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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
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Abstract |
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Introduction |
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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).
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Materials and Methods |
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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.
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Results |
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). Immunodetectable levels of CYP1B1
increased approximately 5-fold from gd18 to gd20 and by a similar
factor to pd1 (Fig. 1B). 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. 1A).
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). 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. 2C.
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). DMBA
activity was proportional to CYP1B1 protein levels (r2
= 0.72; Fig. 3) over the limited subset
of ages examined. Regioselectivity of DMBA metabolism was similar
between adult and neonatal (pd1) adrenal microsomes (Fig. 4) 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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), 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. 5A). Treatment of adult animals with the
same dosage of ACTH resulted in a moderate increase (2-fold) in adrenal
CYP1B1 (Fig. 5B), consistent with previous results (15).
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). CYP1B1-mediated
DMBA metabolism was stimulated more than 3-fold after both ACTH and
8-Br-cAMP treatment (Table 3). 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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), 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 4).
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). 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 4). 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 4). 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 |
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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.
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Acknowledgments |
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Footnotes |
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2 Present address: University of California, San Francisco, California
94143.
3 Present address: Massachusetts General Hospital, Boston,
Massachusetts 02114.
Received June 15, 1998.
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) expression leads to
episodic fetal cortisol production. J Biol Chem 263:16195–16201This article has been cited by other articles:
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