were maintained in DMEM/F12 (Gibco, Paisley, UK) supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma, St. Louis, MO) and 10% fetal bovine serum (BioWhitaker, Walkersville, MD). The cells were seeded in microtiter plates (Costar, Acton, MA) at a density of 5000 cells per well in DMEM/F12 containing 10% charcoal-treated fetal bovine serum (Hyclone, Lo-
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gan, Utah) and incubated at 37°C in a humidified atmosphere of 5% CO2/air. After 24 h, the chemicals to be tested were added, dissolved in culture medium. The test solutions were prepared from 10 mM stock solutions in DMSO (final DMSO concentration in the media was 0.05–0.1%). R1881 (in EtOH) was from NEN (Boston, MA), benzo[a]pyrene (Sigma), benz[a]anthracene (Sigma, 95% pure), dibenzo[a,h]anthracene (Supelco, 99% pure), DMBA (Fluka, 97% pure), chrysene (Supelco, 99% pure), fluoranthene (Supelco, 99% pure), pyrene (Supelco, 99% pure), anthracene (Riedel-de Ha¨en, \ 99% pure), phenanthrene (Riedel-de Ha¨en, 98% pure). Shortly after addition of test compounds, each well was transfected with a total of 50 ng DNA consisting of the expression vector pSVAR0 (Brinkmann et al., 1989) and the MMTV-LUC reporter plasmid (both provided by Dr Albert Brinkmann, Erasmus University, Rotterdam) in a ratio of 1:100 using 0.15 ml of the non-liposomal transfection reagent FuGene (Boehringer Mannheim, Germany). After an incubation period of 24 h, the media was aspirated and the cells were lysed by adding 15 ml per well of a lysis buffer containing 25 mM trisphosphate, pH 7.8, 15% glycerol, 1% Triton X-100, 1 mM DTT and 8 mM MgCl2, followed by shaking at room temperature for 10 min. Five microlitre was transferred to white Dynatech microtiter plates for measurement of luciferase activity in a BioOrbit Galaxy luminometer. Ten microlitre of a substrate containing 1 mM luciferin (Amersham, Buckinghamshire, UK) and 1 mM ATP (Boehringer Mannheim, Germany) in lysis buffer was injected automatically and the chemiluminiscence generated from each well was measured over a 1 s interval after an incubation time of 2 s.
2.2. Cytotoxicity tests Testing for cytotoxicity specifically on cell number was determined by measuring the reduction of AlamarBlue (Serotec, Kidlington, UK). The assay is based on metabolic reduction of the AlamarBlue dye into a fluorescent species, which is detected after excitation of the reduced dye at 560-
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nm and subsequent emission at 590 nm. Each well was added 5× 103 CHO cells in black clear-bottomed microtiter plates (Costar). The following day, compounds dissolved in media were added, and the plates were incubated for 24 h before addition of a 10% solution of AlamarBlue in media. Fluorescence was measured after 3 h at a slit width of 10 nm for both excitation and emission using a PerkinElmer luminiscence spectrophotometer LS50B, equipped with a microtiter plate reader. The fluorescence intensity was found to be linear for these assay conditions including cell numbers and incubation period for CHO cells, which is in agreement with previous reports (Nakayama et al., 1997; Vinggaard et al., 1999). The response is expressed as arbitrary fluorescence units after subtraction of background levels of fluorescence. Testing for cytotoxicity specifically on the transactivation process was performed as described above for the AR assay, except that the hAR expression vector was replaced by the constitutively active AR expression vector, pSVAR13 (a generous gift from Dr Albert Brinkmann), which lacks the ligand binding domain of the receptor.
2.3. Ah receptor assay (CALUX assay) The Chemical-Activated Luciferase Expression assay (CALUX assay) is based on H4IIE cells, stably transfected with a luciferase reporter gene, which were kindly provided by Dr Abraham Brouwer, Waageningen University, The Netherlands. Cells were maintained in MEMa medium supplemented with 5% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. The cells were seeded at a density of 2.21× 104 cells/ well (100 ml/well) into Costar microtiter plates. After an incubation period of 24 h (80 – 90% confluence of cells), media was aspirated, and PAHs (0.001–10 mM) and TCDD (0.003 – 3 nM) were added. The DMSO concentration was maximally 0.4%. The following day, cells were washed twice with PBS and added 20 ml of lysis buffer (as used in the AR assay). Luciferase activity was determined as described above for the AR assay.
2.4. Statistical analysis Data were analysed by One Way Analysis of Variance (SigmaStat®, vers. 2.0). If a statistically significant difference was detected, multiple comparisons were performed using a Bonferroni test. PB0.05 was regarded as statistically significant.
3. Results
3.1. Effects of PAHs on androgen receptor acti6ation The in vitro method used here for determination of antiandrogenic effects has previously been validated and found applicable for this purpose (Vinggaard et al., 1999). We selected nine commonly found PAHs with different structures, including three, four or five aromatic rings (Fig. 1). Among these DB[a,h]A, B[a]P, B[a]A, DMBA and chrysene are considered to be carcinogenic, whereas pyrene, phenanthrene and anthracene are considered not to be carcinogenic. A limited number of recent studies have shown that fluoranthene is an experimental carcinogen (WHO/IPCS, 1998). The compounds were tested for antiandrogenic effects in CHO cells transiently cotransfected with a hAR vector and an MMTV-LUC vector (Fig. 2). B[a]A, B[a]P, fluoranthene, chrysene and DMBA were able to act as antiandrogens in vitro, resulting in IC50 values of 3.2, 3.9, 4.6, 10.3 and 10.4 mM, respectively (Table 1). At the highest concentration tested (10 mM), a slight inhibitory, although not statistically significant, effect by pyrene, phenanthrene, and anthracene was observed. In contrast, DB[a,h]A gave rise to an agonistic effect, which was added upon the effect of 0.1 nM R1881. Data showed that many of the compounds caused a slight increase in luciferase activity at the lowest concentrations, an effect that disappeared at higher concentrations. Some variation between the transfection efficiency in repeated experiments was observed. However, as the transfection reagent FuGene™ results in very efficient transfections, no need for a transfection control has arisen. The protocol used
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implicated the addition of test compounds before the transfections were performed. In order to exclude that R1881 or the PAHs affected the transfection efficiency, experiments were performed, in which transfections were done before or after addition of R1881 and B[a]P and similar results were obtained (data not shown). In order to determine if the antiandrogenic responses by PAHs were reversible, transfected
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cells were incubated in the presence of 10 mM PAH and increasing concentrations of R1881 (Fig. 3). Results showed that the antagonizing effect was totally reversed by the AR agonist. Furthermore, data showed that the degree of inhibition of the hAR activation in general was dependent on the selected androgen concentration, i.e. lower IC50 values were obtained when using 0.01 nM R1881 compared to 0.1 nM, which was chosen in this study on the basis of a R1881 dose–response curve. On the other hand, at the higher R1881 concentrations (1–100 nM) no inhibitory effects were observed in general, but in some cases rather a slightly stimulatory effect. In agreement with the data for DB[a,h]A in Fig. 2, this compound gave rise to an agonistic response at all tested R1881 concentrations, an effect that was additive.
