* Toxicology Research Division, Food Directorate, Health Canada; and
Reproductive Biology Unit, and Departments of Cellular and Molecular Medicine and Obstetrics and Gynecology, University of Ottawa, Sir Frederick G. Banting Research Centre, Ottawa, Ontario, Canada
Received February 23, 1999; accepted August 30, 1999
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ABSTRACT |
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Key Words: androgen; receptor; pesticides; food additives; human.
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INTRODUCTION |
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MATERIALS AND METHODS |
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Cell culture and transfection.
Human PC-3 cells were maintained in 75 cm2 tissue culture flasks containing 15 ml RPMI medium supplemented with 10% fetal bovine serum stripped of steroids by charcoal/dextran (Borras et al., 1994) and gentamycin. Cells were passaged by trypsinization as required.
Cells were transfected using the calcium phosphate:DNA coprecipitation technique of Graham and van der Eb (1973). Briefly, PC-3 cultures were trypsinized, 250,000 PC-3 cells seeded into 60-mm tissue culture dishes, and the cultures allowed to attach overnight. The next day, cultures were cotransfected with pCMV5-hAR (5 µg per plate) and pMAMneo-luc (0.05 µg per plate), the cultures incubated (4 h), and given a 15% glycerol shock. The transfected cells were allowed to recover for 2 days and were then trypsinized and transferred to 100-mm tissue culture dishes. Stable transfectants were selected for G418 (a neomycin analogue) resistance. Resistant colonies were surrounded using cloning cylinders, isolated by trypsinization, and amplified in 24-well tissue culture plates. When confluent, the cells were split into triplicate plates and when again confluent, 1 nM DHT was added to one plate and incubation continued for 24 h. The cells from one of the untreated plates were then lysed in lysis buffer (100 µl, 25 mM Tris-phosphate, pH 7.8, 2mM CDTA, 10% glycerol, 1% Triton-X100) as well as those from the DHT treated plates, and 10-µl aliquots used for luciferase activity. The cells in the remaining plate corresponding to the clone exhibiting a combination of low background fluorescence and high DHT induction ration, designated PC-3 LUCAR+, were chosen for further characterization.
Firefly luciferase assay procedure.
PC-3 LUCAR+ cells (200,000 in 1 ml medium) were cultured in 24-well plates at 200,000 cells per well for 24 h prior to the addition of DHT (50 pM) and/or chemicals of interest at 0, 0.1, 1.0, or 10.0 µM. Chemicals and steroids were added in 1µl DMSO such that cultures were never exposed to greater than 0.1% DMSO. Eighteen hours later, lysis buffer (100 µl) was added to each well and the plates agitated gently prior to being frozen at 20°C until assay (usually within 48 h). For assay, 10-µl aliquots of cell lysate were transferred to 51 x 12 mm polypropylene luminometer cuvettes (Sarstedt) and light emission measured with a BioOrbit (Turku, Finland) luminometer after injection of 40 µl luciferase assay reagent. Each of the chemicals of interest was examined in at least three separate assays.
The kinetic characteristics of the activation of luciferase activity by DHT in cells were investigated using a range of DHT concentrations (0, 5, 25, 50, 75, 100, 250, and 500 pM). Inhibition of the effect of DHT (0, 5, 25, 50, 75, 100, 250, and 500 pM) was examined using flutamide at 0, 0.25, 1.0, and 10.0 µM. Steroid specificity was assessed by replacing DHT with other steroids (progesterone, estradiol, hydrocortisone, dexamethasone, DES, each at 100, 250, 500, 750, 1000progesterone, estradiol, hydrocortisone, dexamethasone, DES, each at 100, 250, 500, 750, 2000, and 5000 pM) and comparing the activation with that seen with DHT.
Androgen receptor assays.
