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Introduction
Cytochrome P450 (CYP) isoforms are key enzymes responsible for the metabolism of xenobiotics and anabolism of steroid
hormones[1]. The human adult adrenals are considered as the main organs in steroid hormone synthesis, CYP isoforms
expressed in human adult adrenals have been demonstrated to provide these
functions[2]. The human fetal adrenal system is
one of the most important endocrine systems and is indispensable in sustaining biological homeostasis. In particular, steroid
hormones play a key role in reproductive systems and influence the growth, differen-tiation, and function of many target
cells. The majority of enzymes needed to produce steroid hormones, including progestins, androgens, estrogens,
glucocorticoids and mineralocorticoids, are members of the CYP
superfamily[3]. Previous research has demonstrated that
steroidogenesis-related CYP11A1, CYP17, CYP21, CYP11B1, and CYP11B2 were expressed in human fetal
adrenals[4]. Our previous study using RT-PCR technique demonstrated the expressions of CYP1A1, 2C8-19, and 3A7 mRNA in human fetal
adrenals[5]. However, few data are available concerning the correlations among xenobiotics, xenobiotic metabolizing-related CYP1, 2, 3
and steroidogenesis in human fetal adrenals during the fetal development with respect to possible toxicological significance.
In the present paper, we study the steroidogenesis and inducibility of CYP1, 2, and 3 isoforms by 3-methylcholanthrene,
phenobarbital, and dexamethasone in human fetal adrenal cells
in vitro, in order to explore the influence and possible
mechanism of xenobiotics on fetal adrenal steroidogenesis.
Materials and methods
Samples Human fetal specimens were obtained by therapeutic and legal abortion from second trimester tissues (20_24
weeks of gestation, females), approved by the Academic Committee and the Ethics Committee of Medical College of Wuhan
University.
Chemicals 3-Methylcholanthrene, phenobarbital, dexamethasone, bovine serum albumin (BSA), 7-ethoxy-resorufin,
isocitric acid, isocitric acid dehydrogenase, collagenase I and dimethyl sulfoxide were purchased from
Sigma-Aldrich Corporation ( St Louis, Missouri). All other chemicals and reagents were of
Acceptance Requirement grade.
Cell preparation, culture and treatment
Primary human fetal adrenal cortical cells, consisting mainly (90%_95%) of fetal
zone cells, were prepared and cultured as described in a previous
study[6,7]. The gland was decapsulated to remove most of
the definitive zone, and the remaining fetal zone was minced and digested with 0.4 mg/mL collagenase I in
phosphate-buffered saline enriched 10% fetal calf serum (ES Cell qualified,
Gibco Company, Los Angeles, USA) at 37 ºC for 15 min.
Nondigested tissues were allowed to settle, the supernatants were diluted with modified McCoy¡¯s 5A medium
(Gibco Company, Los Angeles, USA), which contained 10% fetal calf serum and penicillin/streptomycin. The mixtures were centrifuged at
67×g for 5 min at 4 ºC to pellet the cells. The pelleted cells were suspended in medium and repelleted as described above.
The cells were plated at a density of approximately
5×106 cells/mL on 12-well plates and incubated overnight in a
humidified atmosphere of 5% CO2 at 25 ºC or cell attachment. Three replicates were used. After 48 h, the medium was renewed
(2 mL/well), the administration was carried out with the agents (3-methylcholanthrene 0_2 µmol/L in 1%
Me2SO, phenobarbital 0_1 mmol/L in
H2O or dexamethasone 0_100 µmol/L in
H2O) for a 24 h period. The medium were removed and kept frozen
at -30 ºC until assayed for the steroids. The fetal zone cells were rinsed twice with pre-cold phosphate-buffered solution.
Pre-cold TEN buffer (pH 7.8) 0.4 mL, which contained Tris-HCl 40 mmol/L, EDTA 1 mol/L and NaCl 150 mmol/L, was added and
then the mixture was kept on ice for 5 min. The cells were harvested with a rubber scalpel and centrifuged at
151×g for 5 min, and then suspended in Tris buffer (pH 7.8, 20 mmol/L) and sonicated thrice in ice water for 5 s by a W375 sonicator (Heat
Systems-Ultrasonics. Inc., Plainview, NY). The cell extracts were used for enzyme assay and protein determination.
Analysis of steroids Commercially available, direct competitive radioimmunoassay kits (North Institute of Biological
Technology, Beijing, China) were used for the measurements of medium cortisol, aldosterone, testosterone and progesterone.
The lower limit of quantification of cortisol, aldosterone, testosterone and progesterone was 10 ng/mL, 3.75 pg/mL, 20 pg/mL,
5 pg/mL, respectively. The correlation coefficient was 0.9815, 0.9834, 0.9715, and 0.9834, respectively, for the working curve
of quantification of cortisol, aldosterone, testosterone and progesterone
Enzyme assays Activity of 7-ethoxyresorufin
O-dealkylase (EROD) was assayed using SHIMADZU RF-540
Fluorescence Spectrophotometer(Kyoto Japan). In brief, the total reaction contained cell extract protein (0.2 mg protein), Tris·HCl 50
mmol/L (pH 7.8), MgCl2 2.5 mmol/L, KCl
50
mmol/L, BSA 12 mg/ml, 7-ethoxyresorufin 2
µmol/L and an NADPH generating system
(NADP+ 0.4 mmol/L, isocitric acid 10 mmol/L and isocitric acid dehydrogenase 0.6 units). The reaction was initiated with the NADPH-generating system and
stopped by the addition of ice-methanol 2.5 mL at 30 min. Fluorescent intensity of the supernatants was read at
lem 586 nm and lex 530 nm against a standard of resorufin.