3.2. Cytotoxicity test of PAHs For each environmental compound tested in this reporter gene assay, a cytotoxicity test on non-transfected cells determining the cell number and/or viability was included. The cytotoxicity of the PAHs was assessed by determining the number of CHO cells per well by the AlamarBlue assay, measuring metabolic reduction (Fig. 4) and no cytotoxicity was observed at concentrations of 0.2–20 mM. In addition, the cytotoxicity of the chemicals on the transactivating process was assessed by transfecting the cells with the truncated and constitutively active human pSVAR13 vector. None of the chemicals tested exhibited alterations in this process up to 10 mM (data not shown).
3.3. Effects of PAHs on Ah receptor acti6ation
Fig. 1. Structures of selected PAHs. The compounds were selected in order to embrace compounds with three, four, and five aromatic rings.
The well-known ability of certain PAHs to activate the Ah receptor was assessed in H4IIE liver cancer cells, stably transfected with a luciferase reporter gene system (Murk et al., 1996). The positive control TCDD caused a 13–14-fold induction of luciferase activity reaching maximum activity at 0.1 nM (Fig. 5). TCDD, DB[a,h]A, B[a]P, Chrysene, B[a]A and DMBA gave rise to a 4.5-fold induction of luciferase activity at 2.3× 10 − 6, 0.03, 0.4, 0.89, 3.06, and 9.27 mM, respec-
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Fig. 2. Antiandrogenic effects of PAHs determined in CHO cells transfected with hAR. A total of 5 × 103 CHO cells were seeded into microtiter plates (0.1 ml/well) and the following day media 9 test compounds was added. R1881 0.1 nM was present in all wells in the presence of 0, 0.05, 0.1, 0.5, 1, 5, and 10 mM PAH. Shortly after addition of compounds, cells were cotransfected with pSVAR0 and MMTV-LUC (1:100) as described in Section 2. Fifteen microlitres cell lysis buffer was added 24 h later and luminiscence units were determined in 5 ml cell extract using 10 ml luciferin/ATP as described. Data are presented as luminiscence units in%. The response to 0.1 nM R1881 was set to 100%, which corresponded to 11714 91912 units (mean 9SD, n =3). Data represent the mean 9 SEM of three experiments performed in quadruplicate. An asterisk is indicating PB 0.05.
tively, whereas fluoranthene, pyrene, phenanthrene and anthracene were without effect (Table 1). The EC50 could be calculated for TCDD and DB[a,h]A to be 5 pM and 60 nM, respectively, indicating that DB[a,h]A is 1.2× 104 times less potent than TCDD.
4. Discussion The results obtained show that the position of the aromatic rings in the PAH is very important for the effect on the hAR. Comparing the two compounds containing five rings show that B[a]P
is acting as an antagonist, whereas DB[a,h]A is acting as an agonist at concentrations between 0.05 and 10 mM. Comparing compounds with four aromatic rings show that B[a]A is clearly the most potent antiandrogen, whereas much weaker antiandrogenic effects by chrysene, DMBA, and pyrene are observed. Surprisingly, fluoranthene, containing three aromatic rings and one cyclopentane ring, was antiandrogenic at concentrations of 5 and 10 mM. Fluoranthene is not able to activate the Ah receptor, but is present in the environment in high amounts relative to many other PAHs (Clemons et al., 1998; Arcaro et al., 1999). Thus, it will be very interesting to examine whether this
Fig. 3. Test for reversibility of the PAH-induced antiandrogenic effect. Each PAH was tested either in the presence or absence of increasing concentrations of R1881 added to CHO cells cotransfected with the hAR and MMTV-LUC as described in Section 2. Filled circles represent R1181 dose-response curves without addition of PAH, whereas filled squares represent R1881 dose-response curves in the presence of 10 mM PAH. Responses are depicted as luminiscence units in%. The response to 1 nM R1881 (maximum response) was set to 100%. Values represent the mean 9 SD of a representative experiment of two, performed in quadruplicate.
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Fig. 3. (Continued)
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Table 1 Effects of PAHs on the hAR and the Ah receptora Compound
Inhibitory effects on hAR IC50 (mM)
Activating effects of Ah Rec 4.5-fold induction (mM)
2,3,7,8-TCDD Benz[a] anthracene Benzo[a]pyrene Fluoranthene
n.d. 3.2
2.3×10−6 3.06
Chrysene DMBA Pyrene
10.3b 10.4b \10
Phenanthrene
10
Anthracene
10
Dibenzo[a,h] anthracene
Activation (0.1–10 mM)
3.9 4.6
0.40 No activation (510 mM) 0.89 9.27 No activation (510 mM) No activation (510 mM) No activation (510 mM) 0.03
a Concentrations necessary for 50% inhibition of hAR transactivation induced by R1881 (0.1 nM) in transiently transfected CHO cells compared to concentrations that elicit a 4.5-fold induction of Ah receptor activation in stably transfected H4IIE liver cancer cells. b Estimated value. n.d.: Not determined.
antiandrogenic effect in vitro can be confirmed from in vivo studies. In an early study by Chang and Liao (1987), phenanthrene was reported to bind weakly to the AR of rat prostate (IC50 =800 mM), whereas it did not bind to either the ER or the glucocorticoid receptor. The 9,10-dihydro derivative of phenanthrene was 80-fold more active than the compound itself. In vivo experiments showed that both compounds reduced the androgen-dependent growth of the ventral prostate, seminal vesicles and coagulating gland in castrated rats. These results led to the suggestion that polycyclic aromatic hydrocarbons and some of their metabolites can be active antiandrogens in mammals. In our assay system a slight, but not significant, antiandrogenic effect of phenanthrene was evident at 10 mM, which is in agreement with the binding data. Higher concentrations were not tested here as these were considered to be of less relevance.