The number of specific DHT binding sites per PC-3 LUCAR+ cell was assessed using modifications of both whole cell (Olea-Serrano et al., 1985) and cytosolic (Green et al., 1986
; 1988
) assays with nonspecific binding determined by parallel assays using a PC-3 clone in which only the luciferase reporter had been transfected (PC-3 LUCAR). For the whole cell assays, cells were grown in 24-well plates until confluent, including three wells for the determination of cell number. On the day of the experiment, the wells allocated for cell number determination were trypsinized and the cells counted by haemocytometer. For receptor binding measurement in the remaining wells, the medium was replaced and cells incubated in the presence of 0.26163 nM 3H-DHT for 1 h. After incubation, the medium was removed and the monolayer washed three times with ice-cold PBS. Bound DHT was extracted from the cells by incubation in 250 µl ethanol at room temperature (20 min). Radioactivity in the extracts (200-µl aliquots) was quantified by scintillation counting and specific binding calculated by subtracting the radioactivity per cell associated with PC-3 LUCAR cells from that found with PC-3 LUCAR+ cells. The maximal specific binding per cell was determined by subjecting a plot of the specific radioactivity bound per cell against DHT concentration to one site binding hyperbola nonlinear regression analysis and Scatchard analysis of DHT bound/free against DHT bound (Sigmaplot 4.0, SPSS Inc, 1997, San Rafael, CA) The receptor number was then calculated by taking into account the DHT specific activity and Avogadro's constant.
The second method involved a radioreceptor assay of the cytosol prepared by ultracentrifugation of homogenized PC-3 LUCAR+ as well as PC-3 LUCAR cells. Cells from 10100 mm dishes were trypsinized, combined, counted, and lysed by 50 strokes in a glass homogenizer on ice in 1 ml of 20 mM TrisHCl, pH 7.4, 1 mM EDTA, 2mM DTT, 50 mM NaCl, 0.3 mM PMSF, 10 mM Na molybdate. After the cytosol was cleared by centrifugation (10,000 x g, 15 min, 4°C), glycerol was added to the supernatant to 10% final concentration. Aliquots (50 µl) were taken for hormone binding and incubated with 0100 nM 3H DHT (0°C, 18 h). Dextran-coated charcoal was added (0.5% in 10 mM Tris-HCl, pH 7.5, 100 µl), the samples incubated on ice (15 min) to remove free steroid, and the samples centrifuged to sediment the charcoal. Bound steroid was measured in 100 µl of supernatant by scintillation counting, specific binding calculated after subtraction of nonspecific binding, and the number of sites per cell determined as for the whole cell assay.
Neutral red assay for toxicity.
The effects of the chemicals of interest on cell viability were assessed by staining parallel cultures with neutral red using a modified technique of Borenfreund and Puerner (1985). Briefly, 24 h after chemical addition, neutral red solution (4 µl) was added per well to a final concentration of approximately 52 µg/ml, and the cultures incubated (1 h). The neutral red- containing medium was removed, the wells washed with PBS, and solubilization buffer added (50% ethanol, 1% acetic acid; 100 µl/well). After mixing on an orbital shaker (15 min), the plates were read on a Cytofluor fluorescence plate reader (Millipore, Beford, MA; 485 nm excitation, 645 nm emission).
Statistical analyses.
Kinetic constants for the induction of luciferase expression by DHT and inhibition by flutamide were determined by a nonlinear least-squares method resident in the graphics software (Graph-Pad Prism, version 1.02, 1994, Graphpad Software Incorporated, San Diego, CA). Slope and intercept replots to determine Ki values were obtained from, respectively, Kd/Vmax vs [I] and 1/Vmax vs [I]. Linear regression analyses of the replots were done to determine the Ki values. Significant effects of the chemicals of interest on luciferase activity were determined by one-way ANOVA (Sigmastat 2.0, Jandel Scientific, 1992Sigmastat 2.0, Jandel Scientific, 1995, San Rafael, CA) using arcsine transformed proportionalized data. EC50 values were determined from the equations for hyperbolic decay generated by Sigmaplot (Sigmaplot 4.0, SPSS Inc., 1997, San Rafael, CA).
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RESULTS |
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The Effect of p,p'-DDE on Androgen Receptor Activation
When PC-3 LUCAR+ cells were incubated with p,p'-DDE (50 and 100 µM), luciferase activity was induced, although not as effectively as observed with 50 pM DHT (Fig. 6). However, p,p'-DDE concentrations of 1.0 and 5.0 µM partially antagonized (58%) the DHT- mediated activation of the androgen receptor, and higher concentrations completely antagonized the DHT activation (Fig. 6
and Table 1
); the luciferase activity measured was the same as in the nonstimulated cells (p > 0.05). Cell viability was reduced by p,p'-DDE concentrations of 50 and 100 µM (36% and 24%, respectively, compared with control) but in the presence of 1.0, 5.0, and 10.0 µM p,p'-DDE, the viability was 100, 89, and 76% compared with control.
