Department of Toxicology, North Carolina State University, Raleigh, North Carolina 27695
Received July 9, 1999; accepted November 1, 1999
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: androgen disruption; hepatic biotransformation; ketoconazole; mice; testosterone.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Whether or not chemically mediated changes in hepatic biotransformation of testosterone can impact androgen homeostasis is an important issue. Steroid hormones play decisive and irreversible roles in embryonic development and sex differentiation ( vom Saal et al., 1992). In addition, they influence the acquisition and maintenance of secondary sex characteristics in adults (Grumbach and Conte, 1981
) and are critical in ensuring adult male and female reproductive function (Hadley, 1982). Abnormalities in these processes generally occur when there are changes in the amount or availability of a particular hormone or due to differences in the hormone's pattern of secretion (Wilson and Foster, 1985
). Therefore, chemically induced modulations in hepatic biotransformation capabilities (either inductive or suppressive) that impact circulating steroid hormone levels have the potential to produce dramatic effects on steroid hormone regulated processes.
Circulating testosterone levels are maintained by a dynamic system that ensures androgen homeostasis. In simplest terms, this system can be considered a balancing act between the rate of testosterone synthesis and the rate of its metabolic inactivation/elimination. If this system is truly a balancing act, then both the rate of synthesis and the rate of metabolic inactivation/elimination impact the maintenance of circulating testosterone levels, and chemically induced disruptions in either process can potentially modify circulating testosterone levels.
In the present study, ketoconazole was used as a model compound to further investigate the role of the metabolic inactivation of testosterone on the maintenance of circulating androgen levels. Our goal was to determine whether the modulation of the hepatic biotransformation of testosterone by ketoconazole, in combination with its effects on testosterone synthesis, would contribute to the lowering of serum testosterone levels by this compound. These studies were also used to continue evaluation our recent observation that the ratio of hepatic testosterone 6/15
-hydroxylase activities provides a superior indicator of the androgen status in CD-1 mice (Wilson et al., 1999
). These two hydroxylase activities are differentially regulated by testosterone such that a high ratio is typical of females or indicative of demasculinization, and a low ratio is typical of males or indicative of masculinization. Ketoconazole has been shown to significantly lower serum testosterone levels (Pont et al., 1982
). If so, then it should also dramatically affect the testosterone 6
/15
-hydroxylase ratio. The lowering of serum testosterone levels by ketoconazole treatment has been attributed primarily to the ability of ketoconazole to inhibit gonadal synthesis of testosterone (Pont et al., 1982
). There have been, however, conflicting observations regarding the effects of ketoconazole on hepatic metabolic enzymes, with induction, inhibition, or no effect all being reported by different researchers (Jiritano et al., 1986
; Morita et al., 1988
; Niemegeers et al., 1981
; Rodrigues et al., 1988
; Ronis et al., 1994
; Thomson et al., 1988
).
In CD-1 mice, testosterone is inactivated/metabolized in the liver by four primary pathways. First, cytochrome P450 isozymes can monohydroxylate testosterone in a characteristic manner that is both region specific and stereospecific (Waxman et al., 1983). Second, testosterone can be dehydrogenated to produce androstenedione through P450-mediated pathways (Waxman, 1988
), and also through the action of 17ß-hydroxysteroid dehydrogenase (Bloomquist et al., 1977
). Although the production of androstenedione can contribute to the androgen precursor pool in the body, we focused primarily on its role as an inactivation product. Third and fourth, UDP-glucuronosyltransferase and sulfotransferase can conjugate testosterone directly or subsequent to hydroxylation to either glucuronic acid or sulfate, respectively. These pathways generally produce more polar products that can then be eliminated from the body.
The primary goals of this study were the following: a) assess the temporal effect of ketoconazole on serum testosterone levels; b) establish the relative contribution of gonadal synthesis and hepatic biotransformation of testosterone in altering serum testosterone levels following ketoconazole treatment; and c) evaluate the effects of ketoconazole on the testosterone 6/15
-hydroxylase ratio, an indicator of androgen status.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Serum testosterone.