Only very few in vivo studies on reproductive toxicity of PAHs have been reported. In one study, oral doses of 40 mg/kg of B[a]P were administered to pregnant mice on days 7–16, and caused total sterility in 97% of the F1 generation. Impaired fertility was observed in both male and female mice at a dose of 10 mg/kg and was associated with marked alterations in germ cell development (including impaired gametogenesis and folliculogenesis) and a dramatic decrease in the size of the gonads (Mackenzie and Angevine, 1981). Thus, the fetal gonads are very sensitive to B[a]P. Whether the antiandrogenic effects by PAHs observed in this study are due to (a) a direct binding to the AR (b) binding by one or more metabolites of the PAH to AR or (c) an increase in the metabolism of R1881 remains to be shown. The last possibility, however, seems less likely, as R1881 is generally known as a compound that resists metabolism (Liao et al., 1973). Whether the effects are due to merely a physical blockade of the receptor or the effects are due to the presence of a specific group, arisen after metabolism, that interacts with the receptor remains to be shown. A large number of mono- and dihydroxy metabolites of PAHs may be produced in intact cells or animals, which may serve as ligands with higher binding affinity than the parent compound. Previous results in our AR assay on the antiandrogenic effects of vinclozolin, which requires metabolism to be active (Wong et al., 1995), showed that the CHO cells possess some biotransformation capacity as vinclozolin was active (Vinggaard et al., 1999). Thus, the formation of hydroxylated metabolites of the PAHs during the 24 h incubation period is very likely. The Ah receptor activation obtained by 2,3,7,8TCDD in the CALUX assay is in agreement with the original report by Murk et al. (1996). The detection limit is around 0.5 fmol TCDD and saturation is achieved at 0.1 nM TCDD. The fold induction here is found to be 14, whereas a maximum induction of 30-fold was reported previously, but this may be due to the use of microtiter plates in this study compared to the 24-well plates used by the group of Murk.
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Fig. 4. Test of cytotoxicity of PAHs. PAHs were added at concentrations of 0, 0.2, 2 and 20 mM to CHO cells (5×103 cells/well in microtiter plates) and incubated for 24 h before determination of cell number per well using the AlamarBlue cell proliferation assay as described. Background fluorescence was subtracted from all values. Results represent the mean 9SD of eight determinations from two experiments.
In a recent study, the estrogenic and Ah receptor activating effects by PAHs were compared using in vitro reporter gene assays (Clemons et al., 1998). Three PAHs, namely B[a]P, chrysene, and B[a]A were found to be both estrogenic in transient transfected MCF7 cells with EC50 values of 1.3, 4.0 and 5.5 mM, respectively, and to activate the Ah receptor. Benz[k]fluoranthene, DB[a,h]A and anthracene also exhibited Ah receptor activity. Comparing these results to our data, show that the three estrogenic compounds were also among the most potent antiandrogenic compounds in our assay system, displaying binding affinities in the same order of magnitude. Slightly deviating data on the Ah receptor activation was found in the two reporter gene assays. The greatest discrepancy was the Ah receptor agonism of anthracene found by Clemons et al. but not by us, and furthermore the relative potencies of the other active PAHs was slightly deviating. How-
ever, comparing our Ah receptor data to a recent study in rat hepatocytes using the erthoxyresorufin-O-deethylase (EROD) bioassay as the endpoint (Till et al., 1999) show that exactly the same ranking potency (i.e. DB[a,h]A\ B[a]P\ chrysene\ B[a]A) is observed. Furthermore, the same ranking of the compounds for EROD activation was reported by Willett et al. (1997). In conclusion, this study demonstrates further evidence for the promiscuity of the hAR to bind structurally different compounds, as environmentally prevalent PAHs or their metabolites are capable of interacting in vitro with the hAR and inducing an AR-mediated response. No clear correlation between the antiandrogenic effects and the Ah receptor activation in vitro was seen. However, the Ah receptor agonists containing four or five aromatic rings (B[a]A, B[a]P, chrysene, DMBA) appeared to be the most potent antiandrogens (with the exception of DB[a,h]A),
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by Organon BV. Thanks to Lonnie Sand for excellent technical assistance.
References
Fig. 5. Activation of the Ah receptor by PAHs determined in the CALUX assay. H4IIE cells, stably transfected with a luciferase reporter gene, were seeded at a density of 2.21 × 104 cells/well in microtiter plates. After an incubation period of 24 h, PAHs (0.001 – 10 mM) or TCDD (0.003–3 nM) was added. Luciferase activity was determined the following day as described in Section 2. TCDD, solid triangles (upwards); DB[a,h]A, solid circles; B[a]P, open circles; Chrysene, solid triangles (downwards); B[a]A, open triangles; DMBA, solid squares. Data are presented as luminiscence units relative to control values ( = 1). The actual luminiscence units for a control incubation was 495 9 96 units (mean 9 SD, n= 3). Results represent the mean 9 SD of four independent experiments performed in quadruplicate.
