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Endocrinology Vol. 140, No. 4 1672-1680
Copyright © 1999 by The Endocrine Society

ARTICLES

Developmental Expression and Regulation of Adrenocortical Cytochrome P4501B1 in the Rat1

Paul B. Brake2, Maya Arai3, Suzie As-Sanie, Colin R. Jefcoate and Eric P. Widmaier

Center for Environmental Toxicology and Department of Pharmacology, University of Wisconsin Medical School (P.B.B., C.R.J.), Madison, Wisconsin 53706; and the Department of Biology, Boston University (M.A., S.A.-S., E.P.W.), Boston, Massachusetts 02215

Address all correspondence and requests for reprints to: Dr. Eric P. Widmaier, Department of Biology, Boston University, 5 Cummington Street, Boston, Massachusetts 02215. E-mail: widmaier{at}bio.bu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A 57-kDa protein whose expression in rat adrenocortical microsomes is increased after weaning has been identified as cytochrome P4501B1 (CYP1B1). Levels of CYP1B1 protein were moderately expressed in late gestation fetuses and on postnatal day 1 (pd1), but were nearly undetectable on pd6 and pd10. CYP1B1 expression initially increased in the late preweaning period (pd17–19) and again immediately postweaning (pd21–24). The temporal coincidence of CYP1B1 expression and weaning was not due to transition from suckling to solid food, as neonates that were prematurely weaned showed no increase in adrenal CYP1B1 compared with normally weaned littermates. The pattern of CYP1B1 expression paralleled changes in microsomal metabolism of 7,12-dimethylbenz[a]anthracene (DMBA), a marker of CYP1B1 activity. Twice daily injections of ACTH to rat pups (pd3–10) failed to significantly increase the expression of CYP1B1 in pd10 adrenals, although the injections weakly stimulated steroidogenesis. Adrenocortical cells from pd17 neonates and adult cells, when cultured for 3 days, responded similarly to ACTH induction, although neonates showed more than 4-fold less basal activity. It is concluded that rat adrenal CYP1B1 may be developmentally suppressed, and its expression is independent of diet or the presence of a dam. This suppression is retained in cell culture, but is not due to deficient ACTH signaling. These results may explain the reported resistance of neonatal rat adrenals to the toxic effects of polycyclic aromatic hydrocarbons, which are metabolized by CYP1B1 into mutagenic by-products.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DEVELOPMENT of adrenocortical function is an absolute requirement for adaptation to ex utero life in mammals. Adrenal steroids participate in numerous metabolic and differentiative processes, including those within the developing central nervous system (1, 2, 3). Outside the central nervous system, glucocorticoids are required for adequate pulmonary development (i.e. surfactant production) before the first encounter with an air/liquid interface in the lung (4, 5) as well as numerous other developmental events.

The maturational process of the adrenal is complex and triphasic in certain mammalian species, including the rat (6). For example, in the fetal sheep (and possibly the fetal calf) adrenal responsiveness to its major trophic factor, ACTH, is relatively normal early in gestation, then subsides during the midgestational period. Finally, near term, adrenal gland responsiveness to ACTH is fully restored (7, 8, 9). A similar pattern is observed in the rat, only in this species the process is shifted to late gestation, neonatal life, and weaning (1, 6, 10, 11). The insensitivity of the neonatal rat adrenal is in some ways analogous to the hyporesponsive fetal adrenal of larger mammals, making the neonatal rat a useful model for studying the maturation of adrenal responsiveness.

Pituitary and hypothalamic activities during the neonatal period do not vary as much as adrenocortical activity (12). For instance, basal levels of plasma ACTH and secretion of CRH in vitro are not significantly different between the adult and neonate (13). The biochemical nature of adrenal hyporesponsiveness to ACTH and other stimuli that persists for most of the first 3 postnatal weeks [excluding postnatal day 1 (pd1)] in rats is uncertain, but is related in part to the developmental expression of microsomal steroidogenic enzymes (14).