Blood from each animal was collected by cardiac puncture and allowed to clot at room temperature for 1 h; serum was obtained by centrifugation at 14,000 x g for 10 min (LeBlanc and Waxman, 1988). Serum from each sample was then immediately frozen at 20°C until assayed. Total testosterone was measured in prepared serum by solid-phase radioimmunoassay using commercially available reagents and protocols (Diagnostic Products Corp., Los Angeles, CA).
Hepatic microsome and cytosol preparation.
Livers were excised, weighed, minced in ice-cold buffer, and rapidly frozen in liquid nitrogen. Individual livers were thawed and homogenized on ice in chilled buffer (0.1 M HEPES, pH 7.4, 1mM EDTA, and 10% glycerol) using a glass tissue homogenizer. Microsomes and cytosol were prepared by differential centrifugation (van der Hoeven and Coon, 1974). The cytosolic supernatant was reserved for sulfotransferase assays and the microsomal pellet was resuspended in microsome buffer (0.1 M potassium phosphate, pH 7.4, 0.1 mM EDTA, and 20% glycerol). Protein concentrations were determined (Bradford, 1976
) using commercially prepared reagent (Bio-Rad, Hercules, CA.) and bovine serum albumin (Sigma, St. Louis, MO) as standards. Both microsomes and cytosol were stored at 80°C until assays were performed.
Testosterone hydroxylase activity.
Both the rate of testosterone hydroxylation and the rate of androstenedione production were measured in this assay. These activities were assayed using 400 µg microsomal protein and 40 nmol [14C]testosterone as a substrate (1.8 mCi/mmol, Dupont NEN, Boston, MA) in 0.1 M potassium phosphate buffer (pH 7.4) as previously described (Baldwin and LeBlanc, 1992). Reactions were initiated by the addition of 1 mM NADPH and incubated for 10 min at 37°C. Product formation was shown to be linear over this time period. The total assay volume was 400 µl. After 10 min , the reactions were terminated by the addition of 1 ml ethyl acetate and vortexing. Metabolites were removed from the reaction mixture by differential solvent extraction. The aqueous phase was extracted with ethyl acetate a total of three times (1 ml, 2 ml, 1 ml). After each addition of ethyl acetate, the tubes were vortexed for 1 min, then centrifuged for 10 min to separate the ethyl acetate and aqueous phases. Ethyl acetate fractions derived from each aqueous sample were combined and then dried under a stream of nitrogen. The residues were suspended in 79 µl (35 µl x 2) ethyl acetate and separated by thin-layer chromatography. Individual metabolites and unmetabolized [14C]testosterone were identified as we have previously described (Baldwin and LeBlanc, 1992
) and quantified using InstantImager electronic autoradiography (Packard Instrument Co., Meriden, CT). The metabolite identified in the text and figures as 15ß/7
-hydroxytestosterone comigrated with both standards during TLC and the precise identity of this metabolite could not be determined. Specific activity for the production of each metabolite was calculated as picomoles of metabolite produced per minute of the assay per milligram of microsomal protein.
UDP-glucuronosyltranferase activity.
UDP-glucuronosyltransferase activity towards [14C]testosterone was assayed under conditions previously described (Tedford et al., 1991). In brief, microsomal protein (200 µg) was incubated at 37°C with 40 nmol [14C]testosterone (1.8 mCi/mmol) in 0.1 M potassium phosphate buffer, pH 7.4. Reactions were initiated with 10 µl uridine 5'-diphosoglucuronic acid in buffer (12.9 mg/ml, Sigma) to give a total assay volume of 400 µl. The reactions were terminated after 10 min by the addition of 2 ml ethyl acetate and vortexing. Product formation was shown to be linear over this time period. Each sample was vortexed for 1 min and aqueous and ethyl acetate phases were separated by centrifugation. The ethyl acetate fraction containing the unconjugated [14C]testosterone was removed and extraction of the aqueous phase was repeated a second time with an additional 2 ml ethyl acetate. [14C]Testosterone-glucuronide was quantified by liquid scintillation counting of a 100-µl aliquot of the postextraction aqueous phase. Samples were run with every assay that consisted of all constituents except microsomes. These samples were used to account for any spontaneous conjugation of glucuronic acid to testosterone and for any [14C]testosterone that was not extracted from the aqueous phase. Radioactivity in these aqueous samples following ethyl acetate extraction (typically
0.1% of the total activity in the assay) was subtracted from the total radioactivity associated with the glucuronic acid conjugated testosterone in each assay. Specific activity was calculated as picomoles of conjugate produced per minute per milligram of microsomal protein.