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Acknowledgements The support by the Danish Medical Research grant no. 9700832 is greatly acknowledged. Thanks to Dr Albert Brinkmann, Dept. of Endocrinology and Reproduction, Erasmus University, Rotterdam for providing the pSVAR0, the pSVAR13, and the MMTV-LUC constructs. The MMTV-LUC construct was originally developed
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A.M. Vinggaard et al. / Toxicology 145 (2000) 173–183 ferentiation produces diverse profiles of reproductive malformations in the male rat. Toxicol. Ind. Health 15 (1–2), 94– 118. Heizmann, C.W., Wyss, H.I., 1972. Comparison of the binding of 7,12-dimethylbenz[a]anthracene and estradiol-17b in the calf uterus cytosol. Steroids 20 (4), 499–514. IARC, 1983. Polynuclear aromatic compounds, Part 1, Chemicals, Environmental and Experimental data. IARC Monographs on the Evaluation of the carcinogenic risk of chemicals to humans. Vol. 32. International Agency for Research on Cancer, Lyon, France. IARC, 1987. Overall evaluations of carcinogenicity: An updating of IARC Monographs, Vol 1 to 42. IARC Monographs on the Evaluation of the carcinogenic risk of chemicals to humans. Suppl. 7. International Agency for Research on Cancer, Lyon, France. Keightley, D.D., Okey, A.B., 1973. Effects of dimethylbenz[a]anthracene and dihydrotestosterone on estradiol17b binding in rat mammary cytosol fraction. Cancer Res. 33, 2637 – 2642. Kelce, W.R., Stone, C.R., Laws, S.C., Gray, L.E., Kemppainen, J.A., Wilson, E.M., 1995. Persistent DDT metabolite p,p%-DDE is a potent androgen receptor antagonist. Nature 375, 581 – 585. Kelce, W.R., Wilson, E.M., 1997. Environmental antiandrogens: developmental effects, molecular mechanisms, and clinical implications. J. Mol. Med. 75 (3), 198–207. Liao, S., Liang, T., Fang, S., Castaneda, E., Shao, T.C., 1973. Steroid structure and androgenic activity: specificities involved in the receptor binding and nuclear retention of various androgens. J. Biol. Chem. 248, 6154–6162. Mackenzie, K.M., Angevine, D.M., 1981. Infertility in mice exposed in utero to benzo[a]pyrene. Biol. Reprod. 24, 183 – 191. Murk, J., Legler, J., Denison, M.S., Giesy, J.P., Guchte, C. van den, Brouwer, A., 1996. Chemical-Activated Luciferase gene eXpression (CALUX): a novel in vitro bioassay for Ah-receptor active compounds in sediments and pore water. Fundam. Appl. Toxicol. 33, 149–160. Mylchreest, E., Sar, M., Cattley, R.C., Foster, P.M.D., 1999. Disruption of androgen-regulated male reproductive development by di(n-butyl)phthalate during late gestation in rats is different from flutamide. Toxicol. Appl. Pharmacol. 156, 81 – 95.
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Nakayama, G.R., Caton, M.C., Nova, M.P., Parandoosh, Z., 1997. Assessment of the Alamar Blue assay for cellular growth and viability in vitro. J. Immunol. Methods 204, 205 – 208. Santodonato, J., 1997. Review of the estrogenic and antiestrogenic activity of polycyclic aromatic hydrocarbons: relationship to carcinogenecity. Chemosphere 34 (4), 835 – 848. Schneider, S.L., Alks, V., Morreal, C.E., Sinha, D.K., Dao, T.L., 1976. Estrogenic properties of 3,9-dihydroxybenz[a]anthracene, a potential metabolite of benz[a]anthracene. J. Natl. Cancer Inst. 57 (6), 1351 – 1354. Shen, Y.Y., Kim, S.S., Yun, H.C., 1993. Effect of 3-methylcholanthrene on rat uterus: uterine growth and mechanism of action of 3-methylcholanthrene. Arch. Pharm. Res. 4, 276 – 282. Sohoni, P., Sumpter, J.P., 1998. Several environmental oestrogens are also anti-androgens. J. Endocrinol. 158, 327 – 339. Till, M., Riebinger, D., Schmitz, H.-J., Schrenk, D., 1999. Potency of various polycyclic aromatic hydrocarbons as inducers of CYP1A1 in rat hepatocyte cultures. Chem. Biol. Interact. 117, 135 – 150. Toft, D.O., Spelsberg, T.C., 1972. A distinction between 3methylcholanthrene and estrogen binding in the uterus. Cancer Res. 32, 2743 – 2746. Tran, D.Q., Ide, C.F., McLachlan, J.A., Arnold, S.F., 1996. The antiestrogenic activity of selected polynuclear aromatic hydrocarbons in yeast expressing human estrogen recceptor. Biochem. Biophys. Res. Commun. 229, 102 – 108. Vinggaard, A.M., Jørgensen, E.C.B., Larsen, J.C., 1999. Rapid and sensitive reporter gene assays for detection of antiandrogenic and estrogenic environmental chemicals. Toxicol. Appl. Pharmacol. 155 (2), 150 – 160. WHO/IPCS, 1998. Environmental Health Criteria 202. Selected non-heterocyclic polycyclic aromatic hydrocarbons. World Health Organization, Geneva, Switzerland. Willett, K.L., Gardinali, P.R., Seticano, J.L., Wade, T.L., Safe, S.H., 1997. Characterization of H4IIE rat Hepatoma Cell Bioassay for evaluation of environmental samples containing polynuclear aromatic hydrocarbons (PAH). Arch. Environ. Contam. Toxicol. 32, 442 – 448. Wong, C., Kelce, W.R., Sar, M., Wilson, E.M., 1995. Androgen receptor antagonist versus agonist activities of the fungicide vinclozolin relative to hydroxyflutamide. J. Biol. Chem. 270, 34, 19998 – 20003.