In the course of analyzing adrenocortical microsomes for steroidogenic enzyme activities and contents, it was observed that the expression of microsomal cytochrome P4501B1 (CYP1B1) varied with age in rat adrenal glands in a manner similar to changes in ACTH-inducible steroidogenesis. This report documents the developmental profile and regulation of this protein in the adrenal cortex in vivo and in cultured cells. Furthermore, it compares expression of the protein with the ability of neonatal adrenocortical microsomes to metabolize 7,12-dimethylbenz[a]anthracene (DMBA). The polycyclic aromatic hydrocarbon DMBA is an exogenous substrate for CYP1B1 (15).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
DMBA, 8-bromo-cAMP (8-Br-cAMP), cortisone, corticosterone, deoxyribonuclease II, and dimethylsulfoxide were purchased from Sigma Chemical Co. (St. Louis, MO). Cortrosyn [ACTH-(1–24)] was purchased from Organon, Inc. (West Orange, NJ). Collagenase A was obtained from Boehringer Mannheim (Indianapolis, IN). DMEM-Ham’s F-12 (DMEM:F12; 1:1) was obtained from Life Technologies (Grand Island, NY), and donor horse and fetal bovine sera were obtained from Gemini Bioproducts, Inc. (Calabasas, CA). Solvents for HPLC analyses and tissue culture plates (Falcon) were purchased from Fisher Scientific International, Inc. (Itasca, IL).

Animals and tissues
All animals and animal tissues used in these studies were maintained at and purchased from Harlan Bioproducts for Science, Inc. (Madison, WI). Adult Sprague-Dawley rats (~250 g) were used for studies of in vivo expression of adrenocortical CYPs. Fresh adrenal glands from female Sprague-Dawley rats (~150 g) and rat pups (either sex), pd17, were used for all rat adrenocortical cell preparations. Mixed litters of male and female Sprague-Dawley rat pups were used in the studies concerning the developmental expression of CYP1B1 and the preweaning studies. Adrenal glands were isolated from fetal rats [gestational days (gd) 18 and 20] that were collected from timed pregnant female Sprague-Dawley rats. In those experiments, pregnant females were anesthetized with sodium pentobarbitol, and the fetuses were removed within 30 min. The number of fetuses pooled for gd 18 and gd 20 were 136 and 102, respectively.

Animal treatments
In studies measuring the effect of ACTH on CYP1B1 expression in adult and neonatal rat adrenal glands, neonatal Sprague-Dawley pups from 15 and 11 (2 separate experiments) random litters were injected ip twice per day (1000 and 1800 h; lights on, 0700–1900 h) with 100 µl saline or 10 µg/kg BW porcine ACTH-(1–39) as previously described (16). Injections began on pd3 and continued through pd10. This procedure has been previously documented by one of us (16) to elevate plasma ACTH to high physiological (i.e. stress-induced) levels for 2 h after each injection. Moreover, the daily injections accelerate adrenocortical steroidogenic development and responsiveness to ACTH (16). Thirty minutes after a final injection on the morning of pd10, rats were killed by decapitation. Trunk blood from a random subset of animals from each group was collected into heparinized tubes for determination of corticosterone levels by RIA (ICN Biomedicals, Inc., Costa Mesa, CA), and adrenals were removed and processed for isolation of microsomal protein. Adrenals from approximately 40 pups/treatment group·experiment were pooled for microsomal protein.

In studies measuring dietary and maternal effects on expression of CYPs in the neonatal adrenal, neonatal rat pups were raised normally until pd17, at which time 2 litters (12 pups/litter) were removed from their dams and placed on semisolid food (crushed standard rat chow moistened with water) and water (preweaned); 2 additional litters remained with the dams under normal conditions. On pd17, an additional group of suckling pups randomly selected from 5 litters was killed, and adrenal glands were removed and immediately processed for microsomal protein to be used as controls. The remaining suckling or preweaned pups were killed on pd19 and pd21, and adrenal glands were removed and immediately processed for preparation of microsomal protein. This experiment was also repeated a second time and extended by sacrificing two random pups from suckling or preweaned litters at pd19, -21, -24, and -27. The raising of neonatal rat pups and isolation of adrenal glands in the second experiment was performed at Harlan Bioproducts for Science, Inc. Raising of pups and other procedures for the first experiment were performed at Boston University (Boston, MA). All procedures were approved by the Boston University Institute animal care and use committee.

