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Endocrinology, doi:10.1210/en.2004-1248
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Endocrinology Vol. 146, No. 7 2911-2919
Copyright © 2005 by The Endocrine Society

Potential Role for Human Cytochrome P450 3A4 in Estradiol Homeostasis

Ai-Ming Yu, Katsumi Fukamachi, Kristopher W. Krausz, Connie Cheung and Frank J. Gonzalez

Laboratory of Metabolism (A.-M.Y., K.F., K.W.K., C.C., F.J.G.), National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892; and Department of Pharmaceutical Sciences (A.-M.Y.), State University of New York at Buffalo, Buffalo, New York 14221

Address all correspondence and requests for reprints to: Frank J. Gonzalez, Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Building 37, Room 3106, Bethesda, Maryland 20892. E-mail: fjgonz{at}helix.nih.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previously, a human CYP3A4-transgenic (Tg-CYP3A4) mouse line was reported to exhibit enhanced metabolism of midazolam by cytochrome P450 3A4 (CYP3A4) expressed in small intestine. Here we show that expression of CYP3A4 and murine cyp3a and cyp2b was both age and sex dependent. CYP3A4 was expressed in the livers of male and female Tg-CYP3A4 mice at 2 and 4 wk of age. Since 6 wk, CYP3A4 was undetectable in male livers, whereas it was constitutively expressed in female livers at decreased levels (3- to 5-fold). Pregnenolone 16{alpha}-carbonitrile markedly induced hepatic CYP3A4 expression, and the level was higher in females than males. Induction of intrinsic murine cyp3a and cyp2b was also sex dependent. Tg-CYP3A4 females were found to be deficient in lactation, leading to a markedly lower pup survival. The mammary glands of the Tg-CYP3A4 lactating mothers had underdeveloped alveoli with low milk content. Furthermore, ß-casein and whey acidic protein mRNAs were expressed at markedly lower levels in Tg-CYP3A4 pregnant and nursing mouse mammary glands compared with wild-type mice. This impaired lactation phenotype was associated with significantly reduced serum estradiol levels in Tg-CYP3A4 mice. A pharmacokinetic study revealed that the clearance of iv administrated [3H]estradiol was markedly enhanced in Tg-CYP3A4 mice compared with wild-type mice. These results suggest that CYP3A4 may play an important role in estradiol homeostasis. This may be of concern for treatment of pregnant and lactating women because CYP3A4 gene expression and enzymatic activity can be potentially modified by CYP3A4 inhibitors or inducers in medications, supplements, beverages, and diet.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CYTOCHROME P450 (CYP) enzymes are responsible for the metabolism of a diverse range of xenobiotics, including therapeutic drugs and countless toxins and carcinogens and the biosynthesis of steroid hormones and bile acids (1, 2, 3, 4). Fifty-seven functional CYP genes have been identified in the human genome. Among them, CYP11, CYP17, CYP19, and CYP21 enzymes are involved in steroidogenesis, and CYP7, CYP8, and CYP27 enzymes catalyze the biosynthesis of bile acids (1). Mutations of certain CYP genes result in the disruption of hormone biosynthesis, potentially leading to certain diseases (3, 5). For instance, deficiency of CYP27 causes cerebrotendinous xanthomatosis, an autosomal recessive sterol storage disease characterized by the accumulation of a bile alcohol in diverse tissues (5). By contrast, enzymes belonging to CYP1A, CYP2C, CYP2D, and CYP3A subfamilies are generally recognized as xenobiotic-metabolizing enzymes contributing to the oxidations of numerous chemicals including therapeutic drugs (1, 3, 4, 6). These CYP enzymes play a central role in drug metabolism and disposition. Most CYP enzymes are primarily expressed in liver, whereas some are specifically expressed in extrahepatic tissues. Their gene expression and enzymatic activities are affected by many factors such as induction of gene expression, genetic polymorphisms, and enzymatic inhibition, which may lead to a change in drug clearance.

CYP3A4 is the most abundant CYP isozyme in both the liver and small intestine, contributing to the biotransformations of approximately 50% of marketed drugs including benzodiazepines, HIV antivirals, and macrolide antibiotics (1, 2, 3). In addition, CYP3A4 is involved in the oxidation of a variety of endogenous substrates, such as steroids and bile acids (1, 2). Notably, CYP3A4 gene expression exhibits substantial interindividual variation, which is largely a result of the transcriptional regulation of CYP3A4 by endobiotics and xenobiotics through the nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (7, 8). This variability significantly influences the metabolism of drugs, thus altering their pharmacokinetics and pharmacodynamics. Whether this variability affects the homeostasis of endogenous steroids such as testosterone and estradiol, which are both metabolized by CYP3A4 with high affinity and activity (2, 9, 10, 11), remains unknown.

Transgenic and gene knockout mice have proven to be valuable models for studying the functions of CYP enzymes (4, 12, 13), especially at the systemic, developmental, and physiological levels. For example, CYP1B1 was identified as a major genetic determinant of primary congenital glaucoma (3, 14). This was confirmed by analysis of the cyp1b1-null mouse model (15). Overexpression of CYP19 aromatase resulted in lower testosterone and higher estradiol systemic levels that were associated with female-type mammogenesis and even milk protein gene expression in males (16, 17). Moreover, the CYP2D6-humanized mouse was proven to be a unique model to test the in vivo biotransformation of endogenous substrates for CYP2D6 (18, 19). Previously a CYP3A4-transgenic mouse (Tg-CYP3A4) line was generated using a bacterial artificial chromosome containing the complete gene and PXR-responsive elements, essential factors for its transcriptional regulation (20). The expression of functional CYP3A4 protein in the small intestines of male adult mice led to an increased first-pass metabolism and disposition of midazolam (20), a short-acting 1,4-benzodiazepine widely used in clinical practice for sedation. In the present study, the expression and induction of CYP3A4 transgene and intrinsic murine cyp3a and cyp2b were found to be both sex and age dependent. The data revealed that Tg-CYP3A4 mice exhibited a lactation deficiency. Pharmacokinetic study indicated that estradiol clearance was enhanced in Tg-CYP3A4, probably caused by CYP3A4 expressed in the small intestines of female transgenic mice and to some extent in the livers. Estradiol insufficiency in Tg-CYP3A4 mice resulted in impaired mammary gland function and lower pup survival. These results suggest that CYP3A4 may play an important role in estradiol homeostasis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
[2,4,6,7,16,17-3H(N)]Estradiol was purchased from PerkinElmer Life Sciences, Inc. (Boston, MA). Testosterone and 2-hydroxy- and 4-hydroxyestradiol were bought from Steraloids (Newport, RI). HPLC-grade organic solvents were purchased from Fisher Scientific (Pittsburgh, PA) and were used as received. Pregnenolone 16{alpha}-carbonitrile (PCN), estradiol, estrone, and all other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Pooled human liver microsomes (coded H161) and recombinant CYP3A4 were purchased from BD GenTest (Woburn, MA). Immunoblot polyclonal and monoclonal antibody (MAb) to human CYP3A4 (MAb 275-1-2) and rat cyp3a1 (MAb 2-13-1) (21), cyp2b (22), cyp1a2 (23), cyp2a (24), cyp2c (25), and cyp2d (26) were characterized previously.

Animals
All animals were maintained under controlled temperature (23 ± 1 C) and lighting (lights on 0600–1800 h) with food and water provided ad libitum. Experiments were conducted under National Institutes of Health guidelines for the care and use of laboratory animals, with protocols approved by the National Cancer Institute Animal Care and Use Committee. Tg-CYP3A4 mice were genotyped as described (20). Breeding was set up with one male and two females per cage. Wild-type and Tg-CYP3A4 mice used in these studies were age matched. Virgin mice were 8 wk old, pregnant mice were 18 days postcoitus, and lactation mice (three to five in each group) were 2 d postpartum, respectively. Ninety-nine mice (four to five in each group) were used to examine the influence of sex and age on the expression of CYPs.