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DISCUSSION |
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We have used PC-3 LUCAR+ cells to investigate the possibility that some organochlorine food contaminants and food additives may interact with the androgen receptor. This was first reported for vinclozolin (Gray et al., 1994) and p,p'-DDE ( Kelce et al., 1995
) and more recently for DDT and butyl benzyl phthalate (Sohoni and Sumpter, 1998
). While p,p'-DDE has been found to have poor estrogenic activity (Kelce et al., 1995
) and vinclozolin appeared to act via the androgen receptor (Wong et al., 1995
), the other two chemicals have been shown to have estrogenic activity (Soto et al., 1995
) in addition to androgenic activity. In the present studies, we have confirmed that p,p'-DDE interacts with the androgen receptor both as an agonist in the absence of DHT and as an antagonist in the presence of DHT. The agonistic action was observed at high concentrations and was accompanied by poor cell viability. The antagonistic action of p,p'-DDE was observed at concentrations as low as 1.0 µM and complete antagonism of the DHT effect was seen at 10 µM. In a radioreceptor assay of the rat prostate androgen receptor, p,p'-DDE at 100 µM completely inhibited the binding of DHT but was only partially active in preventing the binding of DHT to epididymal androgen binding protein (Danzo, 1997
). Thus, the action of p,p'-DDE as an endocrine disrupter would appear to be predominantly at the level of the androgen receptor.
Toxaphene is a persistent environmental contaminant that has estrogenic activity. At 10 µM, toxaphene promotes the proliferation of MCF7 cells and increases the progesterone receptor levels in these cells, but at 1.0 µM toxaphene has no effect on these end points. However, at both 1.0 and 10 µM, toxaphene appeared to inhibit the processing of estrogen receptors (Soto et al., 1995). To our knowledge, prior to the present studies, toxaphene has not been examined for interaction with the androgen receptor. We observed that while toxaphene was a poor antagonist, it activated the androgen receptor at 10 µM in the absence of DHT, and although this activation was blocked by flutamide, it was observed when cell viability was reduced. This paradox confounds interpretation, but the present evidence suggests that toxaphene interacts with estrogen receptors and extremely poorly with androgen receptors. As both androgen and estrogen receptors are essential for normal male reproductive development (George and Wilson, 1994
; Layman, 1995
; Luke and Coffey, 1994
; Sharpe, 1995), toxaphene may be able to interfere with estrogen-dependent processes but would be less likely to interfere with androgen-dependent processes.
In contrast to the androgenic effect of toxaphene, TCDD, kepone, BHA, and BHT acted only as partial androgen antagonists in these studies. TCDD has been shown to have endocrine-disruptive effects on the male reproductive system that were considered to be due to antiestrogenic activity or to other mechanisms not related to steroid receptors, such as steroid biosynthesis and gonadotropin responsiveness (Bjerke and Peterson; 1994; Bjerke et al., 1994a,b
; Gray et al., 1995
, 1997
; Mably et al., 1992a
,b
,c
; Moore and Peterson, 1988
; Peterson et al., 1993
). Kepone is considered to have estrogenic activity and is able displace estradiol from the estrogen receptor (Bolger et al., 1998
; Soto et al., 1995
). However, an interaction with the androgen receptor appears to be less important, as in the present study the antagonistic effect of kepone was accompanied by a significant reduction in cell viability and in other studies, the ability of kepone to displace androgen from the androgen receptor was only seen at concentrations higher than 50 µM (Kelce et al., 1995
).
BHA and BHT are used as antioxidants in foods. BHA is considered to be estrogenic due to its promotion of MCF7 cell proliferation (Soto et al., 1995) and its ability to stimulate a response in MCF7 cells transfected with an estrogen-regulated luciferase construct (Jobling et al., 1995
). BHT, on the other hand, had no effect on either of these end points and is not considered estrogenic. It is of interest, therefore, that both BHA and BHT were androgen antagonists in the present studies and that BHT completely inhibited the activation by DHT without having deleterious effects on cell viability. Consequently, BHT could act as an androgen antagonist, but BHA could act as an estrogen and also as an androgen antagonist.