www.elsevier.com/locate/toxicol
Environmental polycyclic aromatic hydrocarbons affect androgen receptor activation in vitro Anne Marie Vinggaard *, Christina Hnida, John Christian Larsen Institute of Food Safety and Toxicology, Di6ision of Biochemical and Molecular Toxicology, Danish Veterinary and Food Administration, Morkhoj Bygade 19, 2860 Soborg, Denmark Received 19 August 1999; accepted 30 December 1999
Abstract Nine structurally different polycyclic aromatic hydrocarbons (PAHs) were tested for their ability to either agonize or antagonize the human androgen receptor (hAR) in a sensitive reporter gene assay based on CHO cells transiently cotransfected with a hAR vector and an MMTV-LUC vector. Benz[a]anthracene (B[a]A), benzo[a]pyrene (B[a]P), fluoranthene, chrysene and 7,12-dimethylbenz[a]anthracene (DMBA) were acting as antiandrogens in vitro, resulting in IC50 values of 3.2, 3.9, 4.6, 10.3 and 10.4 mM, respectively. Only at the highest concentration tested (10 mM), a slight inhibitory effect by pyrene, phenanthrene, and anthracene was observed. In contrast, dibenzo[a,h]anthracene (DB[a,h]A) gave rise to an agonistic effect, which was added upon the effect of the androgen receptor agonist R1881 (0.1 nM). The antiandrogenic responses by PAHs (10 mM) were found to be fully reversible, determined in the presence of increasing concentrations of R1881. No cytotoxic effects of the tested compounds were observed as determined either by metabolic reduction using AlamarBlue (up to 20 mM) or determined in cells transfected with a constitutively active hAR (up to 10 mM). The well-known ability of certain PAHs to activate the Ah receptor was assessed in H4IIE liver cancer cells, stably transfected with a luciferase reporter gene system. The positive control 2,3,7,8-tetrachlorodibenzodioxin (TCDD) caused a 13 – 14-fold induction of luciferase activity reaching maximum activity at 0.1 nM. DB[a,h]A, B[a]P, Chrysene, B[a]A and DMBA gave rise to a 4.5-fold induction of luciferase activity at 0.03, 0.4, 0.89, 3.06, and 9.27 mM, respectively, whereas fluoranthene, pyrene, phenanthrene and anthracene were without effect. In conclusion, no clear correlation between the antiandrogenic effects and the Ah receptor activation in vitro was seen. However, the Ah receptor agonists containing four or five aromatic rings (i.e. B [a] A, B [a] P, chrysene, DMBA) appeared to be the most potent antiandrogens (with the exception of DB [a, h] A), whereas those not able to activate the Ah receptor containing three or four aromatic rings (i.e. pyrene, phenanthrene, anthracene) displayed either very weak or no antiandrogenic effect at concentrations up to 10 mM (with the exception of fluoranthene which blocked the hAR at lower concentrations, but did not activate the Ah receptor). © 2000
Abbre6iations: B[a]A, benz[a]anthracene; B[a]P, benzo[a]pyrene; DB[a,h]A, dibenzo[a,h]anthracene; DES, diethylstilbestrol; DMBA, 7,12-dimethylbenz[a]anthracene; hAR, human androgen receptor; PAH, polycyclic aromatic hydrocarbon; p,p%-DDE, 1,1%(2,2-dichloroethylidene)bis(4-chlorobenzene); p,p%-DDT, 1,1%(2,2,2-trichloroethylidene)bis(4-chlorobenzene); R1881, methyltrienolone; TCDD, 2,3,7,8-tetrachlorodibenzodioxin. * Corresponding author. Tel.: + 45-33-956000; fax: +45-33-956696. E-mail address: [email protected] (A.M. Vinggaard) 0300-483X/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 0 0 ) 0 0 1 4 3 - 8
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Elsevier Science Ireland Ltd. All rights reserved. Keywords: Androgen receptor; Polycyclic aromatic hydrocarbons; Ah receptor; Endocrine disruptors; Antiandrogen; Reporter gene assay
1. Introduction Environmental chemicals may disrupt reproductive development in wildlife and humans by either mimicking or inhibiting the action of the gonadal steroid hormones, estradiol and testosterone. Several pieces of evidence indicate that environmental chemicals, which are able to bind to the androgen receptor (AR) may have an important impact on abnormalities associated with the developing male reproductive system (Kelce and Wilson, 1997; Gray et al., 1999). An inhibition of the action of the AR during the embryonic stage very often leads to alterations in the development of the male external genitalia such as cryptorchidism and hypospadia (Foster, 1997). Thus, environmental antiandrogens may have contributed to the increasing incidence of reproductive abnormalities observed in the human male population and potentially cause demasculinization of males (Kelce and Wilson, 1997). The number of environmental chemicals identified with antiandrogenic properties is steadily growing (Gray et al., 1999). For these chemicals, receptor mediated as well as non-receptor mediated toxicity has been reported as potential mechanisms of actions. Many small molecules that were hitherto considered to be biologically inert have been found to interact with the AR specifically and affect hormonal activities in vivo. Metabolites of the fungicide vinclozolin (Wong et al., 1995), of the insecticide p,p%-DDT (i.e. p,p%DDE) (Kelce et al., 1995), and the fungicide procymidone (Gray et al., 1993) have been found as potent antagonists of the binding and transactivation of the hAR in vitro and in vivo. In contrast, dibutylphthalate has been found to act as an antiandrogen via a non-receptor mediated mechanism (Mylchreest et al., 1999). The compound caused cryptorchidism and degeneration of testicular tissues, but in contrast to the well-known AR antagonist, flutamide, caused only a low incidence
of hypospadias and prostate agenesis. Subsequent in vitro analysis revealed that the compound did not act directly via the receptor. PAHs are a class of toxic organic chemicals comprising hundreds of congeners that are ubiquitous in the environment and in foodstuffs. They are produced and released into the environment by incomplete combustion of fossil fuel, oil spills, and industrial processes. A number of PAHs have shown carcinogenicity in experimental animals following oral, pulmonary, dermal or subcutaneous administration. Tumor formation has been induced in a number of tissues, such as lung, skin, forestomach, liver, the haematopoeitic system, and the mammary gland (IARC, 1983, 1987; WHO/IPCS, 1998). Most of the carcinogenic PAHs are metabolically activated to DNA-binding, reactive diol-epoxides, which are thought to act as the initiators of the carcinogenicity. In addition the more potent carcinogenic PAHs are able to activate the Ah receptor, which might play a role in promoting carcinogenicity. A growing body of literature identifies PAHs as potential environmental endocrine disruptors (Santodonato, 1997). First of all, PAHs may act as antiestrogens by binding to the Ah receptor (Chaloupka et al., 1992, 1993; Clemons et al. 1998; Arcaro et al. 1999) leading to induction of Ah responsive genes that result in a broad spectrum of antiestrogenic responses. Secondly, PAHs may act as antiestrogens by blocking activation of the estrogen receptor (ER) in transfected yeast cells (Tran et al., 1996), whereas agonistic activities were observed in transfected MCF7 cells (Clemons et al., 1998). Thirdly, simple binding to ER of PAHs or their hydroxylated metabolites has been reported to occur in some cases (Schneider et al., 1976; Ebright et al., 1986; Shen et al., 1993; Arcaro et al., 1999) but not in others (Heizmann and Wyss, 1972; Toft and Spelsberg, 1972; Keightley and Okey, 1973). Binding studies in vitro employing PAHs must, however, be
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viewed with caution in light of the large number of mono- and dihydroxy metabolites that are produced in intact cells or animals, which may serve as ligands with higher ER binding affinity than the parent compound. Furthermore, the presence of different ER subtypes within different tissues may explain some of the conflicting findings of PAH binding. Potent aryl hydrocarbon carcinogens bear a remarkable similarity in their molecular architecture to certain steroid hormones (Santodonato, 1997). Especially, the superimposibility of estradiol with certain hydroxy derivatives of carcinogenic PAH is clear, leading to the suggestion that the apparent steric similarities to steroids is important for the capability for interaction and binding with specific cytoplasmic receptor proteins to cause enhanced gene transcription. Natural and synthetic estrogens (like diethylstilbestrol and 17b-estradiol) and very often so called environmental estrogens are able to activate or block the AR (Kelce et al., 1995; Sohoni and Sumpter, 1998) underlining the relevance of testing interaction of environmental chemicals with both receptor types. In this study, eight structurally different and commonly found PAHs, as well as the experimental carcinogen DMBA, were tested for their ability to either agonize or antagonize the human AR in a sensitive reporter gene assay. These data were related to the ability of the compounds to activate the Ah receptor in a stably transfected liver cancer cell line.