Preparation of microsomal protein from tissue and cell sources
Microsomal protein was prepared from tissue samples as previously described (14) or with modifications as follows, with all steps performed on ice or at 4 C. Tissues were washed once with homogenization buffer [0.1 M KHPO4 (pH 7.25), 150 mM KCl, 10 mM EDTA, 0.25 mM phenylmethylsulfonylfluoride (PMSF), and 0.1 mM dithiothreitol (DTT)] and resuspended in 2–3 vol homogenization buffer. Samples were then homogenized twice (Tissuemizer, Tekmar, Cincinnati, OH) at 6,000 rpm for 10 sec. The homogenate was centrifuged at 15,000 x g for 20 min, and the postmitochondrial supernate was collected. This supernate was then centrifuged at 105,000 x g for 90 min. The resulting cytosolic fraction (supernatant) was collected for later use, and the microsomal pellet was resuspended in wash buffer (0.1 M KPO4, 10 mM EDTA, 0.25 mM PMSF, and 0.1 mM DTT) and centrifuged at 105,000 x g for 60 min. After washing, which lyses heme-containing contaminating red blood cells, the microsomal pellet was resuspended in 2–3 vol dilution buffer [0.1 M KHPO4 (pH 7.25), 10 mM EDTA, 0.25 mM PMSF, 0.1 mM DTT, and 20% glycerol] and kept at -70 C until further use.

Preparation of rat adrenocortical cells and cell culture
Adrenocortical cells were prepared as previously described (17) with minor modifications. Briefly, 40–50 adrenal glands were decapsulated, minced, and placed in 25 ml DMEM:F12 (2 mM HEPES, pH 7.2) containing 2.5 mg/ml collagenase A and 0.1 mg/ml deoxyribonuclease II. Tissue suspensions were then incubated for 30 min in a shaking water bath at 37 C. Dissociated cells were collected and placed into separate sterile polyethylene tubes. Undissociated tissues were washed with 15 ml DMEM:F12 (2 mM HEPES, pH 7.2) to release adherent cells, which were pooled with their respective first cell fractions. The remaining tissues were then subjected to another round of collagenase treatment and washing. Resulting cell suspensions were centrifuged at 500 x g for 5 min. Cell pellets were resuspended and washed in fresh cell medium (DMEM:F12, pH 7.2, supplemented with 10% donor horse serum, 2.5% FBS, 1 x MEM amino acids, 1 x MEM vitamins, 1 µM vitamin E, 100 µM vitamin C, and 50 nM selenium), centrifuged at 500 x g for 5 min, and repeated. Cells were counted with a hemocytometer (American Optical, Buffalo, NY), plated at a cell density of approximately 1 x 105 cells/cm2, and maintained in a humidified atmosphere of 5% CO2-95% air at 37 C. This preparation results in predominantly zonae fasciculata cells with readily visible vacuoles consistent with the appearance of lipid droplets. As rat adrenocortical cells do not proliferate in culture (17), experiments were performed on cell monolayers after 2–3 days in culture. For cell suspension assays, isolated RAC cells were placed in conditioned cell medium (incubated for 24 h in humidified atmosphere of 5% CO2-95% air at 37 C) at a density of approximately 1 x 105 cells/ml and placed in a shaking water bath at 37 C for the specified times.

In vitro DMBA metabolism assay and steroid analysis
RAC cells grown in 30-cm2 plates and treated with 100 nM ACTH, 500 µM 8-Br-cAMP, or saline (control) for 24 h were incubated with medium containing 5 µM DMBA for 1 h. At the end of the incubation period, the medium was removed, placed into individual 10-ml glass borosilicate tubes, and treated with ß-glucuronidase solution [2000 IU ß-glucuronidase/ml, 0.5 M sodium acetate (pH 5.0), and 0.5 mg/ml ascorbate] for 4–5 h at 37 C to hydrolyze the polar glucuronidated DMBA metabolites. Cell number was determined for each sample by brief trypsinization of remaining cells at 37 C and counting on a hemocytometer. Cortisone, which is not produced by RAC cells, was added as an internal standard before DMBA metabolites were extracted with ethyl acetate/acetone containing DTT (2:1:0.003). The solvent phases containing the DMBA metabolites, the cortisone standard, and steroids produced by the cells (corticosterone) were removed, dried down under nitrogen gas, and resuspended in 100 µl methanol for HPLC analysis. Separation of DMBA metabolites by C18 reverse phase HPLC and quantitation relative to cortisone were carried out as previously described (18, 19). Corticosterone was detected in the same elutions by UV absorption and was quantitated with corticosterone standards as described previously (20).