In MCF7 cell proliferation assays, lindane (-HCH) was considered to be nonestrogenic (Soto et al., 1995
). Lindane has the potential to be toxic to the male reproductive system (Dalsenter et al., 1996
; Prasad et al., 1995
; Silvestroni et al., 1997
). Toxicity does not appear to be due to any interaction with either the androgen receptor (these studies) or with androgen- binding protein, but could possibly arise through its weak affinity for the human SHBG (Danzo, 1997
). On the other hand,
-HCH, which was effective in displacing androgen from androgen- binding protein, had little affinity for the androgen receptor (Danzo, 1997
). Of the HCHs, only
-HCH appeared to have any effect on the androgen receptor, and only at a concentration of 10 µM.
Mirex, photomirex, oxychlordane, cis- and trans-nonachlor exhibited no interaction with the androgen receptor in our system. Mirex is not considered to be estrogenic (Soto et al., 1995), nor is technical chlordane, the chlordane constituents cis- and trans-nonachlor, or the metabolite oxychlordane (Soto et al., 1995
). To our knowledge, the possibility that these compounds might interact with the androgen receptor has not been investigated previously.
The mechanism of androgen receptor-mediated responses has been the subject of several recent studies. The androgen receptor binds to DNA as a dimer (Langley et al., 1995; Zhou et al., 1995
). The possibility that mixed-ligand dimers (e.g., a DHT-AR dimerized with progesterone-AR complex) could lead to blocked gene expression has been proposed to explain mixed agonist/antagonist activity (Maness et al., 1998
). In other studies, the type of conformational change in the androgen receptor that occurs on binding ligand has been related to the stability of the complex, which in turn dictates the extent of androgen receptor-dependent gene expression (Doesburg et al., 1997
; Kemppainen and Wilson, 1996
; Kuil and Mulder, 1994
Kuil and Mulder, 1995; Zhou et al., 1995
). However, other possibilities exist for ligands to affect androgen receptor-mediated function that may not be directly related to androgen-receptor binding. Chemicals that interfere with androgen receptor coactivators such as ARA55 and ARA70 (Fujimoto et al., 1999
; Yeh and Chang, 1996
) or with the vitamin D or retinoic acid receptors (Zhao et al., 1999
) could also result in altered androgen responses. In addition, activation of the protein kinase A pathway can lead to the expression of androgen receptor-dependent genes in the absence of androgen (Sadar, 1999
), indicating that interference with the cross-talk between the PKA signal transduction pathway and the androgen receptor could lead to altered gene expression. Consequently, there are several possibilities for xenobiotics to affect the expression of androgen-dependent genes. Some would be related to the nature of their binding to the androgen receptor, but other mechanisms are possible that would not require direct interaction with the ligand-binding domain of the androgen receptor.
When considering the physiologic consequences of exposure to xenobiotics, it is important to relate the findings of research activities to the levels of human exposure and recommended daily intake levels. In terms of androgen-receptor activation, these studies have shown that p,p'-DDE, BHT, kepone, and - and
-HCHs exhibited androgen antagonistic actions. For p,p'-DDE, estimates of greater than 1 µg/g breast milk fat have been determined in certain indigenous populations (Dewailly et al., 1993
; Mes, 1993; Newsome et al., 1995
) and levels greater than 1 µg/l in the cord blood of Inuit newborns have been detected (Canadian Arctic Contaminants Report, 1997
). An acceptable daily intake of 0.5 mg/kg/day has been recommended for BHT and no reproductive impairment has been observed in animal studies with doses equivalent or greater than the TDI (Smith, 1984
). The chemical structures of mirex, photomirex, and kepone are closely related; whereas mirex and photomirex had no effect on androgen receptor activation, kepone (10 µM) was antagonistic. Environmental levels of kepone have been diminishing since cessation of usage in the late 1970s. Seafoods containing less than 400 ng/g are considered unlikely to cause harm (Faroon and Kueberuwa, 1995
). Tolerable daily intake levels of 300 ng/kg/day for
- and
-HCHs have been recommended in the United States and Canada (Choudhary et al., 1994
). An estimated intake of 5 ng/kg/day for
-HCH was obtained and human breast milk contains 1040 ng/g lipid (Canadian Arctic Contaminants Report, 1997
). Data for
-HCH are sparse, but in one study the levels in human breast milk were undetectable (Keewatin Environmental Health Project, 1998
). It would appear that, with the possible exception of p,p'-DDE, normal human exposure to the chemicals investigated in these studies would be unlikely to cause reproductive dysfunction through antagonism of androgen receptor-mediated events, although other mechanisms could be possible targets for these chemicals.