Sulfotransferase activity.
Cytosolic protein (200 µg) was incubated with 40 nmol [14C]testosterone (1.8 mCi/mmol) in 0.1 M potassium phosphate buffer (pH 6.5) to assay for the activity of sulfotransferase enzymes toward testosterone. Reactions were initiated with 10 µl adenosine 5'-phosphosulfate (10.1 mg/ml) for a total assay volume of 400 µl. Assay tubes were covered and incubated in a 37°C water bath for 20 h. Product formation was linear over this time period. Reactions were terminated by addition of 2 ml ethyl acetate and vortexing. Unconjugated testosterone was removed by ethyl acetate extraction (two times, 2 ml each). Sulfate-conjugated [14C]testosterone was identified and quantified as we have previously described (Wilson and LeBlanc, 1998). Specific activity was calculated as picomoles of metabolite produced per minute per milligram of cytosolic protein.
Testosterone hydroxylase inhibition assay.
Testosterone hydroxylase activities were measured as described above except that a series of concentrations of ketoconazole were added to the reaction mixture. A 5 mM stock solution of ketoconazole in 100% ethanol was prepared. Working solutions (100x) for each ketoconazole concentration were prepared by diluting this stock solution in ethanol so that 4 µl of ethanol was added to each 400-µl assay. Concentrations of ketoconazole in individual assays were 0 (vehicle only), 0.010, 0.030, 0.10, 0.30, 1.0, 3.0, 10, and 30 µM. Assays were run at each ketoconazole concentration in triplicate.
Testicular synthesis of testosterone.
Four h after treatment, the effects of ketoconazole on testicular synthesis of testosterone were evaluated ex vivo with and without human chorionic gonadotropin (hCG, Sigma) stimulation. Both testes were removed from each animal in the control and 80 and 160 mg/kg/day treatment groups. Each testis was immediately transferred into 5 ml of phenol red free Dulbecco's Modified Eagle Medium (Life Technologies, Gaithersburg, MD) supplemented with 10% charcoal-dextrantreated fetal bovine serum (Hyclone, Logan, UT). The right testis from each animal was incubated in medium without hCG. The left testis was incubated in medium with 100 mIU hCG per ml of medium. Testes were incubated in an atmosphere of 95% air/5% CO2 for exactly 4 h. Testosterone secreted into the medium was measured by RIA as described for serum testosterone. Preliminary experiments had demonstrated that the testicular secretion of testosterone under these conditions was linear for at least 22 h and that there were no detectable levels of testosterone in medium incubated without a testis.
Immunoblotting procedure.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of 10 µg microsomal protein was performed using a 10% separating gel with a 4% stacking gel. Dexamethasone induced rat hepatic microsomal protein (Oxford Biomedical Research, Oxford, MI) was included in one lane of each gel as a positive control. Proteins were then electrophoretically transferred onto nitrocellulose at 120 V for 1 h in cold buffer (25 mM Tris base, 192 mM glycine, 20% v/v methanol, 0.015% w/v sodium dodecyl sulfate). The nitrocellulose was blocked overnight at 4°C in phosphate-buffered saline containing 0.3% Tween 20 (Sigma). After rinsing, the nitrocellulose membrane was incubated with monoclonal rat anti-CYP3A1 antibody (Oxford Biomedical Research) at a dilution of 1:1000 for 2 h at room temperature. The nitrocellulose was again rinsed and then incubated with anti-mouse IgG secondary antibody conjugated to alkaline phosphatase for 2 h at a 1:1000 dilution. Immunoblotted protein was visualized colorimetrically using 5-bromo-4-chloro-indolyl phosphate/nitro blue tetrazolium (Waxman et al., 1988). Relative band intensities were determined using a scanning laser densitometer (Zeineh, Fullerton, CA).