Induction of CYP3A4 transgene by PCN
PCN was dissolved in corn oil at a concentration of 10 mg/ml. Mice (wild-type or Tg-CYP3A4, male or female, 4 or 8 wk old, three to five in each group) were administrated PCN (100 mg/kg) or corn oil ip for 2 d. Mice were killed on d 3 after the first injection, and livers were collected and kept at –80 C for future use.

Western blot analyses
Preparation of intestinal microsomes was performed according to a published method (20). Liver and other tissue microsomes were prepared as described (19). Protein concentrations of tissue microsomes were determined using a BCA protein assay kit (Pierce Chemical Co., Rockford, IL), following the manufacturer’s instructions. Microsomal proteins (20 µg per well) were separated by SDS-PAGE with a 4% stacking and 12% resolving gel and transferred onto nitrocellulose membrane. Immunoblot analysis was carried out using monoclonal or polyclonal antibody as the primary antibody. The secondary antibody, a phosphatase-labeled goat antimouse IgM, antimouse IgG, or antirabbit IgG (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) was detected using BCIP/NBT phosphatase substrate (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD). The primary and secondary antibodies were used as reported (20, 23, 24, 25, 26). Blots were scanned, and relative intensity of each band was analyzed using Kodak 1D (version 3.6.3) Scientific Imaging Systems software (New Haven, CT).

Whole mounts and histology of mammary glands
Fourth inguinal mammary glands were excised, spread onto glass slides, and fixed in Carnoy’s fixative (ethanol/chloroform/glacial acetic acid 6/3/1, vol/vol) for 2–4 h at room temperature. The samples were then washed in 70% ethanol for 15 min and changed gradually to distilled water. Once hydrated, the mammary squashes were stained overnight in carmine alum (1 g carmine and 2.5 g aluminum potassium sulfate in 500 ml distilled water). The samples were then dehydrated using stepwise ethanol concentrations, defatted in xylene, and mounted in Permount (Fisher Scientifics, Fair Lawn, NJ). For histological analyses, tissues were fixed in formalin. After fixation, the tissues were placed in 70% ethanol, dehydrated, cleared in xylene, embedded in paraffin, and sectioned at 5 µm. Hematoxylin and eosin staining was performed by standard procedures.

RT-PCR
Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. First-strand cDNA was synthesized from total RNA using the Superscript first-strand synthesis system (Invitrogen). Forward and reverse primers specific for human CYP3A4 (27) and mouse whey acidic protein (WAP) (28), ß-casein (29), and ß-actin (30) were purchased from Integrated DNA Technologies Inc. (Coralville, IA). PCR amplifications were run for 5 min at 90 C, then 25–35 cycles of 1 min at 95 C, 1 min at 60 C, and 2 min at 72 C, followed by a 5-min extension at 70 C. PCR products were 187, 527, 538, and 194 bp for human CYP3A4, mouse WAP, ß-casein, and ß-actin, respectively.

Quantitation of serum estradiol
Blood was collected from mouse suborbital veins into amber tubes with a serum separator (Becton Dickinson and Co., Franklin Lakes, NJ) following the manufacturer’s instructions. Serum samples were separated by centrifugation, transferred, and stored at –80 C until analysis. The concentration of serum estradiol was determined using the commercial ELISA kit (Alpha Diagnostic, San Antonio, TX).

Estradiol hydroxylation in wild-type and Tg-CYP3A4 mouse intestinal microsomes
Incubation reactions were carried out in 100 mM potassium phosphate (pH 7.4) containing pooled intestinal microsomes (from four 8-wk-old female mice) with 200 µg protein and estradiol at a final concentration of 100 µM in a final volume of 500 µl. Reaction mixtures were preincubation at 37 C for 5 min, then initiated by the addition of reduced nicotinamide adenine dinucleotide phosphate at a final concentration of 1 mM. After incubation for 15 min, reactions were terminated by the addition of 6 ml ethyl acetate. Internal standard estrone (50 µl of 50 µM) was then added in each reaction. After extraction and separation, the organic phase was dried under nitrogen gas. The residue was reconstituted in 60 µl acetonitrile and derivatized with 40 µl N,O-bis(tri-methylsilyl)trifluoroacetamide containing 1% trimethylchlorosilane (Pierce) at 60 C for 30 min. One microliter of the solution was injected for gas chromatography mass spectrometry (GC-MS) analysis. All reactions were performed in duplicate.

The instrument contained an Agilent 6890N gas chromatograph and a 5973N mass spectrometry equipped with a 0.25-mm x 30-m, 0.25-µm film thickness RTX-5 capillary column (Restek Corp., Bellefonte, PA). Helium was used as carrier gas. The reported GC-MS condition (31) was applied in the study, and 2- and 4-hydoxylated estradiol was eluted at 22.5 and 23.5 min, respectively. The calibration curve was linear for the two metabolites ranging from 0.25–50 µM.

Clearance and disposition of [3H]estradiol in wild-type and Tg-CYP3A4 mice
Female Tg-CYP3A4 and wild-type mice (8 wk old, four in each group) were administered [3H]estradiol (300 µCi/kg) iv. Blood samples were collected from suborbital veins using heparinized tubes at 2, 5, 8, 10, 12, 15, 20, 30, 45, and 60 min after administration of estradiol. Plasma was separated by centrifugation at 13,000 x g for 10 min and stored at –80 C until analysis.

A similar experiment was carried out to confirm estradiol enterohepatic circulation. Female wild-type and Tg-CYP3A4 mice (8 wk old, three in each group) were administered [3H]estradiol (150 µCi/kg) iv. Immediately after the dosage, mice were transferred into metabolic chambers (Jencons, Leighton Buzzard, UK). Total urine and feces from each individual mouse were collected for 24 h. Radioactivity of [3H]estradiol disposition in urine was directly analyzed by an LS 6500 scintillation counter (Beckman/Coulter, Fullerton, CA). Feces were incubated in 10-fold volume of 80% methanol at 50 C for 30 min, and the supernatant was analyzed with the scintillation counter.

HPLC analysis was carried out with an Agilent 1050 system consisting of a quaternary pump, autosampler, diode array detector, and Radiomatic Flow-one ßII radioactivity detector. Samples were separated on a Luna 5 µC18 250 x 4.6 mm id Phenomenex column (Torrance, CA). [3H]Estradiol and internal standard, regular testosterone, were monitored by radioactivity and diode array detector, respectively. Identification and quantitation of radioactive estradiol in plasma was achieved according to a published method (11) with a slight modification. The flow rate through the column at ambient temperature was 1.0 ml/min with a gradient elution: 60% methanol (A) and 40% water containing 0.1% trifluoroacetic acid (B) for 2 min followed by 70% A and 30% B for 13 min. The HPLC/Radiomatic detector flow was mixed at a ratio of 1:3 using Ultima Flo-M scintillation cocktail (PerkinElmer, Wellesley, MA).

Pharmacokinetics parameters were estimated from the plasma concentration vs. time data by a noncompartmental approach using the WinNonLin software (Pharsight, Mountain View, CA). The area under the curve from zero to infinity (AUC0–{infty}) was calculated by the trapezoidal rule. The systemic clearance (CLiv) of estradiol was calculated as the dose divided by the AUC0–{infty} (Div/AUC0–{infty}).