Activities of benzphetamine, aminopyrine and erythromycin
N-demethylase were determined as previously described. In
brief, the assay mixture contained cell extract protein (0.2 mg protein), Tris·HCl 50 mmol/L (pH 7.8),
MgCl2 10 mmol/L, KCl 150 mmol/L, and substrate. The final concentration of erythromycin, benzphetamine and aminopyrine was 0.4 mmol/L, 2 mmol/L,
and 8 mmol/L, respectively. The reaction was initiated with the NADPH-generating system and stopped by the addition of
25% ZnSO4 and saturated Ba(OH)2
at 60 min. The supernatant, after centrifugation, was incubated with the Nash reagent
(Wiltshire SP4 0JG. England) at 60 ºC for 20 min and its color was measured at 415 nm spectrophotometrically.
Statistical analysis Data were analyzed using one-way ANOVA test and differences were considered statistically
significant if the P value was less than 0.05.
Results
Inducibility of CYP isoforms The activities of benzphetamine and aminopyrine
N-demethylase were
increased in the cultural fetal adrenal cortical cells treated with phenobarbital (0.25_1 mmol/L) for 24 h, and the maximal
induction was 1.96- and 3.40-fold, respectively
(P<0.05, P<0.01; Figure 1). Dexamethasone (25_100 µmol/L) also
increased the activity of erythromycin N-demethylase, and this increase showed a concentration-effect relationship with a
maximal induction of 4.74-fold (P<0.01; Figure 2). The activity of 7-ethoxyresorufin
O-dealkylase was undetected in the cells treated without and with 3-methylcholanthrene (0.5_2 µmol/L).
Steroidogenesis The results (Table 1) showed that the contents of medium cortisol, aldosterone and progesterone have
decreased to 51%, 21% (P<0.01) and 13%
(P<0.05) of control, respectively after treatment with 3-methylcholanthrene (0.5_2
µmol/L). Cortisol, aldosterone and progesterone concentrations have also decreased to 32%, 30%, and
39% of control respectively, with phenobarbital (0.25_1
mmol/L). Dexamethasone (25_100 µmol/L) had enhanced the
production of cortisol and progesterone remarkably, almost 14.90- and 3.62-fold higher than their controls
(P<0.01). However, the trend of testosterone concentration was uncertain after 3-methylcholanthrene, phenobarbital or
dexamethasone treatment.
Discussion
CYP1, 2, and 3 families are the most important CYP isoforms in xenobiotic metabolism. 3-Methylcholanthrene,
phenobarbital and dexamethasone are considered as the classical inducers of CYP1A, CYP2B/2C, and CYP3A, respec-tively.
7-Ethoxyresorufin, benzphetamine, aminopyrine and erythromycin are classical substrates of CYP1, 2, and 3. Our previous
results obtained by RT-PCR technique demonstrated the expression of CYP1A1
mRNA[5]. However, in this study, whether
3-methylcholanthrene was administrated or not, EROD activity was undetected in the cultured fetal zone cells. This might be
attributed to a lower enzyme activity of CYP1A in human fetal adrenal cortical cells. Meanwhile, both
N-demethylations of benzphetamine and aminopyrine had increased when phenobarbital was added, erythromycin
N-demethylation had also increased by the treatment with dexamethasone. All these observations suggested that the fetal adrenal CYP isoforms were
capable of catalyzing the metabolism of xenobiotics, and possess inducibility.
During pregnancy, cortisol may influence placental function and uterine blood flow. If cortisol concentration was
changed, it would affect the growth and development of the fetus or induce the onset of preterm
labor[8]. Progesterone has suppressive actions on lymphocyte proliferation and on the immune system to prompt the developing fetus and placenta. A
higher progesterone level was reported to display a delay in parturition of several
days[9]. Aldosterone can sustain a homeostatic mechanism for the conservation of sodium for both mother and
fetus[10]. Therefore, it can be presumed that any
changes of steroid hormone level caused by xenobiotics might affect the growth and development of the fetus. The adrenal
steroid hormones are produced in the multi-step pathways involving different CYP isoforms, such as CYP11A1, CYP17,
CYP21, CYP11B1, and CYP11B2[4], and steroidogenic acute regulatory (StAR) protein and CYP11A1 is a rate-limiting step in
steroidogenesis. It was found that steroidogenesis of fetal adrenals could be affected by multiple factors, such as
estrogen[11], nicotine[12] and
ethanol[13], which are the substrates and inducers of CYP1, 2, and 3. In the present work, we observed the
steroidogenesis in human fetal adrenal cortical cells, and paid more attention to the treatments of 3-methylcholanthrene,
phenobarbital and dexamethasone, which have not been reported in the published literature. As shown in the results, the
production of cortisol, aldosterone and progesterone were decreased to some extent after 3-methylcholanthrene and
phenobarbital treatments. Meanwhile, dexamethasone remarkably increased the levels of cortisol and progesterone. Recently,
our research further demonstrated that exposure to nicotine (CYP1A1 inducer) in utero during the middle and last gestation
decreased the expression of StAR protein and CYP11A1 mRNA of fetal adrenals in rats (data not shown). These results
suggested that xenobiotics could interfere with adrenal steroidogenesis during the fetal development, and a possible
pathway may be CYP isoform activation induced by xenobiotics. However, few investigations were focused on the interfering
mechanism of xenobiotics on the steroido-genesis. The latest research of Aluru
et al (2005) found that b-naphthoflavone, another specific inducer of CYP1A, significantly elevated the expression of CYP1A1 gene and protein in the kidney, and
steroidogenesis[14].