Western immunoblot analysis
Microsomal proteins were prepared for immunoblot analysis by suspension in sample loading buffer, heated at 100 C for 5 min, and separated by SDS-PAGE (8% acrylamide). After separation, the proteins were transferred to nitrocellulose membranes (Schleicher & Schuell, Inc., Keene, NH) and blocked in Tris-buffered saline/Tween-20 (TBST) containing 5% milk overnight at 4 C (or for 1 h at room temperature). The membranes were washed in TBST for 20 min before addition of the primary antibodies. Antibodies used in these studies include affinity-purified polyclonal antibodies to rat CYP1B1 (15), recombinant mouse CYP1B1 (21), and CYP21 (Otto, S., and C. Jefcoate, unpublished). After incubation with primary antibodies, the membranes were washed with TBST for 20 min, then incubated with, antirabbit secondary antibody with horseradish peroxidase (Promega Corp., Madison, WI). After washing the membranes, immunoreactive proteins were visualized by the enhanced chemiluminescence method (Amersham, San Diego, CA) according to the manufacturer’s instructions.

Analytical methods
Quantitation and densitometry of the immunoblots were performed using a Zeineh soft laser scanning densitometer (model SL-504-XL, Bio-Medical Instruments, Inc., Fullerton, CA) and by analysis of electronically scanned images on a Power Macintosh 6100/60 using the public domain NIH Image (version 1.56) program (written by Wayne Rasband at the NIH and available from the internet by anonymous FTP from zippy.nimh.nih.gov).

Statistics
For comparison among several groups, statistical analysis of results was carried out using one-way ANOVA and Student’s t test where appropriate. Significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CYP1B1 expression in adrenocortical microsomes
Previous work has shown that Western immunoblot analysis of rat adrenocortical microsomal membranes with antibodies raised against rat CYP1B1 reveal a single immunoreactive band corresponding to 57 kDa (15). Here we show that the level of CYP1B1 is age dependent and corresponds to a major band that exhibited the same age-dependent expression in Coomassie-stained adrenocortical microsomes (not shown). CYP1B1 displayed a pronounced triphasic pattern of expression in the adrenal gland of the developing rat (Fig. 1AGo). Immunodetectable levels of CYP1B1 increased approximately 5-fold from gd18 to gd20 and by a similar factor to pd1 (Fig. 1BGo). CYP1B1 levels declined to nearly undetectable levels from pd6 to pd10 and began to rise by pd21, reaching adult levels by pd31. A strongly expressed steroidogenic P450 cytochrome, CYP21, did not follow this developmental profile, and its expression was only slightly altered during this period (Fig. 1AGo).



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  		Figure 1. CYP1B1 protein displays a triphasic pattern of expression in adrenocortical microsomes from fetal and neonatal rats. Microsomal protein isolated from adrenal glands of neonatal (A) and both fetal and neonatal (B) rats was analyzed for CYP1B1 and CYP21 by Western immunoblot. Each microsomal preparation was from pools of approximately 10 adult (male) to approximately 100 fetal or neonatal (both male and female) animals. Loadings: A, adult (Adt), 1 µg; pd1–31, 1 µg; B, gd18 and gd20, 10 µg; pd1–10, 10 µg.

 
Effect of preweaning on expression of CYP1B1
The temporal coincidence of weaning and the increased expression of immunoreactive and bioactive CYP1B1 prompted us to test the hypothesis that a suckling diet and/or the presence of the dam were responsible for suppression of this protein. All litters of rat pups were raised with dams until pd17. On this day, one group of pups was removed from their dams and placed on a semisolid chow diet (preweaned); the other group remained with the dams until being weaned at 21 days of age (normal weaning). Adrenals were removed from pups in both groups on pd19, -21, -24, and -27 and compared with those obtained on pd17 for expression of microsomal CYP1B1 (Fig. 2Go). Levels of immunodetectable CYP1B1 were increased more than 6-fold from pd17 to pd19 and pd21 and about 15-fold from pd21 to pd24 and pd27; however, preweaning the pups did not increase the expression of adrenocortical CYP1B1 on pd19 through pd27 compared with that in pups left with the dam for normal weaning on pd21. The temporal profile of CYP1B1 expression is summarized in Fig. 2CGo.



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  		Figure 2. Effect of preweaning on expression of CYP1B1 protein in neonatal rat adrenals. Neonatal rat pups were removed from the dam at 17 days of age (preweaned, +) or were left with the dam (normal weaning, -). On the days indicated, adrenal glands were removed, and microsomal protein was isolated and prepared for Western immunoblot analysis for CYP1B1 (A and B). Protein loadings were as follows: A, adult untreated (Adt), 0.1 µg; pd, preweaned or normal weaning, 2.5 µg; and B, pd, preweaned or normal weaning, 1.0 µg. C, Densitometric summary of changes in CYP1B1 expression as a function of gestational (g; solid bar) and postnatal (p; stippled bar) age (data from Figs. 1Go and 2Go, combined). A, Adult rats of approximately 70–80 days of age.