Statistics
Values were expressed as mean ± SD. All data were compared with unpaired Student’s t test (GraphPad Prism version 3.02; GraphPad, San Diego, CA), and the difference was considered significant if the probability (P value) was less than 5%.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of CYP3A4 transgene and murine cyp3a and cyp2b is sex and age dependent
Male and female Tg-CYP3A4 mice were genotyped using a PCR method as previously described (20). A 406-bp product was amplified specifically from Tg-CYP3A4 mice, which was absent in wild-type mice as expected. By contrast, a 341-bp microsomal epoxide hydrolase (mEH) fragment was produced in both wild-type and Tg-CYP3A4 mice (data not shown). To quantify expression of CYP3A4, a specific MAb that reacts only with the human CYP3A4 and not with the corresponding mouse cyp3a proteins was used. Levels of expression were determined using a recombinant CYP3A4 as a standard. Interestingly, CYP3A4 was expressed in both the small intestines and livers of female adult Tg-CYP3A4 mice (Fig. 1AGo) but detected only in the small intestines of male adult mice as shown previously (20). The level of CYP3A4 expressed in female liver was markedly lower (about 2.5 pmol/mg protein) compared with small intestine (around 35 pmol/mg protein). This observation indicates that hepatic CYP3A4 expression is sex dependent in the Tg-CYP3A4 mouse. Therefore, a comprehensive study was carried out to examine whether CYP3A4 transgene expression is also age dependent. Consistent with these observations, CYP3A4 was not detected in the livers of 8-wk-old male Tg-CYP3A4 mice but only in female mice (Fig. 1BGo). In the 6- to 16-wk-old females, CYP3A4 was constitutively expressed at comparable levels. Surprisingly, CYP3A4 was expressed not only in the livers of females but also in the 2- and 4-wk-old males, and their expression levels were about 3- to 5-fold higher than those in the older female mice. Because all the Tg-CYP3A4 mice were maintained under the same conditions of bedding and food, these results suggest that CYP3A4 expression is not only sex but also age dependent.



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  		FIG. 1. Immunoblot analyses indicate that the expression of human CYP3A4 transgene and murine cyp2b and cyp3a is both age and sex dependent in mice. A, CYP3A4 is expressed in both the liver and small intestine of 8-wk-old female transgenic mice but only in the small intestine of males. Recombinant CYP3A4 was used for quantitative analysis, and pooled human liver microsomes (HLM) were positive controls. B, Expression of the CYP3A4 transgene and murine cyp3a and cyp2b is dependent not only on sex but also on age. Pooled samples (four to five in each group) of microsomes were prepared from livers from 2-wk-old (2W) to 16-wk-old (16W) male or female mice. Microsomal proteins (20 µg per well) were subjected to electrophoresis on 12% SDS-PAGE and transferred to a nitrocellulose membrane. Blotting was performed using a specific MAb against CYP3A4 (MAb 275-1-2) (21 ), which reacts only with CYP3A4 and does not recognize murine cyp3a or other proteins. The antibody against rat CYP3A1 (MAb 2-13-1) (21 ) reacts strongly with mouse cyp3a and very weakly with human CYP3A4 protein.

 
Sex- and age-dependent expression was also observed for intrinsic murine cyp3a and cyp2b (Fig. 1BGo). The precise levels of expression of these CYPs could not be determined because of the absence of specific antibodies and recombinant protein standards. In any case, the levels of mouse cyp3a expression decreased with age in wild-type males, although they increased with age in females. This opposite developmental expression pattern resulted in a 62% decrease for cyp3a in males and a 50% increase in females of 16 wk of age compared with their counterparts of 2 wk of age, respectively. Cyp2b expression also increased with age in wild-type females, resulting in a level about 6-fold higher in 16-wk-old compared with 2-wk-old mice. By contrast, the expression levels of mouse cyp1a2, cyp2a, cyp2c, and cyp2d remained unchanged in wild-type mice between 4 and 16 wk of age. In 2-wk-old wild-type mice, they were all expressed at relatively lower levels compared with the older mice.

Introduction of human CYP3A4 in the Tg-CYP3A4 mice did not affect the expression of intrinsic murine cyp1a2, cyp2b, cyp2a, cyp2c, and cyp2d (Fig. 1BGo). However, the expression level or developmental expression trends of murine cyp3a were different in Tg-CYP3A4 mice. Murine cyp3a levels in Tg-CYP3A4 females were about 50% lower than wild-type females of the same age, although they showed the same developmental trend. In males, murine cyp3a increased with age in the Tg-CYP3A4 mice, resulting in a relatively higher level of cyp3a in Tg-CYP3A4 than in 12- and 16-wk-old wild-type mice. The underlying mechanism of the altered regulation of cyp3a in Tg-CYP3A4 mice is currently unknown.

Induction of CYP3A4 and murine cyp3a and cyp2b by PCN
The CYP3A4 transgene was inducible by PXR activator in transgenic mice (Fig. 2Go). In 4-wk-old Tg-CYP3A4 mice, CYP3A4 was induced by PCN to a level of about 13-fold and 20-fold in males and females, respectively, compared with male controls. This was associated with significantly elevated CYP3A4 mRNA levels (data not shown). Induction of human CYP3A4 by PCN was also observed in 8-wk-old Tg-CYP3A4 mice. Similar to the results obtained with 4-wk-old mice, CYP3A4 was induced to about 68% higher levels in 8-wk-old females than males. As expected, murine cyp3a and cyp2b were also markedly induced by PCN in both wild-type and Tg-CYP3A4 mice, whereas cyp2d expression was not affected. PCN elevated cyp3a to a level about 1-fold higher in 8-wk-old wild-type females than males but to a similar level in 4-wk-old males and females. Cyp2b was also induced by PCN to significantly higher levels in 8-wk-old females than males. Elevated CYP3A4 and cyp3a levels consequently led to a significantly increased enzymatic activity (our unpublished results).



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  		FIG. 2. Immunoblot analyses demonstrate that the induction of CYP3A4 transgene and murine cyp3a and cyp2b by PCN is dependent on sex and age. After PCN treatment, CYP3A4 level was about 50% higher in 4-wk-old (4W) female than male Tg-CYP3A4 mice. Murine cyp3a and cyp2b were also induced by PCN in both wild-type and Tg-CYP3A4 mice. CYP3A4, cyp3a, and cyp2b were all induced to higher levels in 8-wk-old (8W) female than male mice. Pooled samples (three to five in each group) of microsomes prepared from livers of mice treated with PCN or corn oil (Cont) were used in the study. Pooled human liver microsomes (HLM) from BD GenTest were used as positive control. Twenty micrograms of hepatic proteins were separated by 12% SDS-PAGE and transferred to a nitrocellulose membrane. The CYP3A4 MAb (MAb 275-1-2) (21 ) reacts only with CYP3A4 and does not recognize mouse cyp3a proteins. Antibodies against rat cyp3a1 (MAb 2-13-1) (21 ) and cyp2b (22 ) were used to detect murine cyp3a and cyp2b isozymes, respectively.

 
Deficient lactation phenotype of Tg-CYP3A4 mice
During the course of colony expansion of the Tg-CYP3A4 mice, it was noted that the litter size was significantly lower than expected (Fig. 3AGo). Moreover, most pups were found dead within 2 d after birth. This resulted in a markedly lower pup survival rate of the Tg-CYP3A4 mice (Fig. 3BGo). The growth of the Tg-CYP3A4 mice was also significantly slower compared with wild-type mice (Fig. 3CGo).



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  		FIG. 3. Impaired fertility phenotype of transgenic mice. Tg-CYP3A4 and wild-type breeders were set up in the same manner and housed under identical environments. A, Litter size from Tg-CYP3A4 breeders (n = 5, one male with two females) is significantly decreased (***, P < 0.0001). B, Pup survival rate is markedly lower (***, P < 0.0001) from Tg-CYP3A4 breeders (n = 5). Shown are averaged values at 4 months from commencement of breeding. C, The growth of transgenic male (n = 6) and female (n = 4) mice are slower than wild-type male (n = 10) and female (n = 6) mice.