Statistics.
Statistical significance was determined using ANOVA and Dunnett's Multiple Comparison Test. These statistical analyses were performed using JMP statistical software (SAS Institute, Cary, NC). IC50 calculations and linear regression analyses were performed using Origins statistical/graphing software (Microcal, Northhampton, MA).
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
Mice were dosed with 0, 80, or 160 mg/kg ketoconazole and the effects on serum testosterone levels, gonadal testosterone secretion, and hepatic testosterone hydroxylation were evaluated 4 h later. Serum testosterone levels, gonadal testosterone secretion, and hepatic testosterone hydroxylation were all significantly reduced in both the 80 and 160 mg/kg ketoconazole treatment groups (Fig. 6). hCG stimulation increased overall testosterone secretion by isolated testes, but did not overcome the inhibitory effect of ketoconazole on testicular testosterone secretion. With hCG stimulation, testicular secretion of testosterone was decreased by 27% and 49% of control levels in the 80 and 160 mg/kg ketoconazole treatment groups, respectively.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mechanism responsible for the transient decrease in some hepatic hydroxylase activities following ketoconazole treatment became apparent during the in vitro assessment of the effects of ketoconazole on hepatic testosterone hydroxylase activities. Following in vivo treatment of mice with ketoconazole, 6ß- and 15-hydroxylase activities were significantly decreased and 15ß/7
-hydroxylase activity was slightly decreased. Testosterone 16ß-, 6
-, and 16
-hydroxylase activities were not affected by treatment with ketoconazole. In vitro experiments with liver microsomal preparations demonstrated that testosterone 6ß- and 15
-hydroxylase activities were highly susceptible to direct inhibition by ketoconazole, 15ß/7
-hydroxylase activity was moderately susceptible to inhibition, and 16ß-, 6
-, and 16
-hydroxylase activities were rather insensitive to inhibition by ketoconazole. Thus, the transient decrease in some of the testosterone hydroxylase activities may be attributed to direct inhibition by ketoconazole.
Hepatic testosterone 2-hydroxylase activity was also transiently reduced following treatment of animals with ketoconazole. However, this enzymatic activity was not inhibited during in vitro incubations with ketoconazole. The rapid and transient nature of this reduction in activity after in vivo treatment suggests that this enzyme also was inhibited. These observations raise the possibility that a metabolite of ketoconazole generated in vivo was responsible for the inhibition of this enzyme.
Ketoconazole treatment resulted in an increase in the testosterone 6/15
-hydroxylase ratio. Normally, an increase in this ratio is indicative of demasculinization because of the negative effect of testosterone on 6
-hydroxylase activity and its positive effect on 15
-hydroxylase activity (Wilson et al., 1999
). Although the increase in the ratio was coincidental with the decrease in serum testosterone levels following ketoconazole treatment, altered serum testosterone levels did not mediate this effect. Rather, ketoconazole differentially inhibited testosterone 15
-hydroxylase activity, causing an increase in the ratio. Thus, care must be exercised when judging whether an increase in the testosterone 6
/15
-hydroxylase ratio is indicative of altered testosterone levels.
We demonstrated that ketoconazole had an inhibitory effect on both testicular testosterone secretion and hepatic cytochrome P450 enzymes and that this inhibition was transient. If synthesis and inactivation of testosterone were a true balancing act, then decreased hepatic biotransformation might compensate for the decrease in gonadal testosterone synthesis, resulting in an attenuated effect on circulating testosterone levels. Results of the present study, however, demonstrated that changes in the hepatic biotransformation of testosterone had no significant effect on serum testosterone levels. Rather, changes in serum testosterone levels could be explained by changes in the rate of gonadal testosterone secretion. These results are consistent with our previous study demonstrating that endosulfan treatment resulted in a significant increase in hepatic biotransformation and elimination of testosterone, but had no significant effect on serum testosterone levels (Wilson and LeBlanc, 1998).