In conclusion, we report here the development and use of a system to evaluate the interaction between environmental food contaminants and the human androgen receptor in a human prostate cancer cell line. This assay can be used to determine whether a chemical can activate the receptor or antagonize the activation of the receptor by DHT. It is anticipated that this assay will prove useful in the screening of chemicals that are possible endocrine disrupters.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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Bjerke, D. L., and Peterson, R. E. (1994). Reproductive toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in male rats: Different effects of in utero versus lactational exposure. Toxicol. Appl. Pharmacol. 127, 241249.[ISI][Medline]
Bjerke, D. L., Sommer, R. J., Moore, R. W., and Peterson, R. E. (1994b). Effects of in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure on responsiveness of the male reproductive system to testosterone stimulation in adulthood. Toxicol. Appl. Pharmacol. 127, 250257.[ISI][Medline]
Bolger, R., Wiese, T. E., Ervin, K., Nestich, S., and Checovich, W. (1998). Rapid screening of environmental chemicals for estrogen receptor binding capacity. Environ. Health Perspect. 106, 551557.[ISI][Medline]
Borenfreund, E., and Puerner, J. A. (1985). Toxicity determined in vitro by morphological alterations and neutral red absorption. Toxicol. Lett. 24, 119124.[ISI][Medline]
Borras, M., Hardy, L., Lempereur, F., el Khissiin, A. H., Legros, N., Gol-Winkler, R., and Leclercq, G. (1994). Estradiol-induced down-regulation of estrogen receptor: effect of various modulators of protein synthesis and expression. J. Steroid Biochem. Mol. Biol. 48, 325336.[ISI][Medline]
Canadian Arctic Contaminants Report (1997). Chapter 4, Human Health: Environmental Contaminants (J. Jensen, K. Adove, and R. Shearer, Eds) pp 318377. Indian and Northern Affairs, Canada.
Choudhary, G., O'Connor, R., Wedge, R., and Anderson, E. (1994). In: Toxicological profile for hexachlorocyclohexanes. U.S. Department of Health and Human Services, pp 111116.
Colborn, T., vom Saal, F. S., and Soto, A. M. (1993). Developmental effects of endocrine-disrupting chemicals in wildlife and humans. Environ. Health Perspect. 101, 378384.[ISI][Medline]
Cooper, R. L., and Kavlok, R. J. (1997). Endocrine disrupters and reproductive development: a weight-of-evidence overview. J. Endocrinol. 152, 159166.
Dai, J. L., Maiorino, C. A., Gkonos, P. J., and Burnstein, K. L. (1996). Androgenic up-regulation of androgen receptor cDNA expression in androgen-independent prostate cancer cells. Steroids 61, 531539.[ISI][Medline]
Dalsenter, P. R., Faqi, A. S., Webb, J., Merker, H-J., and Chahoud, I. (1996). Reproductive toxicity and tissue concentrations of lindane in adult male rats. Hum. Exp. Toxicol. 15, 406410.[ISI][Medline]
Danzo, B. J. (1997). Environmental xenobiotics may disrupt normal endocrine function by interfering with the binding of physiological ligands to steroid receptors and binding proteins. Environ. Health Perspect. 105, 294301.[ISI][Medline]
Dewailly, E., Ayotte, P., Bruneau, S., Laliberté, C., Muir, D. C. G., and Norstrom, R. (1993). Inuit exposure to organochlorines through the aquatic food chain in Arctic Québec. Environ. Health Perspect. 101, 618620.[ISI][Medline]
Doesburg, P., Kuil, C. W., Berrevoets, C. A., Steketee, K., Faber, P. W., Mulder, E., Brinkmann, A. O., and Trapman, J. (1997). Functional in vivo interaction between the amino-terminal, transactivation domain and the ligand binding domain of the androgen receptor. Biochemistry 36, 10521064.[ISI][Medline]
Faroon, O., and Kueberuwa, S. (1995) In: Toxicological profile for mirex and chlordecone. U.S. Department of Health and Human Services, pp 8 and 214231.
Fujimoto, N., Yeh, S., Kang, H-Y., Inui, S., Chang, H-C., Mizokami, A., and Chang, C. (1999). Cloning and characterization androgen receptor coactivator, ARA55 in human prostate. J. Biol. Chem. 274, 83168321.