 
Lower pup survival and slower pup growth rate was presumably a result of starvation because close examination revealed the absence of milk in the stomach of the Tg-CYP3A4 pups. Because Tg-CYP3A4 mothers showed normal nursing behavior, a lactation defect was suspected. Therefore, cross-fostering experiments were performed, revealing that 16 of 17 Tg-CYP3A4 pups survived after fostering with wild-type mothers, whereas only six of 37 wild-type pups nursed by Tg-CYP3A4 mothers survived. Moreover, the surrogated transgenic pups exhibited a greater increased body weight compared with the littermates nursed by Tg-CYP3A4 mothers, and their stomachs were full of milk (data not shown). These observations further indicated that Tg-CYP3A4 mothers have a lactation defect, which led to further examination of mammary gland development.

Examination of mammary gland structure
Mammary gland development is classified into four distinct stages: virgin, pregnancy, lactation, and involution (32, 33, 34). The functional regulation of these processes requires a complex interplay of steroid and peptide hormones through their cognate receptors (32, 35). The mammary glands from virgin (8-wk-old), pregnant (18-d postcoitus), and lactating (2-d postpartum) Tg-CYP3A4 mice were smaller than those from wild-type mice in each group. However, it was not significant when normalized with their body weights (Tg-CYP3A4 vs. wild-type: 7.82 ± 1.89 vs. 6.78 ± 0.51 for virgin; 7.78 ± 1.76 vs. 7.66 ± 0.40 for pregnant; 10.6 ± 3.56 vs. 12.8 ± 1.14 for lactation; n = 3–5).

Stained whole mounts of mammary glands revealed that mammary gland development was impaired in Tg-CYP3A4 nursing mice (Fig. 4Go). Tg-CYP3A4 virgin mice developed ductal trees, but their secondary and tertiary ducts were not completely elongated but more branched in fat pads. Although lobuloalveolar development took place in pregnant and lactating Tg-CYP3A4 mouse stroma, they were less expanded in transgenic nursing mice, indicating deficient epithelial proliferation. Histological analysis was then carried out with the results further confirming the extent of differentiation and abundance of alveoli in wild-type mouse mammary glands, whereas Tg-CYP3A4 mouse lactating mammary glands had sparsely filled, underdeveloped alveoli (Fig. 5Go). Moreover, the accumulation of milk fully distended the alveoli in wild-type mice with a relatively small volume of adipose tissue being present. In Tg-CYP3A4 mice, a minimal volume of milk was present with large lipid droplets remaining trapped within the epithelial cells, and large areas of adipose tissue were obviously visible (Fig. 5Go).



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  		FIG. 4. Representative whole mounts of mammary glands from wild-type and Tg-CYP3A4 mice. Inguinal mammary glands from virgin (8-wk-old), pregnant [18 d postcoitus (d.p.c.)] and lactating [2 d postpartum (d.p.p.)] mice were fixed in Carnoy’s and stained with carmine alum to visualize ductal development. White arrows point to terminal end buds, and blue arrows point to ducts. Note that the alveoli (black arrows) are poorly developed in Tg-CYP3A4 lactating mice compared with wild-type mice with highly branched alveolar structure filled in fat pad. Scale bar, 500 µm. Tg-CYP3A4 and wild-type mice used for the studies were age matched in each group.

 


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  		FIG. 5. Histological analyses indicate that mammary glands of transgenic nursing mothers are sparsely filled with underdeveloped alveoli. Paraffin wax-embedded sections of mammary glands were stained with hematoxylin and eosin. Note the uniform size of alveolus (A) and the extent of differentiation in wild-type mammary glands vs. the mixture of collapsed, nondifferentiated and differentiated alveoli in the Tg-CYP3A4 glands. In wild-type mice, the alveoli were fully distended by the accumulation of milk, and a minimal volume of adipose tissue (AD) was present. On the contrary, a relatively smaller volume of milk was accumulated, and large areas of adipose tissue were obviously visible in Tg-CYP3A4 mice. At higher magnification, the lumen (Lu) and epithelial cells (black arrow) of the alveolus are indicated. Scale bar, 50 µm.

 
The number of live fetuses was significantly decreased in the Tg-CYP3A4 pregnant mice (6.6 ± 0.4, n = 9) compared with wild-type (9.2 ± 0.3, n = 9), a result that is consistent with the initial observations of lower litter size (Fig. 3AGo). The underlying mechanism for the developmental defect is unknown and needs further investigation.

Analysis of milk protein gene expression in mammary glands
To further evaluate the maturation status of Tg-CYP3A4 mouse mammary glands, expression of some milk protein genes was examined. As expected, neither WAP nor ß-casein (36) mRNA were detected in virgin mouse mammary glands (Fig. 6Go). In contrast to the abundant expression of both WAP and ß-casein genes in wild-type pregnant and lactating mice, ß-casein mRNA was not detectable and WAP mRNA was weakly detected in Tg-CYP3A4 mouse mammary glands (Fig. 6Go). These results were consistent with those obtained from the morphological and histological analyses (Figs. 4Go and 5Go), indicating deficient lactation in the Tg-CYP3A4 mice.



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  		FIG. 6. Expression of milk protein genes in mammary glands as examined by RT-PCR. Total RNA was isolated using Trizol reagent, and first-strand cDNA was synthesized using the Superscript system with specific primers for each gene. In both wild-type and Tg-CYP3A4 virgin mouse mammary glands, WAP and ß-casein were not detectable. During pregnancy and lactation, WAP and ß-casein were abundant in wild-type mice, whereas they were reduced or undetectable in transgenic mice.

 
The expression of CYP3A4 was measured in these mammary glands by RT-PCR. However, CYP3A4 mRNA was not detected in the mammary glands (data not shown). These results suggest that the deficient lactogenesis is likely influenced by CYP3A4 expressed in other tissues that may dramatically alter the levels of systemic hormones.

Role of CYP3A4 in estrogen homeostasis
It is known that estradiol is required for both mammary ductal growth and lobuloalveolar development, acting as an inducer of mammary epithelial cell proliferation (33, 37, 38, 39, 40). Because estradiol is predominately produced from testosterone, and both are oxidized by CYP3A4 (2, 9, 11, 31, 41, 42, 43), overexpression of CYP3A4 could potentially lead to altered systemic estradiol levels in female Tg-CYP3A4 mice by diverting its biosynthesis and enhancing its metabolism.

To test this hypothesis, serum estradiol levels were measured and compared between Tg-CYP3A4 and wild-type mice. As expected, estradiol levels were significantly lower in Tg-CYP3A4 mice during both pregnancy and lactation compared with wild-type mice (Fig. 7Go). To further determine the role of metabolism in estradiol levels, a pharmacokinetic study was carried out using [3H]estradiol. Indeed, plasma [3H]estradiol concentrations were lower in Tg-CYP3A4 mice than wild-type mice, and the clearance of the iv administrated estradiol was markedly enhanced by 59% in Tg-CYP3A4 mice (Fig. 8Go). It should be noted that CYP3A4 is predominantly expressed in the small intestine and at lower levels in the liver of female Tg-CYP3A4 mouse (Fig. 1AGo). Therefore, enhanced metabolism of estradiol is presumably performed in small intestines during enterohepatic circulation (44, 45). This is further suggested by the results that the iv dosed [3H]estradiol was excreted almost equally in feces and urine in both wild-type and Tg-CYP3A4 mice. In addition, 2- and 4-hydroxylation of estradiol were markedly elevated in Tg-CYP3A4 mouse intestinal microsomes in vitro (Fig. 9Go). These results suggest that the estradiol insufficiency was likely caused by CYP3A4 expressed in small intestine, resulting in deficient lactogenesis and leading to starvation and death of newborn pups.