2. Materials and methods
2.1. Androgen receptor assay Chinese Hamster Ovary cells (CHO K1) were maintained in DMEM/F12 (Gibco, Paisley, UK) supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin (Sigma, St. Louis, MO) and 10% fetal bovine serum (BioWhitaker, Walkersville, MD). The cells were seeded in microtiter plates (Costar, Acton, MA) at a density of 5000 cells per well in DMEM/F12 containing 10% charcoal-treated fetal bovine serum (Hyclone, Lo-
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gan, Utah) and incubated at 37°C in a humidified atmosphere of 5% CO2/air. After 24 h, the chemicals to be tested were added, dissolved in culture medium. The test solutions were prepared from 10 mM stock solutions in DMSO (final DMSO concentration in the media was 0.05–0.1%). R1881 (in EtOH) was from NEN (Boston, MA), benzo[a]pyrene (Sigma), benz[a]anthracene (Sigma, 95% pure), dibenzo[a,h]anthracene (Supelco, 99% pure), DMBA (Fluka, 97% pure), chrysene (Supelco, 99% pure), fluoranthene (Supelco, 99% pure), pyrene (Supelco, 99% pure), anthracene (Riedel-de Ha¨en, \ 99% pure), phenanthrene (Riedel-de Ha¨en, 98% pure). Shortly after addition of test compounds, each well was transfected with a total of 50 ng DNA consisting of the expression vector pSVAR0 (Brinkmann et al., 1989) and the MMTV-LUC reporter plasmid (both provided by Dr Albert Brinkmann, Erasmus University, Rotterdam) in a ratio of 1:100 using 0.15 ml of the non-liposomal transfection reagent FuGene (Boehringer Mannheim, Germany). After an incubation period of 24 h, the media was aspirated and the cells were lysed by adding 15 ml per well of a lysis buffer containing 25 mM trisphosphate, pH 7.8, 15% glycerol, 1% Triton X-100, 1 mM DTT and 8 mM MgCl2, followed by shaking at room temperature for 10 min. Five microlitre was transferred to white Dynatech microtiter plates for measurement of luciferase activity in a BioOrbit Galaxy luminometer. Ten microlitre of a substrate containing 1 mM luciferin (Amersham, Buckinghamshire, UK) and 1 mM ATP (Boehringer Mannheim, Germany) in lysis buffer was injected automatically and the chemiluminiscence generated from each well was measured over a 1 s interval after an incubation time of 2 s.
2.2. Cytotoxicity tests Testing for cytotoxicity specifically on cell number was determined by measuring the reduction of AlamarBlue (Serotec, Kidlington, UK). The assay is based on metabolic reduction of the AlamarBlue dye into a fluorescent species, which is detected after excitation of the reduced dye at 560-
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nm and subsequent emission at 590 nm. Each well was added 5× 103 CHO cells in black clear-bottomed microtiter plates (Costar). The following day, compounds dissolved in media were added, and the plates were incubated for 24 h before addition of a 10% solution of AlamarBlue in media. Fluorescence was measured after 3 h at a slit width of 10 nm for both excitation and emission using a PerkinElmer luminiscence spectrophotometer LS50B, equipped with a microtiter plate reader. The fluorescence intensity was found to be linear for these assay conditions including cell numbers and incubation period for CHO cells, which is in agreement with previous reports (Nakayama et al., 1997; Vinggaard et al., 1999). The response is expressed as arbitrary fluorescence units after subtraction of background levels of fluorescence. Testing for cytotoxicity specifically on the transactivation process was performed as described above for the AR assay, except that the hAR expression vector was replaced by the constitutively active AR expression vector, pSVAR13 (a generous gift from Dr Albert Brinkmann), which lacks the ligand binding domain of the receptor.
2.3. Ah receptor assay (CALUX assay) The Chemical-Activated Luciferase Expression assay (CALUX assay) is based on H4IIE cells, stably transfected with a luciferase reporter gene, which were kindly provided by Dr Abraham Brouwer, Waageningen University, The Netherlands. Cells were maintained in MEMa medium supplemented with 5% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. The cells were seeded at a density of 2.21× 104 cells/ well (100 ml/well) into Costar microtiter plates. After an incubation period of 24 h (80 – 90% confluence of cells), media was aspirated, and PAHs (0.001–10 mM) and TCDD (0.003 – 3 nM) were added. The DMSO concentration was maximally 0.4%. The following day, cells were washed twice with PBS and added 20 ml of lysis buffer (as used in the AR assay). Luciferase activity was determined as described above for the AR assay.
2.4. Statistical analysis Data were analysed by One Way Analysis of Variance (SigmaStat®, vers. 2.0). If a statistically significant difference was detected, multiple comparisons were performed using a Bonferroni test. PB0.05 was regarded as statistically significant.