A high degree of redundancy exists in the hepatic biotransformation of endogenous ligands such as testosterone. Testosterone is susceptible to hydroxylation by multiple P450 enzymes, oxidoreduction to androstenedione and androstanediols, and conjugation to either glucuronic acid or sulfate. All of these metabolic conversions contribute to the inactivation of the hormone. The present study demonstrated that the inhibition of certain biotransformation enzymes did not significantly impact changes in serum testosterone levels. The redundancy in biotransformation processes may be responsible for ensuring that endogenous ligands such as testosterone are efficiently cleared despite the selective inhibition of some biotransformation enzymes.
Exposure to some xenobiotics can result in increased activity of some hepatic biotransformation enzymes, leading to increased clearance of testosterone (Wilson and LeBlanc, 1998). Again, this effect on biotransformation and elimination did not impact serum testosterone levels. Gonadal testosterone secretion is regulated through serum testosterone levels by feedback control processes (Wilson and Foster, 1985
). Although increased clearance of testosterone due to induction of biotransformation elimination processes may result in an initial decrease in serum testosterone levels, this decline would be detected and compensated for by an increase in gonadal testosterone secretion.
The modulation of hepatic testosterone biotransformation processes would likely impact serum testosterone levels only if the feedback control of testosterone synthesis was also adversely affected. For example, chronic exposure to polychlorinated biphenyls was shown to increase hepatic 6ß-hydroxylation of testosterone and to lower serum testosterone levels (Machala et al., 1998). However, this chemical treatment also inhibited gonadal CYP11A activity, the rate-limiting enzyme of steroidogenesis. Thus, the reduced serum testosterone levels were either due exclusively to the inhibition of testosterone synthesis or the combination of reduced synthesis and increased clearance.
In conclusion, the results from this study indicate that testosterone levels are controlled primarily by the rate of synthesis. Chemically induced effects on hepatic steroid hormone biotransformation processes do not necessarily imply altered steroid hormone homeostasis. As noted in the introduction, there are many instances in the scientific literature where xenobiotic-mediated changes in the rate of steroid hormone biotransformation and inactivation are presumed to lead to alterations in circulating hormone levels. Our results indicate that this assertion should not be made without careful investigation.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bammel, A., van der Mee, K., Ohnhaus, E. E., and Kirch, W. (1992). Divergent effects of different enzyme-inducing agents on endogenous and exogenous testosterone. Eur. J. Clin. Pharmacol. 42, 641644.[ISI][Medline]
Bloomquist, C. H., Kotts, C. E., and Hakanson, E. Y. (1977). Microsomal 17ß-hydroxysteroid dehydrogenase of guinea pig liver: Detergent solubilization and a comparison of kinetic and stability properties of bound and solubilized forms. J. Steroid Biochem. 8, 193198.[ISI][Medline]
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein dye binding. Anal. Biochem. 72, 248254.[ISI][Medline]
den Besten, P. J., Elenbass, J. M. L., Maas, J. R., Dieleman, S. J., Herwig, H. J., and Voogt, P. A. (1991). Effects of cadmium and polychlorinated biphenyls (Clophen A50) on steroid metabolism and cytochrome P-450 monooxygenase system in the sea star Asterias rubens L. Aquat. Toxicol. 20, 95110.[ISI]
Gagnon, M. M., Dodson, J. J., and Hodson, P. V. (1994). Ability of BKME (bleached kraft mill effluent) exposed whit suckers (Catostomus commersoni) to synthesize steroid hormones. Comp. Biochem. Physiol. 107C, no. 2, 265273.
Gonzalez, F. J. (1989). The molecular biology of cytochrome P-450s. Pharmaceut. Rev. 40, 243288.