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  		FIG. 7. Serum estradiol levels are significantly (*, P < 0.05; n = 5 in each group) decreased in pregnant and lactating Tg-CYP3A4 mice, compared with wild-type mice, respectively. Tg-CYP3A4 and wild-type mice were age matched in each group. Samples were collected with a serum separator, and the concentrations of estradiol were measured by using an ELISA kit and the manufacturer’s instructions.

 


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  		FIG. 8. Serum [3H]estradiol radioactivity vs. time curves in wild-type (n = 4) and Tg-CYP3A4 (n = 4) female mice after iv administration of [3H]estradiol. Estradiol radioactivity counts per minute (CPM) was normalized with an internal standard (I.S.), regular testosterone that was monitored by an online UV detector. The relative values of area under the curve from zero to infinity (AUC0–{infty}) are significantly different between wild-type (101 ± 18.7) and Tg-CYP3A4 (63.5 ± 12.1) mice. Clearance of estradiol is markedly (P < 0.05) enhanced about 59% in Tg-CYP3A4 (160 ± 27.3) mice, compared with wild-type (101 ± 16.8).

 


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  		FIG. 9. Estradiol hydroxylations were markedly enhanced in Tg-CYP3A4 female mouse intestinal microsomes. Pooled small intestinal microsomes, prepared from four 8- to 10-wk-old female mice, were incubated for 15 min with estradiol at a final concentration of 100 µM. After extraction with ethyl acetate and derivatization with N,O-bis-(trimethyl-silyl)trifluoroacetamide containing 1% trimethylchlorosilane, the metabolites were separated and quantified with GC-MS analyses.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Comprehensive study of the CYP3A4-transgenic mouse line revealed that expression and induction of the CYP3A4 transgene, as well as the endogenous murine cyp3a and cyp2b, were both sex and age dependent. Pregnant and lactating transgenic mice exhibited estradiol insufficiency, presumably caused by enhanced metabolism of estradiol and its precursor testosterone. Low estradiol resulted in deficient lactogenesis with underdeveloped alveoli and deficient milk protein gene expression. Impaired lactation led to low pup survival and slower pup growth rates. These observations are consistent with the notion that estradiol plays a major role in promoting functional mammary development. Most importantly, these results indicate a possible important role for CYP3A4 in the regulation of estradiol homeostasis.

Steroid hormones, especially estrogen and progesterone, are known to have crucial roles in the development and maintenance of the normal function of mammary glands, acting via their specific receptors (33, 37, 38, 46). Estrogen is a known inducer of mammary epithelial cell proliferation and is required for both ductal growth and lobuloalveolar development (33, 37). This was further demonstrated in vivo to be a direct action using antiestrogens (47). Estradiol is the most potent estrogen and the form mainly responsible for estrogen action in women. Estradiol is produced from testosterone through sequential oxidation by CYP19 (Fig. 10Go). Recently, a transgenic mouse strain was generated bearing the human ubiquitin C promoter/human CYP19 fusion gene resulting in significantly reduced testosterone and elevated estradiol levels (16), which was associated with ductal and alveolar development in male mammary glands (17), a morphogenesis that normally occurs only in females. These mammary glands in male transgenic mice also expressed milk protein gene (ß-casein) and multiple hormone receptors (estrogen receptors {alpha} and ß, progesterone receptor, and prolactin receptor) typical for female mammary glands (17). By using this transgenic animal model, a critical role of CYP19 in estradiol homeostasis was demonstrated.



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  		FIG. 10. Biosynthesis of estradiol from testosterone is catalyzed by CYP19 (aromatase), and their metabolic hydroxylations are mediated by CYP3A4. Altered CYP3A4 gene expression and enzymatic activity may markedly influence the homeostasis of estradiol through the metabolism of estradiol and its precursors as supported by this study.

 
CYP3A4 catalyzes testosterone 6ß-hydroxylation with high affinity and activity, a pathway that is widely accepted and used as an in vitro index for CYP3A4 enzymatic activity (Fig. 10Go). CYP3A4 also mediates the 2ß-, 15-, and 16ß-hydroxylation of testosterone with appreciable activities (48). In addition, CYP3A4 catalyzes 2-, 4-, and 16-hydroxylation of estradiol (Fig. 10Go) with high enzymatic activity (9, 11, 31, 41, 42, 43, 49). However, a role of CYP3A4 in the homeostasis of the sex steroids in vivo remains inconclusive. The data presented in the present study indicate that excessive expression of CYP3A4 is able to alter systemic estradiol levels. Lower serum estradiol levels in Tg-CYP3A4 mice are likely caused by a combination of the enhanced metabolism of estradiol and its precursor, testosterone. Indeed, serum testosterone levels in transgenic males were lower compared with wild-type males (our unpublished results). It will be of interest to further investigate which pathway would be more significant for estradiol homeostasis and its possible role in maintaining testosterone levels.

The data shown in this study are also in agreement with estradiol being an important hormone to stimulate casein synthesis in mammary glands as previously demonstrated (40, 50). Actually, a specific role for estradiol after mammary ductal morphogenesis is less understood, although the hormone is also thought to be responsible for the induction of progesterone receptor in luminal epithelial cells and in alveoli development (35). It is generally accepted that progesterone, prolactin, and cortisol are the major hormones controlling alveolar morphogenesis and lactation. Nevertheless, both progesterone and cortisol are also metabolized by CYP3A4 with high affinity and high turnover (2, 10, 48). The ratio of 6ß-cortisol, the major metabolite produced by CYP3A4, over substrate cortisol in human urine has been suggested and used as a marker for CYP3A4 activity (51, 52). Cortisol enhances full differentiation of the lobuloalveolar system and remarkably extends the half-life of casein mRNA (53). It also affects the lactogenic response of mammary tissue by regulating prolactin binding to the epithelial cells (54). Whether the deficient lactation found in the Tg-CYP3A4 mice is also influenced by these hormones and the significance of CYP3A4 in their homeostasis need to be further studied.

Estrogens are implicated in breast carcinogenesis, a leading cancer and cause of mortality among females in western countries (14). CYP3A4 was shown to be expressed in estrogen and testosterone target cells, and its expression is tightly regulated (9). Therefore, significantly altered CYP3A4 expression and enzymatic activity may change by ingestion of various chemical inducers and inhibitors in medications, supplements, beverages, and diet (1, 2, 55). CYP3A4 was reported to be associated with prostate cancer (56) and was investigated as a risk factor for breast cancer, in particular higher-grade tumors, and possibly childhood leukemias (57, 58, 59, 60, 61, 62). The CYP3A4*1B allele was shown in vitro to exhibit about a 2-fold higher activity compared with the wild-type CYP3A4*1A variant, although its function has not yet been established in vivo. After examining the relationship between CYP3A4 allelic variants and the onset of breast puberty, 90% of girls with the CYP3A4*1B/CYP3A4*1B were found to have Tanner breast stage 2 or higher, compared with 56% of the heterozygotes and 41% of wild-type homozygotes (57). These observations might be explained by an altered estradiol over testosterone ratio caused by high-activity CYP3A4*1B allelic variant because androgens have also been known to influence the development and growth of mammary glands in women (63). By contrast, estradiol is also known to play an important physiological and pathological role in men, such as in bone growth and metabolism (64, 65). Estrogen insufficiency in males has also been associated with a mutation of CYP19 (66). However, studies of human and gene knockout mouse models have failed to clarify all the roles and interactions of CYP19, estrogens, and estrogen receptors (64, 66). It is very possible that estrogen-metabolizing enzymes including CYP3A4 participate in estrogen physiology by altering systemic and even local levels of estradiol. There also exists the possibility that metabolites produced from estrogens by CYP enzymes may be functionally significant. The evidence that enhanced metabolism of estradiol by CYP3A4, decreased estradiol levels, suggest that CYP3A4 may have an important role in the homeostasis of sex steroids and thus may further influence physiological and pathological conditions in men and women.