 
DMBA metabolism
The ability of neonatal rat adrenal microsomes to metabolize DMBA, a marker of CYP1B1 activity (19, 22), paralleled the developmental profile of CYP1B1 protein (Table 1Go). DMBA activity was proportional to CYP1B1 protein levels (r2 = 0.72; Fig. 3Go) over the limited subset of ages examined. Regioselectivity of DMBA metabolism was similar between adult and neonatal (pd1) adrenal microsomes (Fig. 4Go) and was similar, but not identical, to metabolic profiles catalyzed by recombinant mouse CYP1B1 protein, where CYP1B1 specifically catalyzes the formation of 10,11- and 3,4-dihydrodiols (21). Slight differences in the metabolic profiles observed with the recombinant protein (21) and whole cell assays (this study) may reflect differences in the in vitro milieu of the two preparations as well as differences in extraction efficiency in different media. The only difference between adult and neonatal cells was the slightly smaller production of the 3,4-dihydrodiol by neonatal adrenocortical microsomes compared with adult microsomes (P < 0.05). Therefore, we conclude that CYP1B1 is the species of P450 cytochrome detected in the neonatal adrenal and that it displays a triphasic pattern of expression in the adrenal gland of the developing rat.


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  		Table 1. CYP1B1 activity in fetal and neonatal adrenal microsomes as a function of age

 


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  		Figure 3. Correlation of neonatal adrenocortical CYP1B1 protein and activity in a subset of animals for which both determinations were available. Immunodetectable CYP1B1 from Fig. 1Go was quantified and contrasted against levels of CYP1B1 activity, as measured by DMBA metabolism (as picomoles of total DMBA per mg microsomal protein/h) from the corresponding age groups from Table 1Go.

 


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  		Figure 4. Comparison of regioselective DMBA metabolism between adult and day 1 neonatal rat adrenocortical microsomes. Regioselective DMBA metabolites (data not shown) were used to calculate the percentage of the total DMBA metabolism from Table 1Go. Phenol A (9-OH, 3-OH) and phenol B (4-OH) represent chemical reduction of diol metabolites of DMBA. Values represent the mean ± SEM of the percentage of total DMBA metabolites from triplicate analyses (n = 3). The number of animals varied depending on the age group, from 2–3 adult animals to 10 or more neonatal animals.

 
Responsiveness of CYP1B1 to ACTH in neonatal rats
The triphasic pattern of CYP1B1 expression is similar to the developmental pattern of adrenocortical steroidogenic response to ACTH. Therefore, we tested whether suppression of CYP1B1 was due to a lack of stimulation by ACTH in the pup. When rat pups were pretreated with twice daily injections of ACTH from pd3–10, the adrenocortical response to a final ACTH injection, as measured by corticosterone production, was moderately, but significantly, potentiated 2.6-fold (P < 0.05) relative to that in saline-pretreated controls (Table 2Go), although remaining much lower than the 10- to 20-fold increases typically observed in adults (23). However, ACTH treatment completely failed to stimulate the expression of immunodetectable CYP1B1 in neonates (Fig. 5AGo). Treatment of adult animals with the same dosage of ACTH resulted in a moderate increase (2-fold) in adrenal CYP1B1 (Fig. 5BGo), consistent with previous results (15).


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  		Table 2. Plasma corticosterone levels in day 10 neonatal rats after ACTH treatments

 


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  		Figure 5. Effect of ACTH on CYP1B1 expression in neonatal rats. Neonatal rats were injected with saline (Sal) or ACTH twice daily from pd3–10. Thirty minutes after the final injections on pd10, microsomal protein was isolated from the adrenal glands of approximately 40 pups/treatment group and prepared for immunoblot analysis of CYP1B1 (A). The process was repeated in a separate experiment with an additional approximately 40 pups/group from different litters. Thus, each lane in A represents a separate experiment within a treatment (saline or ACTH). An aliquot of microsomal protein from unstimulated adults (Adt) is included for comparison. In a third experiment, 1 litter of neonatal animals and 2 adults were similarly injected with saline or ACTH, and microsomal protein was isolated from adrenal glands and prepared for Western immunoblot analysis of CYP1B1 (B). Protein loadings: A, pd10, saline and ACTH treated, 10 µg; adult untreated (Adt), 1 µg; B, pd10 and adult (Adt), saline and ACTH treated, 1 µg.