George, F. W., and Wilson, J. D. (1994). Sex determination and differentiation. In The Physiology of Reproduction, Second Edition (E. Knobil and J.D. Neill, Eds.) Vol 1, pp 328. Raven Press Ltd., New York.
Graham, F. L., and van der Eb, A. J. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456467.[ISI][Medline]
Gray, L. E. Jr., Kelce, W. R., Monosson, E., Ostby, J. S., and Birnbaum L. S. (1995). Exposure to TCDD during development permanently alters reproductive function in male Long Evans rats and hamsters: Reduced ejaculated and epididymal sperm numbers and sex accessory gland weights in offspring with normal androgenic status. Toxicol. Appl. Pharmacol. 131, 108118.[ISI][Medline]
Gray, L. E. Jr., Ostby, J. M., and Kelce, W. R. (1994). Developmental effects of an environmental antiandrogen: The fungicide vinclozolin alters sex differentiation of the male rat. Toxicol. Appl. Pharmacol. 129, 4652.[ISI][Medline]
Gray, L. E. Jr., Ostby, J. S., and Kelce, W. R. (1997). A dose-response analysis of the reproductive effects of a single gestational dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin in male Long Evans hooded rat offspring.Toxicol. Appl. Pharmacol. 146, 1120.[ISI][Medline]
Green, S., Kumar, V., Theulaz, I., Wahli, W. and Chambon, P. (1988). The N-terminal DNA-binding `zinc finger' of the oestrogen and glucocorticoid receptors determines target gene specificity. EMBO J. 7, 30373044.[Abstract]
Green, S., Walter, P., Kumar, V., Krust, A., Bornert, J-M., Argos, P., and Chambon, P. (1986). Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature 320, 134139.[ISI][Medline]
Husmann, D. A and McPhaul, M. J. (1991). Time-specific androgen blockade with flutamide inhibits testicular descent in the rat. Endocrinology (Baltimore) 129, 14091416.[Abstract]
Jobling, S., Reynolds, T., White, R., Parker, M. G., and Sumpter, J. P. (1995). A variety of environmentally persistent chemicals, including some phthalate plasticizers, are weakly estrogenic. Environ. Health Perspect. 103, 582587.[ISI][Medline]
Keewatin Environmental Health Project: Contaminants and People. Breast Milk Report. (1998). Keewatin Regional Health and Social Services Board, pp 26.
Kelce, W. R., Lambright, C. R., Gray, L. E. Jr., and Roberts, K. P. (1997). Vinclozolin and p,p'-DDE alter androgen-dependent gene expression: in vivo confirmation of an androgen receptor-mediated mechanism. Toxicol. Appl. Pharmacol. 142, 192200.[ISI][Medline]
Kelce, W. R., Stone, C. R., Laws, S. C., Gray, L. E. Jr., Kemppainen, J. A., and Wilson, E. M. (1995). Persistent DDT metabolite p,p'-DDE is a potent androgen receptor antagonist. Nature (London) 375, 581585.[ISI][Medline]
Kemppainen, J. A., and Wilson, E. M. (1996) Agonist and antagonist activities of hydroxyflutamide and casodex relate to androgen receptor stabilization. Urology 48, 157163.[ISI][Medline]
Kokontis, J., Ito, K., Hiipakka, R. A., and Liao, S. (1991). Expression and function of normal and LNCaP androgen receptors in androgen-insensitive human prostatic cancer cells. Altered hormone and antihormone specificity in gene transactivation. Receptor 1, 271279.[Medline]
Kuil, C. W., Berrevoets, C. A. and Mulder, E. (1995). Ligand-induced conformational alterations of the androgen receptor analyzed by limited trypsinization. Studies on the mechanism of antiandrogen action. J. Biol. Chem. 270, 2756927576.
Kuil, C. W., and Mulder, E. (1994). Mechanism of antiandrogen action: conformational changes of the receptor. Mol. Cell. Endocrinol. 102, R1R5.[ISI][Medline]
Langley, E., Zhou, Z. X., and Wilson, E. (1995). Evidence for an anti-parallel orientation of the ligand-activated human androgen receptor dimer. J. Biol. Chem. 270, 2998329990.