Low pup survival rates and slow pup growth rates may be caused by not only milk insufficiency but also the lack of nutrition in the milk. The significance of milk protein insufficiency has also been demonstrated using gene knockout mouse models. For instance, when the ß-casein gene is disrupted, mice grow much slower than wild-type control mice (67). Indeed, the Tg-CYP3A4 lactating mice not only produced less milk but also expressed less milk proteins, thus accounting for the slower pup growth rates and lower pup survival rates. The number of Tg-CYP3A4 newborn per litter is consistent with the number of fetuses in pregnant mice, but the underlying mechanism is unknown and needs further investigation.

Our observations also indicate that both sex and age are major determinants for the expression of CYP3A4 transgene and murine cyp3a and cyp2b in livers. Whether CYP3A4 expression in human livers is dependent on age and sex has been debated because of the controversial reports summarized in recent reviews (68, 69, 70, 71). It should be noted that CYP3A4 transcriptional induction and enzymatic activity are easily altered by a vast number of chemicals in diet, beverages, and supplements as well as medications (2, 55). It is extremely difficult to obtain, evaluate, and process the true information of human subjects and liver samples. The lack of specific antibody against or probe drug for the CYP of interest is another difficulty that needs to overcome. Actually, most studies examined the effect of either only sex or only age instead of both at the same time. Although subdivision with narrow range of ages was warranted, it was not achieved as acceptable numbers of samples was expected in each group. All of these factors make it extremely difficult to properly interpret the data obtained from the studies, which might result in the controversy. Because Tg-CYP3A4 male and female litters are identical in genotype and maintained under the same environment, the differential expression pattern should be caused by age and sex. It is of interest that our finding of higher constitutive expression of CYP3A4 in female Tg-CYP3A4 mice is in agreement with a recent study using human liver surgical specimens (72). Generally, CYP3A4 transcriptional regulation is determined through the PXR and constitutive androstane receptor pathways (8). Induction of CYP3A4 transcription through the nuclear receptor pathways was demonstrated to alter the metabolism and clearance of drugs. Various endogenous compounds are CYP3A4 inducers, and the cholesterol-derived 5ß-cholestane-3{alpha},7{alpha},12{alpha}-tiol has been identified as a potent endogenous ligand for mouse PXR (73, 74). Studies also revealed the up-regulation of CYP3A by GH (75) and characterized the involvement of the signal transducer and activator of transcription factor-5 in this pathway (76). It is highly warranted to study this relatively less understood pathway and to investigate the involvement of other genes, endobiotics, and factors.

In conclusion, the observation of low estradiol as a result of enhanced metabolism suggests that CYP3A4 may play an important role in estradiol homeostasis. Altered CYP3A4 gene expression and enzymatic activity could significantly influence mammogenesis and lactogenesis, which are potentially achieved by various chemicals that are present in orally administered drugs, supplements, beverages, and diet. These results also suggest that caution should be observed when prescribing CYP3A4 inducer or inhibitor drugs to women during pregnancy and lactation.


   Acknowledgments
 
We thank Drs. Jeffery Idle, Linda Byrd, Yong-Zhi Cui, and Gertraud Robinson for their helpful suggestions and discussions and John Buckley for his technical assistance.


   Footnotes
 
First Published Online April 7, 2005

Abbreviations: CYP, Cytochrome P450; GC-MS, gas chromatography mass spectrometry; MAb, monoclonal antibody; PCN, pregnenolone 16{alpha}-carbonitrile; PXR, pregnane X receptor; Tg, transgenic.

Received September 21, 2004.