Gradowska-Olszewska, I., Brzezinski, J. and Rusiecki, W. (1984). Excretion and peripheral metabolism of 1,23H-testosterone and androgens in rats following intoxication with organophosphorous insecticides: acute exposure. J. Appl. Toxicol. 4, 261264.[ISI][Medline]
Grumbach, M. M., and Conte, F. A. (1981). Disorders of sexual differentiation. In Textbook of Endocrinology, 7th ed. ( J. D. Wilson and D. W. Foster, Eds.). W.B. Saunders Co., Philadelphia, PA.
Guengerich, F. P., and Shimada, T. (1991). Oxidation of toxic and carcinogenic chemicals by human cytochrome P-450 enzymes. Chem. Res. Toxicol. 4, 391407.[ISI][Medline]
Haake, J., Kelley, M., Keys, B., and Safe, S. (1987). The effects of organochlorine pesticides as inducers of testosterone and benzo[a]pyrene hydroxylases. Gen. Pharmacol. 18, 165169.[Medline]
Hadley, M. E. (1992). Endocrinology. Simon & Schuster, Upper Saddle River, NJ.
Heckman, W. R., Kane, B. R., Pakyz, R. E., and Consentino, J. (1992). The effect of ketoconazole on endocrine and reproductive parameters in male mice and rats. J. Androl. 13, 191198.[Abstract]
Jiritano, L., Bortolotti, A., and Bonati, M. (1986). The effect of chronic oral ketoconazole on in vivo drug metabolism in the rat. Res. Commun. Chem. Pathol. Pharmacol. 54, 173180.[ISI][Medline]
Kulkarni, A. P. and Hodgson, E. (1984). The metabolism of insecticides: the role of monooxygenase enzymes. Ann. Rev. Pharmacol. Toxicol. 24, 1942.[ISI][Medline]
LeBlanc, G. A. and Waxman, D. J. (1988). Feminization of rat hepatic P450 expression by cisplatin. J. Biol. Chem. 263, 1573215739.
Machala, M., Neca, J., Drabek, P., Ulrich, R., Sabatova, V., Nezveda, K., Raszyk, J., and Gajduskova, V. (1998). Effects of chronic exposure to PCBs on cytochrome P450 systems and steroidogenesis in liver and testis of bulls (Bos taurus). Comp. Biochem. Physiol. A Mol. Integr. Physiol. 120, 6570.[ISI][Medline]
McMaster, M. E., van der Kraak, G. J., Portt, C. B., Munkittrick, K. R., Sibley, P. K., Smith, I. R., and Dixon, D. G. (1991). Changes in hepatic mixed-function oxygenase (MFO) activity, plasma steroid levels and age at maturity of a white sucker (Catostomus commersoni) population exposed to bleached kraft pulp mill effluent. Aquat. Toxicol. 21, 199218.[ISI]
Morita, K., Ono, T., and Shimakawa, H. (1988). Inhibitory effects of ketoconazole and miconazole on cytochrome P-450 mediated oxidative metabolism of testosterone and xenobiotics in mouse hepatic microsomescomparative study with cimetidine. J. Pharmacobiodyn. 11, 106114.[Medline]
Niemegeers, C. J. E., Levron, J. C., Awouters, F., and Janssen, P. A. J. (1981). Inhibition and induction of microsomal enzymes in the rat. A comparative study of four antimycotics: miconazole, econazole, clotrimazole and ketoconazole. Arch. Int. Pharmacodyn. Ther. 251, 2638.[ISI][Medline]
Pont, A., Williams, P. L., Azhar, S., Reitz, R. E., Bochra, C., Smith, E. R., and Stevens, D. A. (1982). Ketoconazole blocks testosterone synthesis. Arch. Intern. Med. 142, 21372140.[Abstract]
Rodrigues, A. D., Waddell, P. R., Ah-Sing, E., Morris, B. A., Wolf, C. R., and Ioannides, C. (1988). Induction of the rat hepatic microsomal mixed-function oxidases by 3 imidazole-containing antifungal agents: selectivity for the cytochrome P-450IIB and P-450III families of cytochrome P450. Toxicology 50, 283301.[ISI][Medline]