Layman, L. C. (1995). Molecular biology in reproductive endocrinology. Curr. Opin. Obstet. Gynecol. 7, 328329.[ISI][Medline]
Luke, M. C., and Coffey, D. S. (1994) The male sex accessory tissues. In The Physiology of Reproduction, Second Edition (E. Knobil and J.D. Neill, Eds.) Vol 1, pp 14351487. Raven Press Ltd., New York.
Lustig, R. H., Hua, P., Smith, L. S., Wang, C., and Chang, C. (1994). An in vitro model for the effects of androgen on neurons employing androgen receptor-transfected PC12 cells. Mol. Cell. Neurosci. 5, 587596.[ISI][Medline]
Mably, T. A., Bjerke, D. L., Moore R. W., Gendron-Fitzpatrick, A., and Peterson, R. E. (1992a). In utero and lactational exposure of male rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin. 3. Effects on spermatogenesis and reproductive capability. Toxicol. Appl. Pharmacol. 114, 118126.[ISI][Medline]
Mably, T. A., Moore R. W., Goy, R. W., and Peterson, R. E. (1992b). In utero and lactational exposure of male rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin. 2. Effects on sexual behaviour and the regulation of luteinizing hormone secretion in adulthood. Toxicol. Appl. Pharmacol. 114, 108117.[ISI][Medline]
Mably, T. A., Moore R. W., and Peterson R. E. (1992c). In utero and lactational exposure of male rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin.1. Effects on androgenic status. Toxicol. Appl. Pharmacol. 114, 97107.[ISI][Medline]
Maness, S. C., McDonnell, D. P., and Gaido, K. W. (1998). Inhibition of androgen receptor-dependent transcriptional activity by DDT isomers and methoxychlor in HepG2 human hepatoma cells. Toxicol. Appl. Pharmacol. 151, 135142.[ISI][Medline]
Mes, J., Davies, D. J., Doucet, J., Weber, D., and McMullen, E. (1993). Levels of chlorinated hydrocarbon residues in Canadian human breast milk and their relationship to some characteristics of the donors. Food Addit. Contam. 10, 429441.[ISI][Medline]
Moore, R.W., and Peterson R. E. (1988) Androgen catabolism and excretion in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats. Biochem. Pharmacol. 37, 560562.[ISI][Medline]
Newsome, W. H., Davies, D., and Doucet, J. (1995). PCB and organochlorine pesticides in Canadian human milk 1992. Chemosphere 30, 21432153.[ISI][Medline]
Olea-Serrano, N., Develeeschouwer, N., Leclercq, G., and Heuson, J. C. (1985). Assay for estrogen and progesterone receptors of breast cancer cell lines in monolayer culture. Eur. J. Cancer Clin. Oncol. 21, 965973.[ISI][Medline]
Peterson, R. E., Moore R. W., Mably T. A., Bjerke D. L., and Goy, R. W. (1993). Male reproductive system ontogeny: Effects of perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. In Advances in Modern Environmental Toxicology (T. Colborn and C. Clement, Eds.), pp. 175193. Princeton Scientific Publishing Co. Inc., New Jersey.
Prasad, A. K., Pant, N., Srivastava, S. C., Kumar, R., and Srivastava, S. P. (1995) Effect of dermal application of hexachlorocyclohexane (HCH) on male reproductive system of Rat. Human Exp. Toxicol. 14, 484488.[ISI][Medline]
Ramamoorthy, K., Wang, F., Chen, I-C., Norris, J. D., McDonnell, D. P., Leonard, L. S., Gaido, K. W., Bocchinfuso, W. P., Korach, K. S., and Safe, S. (1997) Estrogenic activity of a dieldrin/toxaphene mixture in the mouse uterus, MCF-7 human breast cancer cells, and yeast-based estrogen receptor assays: no apparent synergism. Endocrinology 138, 15201527.
Sadar, M. D. (1999) Androgen-independent induction of prostate-specific antigen gene expression via cross-talk between the androgen receptor and protein kinase A signal transduction pathways. J. Biol. Chem. 274, 77777783.
Safe, S. (1995). Enviromental and dietary estrogens and human health: is there a problem? Environ. Health Perspect. 103: 346351.[ISI][Medline]
van der Schoot, P. (1992). Disturbed testicular descent in the rat after prenatal exposure to the antiandrogen flutamide. J. Reprod. Fertil. 96, 483496[Abstract]
Sharpe, R. M. (1994). Regulation of spermatogenesis. In The Physiology of Reproduction, Second Edition (E. Knobil and J.D. Neill, Eds.) Vol 1, pp 13631434. Raven Press Ltd. NY.