Accepted for publication March 24, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Nebert DW, Russell DW 2002 Clinical importance of the cytochromes P450. Lancet 360:1155–1162[CrossRef][Medline]
  2. Guengerich FP 1999 Cytochrome P-450 3A4: regulation and role in drug metabolism. Annu Rev Pharmacol Toxicol 39:1–17[CrossRef][Medline]
  3. Guengerich FP 2003 Cytochromes P450, drugs, and diseases. Mol Interv 3:194–204[Abstract/Free Full Text]
  4. Gonzalez FJ 2003 Role of gene knockout and transgenic mice in the study of xenobiotic metabolism. Drug Metab Rev 35:319–335[CrossRef][Medline]
  5. Leitersdorf E, Meiner V 1994 Cerebrotendinous xanthomatosis. Curr Opin Lipidol 5:138–142[CrossRef][Medline]
  6. Evans WE, Relling MV 1999 Pharmacogenomics: translating functional genomics into rational therapeutics. Science 286:487–491[Abstract/Free Full Text]
  7. Akiyama TE, Gonzalez FJ 2003 Regulation of P450 genes by liver-enriched transcription factors and nuclear receptors. Biochim Biophys Acta 1619:223–234[Medline]
  8. Willson TM, Kliewer SA 2002 PXR, CAR and drug metabolism. Nat Rev Drug Discov 1:259–266[CrossRef][Medline]
  9. Lee AJ, Cai MX, Thomas PE, Conney AH, Zhu BT 2003 Characterization of the oxidative metabolites of 17ß-estradiol and estrone formed by 15 selectively expressed human cytochrome p450 isoforms. Endocrinology 144:3382–3398[Abstract/Free Full Text]
  10. Guengerich FP, Muller-Enoch D, Blair IA 1986 Oxidation of quinidine by human liver cytochrome P-450. Mol Pharmacol 30:287–295[Abstract]
  11. Aoyama T, Korzekwa K, Nagata K, Gillette J, Gelboin HV, Gonzalez FJ 1990 Estradiol metabolism by complementary deoxyribonucleic acid-expressed human cytochrome P450s. Endocrinology 126:3101–3106[Abstract/Free Full Text]
  12. Gonzalez FJ, Kimura S 2003 Study of P450 function using gene knockout and transgenic mice. Arch Biochem Biophys 409:153–158[CrossRef][Medline]
  13. Buters JT, Doehmer J, Gonzalez FJ 1999 Cytochrome P450-null mice. Drug Metab Rev 31:437–447[CrossRef][Medline]
  14. Huber JC, Schneeberger C, Tempfer CB 2002 Genetic modelling of the estrogen metabolism as a risk factor of hormone-dependent disorders. Maturitas 42:1–12[CrossRef][Medline]
  15. Libby RT, Smith RS, Savinova OV, Zabaleta A, Martin JE, Gonzalez FJ, John SW 2003 Modification of ocular defects in mouse developmental glaucoma models by tyrosinase. Science 299:1578–1581[Abstract/Free Full Text]
  16. Li X, Nokkala E, Yan W, Streng T, Saarinen N, Warri A, Huhtaniemi I, Santti R, Makela S, Poutanen M 2001 Altered structure and function of reproductive organs in transgenic male mice overexpressing human aromatase. Endocrinology 142:2435–2442[Abstract/Free Full Text]
  17. Li X, Warri A, Makela S, Ahonen T, Streng T, Santti R, Poutanen M 2002 Mammary gland development in transgenic male mice expressing human P450 aromatase. Endocrinology 143:4074–4083[Abstract/Free Full Text]
  18. Yu AM, Idle JR, Gonzalez FJ 2004 Polymorphic cytochrome P450 2D6: humanized mouse model and endogenous substrates. Drug Metab Rev 36:243–277[CrossRef][Medline]
  19. Yu AM, Idle JR, Byrd LG, Krausz KW, Kupfer A, Gonzalez FJ 2003 Regeneration of serotonin from 5-methoxytryptamine by polymorphic human CYP2D6. Pharmacogenetics 13:173–181[CrossRef][Medline]
  20. Granvil CP, Yu AM, Elizondo G, Akiyama TE, Cheung C, Feigenbaum L, Krausz KW, Gonzalez FJ 2003 Expression of the human CYP3A4 gene in the small intestine of transgenic mice: in vitro metabolism and pharmacokinetics of midazolam. Drug Metab Dispos 31:548–558[Abstract/Free Full Text]
  21. Gelboin HV, Krausz KW, Goldfarb I, Buters JT, Yang SK, Gonzalez FJ, Korzekwa KR, Shou M 1995 Inhibitory and non-inhibitory monoclonal antibodies to human cytochrome P450 3A3/4. Biochem Pharmacol 50:1841–1850[CrossRef][Medline]
  22. Yamano S, Nhamburo PT, Aoyama T, Meyer UA, Inaba T, Kalow W, Gelboin HV, McBride OW, Gonzalez FJ 1989 cDNA cloning and sequence and cDNA-directed expression of human P450 IIB1: identification of a normal and two variant cDNAs derived from the CYP2B locus on chromosome 19 and differential expression of the IIB mRNAs in human liver. Biochemistry 28:7340–7348[CrossRef][Medline]
  23. Aoyama T, Gonzalez FJ, Gelboin HV 1989 Mutagen activation by cDNA-expressed P(1)450, P(3)450, and P450a. Mol Carcinog [Erratum (1990) 3:319] 1:253–259
  24. Nagata K, Matsunaga T, Gillette J, Gelboin HV, Gonzalez FJ 1987 Rat testosterone 7{alpha}-hydroxylase. Isolation, sequence, and expression of cDNA and its developmental regulation and induction by 3-methylcholanthrene. J Biol Chem 262:2787–2793[Abstract/Free Full Text]
  25. Matsunaga T, Watanabe K, Yamamoto I, Negishi M, Gonzalez FJ, Yoshimura H 1994 cDNA cloning and sequence of CYP2C29 encoding P-450 MUT-2, a microsomal aldehyde oxygenase. Biochim Biophys Acta 1184:299–301[Medline]
  26. Gonzalez FJ, Vilbois F, Hardwick JP, McBride OW, Nebert DW, Gelboin HV, Meyer UA 1988 Human debrisoquine 4-hydroxylase (P450IID1): cDNA and deduced amino acid sequence and assignment of the CYP2D locus to chromosome 22. Genomics 2:174–179[CrossRef][Medline]
  27. Krusekopf S, Roots I, Kleeberg U 2003 Differential drug-induced mRNA expression of human CYP3A4 compared to CYP3A5, CYP3A7 and CYP3A43. Eur J Pharmacol 466:7–12[CrossRef][Medline]
  28. Dupont J, Renou JP, Shani M, Hennighausen L, LeRoith D 2002 PTEN overexpression suppresses proliferation and differentiation and enhances apoptosis of the mouse mammary epithelium. J Clin Invest 110:815–825[CrossRef][Medline]
  29. Gallego MI, Binart N, Robinson GW, Okagaki R, Coschigano KT, Perry J, Kopchick JJ, Oka T, Kelly PA, Hennighausen L 2001 Prolactin, growth hormone, and epidermal growth factor activate Stat5 in different compartments of mammary tissue and exert different and overlapping developmental effects. Dev Biol 229:163–175[CrossRef][Medline]
  30. Yamada H, Gohyama N, Honda S, Hara T, Harada N, Oguri K 2002 Estrogen-dependent regulation of the expression of hepatic Cyp2b and 3a isoforms: assessment using aromatase-deficient mice. Toxicol Appl Pharmacol 180:1–10[CrossRef][Medline]
  31. Lee AJ, Kosh JW, Conney AH, Zhu BT 2001 Characterization of the NADPH-dependent metabolism of 17ß-estradiol to multiple metabolites by human liver microsomes and selectively expressed human cytochrome P450 3A4 and 3A5. J Pharmacol Exp Ther 298:420–432[Abstract/Free Full Text]
  32. Hennighausen L, Robinson GW 2001 Signaling pathways in mammary gland development. Dev Cell 1:467–475[CrossRef][Medline]
  33. Topper YJ, Freeman CS 1980 Multiple hormone interactions in the developmental biology of the mammary gland. Physiol Rev 60:1049–1106[Free Full Text]
  34. Neville MC, Daniel CW 1987 The mammary gland: development, regulation, and function. New York: Plenum Press
  35. Neville MC, McFadden TB, Forsyth I 2002 Hormonal regulation of mammary differentiation and milk secretion. J Mammary Gland Biol Neoplasia 7:49–66[CrossRef][Medline]
  36. Robinson GW, McKnight RA, Smith GH, Hennighausen L 1995 Mammary epithelial cells undergo secretory differentiation in cycling virgins but require pregnancy for the establishment of terminal differentiation. Development 121:2079–2090[Abstract]
  37. Bocchinfuso WP, Korach KS 1997 Mammary gland development and tumorigenesis in estrogen receptor knockout mice. J Mammary Gland Biol Neoplasia 2:323–334[CrossRef][Medline]
  38. Pepe GJ, Albrecht ED 1995 Actions of placental and fetal adrenal steroid hormones in primate pregnancy. Endocr Rev 16:608–648[Abstract/Free Full Text]
  39. Russo J, Gusterson BA, Rogers AE, Russo IH, Wellings SR, van Zwieten MJ 1990 Comparative study of human and rat mammary tumorigenesis. Lab Invest 62:244–278[Medline]
  40. Borellini F, Oka T 1989 Growth control and differentiation in mammary epithelial cells. Environ Health Perspect 80:85–99[Medline]
  41. Guengerich FP, Martin MV, Beaune PH, Kremers P, Wolff T, Waxman DJ 1986 Characterization of rat and human liver microsomal cytochrome P-450 forms involved in nifedipine oxidation, a prototype for genetic polymorphism in oxidative drug metabolism. J Biol Chem 261:5051–5060[Abstract/Free Full Text]