Ronis, M. J. J., Ingelman-Sundberg, M., and Badger, T. M. (1994). Induction, suppression and inhibition of multiple hepatic cytochrome P450 isozymes in the male rat and bobwhite quail (Colinus Virginianus) by ergosterol biosynthesis inhibiting fungicides (EBIFs). Biochem. Pharmacol. 48, 19531965.[ISI][Medline]
Sanderson, J. T., Janz, D. M., Bellward, G. D., and Giesy, J. P. (1997). Effects of embryonic and adult exposure to 2,3,7,8-tetrchlorodibenzo-p-dioxin on hepatic microsomal testosterone hydroxylase activities in Great Blue Herons (Ardea Herodias). Environ. Toxicol. Chem. 16, 13041310.[ISI]
Santen, R. J., Van den Bossche, H., Symoens, J., Brugmans, J., and DeCoster, R. (1983). Site of action of low dose ketoconazole on androgen biosynthesis in men. J. Clin. Endocrinol. Metab. 57, 732736.[Abstract]
Sivarajah, K., Franklin, C. S., and Williams, W. P. (1978). The effects of polychlorinated biphenyls on plasma steroid levels and hepatic microsomal enzymes in fish. J. Fish Biol. 13, 401409.[ISI]
Tedford, C. E., Ruperto, V. B., and Barnett, A. (1991). Characterization of rat liver glucuronosyltransferase that glucuronidates the selective D1 antagonist, SCH23390, and other benzazepines. Drug Metab. Dispos. 19, 11521159.[Abstract]
Thomson, R. G., Rawlins, M. D., James, O. F. W., Wood, P., and Williams, F. M. (1988). The acute and subchronic effects of ketoconazole on hepatic microsomal monooxygenase in the rat. Biochem. Pharmacol. 37, 39753980.[ISI][Medline]
Tredger, J. M., Smith, H. M., and Williams, R. (1984). Effects of ethanol and enzyme-inducing agents on the monooxygenation of testosterone and xenobiotic in rat liver microsomes. J. Pharmacol. Exp. Ther. 229, 292298.[Abstract]
van der Hoeven, T. A., and Coon, M. J. (1974). Preparation and properties of partially purified cytochrome P-450 and NADPH-cytochrome P-450 reductase from rabbit liver microsomes. J. Biol. Chem. 249, 63026310.
vom Saal, F. S., Montano, M. M., and Wang, M. H. (1992). Sexual differentiation in mammals. In Chemically-Induced Alterations in Sexual and Functional Development: The Wildlife, Human Connection. (T. Colborn and C. Clement, Eds.), pp. 1784. Princeton Scientific Publishing, Princeton, NJ.
Waxman, D. J. (1988). Interactions of hepatic cytochromes P-450 with steroid hormones: Regioselectivity and stereoselectivity of steroid metabolism and hormonal regulation of rat P-450 enzyme expression. Biochem. Pharmacol. 37, 7184.[ISI][Medline]
Waxman, D. J., Atisano, C., Guengerich, F. P., and Lapenson, D. (1988). Human liver microsomal steroid metabolism: identification of the major microsomal steroid hormone 6b-hydroxylase cytochrome P-450 enzyme. Arch. Biochem. Biophys. 263, 424436.[ISI][Medline]
Waxman, D. J., Ko, A., and Walsh, C. (1983). Regioselectivity and stereoselectivity of androgen hydroxylation catalyzed by cytochrome P-450 isozymes purified from phenobarbital-induced rat liver. J. Biol. Chem. 10, 1193711947.
Wilson, J. D., and Foster, D. W. (1985). Textbook of Endocrinology. W.B. Saunders Company, Philidelphia.
Wilson, V. S., and LeBlanc, G. A. (1998). Endosulfan elevates testosterone biotransformation and clearance in CD-1 mice. Toxicol. Appl. Pharmacol. 148, 158168.[ISI][Medline]
Wilson, V. S., McLachlan, J. B., Falls, J. G., and LeBlanc, G. A. (1999). Alteration in sexually dimorphic testosterone biotransformation profiles as a biomarker of chemically induced androgen disruption. Environ. Health Perspect. 107, 377384.[ISI][Medline]