3. Results
3.1. Effects of PAHs on androgen receptor acti6ation The in vitro method used here for determination of antiandrogenic effects has previously been validated and found applicable for this purpose (Vinggaard et al., 1999). We selected nine commonly found PAHs with different structures, including three, four or five aromatic rings (Fig. 1). Among these DB[a,h]A, B[a]P, B[a]A, DMBA and chrysene are considered to be carcinogenic, whereas pyrene, phenanthrene and anthracene are considered not to be carcinogenic. A limited number of recent studies have shown that fluoranthene is an experimental carcinogen (WHO/IPCS, 1998). The compounds were tested for antiandrogenic effects in CHO cells transiently cotransfected with a hAR vector and an MMTV-LUC vector (Fig. 2). B[a]A, B[a]P, fluoranthene, chrysene and DMBA were able to act as antiandrogens in vitro, resulting in IC50 values of 3.2, 3.9, 4.6, 10.3 and 10.4 mM, respectively (Table 1). At the highest concentration tested (10 mM), a slight inhibitory, although not statistically significant, effect by pyrene, phenanthrene, and anthracene was observed. In contrast, DB[a,h]A gave rise to an agonistic effect, which was added upon the effect of 0.1 nM R1881. Data showed that many of the compounds caused a slight increase in luciferase activity at the lowest concentrations, an effect that disappeared at higher concentrations. Some variation between the transfection efficiency in repeated experiments was observed. However, as the transfection reagent FuGene™ results in very efficient transfections, no need for a transfection control has arisen. The protocol used
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implicated the addition of test compounds before the transfections were performed. In order to exclude that R1881 or the PAHs affected the transfection efficiency, experiments were performed, in which transfections were done before or after addition of R1881 and B[a]P and similar results were obtained (data not shown). In order to determine if the antiandrogenic responses by PAHs were reversible, transfected
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cells were incubated in the presence of 10 mM PAH and increasing concentrations of R1881 (Fig. 3). Results showed that the antagonizing effect was totally reversed by the AR agonist. Furthermore, data showed that the degree of inhibition of the hAR activation in general was dependent on the selected androgen concentration, i.e. lower IC50 values were obtained when using 0.01 nM R1881 compared to 0.1 nM, which was chosen in this study on the basis of a R1881 dose–response curve. On the other hand, at the higher R1881 concentrations (1–100 nM) no inhibitory effects were observed in general, but in some cases rather a slightly stimulatory effect. In agreement with the data for DB[a,h]A in Fig. 2, this compound gave rise to an agonistic response at all tested R1881 concentrations, an effect that was additive.
3.2. Cytotoxicity test of PAHs For each environmental compound tested in this reporter gene assay, a cytotoxicity test on non-transfected cells determining the cell number and/or viability was included. The cytotoxicity of the PAHs was assessed by determining the number of CHO cells per well by the AlamarBlue assay, measuring metabolic reduction (Fig. 4) and no cytotoxicity was observed at concentrations of 0.2–20 mM. In addition, the cytotoxicity of the chemicals on the transactivating process was assessed by transfecting the cells with the truncated and constitutively active human pSVAR13 vector. None of the chemicals tested exhibited alterations in this process up to 10 mM (data not shown).
3.3. Effects of PAHs on Ah receptor acti6ation
Fig. 1. Structures of selected PAHs. The compounds were selected in order to embrace compounds with three, four, and five aromatic rings.
The well-known ability of certain PAHs to activate the Ah receptor was assessed in H4IIE liver cancer cells, stably transfected with a luciferase reporter gene system (Murk et al., 1996). The positive control TCDD caused a 13–14-fold induction of luciferase activity reaching maximum activity at 0.1 nM (Fig. 5). TCDD, DB[a,h]A, B[a]P, Chrysene, B[a]A and DMBA gave rise to a 4.5-fold induction of luciferase activity at 2.3× 10 − 6, 0.03, 0.4, 0.89, 3.06, and 9.27 mM, respec-
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Fig. 2. Antiandrogenic effects of PAHs determined in CHO cells transfected with hAR. A total of 5 × 103 CHO cells were seeded into microtiter plates (0.1 ml/well) and the following day media 9 test compounds was added. R1881 0.1 nM was present in all wells in the presence of 0, 0.05, 0.1, 0.5, 1, 5, and 10 mM PAH. Shortly after addition of compounds, cells were cotransfected with pSVAR0 and MMTV-LUC (1:100) as described in Section 2. Fifteen microlitres cell lysis buffer was added 24 h later and luminiscence units were determined in 5 ml cell extract using 10 ml luciferin/ATP as described. Data are presented as luminiscence units in%. The response to 0.1 nM R1881 was set to 100%, which corresponded to 11714 91912 units (mean 9SD, n =3). Data represent the mean 9 SEM of three experiments performed in quadruplicate. An asterisk is indicating PB 0.05.
tively, whereas fluoranthene, pyrene, phenanthrene and anthracene were without effect (Table 1). The EC50 could be calculated for TCDD and DB[a,h]A to be 5 pM and 60 nM, respectively, indicating that DB[a,h]A is 1.2× 104 times less potent than TCDD.
4. Discussion The results obtained show that the position of the aromatic rings in the PAH is very important for the effect on the hAR. Comparing the two compounds containing five rings show that B[a]P
is acting as an antagonist, whereas DB[a,h]A is acting as an agonist at concentrations between 0.05 and 10 mM. Comparing compounds with four aromatic rings show that B[a]A is clearly the most potent antiandrogen, whereas much weaker antiandrogenic effects by chrysene, DMBA, and pyrene are observed. Surprisingly, fluoranthene, containing three aromatic rings and one cyclopentane ring, was antiandrogenic at concentrations of 5 and 10 mM. Fluoranthene is not able to activate the Ah receptor, but is present in the environment in high amounts relative to many other PAHs (Clemons et al., 1998; Arcaro et al., 1999). Thus, it will be very interesting to examine whether this
Fig. 3. Test for reversibility of the PAH-induced antiandrogenic effect. Each PAH was tested either in the presence or absence of increasing concentrations of R1881 added to CHO cells cotransfected with the hAR and MMTV-LUC as described in Section 2. Filled circles represent R1181 dose-response curves without addition of PAH, whereas filled squares represent R1881 dose-response curves in the presence of 10 mM PAH. Responses are depicted as luminiscence units in%. The response to 1 nM R1881 (maximum response) was set to 100%. Values represent the mean 9 SD of a representative experiment of two, performed in quadruplicate.
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Fig. 3. (Continued)
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Table 1 Effects of PAHs on the hAR and the Ah receptora Compound
Inhibitory effects on hAR IC50 (mM)
Activating effects of Ah Rec 4.5-fold induction (mM)
2,3,7,8-TCDD Benz[a] anthracene Benzo[a]pyrene Fluoranthene
n.d. 3.2
2.3×10−6 3.06
Chrysene DMBA Pyrene
10.3b 10.4b \10
Phenanthrene
10
Anthracene
10
Dibenzo[a,h] anthracene
Activation (0.1–10 mM)
3.9 4.6
0.40 No activation (510 mM) 0.89 9.27 No activation (510 mM) No activation (510 mM) No activation (510 mM) 0.03
a Concentrations necessary for 50% inhibition of hAR transactivation induced by R1881 (0.1 nM) in transiently transfected CHO cells compared to concentrations that elicit a 4.5-fold induction of Ah receptor activation in stably transfected H4IIE liver cancer cells. b Estimated value. n.d.: Not determined.
antiandrogenic effect in vitro can be confirmed from in vivo studies. In an early study by Chang and Liao (1987), phenanthrene was reported to bind weakly to the AR of rat prostate (IC50 =800 mM), whereas it did not bind to either the ER or the glucocorticoid receptor. The 9,10-dihydro derivative of phenanthrene was 80-fold more active than the compound itself. In vivo experiments showed that both compounds reduced the androgen-dependent growth of the ventral prostate, seminal vesicles and coagulating gland in castrated rats. These results led to the suggestion that polycyclic aromatic hydrocarbons and some of their metabolites can be active antiandrogens in mammals. In our assay system a slight, but not significant, antiandrogenic effect of phenanthrene was evident at 10 mM, which is in agreement with the binding data. Higher concentrations were not tested here as these were considered to be of less relevance.