  42. Stresser DM, Kupfer D 1997 Catalytic characteristics of CYP3A4: requirement for a phenolic function in ortho hydroxylation of estradiol and mono-O-demethylated methoxychlor. Biochemistry 36:2203–2210[CrossRef][Medline]
  43. Badawi AF, Cavalieri EL, Rogan EG 2001 Role of human cytochrome P450 1A1, 1A2, 1B1, and 3A4 in the 2-, 4-, and 16{alpha}-hydroxylation of 17ß-estradiol. Metabolism 50:1001–1003[CrossRef][Medline]
  44. Adlercreutz H, Martin F 1980 Biliary excretion and intestinal metabolism of progesterone and estrogens in man. J Steroid Biochem 13:231–244[CrossRef][Medline]
  45. Goldzieher JW 1989 Pharmacology of contraceptive steroids: a brief review. Am J Obstet Gynecol 160:1260–1264[Medline]
  46. Hovey RC, Trott JF, Vonderhaar BK 2002 Establishing a framework for the functional mammary gland: from endocrinology to morphology. J Mammary Gland Biol Neoplasia 7:17–38[CrossRef][Medline]
  47. Silberstein GB, Van Horn K, Shyamala G, Daniel CW 1994 Essential role of endogenous estrogen in directly stimulating mammary growth demonstrated by implants containing pure antiestrogens. Endocrinology 134:84–90[Abstract/Free Full Text]
  48. Yamazaki H, Shimada T 1997 Progesterone and testosterone hydroxylation by cytochromes P450 2C19, 2C9, and 3A4 in human liver microsomes. Arch Biochem Biophys 346:161–169[CrossRef][Medline]
  49. Cheng ZN, Shu Y, Liu ZQ, Wang LS, Ou-Yang DS, Zhou HH 2001 Role of cytochrome P450 in estradiol metabolism in vitro. Acta Pharmacol Sin 22:148–154[Medline]
  50. Sankaran L, Qasba P, Topper YJ 1984 Effects of estrogen-depletion on rat casein gene expression. Biochem Biophys Res Commun 125:682–689[CrossRef][Medline]
  51. Galteau MM, Shamsa F 2003 Urinary 6ß-hydroxycortisol: a validated test for evaluating drug induction or drug inhibition mediated through CYP3A in humans and in animals. Eur J Clin Pharmacol 59:713–733[CrossRef][Medline]
  52. Furuta T, Suzuki A, Mori C, Shibasaki H, Yokokawa A, Kasuya Y 2003 Evidence for the validity of cortisol 6ß-hydroxylation clearance as a new index for in vivo cytochrome P450 3A phenotyping in humans. Drug Metab Dispos 31:1283–1287[Abstract/Free Full Text]
  53. Banerjee MR, Terry PM, Sakai S, Lin FK, Ganguly R 1978 Hormonal regulation of casein messenger RNA (mRNA). In Vitro 14:128–139[Medline]
  54. Sakai S, Bowman PD, Yang J, McCormick K, Nandi S 1979 Glucocorticoid regulation of prolactin receptors on mammary cells in culture. Endocrinology 104:1447–1449[Abstract/Free Full Text]
  55. Zhou S, Gao Y, Jiang W, Huang M, Xu A, Paxton JW 2003 Interactions of herbs with cytochrome P450. Drug Metab Rev 35:35–98[CrossRef][Medline]
  56. Rebbeck TR, Jaffe JM, Walker AH, Wein AJ, Malkowicz SB 1998 Modification of clinical presentation of prostate tumors by a novel genetic variant in CYP3A4. J Natl Cancer Inst 90:1225–1229[Abstract/Free Full Text]
  57. Kadlubar FF, Berkowitz GS, Delongchamp RR, Wang C, Green BL, Tang G, Lamba J, Schuetz E, Wolff MS 2003 The CYP3A4*1B variant is related to the onset of puberty, a known risk factor for the development of breast cancer. Cancer Epidemiol Biomarkers Prev 12:327–331[Abstract/Free Full Text]
  58. Spurdle AB, Goodwin B, Hodgson E, Hopper JL, Chen X, Purdie DM, McCredie MR, Giles GG, Chenevix-Trench G, Liddle C 2002 The CYP3A4*1B polymorphism has no functional significance and is not associated with risk of breast or ovarian cancer. Pharmacogenetics 12:355–366[CrossRef][Medline]
  59. Zheng W, Jin F, Dunning LA, Shu XO, Dai Q, Wen WQ, Gao YT, Holtzman JL 2001 Epidemiological study of urinary 6ß-hydroxycortisol to cortisol ratios and breast cancer risk. Cancer Epidemiol Biomarkers Prev 10:237–242[Abstract/Free Full Text]
  60. Jernstrom H, Chu W, Vesprini D, Tao Y, Majeed N, Deal C, Pollak M, Narod SA 2001 Genetic factors related to racial variation in plasma levels of insulin-like growth factor-1: implications for premenopausal breast cancer risk. Mol Genet Metab 72:144–154[CrossRef][Medline]
  61. Felix CA, Walker AH, Lange BJ, Williams TM, Winick NJ, Cheung NK, Lovett BD, Nowell PC, Blair IA, Rebbeck TR 1998 Association of CYP3A4 genotype with treatment-related leukemia. Proc Natl Acad Sci USA 95:13176–13181[Abstract/Free Full Text]
  62. Stoll BA 1999 Western nutrition and the insulin resistance syndrome: a link to breast cancer. Eur J Clin Nutr 53:83–87[CrossRef][Medline]
  63. Liao DJ, Dickson RB 2002 Roles of androgens in the development, growth, and carcinogenesis of the mammary gland. J Steroid Biochem Mol Biol 80:175–189[CrossRef][Medline]
  64. Khosla S, Melton 3rd LJ, Riggs BL 2002 Estrogen and the male skeleton. J Clin Endocrinol Metab 87:1443–1450[Abstract/Free Full Text]
  65. Cutler Jr GB 1997 The role of estrogen in bone growth and maturation during childhood and adolescence. J Steroid Biochem Mol Biol 61:141–144[CrossRef][Medline]
  66. Simpson ER 1998 Genetic mutations resulting in estrogen insufficiency in the male. Mol Cell Endocrinol 145:55–59[CrossRef][Medline]
  67. Kumar S, Clarke AR, Hooper ML, Horne DS, Law AJ, Leaver J, Springbett A, Stevenson E, Simons JP 1994 Milk composition and lactation of ß-casein-deficient mice. Proc Natl Acad Sci USA 91:6138–6142[Abstract/Free Full Text]
  68. Hines RN, McCarver DG 2002 The ontogeny of human drug-metabolizing enzymes: phase I oxidative enzymes. J Pharmacol Exp Ther 300:355–360[Abstract/Free Full Text]
  69. Gandhi M, Aweeka F, Greenblatt RM, Blaschke TF 2004 Sex differences in pharmacokinetics and pharmacodynamics. Annu Rev Pharmacol Toxicol 44:499–523[CrossRef][Medline]
  70. McLean AJ, Le Couteur DG 2004 Aging biology and geriatric clinical pharmacology. Pharmacol Rev 56:163–184[Abstract/Free Full Text]
  71. Parkinson A, Mudra DR, Johnson C, Dwyer A, Carroll KM 2004 The effects of gender, age, ethnicity, and liver cirrhosis on cytochrome P450 enzyme activity in human liver microsomes and inducibility in cultured human hepatocytes. Toxicol Appl Pharmacol 199:193–209[CrossRef][Medline]
  72. Wolbold R, Klein K, Burk O, Nussler AK, Neuhaus P, Eichelbaum M, Schwab M, Zanger UM 2003 Sex is a major determinant of CYP3A4 expression in human liver. Hepatology 38:978–988[CrossRef][Medline]
  73. Dussault I, Yoo HD, Lin M, Wang E, Fan M, Batta AK, Salen G, Erickson SK, Forman BM 2003 Identification of an endogenous ligand that activates pregnane X receptor-mediated sterol clearance. Proc Natl Acad Sci USA 100:833–838[Abstract/Free Full Text]
  74. Goodwin B, Gauthier KC, Umetani M, Watson MA, Lochansky MI, Collins JL, Leitersdorf E, Mangelsdorf DJ, Kliewer SA, Repa JJ 2003 Identification of bile acid precursors as endogenous ligands for the nuclear xenobiotic pregnane X receptor. Proc Natl Acad Sci USA 100:223–228[Abstract/Free Full Text]
  75. Liddle C, Goodwin BJ, George J, Tapner M, Farrell GC 1998 Separate and interactive regulation of cytochrome P450 3A4 by triiodothyronine, dexamethasone, and growth hormone in cultured hepatocytes. J Clin Endocrinol Metab 83:2411–2416[Abstract/Free Full Text]
  76. Choi HK, Waxman DJ 1999 Growth hormone, but not prolactin, maintains, low-level activation of STAT5a and STAT5b in female rat liver. Endocrinology 140:5126–5135[Abstract/Free Full Text]



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M. A. Felmlee, H.-K. Lon, F. J. Gonzalez, and A.-M. Yu
Cytochrome P450 Expression and Regulation in CYP3A4/CYP2D6 Double Transgenic Humanized Mice
Drug Metab. Dispos., February 1, 2008; 36(2): 435 - 441.
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 Mol. Endocrinol.Home page
D. J. Waxman and C. O'Connor
Growth Hormone Regulation of Sex-Dependent Liver Gene Expression
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M. G. Holloway, E. V. Laz, and D. J. Waxman
Codependence of Growth Hormone-Responsive, Sexually Dimorphic Hepatic Gene Expression on Signal Transducer and Activator of Transcription 5b and Hepatic Nuclear Factor 4{alpha}
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