 
Responsiveness of CYP1B1 to ACTH in adrenocortical cells
The diminished responsiveness of CYP1B1 to ACTH in vivo may be due to a developmental suppression inherent in RAC cells that express CYP1B1. Similarly, diminished responsiveness of CYP1B1 to ACTH may be due to loss or suppression of the signal transduction pathway of ACTH, which is well known to be primarily mediated by cAMP. These cells are responsive to ACTH stimulation when isolated from adult rat adrenals, and levels of CYP1B1 protein are elevated 4-fold by ACTH via cAMP (24). Responsiveness of CYP1B1 to hormonal stimulation in neonatal RAC cells via cAMP was tested to determine whether the lack of responsiveness of CYP1B1 to ACTH was at the level of cAMP transduction. Neonatal (pd17) RAC cells responded to both ACTH and 8-Br-cAMP treatment by increased corticosterone production (Table 3Go). CYP1B1-mediated DMBA metabolism was stimulated more than 3-fold after both ACTH and 8-Br-cAMP treatment (Table 3Go). We conclude that diminished responsiveness of CYP1B1 to ACTH in neonatal adrenals in vivo is not due to cellular insensitivity to ACTH and cAMP.


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  		Table 3. Neonatal RAC cells respond to hormonal stimulation with increased corticosterone synthesis and CYP1B1-mediated DMBA metabolism

 
Responsiveness to ACTH was then compared in RAC cells isolated from adult and pd17 rat pups. RAC cells were maintained for 2–3 days in culture and then stimulated with 10-7 M ACTH for 24 h. Although CYP1B1-mediated DMBA metabolism was significantly increased 2- to 3-fold (P < 0.05) by ACTH in both neonatal and adult RAC cells (Table 4Go), basal activities were 5-fold lower in the neonatal RAC cells. By contrast, although adult and neonatal RAC cells exhibited equal basal levels of corticosterone production, ACTH responsiveness remained low in neonatal RAC cells, with an 8-fold increase in corticosterone production compared with the more than 40-fold increase seen in adult RAC cells (Table 4Go).


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  		Table 4. Responsiveness of neonatal and adult RAC cells to hormonal stimulation of CYP1B1 activity and corticosterone production

 
Stimulation by ACTH immediately after isolation provides the means to assess the response to ACTH without the opportunity for the cells to adapt to culture conditions. In suspension cultures where there is no serum, RAC cells from adult and neonatal animals were tested, in turn, for their direct response to ACTH stimulation. Adult and neonatal RAC cells in suspension did not exhibit significant increases in CYP1B1 activity after 6 h of ACTH treatment (Table 4Go). However, as seen with the cells cultured for 3 days, the adult RAC cells in suspension expressed significantly (P < 0.05) higher basal levels of activity compared with the neonatal RAC cells, although adult RAC cells display a decrease in DMBA metabolism when placed in culture (Table 4Go). Neonatal RAC cells appear to recover some responsiveness to ACTH when placed in culture for a few days as corticosterone production is higher basally in cultured cells compared with cells in suspension (Table 4Go). Therefore, it would appear that isolated neonatal RAC cells behave in a similar manner as adrenocortical cells in vivo, with diminished steroidogenesis and CYP1B1 responsiveness to ACTH.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The 57-kDa CYP1B1 protein is shown to undergo dramatic developmental changes in expression in the rat adrenal cortex. This P450 cytochrome is under hormonal control in the adrenals and gonads of rats and is the major polycyclic aromatic hydrocarbon-metabolizing species in these steroidogenic tissues (15, 25). Decreased expression of CYP1B1 in neonatal rats corresponds to a period of steroidogenic hyporesponsiveness in the developing adrenal gland. CYP1B1 appears to be even more tightly restricted in this period by a developmental program.