In conclusion, the present study found the influences of 3-methylcholanthrene, phenobarbital, and dexamethasone on
steroidogenesis in human fetal adrenal cortical cells, which might be mediated by adrenal xenobiotic metabolizing-
related CYP isoform activation. Our results provide an important clue for elucidating the pathogenesis of some diseases and
for guiding clinical medication. For example, phenobarbital and dexamethasone applied in human neonatal
hyperbilirubinemia[15] and fetal lung
fibroblasts[16], respec-tively, might interfere with steroidogenesis and affect the growth and
development of the fetus. Further studies are required to explore the molecular mechanism of xenobiotics on fetal adrenal StAR and
CYP11A1 expressions.
depressed StAR protein and CYP11A1 in rainbow trout, which suggests an Ah receptor-mediated impairment of inter-renal
References
1 Guengerich FP. Human cytochrome P-450 enzymes. Life Sci 1992; 50: 1471_8.
2 Sanderson T, Vandenberg M. Interactions of xenobiotics with the steroid hormone biosynthesis pathway. Pure Appl Chem 2003; 75:
1957_71.
3 Suzuki T, Sasano H, Takeyama J. Developmental changes in steroidogenic enzymes in human postnatal adrenal cortex:
immunohistochemical studies. Clin Endocrinol 2000; 53: 739_47.
4 Rehman KS, Carr BR, Rainey WE. Profiling the steroidogenic pathway in human fetal and adult adrenals. J Soc Gynecol Investing 2003;
10: 372_80.
5 Wang H, Peng RX, Yu JP, Zhang YH. Expression and characteristics of cytochrome P-450 1, 2 and 3 in human fetal adrenals. Asia Pac
J Pharmacol 2000; 14: 33_8.
6 Mesiano S, Jaffe RB. Interaction of insulin-like growth factor-II and estradiol directs steroidogenesis in the human fetal adrenal toward
dehydroepiandrosterone sulfate production. J Clin Endocri Metab 1993; 77: 754_8.
7 Parker CR, Stankovic AK, Falany CN, Faye PO, Grizzle WE. Immunocytochemical analyses of dehydroepiandrosterone sulfotransferase
in cultured human fetal adrenal cells. J Clin Endocrinol Metab 1995; 80: 1027_31.
8 McLean M, Smith R. Corticotropin-releasing hormone in human pregnancy and parturition. Trends Endocrinol Metab 1999; 10:
174_8.
9 Piekorz RP, Gingras S, Hoffmeyer A. Regulation of progesterone levels during pregnancy and parturition by stat5 and
20a-hydroxysteroid dehydrogenase. Mol Endocrinol 2005; 19: 431_7.
10 Lisurek M, Bernhardt R. Modulation of aldosterone and cortisol synthesis on the molecular level. Mol Cell Endocrinol 2004; 215:
149_59.
11 Albrecht ED, Henson MC, Walker ML, Pepe GJ. Modulation of adrenocorticotropin-treated baboon fetal adrenal dehydroepian-drosterone
formation in vitro by estrogen at mid- and late-gestation. Endocrinology 1990; 126: 3083_8.
12 Sarasin A, Schlumpf M, Muller M. Adrenal-mediated rather than direct effects of nicotine as a basis of altered sex steroid synthesis in fetal
and neonatal rat. Reprod Toxicol 2003; 17: 153_62.
13 Khisti RT, Kumar S, Morrow AL. Ethanol rapidly induces steroidogenic acute regulatory protein expression and translocation in rat
adrenal gland. Eur J Pharmacol 2003; 473: 225_7.
14 Aluru N, Renaud R, Leatherland JF, Vijayan MM. Ah receptor-mediated impairment of interrenal steroidogenesis involves StAR protein
and P450scc gene attenuation in rainbow trout. Toxicol Sci 2005; 84: 260_9.
15 Dennery PA, Seidman DS, Stevenson DK. Neonatal hyperbiliru-binemia. N Engl J Med 2001; 344: 581_90.
16 Wen FQ, Kohyama T, Skold CM. Glucocorticoids modulate TGF-beta production by human fetal lung fibroblasts. Inflammation 2003; 27:
9_19.
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