Only very few in vivo studies on reproductive toxicity of PAHs have been reported. In one study, oral doses of 40 mg/kg of B[a]P were administered to pregnant mice on days 7–16, and caused total sterility in 97% of the F1 generation. Impaired fertility was observed in both male and female mice at a dose of 10 mg/kg and was associated with marked alterations in germ cell development (including impaired gametogenesis and folliculogenesis) and a dramatic decrease in the size of the gonads (Mackenzie and Angevine, 1981). Thus, the fetal gonads are very sensitive to B[a]P. Whether the antiandrogenic effects by PAHs observed in this study are due to (a) a direct binding to the AR (b) binding by one or more metabolites of the PAH to AR or (c) an increase in the metabolism of R1881 remains to be shown. The last possibility, however, seems less likely, as R1881 is generally known as a compound that resists metabolism (Liao et al., 1973). Whether the effects are due to merely a physical blockade of the receptor or the effects are due to the presence of a specific group, arisen after metabolism, that interacts with the receptor remains to be shown. A large number of mono- and dihydroxy metabolites of PAHs may be produced in intact cells or animals, which may serve as ligands with higher binding affinity than the parent compound. Previous results in our AR assay on the antiandrogenic effects of vinclozolin, which requires metabolism to be active (Wong et al., 1995), showed that the CHO cells possess some biotransformation capacity as vinclozolin was active (Vinggaard et al., 1999). Thus, the formation of hydroxylated metabolites of the PAHs during the 24 h incubation period is very likely. The Ah receptor activation obtained by 2,3,7,8TCDD in the CALUX assay is in agreement with the original report by Murk et al. (1996). The detection limit is around 0.5 fmol TCDD and saturation is achieved at 0.1 nM TCDD. The fold induction here is found to be 14, whereas a maximum induction of 30-fold was reported previously, but this may be due to the use of microtiter plates in this study compared to the 24-well plates used by the group of Murk.
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Fig. 4. Test of cytotoxicity of PAHs. PAHs were added at concentrations of 0, 0.2, 2 and 20 mM to CHO cells (5×103 cells/well in microtiter plates) and incubated for 24 h before determination of cell number per well using the AlamarBlue cell proliferation assay as described. Background fluorescence was subtracted from all values. Results represent the mean 9SD of eight determinations from two experiments.
In a recent study, the estrogenic and Ah receptor activating effects by PAHs were compared using in vitro reporter gene assays (Clemons et al., 1998). Three PAHs, namely B[a]P, chrysene, and B[a]A were found to be both estrogenic in transient transfected MCF7 cells with EC50 values of 1.3, 4.0 and 5.5 mM, respectively, and to activate the Ah receptor. Benz[k]fluoranthene, DB[a,h]A and anthracene also exhibited Ah receptor activity. Comparing these results to our data, show that the three estrogenic compounds were also among the most potent antiandrogenic compounds in our assay system, displaying binding affinities in the same order of magnitude. Slightly deviating data on the Ah receptor activation was found in the two reporter gene assays. The greatest discrepancy was the Ah receptor agonism of anthracene found by Clemons et al. but not by us, and furthermore the relative potencies of the other active PAHs was slightly deviating. How-
ever, comparing our Ah receptor data to a recent study in rat hepatocytes using the erthoxyresorufin-O-deethylase (EROD) bioassay as the endpoint (Till et al., 1999) show that exactly the same ranking potency (i.e. DB[a,h]A\ B[a]P\ chrysene\ B[a]A) is observed. Furthermore, the same ranking of the compounds for EROD activation was reported by Willett et al. (1997). In conclusion, this study demonstrates further evidence for the promiscuity of the hAR to bind structurally different compounds, as environmentally prevalent PAHs or their metabolites are capable of interacting in vitro with the hAR and inducing an AR-mediated response. No clear correlation between the antiandrogenic effects and the Ah receptor activation in vitro was seen. However, the Ah receptor agonists containing four or five aromatic rings (B[a]A, B[a]P, chrysene, DMBA) appeared to be the most potent antiandrogens (with the exception of DB[a,h]A),
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by Organon BV. Thanks to Lonnie Sand for excellent technical assistance.
References
Fig. 5. Activation of the Ah receptor by PAHs determined in the CALUX assay. H4IIE cells, stably transfected with a luciferase reporter gene, were seeded at a density of 2.21 × 104 cells/well in microtiter plates. After an incubation period of 24 h, PAHs (0.001 – 10 mM) or TCDD (0.003–3 nM) was added. Luciferase activity was determined the following day as described in Section 2. TCDD, solid triangles (upwards); DB[a,h]A, solid circles; B[a]P, open circles; Chrysene, solid triangles (downwards); B[a]A, open triangles; DMBA, solid squares. Data are presented as luminiscence units relative to control values ( = 1). The actual luminiscence units for a control incubation was 495 9 96 units (mean 9 SD, n= 3). Results represent the mean 9 SD of four independent experiments performed in quadruplicate.
whereas those not able to activate the Ah receptor containing three or four aromatic rings (pyrene, phenanthrene, anthracene) had either very weak or no antiandrogenic effect at concentrations up to 10 mM. Fluoranthene, which blocked the hAR, did not activate the Ah receptor. It remains to be shown in future experiments whether these in vitro data on the PAHs are predictive of any adverse effects in wildlife or mammals.
Acknowledgements The support by the Danish Medical Research grant no. 9700832 is greatly acknowledged. Thanks to Dr Albert Brinkmann, Dept. of Endocrinology and Reproduction, Erasmus University, Rotterdam for providing the pSVAR0, the pSVAR13, and the MMTV-LUC constructs. The MMTV-LUC construct was originally developed
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