The biochemical nature of the hyporesponsiveness of steroidogenesis in the developing adrenal to ACTH appears to be partly related to the developmental expression of one steroidogenic enzyme, 3ß-hydroxysteroid dehydrogenase (7, 14, 26) and possibly to changes in ACTH receptor activity (27). Although the expression of immunoreactive 3ß-hydro-xysteroid dehydrogenase roughly parallels that of ACTH-inducible steroidogenesis, activities of this enzyme do not significantly increase between pd10 and adulthood (14), unlike the activity of CYP1B1. The expression of several steroidogenic P450 cytochromes during this period of adrenal development has also been studied; the findings show that CYP11A1, CYP11B1, and CYP21 do not display this ontogeny of expression (Ref. 14 and this study). Recently, it was determined that expression of the peripheral-type mitochondrial benzodiazepine receptor during the neonatal period in rats is greatly reduced compared with that in adults, suggesting that ACTH hyporesponsiveness may also reflect reduced cholesterol delivery to mitochondrial enzymes (23).

Thus, it would appear that CYP1B1 is the only adrenal CYP that displays this triphasic pattern of both its expression and its activity in the postnatal developing rat adrenal. This suggests a developmental function for CYP1B1 and raises the interesting possibility that CYP1B1 may be another factor involved in the maturation of adrenal responsiveness in the developing neonatal rat. Interestingly, recent work shows that CYP1B1 deficiency has been linked to abnormal fetal development of the human eye (28) and to aberrant differentiation of mouse embryo fibroblasts (Alexander, D., L. Granem, and C. R. Jefcoate, unpublished observations). The coincidence of the increased expression of CYP1B1 and the development of maximal responsiveness of rat adrenal glands to ACTH in vitro could argue for a role of the protein in the developmental regulation of steroidogenic activity.

The process of adrenocortical maturation in mammals has been intensively studied, in large part because of the clinical importance of glucocorticoids for the developing fetus (1, 2, 3, 4, 5). It is clear that ACTH and possibly glucocorticoids themselves are important maturational cues for the mammalian adrenal gland (7, 8, 16, 29, 30, 31, 32). However, ACTH treatment during the first postnatal week did not induce an appreciable increase in the level of immunodetectable CYP1B1 in day 10 neonates. The dose of ACTH used in these studies is sufficient to elevate plasma ACTH to the high physiological concentrations that are achieved in the plasma of suckling pups (16, 23, 33). The effectiveness of the injection procedure and the ACTH preparations was verified by the moderate steroidogenic response to ACTH seen in pd10 pups, consistent with earlier observations (16). Thus, even though these glands respond (albeit weakly) to ACTH, it appears unlikely that this is a major regulatory molecule for CYP1B1 expression in the neonatal adrenal gland, as ACTH did not significantly elevate expression of the protein even after 7 days of exposure to the peptide. Nonetheless, it is possible that the developmental period determines the responsiveness of the adrenal cortex to ACTH with respect to induction of CYP1B1. ACTH responsiveness may commence later than the first 10 postnatal days. Nevertheless, this still implies that a developmental program determines the expression of this enzyme.

ACTH regulates CYP1B1 in the adult adrenal in vivo (15) and in vitro via cAMP (24). The hyporesponsiveness of the adrenal during the development of neonatal adrenal gland function may be due to the loss or suppression of some part of the ACTH signal transduction pathway, such as the cAMP pathway. However, 2- to 3-day cultures of neonatal RAC cells (pd17) responded to cAMP and ACTH stimulation equally with increased CYP1B1 activity. Cultured neonatal RAC cells are also still significantly suppressed in their steroidogenic response to ACTH and reflect the in vivo phenotype in their responsiveness. The suppression of CYP1B1 was also maintained in freshly isolated suspensions of neonatal RAC cells where the possibility of chronic adaptation or differentiation in vitro is excluded. Neonatal RAC cells contained lowered CYP1B1 activity compared with adult RAC cells. As with 3-day cultures, steroidogenesis was only slightly increased by ACTH in these cells, with a response that was about 15-fold less than that of similarly treated adult RAC cells. This indicates that hyporesponsiveness to ACTH with respect to steroidogenesis in adrenocortical cells during this developmental period is inherent to the phenotype of the cell. By contrast, the difference between neonatal and adult CYP1B1 expression does not arise from a difference in cAMP or ACTH inducibility, but, rather, to differences in basal activity. This raises the possibility that other factors determined by the developmental program regulate CYP1B1 expression in the neonatal adrenal. The selectivity of this CYP1B1 program further suggests that this gene may play a role in adrenal regulation in the developmental period.