Sharpe, R. M., Fisher, J. S., Millar, M. M., Jobling, S., and Sumpter, J. P. (1995). Gestational and lactational exposure of rats to xenoestrogens results in reduced testicular size and sperm production. Environ. Health Perspect. 103, 11361143.[ISI][Medline]
Shekhar, P. V. M., Werdell, J., and Basrur, V. S. (1997). Environmental estrogen stimulation of growth and estrogen receptor function in preneoplastic and cancerous human breast cell lines. J. Natl. Cancer Inst. 89, 17741782.[Abstract]
Silversides, D. W., Price, C. A., and Cooke, G. M. (1995). Effects of short-term exposure to hydroxyflutamide in utero on the development of the reproductive tract in male mice. Can. J. Physiol. Pharmacol. 73, 15821588.[ISI][Medline]
Silvestroni, L., Fiorini, R., and Palleschi, S. (1997). Partition of the organochlorine insecticide lindane into the human sperm surface induces membrane depolarization and Ca2+ influx. Biochem. J. 321, 691698.[ISI][Medline]
Smith, J. G. (1984). Draft report Chemical Hazards Information Profile: Butylated Hydroxytoluene. Oak Ridge National Laboratory, Oak Ridge, Tennessee.
Sohoni, P., and Sumpter, J. P. (1998). Several environmental oestrogens are also anti-androgens. J. Endocrinol. 158, 327339.
Soto, A. M., Chung, K. L., and Sonnenschein, C. (1994). The pesticides endosulphan, toxaphene and dieldrin have estrogenic effects on human estrogen-sensitive cells. Environ. Health Perspect. 102, 380383.[ISI][Medline]
Soto, A. M., Sonnenschein, C., Chung, K. L., Fernandez, M. F., Olea, N., and Serrano, F. O. (1995). The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic environmental pollutants. Environ. Health Perspect. 103, Suppl. 7. 113122.[ISI][Medline]
Spencer, J. R., Torrado, T., Sanchez, R. S., Vaughn, E. D. Jr., and Imerato-McGinley, J. (1991). Effects of flutamide and finasteride on rat testicular descent. Endocrinology (Baltimore) 129, 741748.[Abstract]
Turner, K. J. and Sharpe, R. M. (1997). Environmental oestrogens present understanding. Rev. Reprod. 2, 6973.
Veldscholte, J., Berrevoets, C. A., Ris-Stalpers, C., Kuiper, G. G., Jenster, G., Trapman, J., Brinkmann, A. O., and Mulder, E. (1992). The androgen receptor in LNCaP cells contains a mutation in the ligand binding domain which affects steroid binding characteristics and response to antiandrogens. J. Steroid Biochem. Mol. Biol. 41, 665669.[ISI][Medline]
Wong, C-I., Kelce, W. R., Sar, M., and Wilson, E. M. (1995). Androgen receptor antagonist versus agonist activities of the fungicide vinclozolin relative to hydroxyflutamide. J. Biol. Chem. 270, 1999820003.
Yeh, S., and Chang, C. (1996).Cloning and characterization of a specific coactivator, ARA70, for the androgen receptor in human prostate cells. Proc. Natl. Acad. Sci. USA 93, 55175521.
Yuan, S., Trachtenberg, J., Mills, G. B., Brown T. J., Xu, F., and Keating, A. (1993). Androgen induced inhibition of cell proliferation in an androgen-insensitive prostate cancer cell line (PC-3) transfected with a human androgen receptor complementary DNA. Cancer Res. 53, 13041311.[Abstract]
Zhao, X-Y., Ly, L. H., Peehl, D. M., and Feldman, D. (1999). Induction of androgen receptor by 1,25-dihydroxyvitamin D3 and 9-cis retinoic acid in LNCaP human prostate cancer cells. Endocrinology 140, 12051212.
Zhou, Z-X., Lane, M. V., Kemppainen, J. A., French, F. S., and Wilson, E. (1995). Specificity of ligand-dependent androgen receptor stabilization: receptor domain interactions influence ligand dissociation and receptor stability. Mol. Endocrinol. 9, 208218.[Abstract]