Weaning produces a major change in the nutrition of the pup as maternal milk is replaced by lab chow. We postulated that factors present in maternal milk may act as suppressors of CYP1B1 expression, given the temporal relationship between weaning and increased enzyme expression. However, neonates that were weaned and removed from the dam on pd17 showed no difference in adrenal CYP1B1 protein over 4 days from the levels in their littermates weaned on pd21. The two-step increase between pd17–19 and after pd21 takes place regardless of the time of weaning. Thus, it would appear that the mechanisms that exist in the neonatal rat adrenal to reverse the suppressed expression of CYP1B1 during this stage of development are not affected by weaning, but, rather, are determined by an age-dependent set of developmental signals.

Although glucocorticoids have been shown to suppress constitutive and polycyclic aromatic hydrocarbon-inducible CYP1B1 in rat mammary fibroblasts (34, 35), it is unlikely that they contribute to the pattern of CYP1B1 expression observed in the present study. Plasma corticosterone levels in the developing rat follow a pattern similar to that of CYP1B1, but with salient differences. The fetal rat adrenal begins producing glucocorticoids by gestational day 16, and corticosterone levels are significantly higher at birth than at subsequent ages (32). Shortly after birth, corticosterone levels drop to very low levels and stay at these levels until about pd14, when the adrenal begins to regain responsiveness and starts producing glucocorticoids at a higher rate. By pd20, plasma corticosterone levels approach those seen in the adult rat. As noted above, CYP1B1 begins a final surge of expression only after pd20.

Metabolism of polycyclic aromatic hydrocarbons results in the generation of more potent mutagenic compounds, notably dihydrodiol epoxides (36). CYP1B1 is particularly effective in producing the most toxic metabolites of DMBA, the 3,4-dihydrodiol-1,2 epoxide (21). Thus, adrenal glands expressing CYP1B1 are likely to be susceptible to the toxic effects of DMBA and other polycyclics. For example, the ability of rat adrenal microsomes to metabolize DMBA is nearly absent in 10- to 15-day-old neonatal rats (37), suggesting probable resistance to DMBA toxicity, but by pd30 the rate of adrenal DMBA metabolism approaches adult levels, and the adrenal glands of these animals are then fully sensitive to the toxic effect of polycyclic aromatic hydrocarbons (38, 39). Moreover, DMBA metabolism and adrenal toxicity are increased after repeated injections of pharmacological doses of ACTH during the neonatal period (39). Each of these observations can now be explained by the developmental pattern of CYP1B1 expression. However, the mechanism of adrenal toxicity is far from clear, as cultured adult rat adrenal cortex cells with high levels of CYP1B1 and DMBA metabolism are fully resistant to toxicity (24). One possibility is that the adjacent endothelial cells that support the rich vascularization of the adrenal are the primary site of toxicity.

In summary, a developmentally sensitive integral microsomal protein of 57 kDa has been identified in neonatal rat adrenal glands as CYP1B1. Expression of CYP1B1 protein and enzymatic activity are correlated with age and do not reach adult-like levels until sometime after weaning, a time that roughly coincides with the advent of mature steroidogenesis. Although no role of CYP1B1 in the regulation of steroidogenesis in the rat adrenal has been reported, its coincident expression with steroidogenic responses to ACTH suggests that it could conceivably function in that capacity in addition to its effects on xenobiotic metabolism. Nonetheless, ACTH-induced steroidogenesis and CYP1B1 activity are clearly dissociable, as physiological levels of ACTH slightly increased steroid production in vivo without increasing CYP1B1 expression, and ACTH induced CYP1B1, but not steroidogenesis, to similar extents in cultured adult and neonatal adrenal cells. Regardless of whether CYP1B1 is ultimately found to have a role in neonatal steroidogenesis, the clear developmental pattern of expression of CYP1B1 in the rat should prove valuable in furthering our understanding of the physiology and pathophysiology of this protein in the intact animal.


   Acknowledgments
 
We thank Rene McCray at Harlan Bioproducts for Science, Inc. (Madison, WI), for maintaining and isolating the rat adrenal glands used for part of these studies.


   Footnotes
 
1 This work was supported by NIH Grant DK-41263 and NSF Grant IBN-9513926 (to E.P.W.), National Research Service Award T32-ES-07015 from the NIEHS (to P.B.B.), NIH Grant CA-16265 (to C.R.J.), and a grant from the Howard Hughes Medical Institute (Biology Education Program) to Boston University. Back

2 Present address: University of California, San Francisco, California 94143. Back

3 Present address: Massachusetts General Hospital, Boston, Massachusetts 02114. Back

Received June 15, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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