Developmental expression and regulation of adrenocortical cytochrome P4501B1 in the rat.

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 (pdl), but were nearly undetectable on pd6 and pd1O. 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 pd 10 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.

Aryl hydrocarbon receptor regulation of cytochrome P4501B1 in rat mammary fibroblasts: evidence for transcriptional repression by glucocorticoids.

Cytochrome P450 1B1 (CYP1B1), which actively metabolizes polycyclic aromatic hydrocarbons, is regulated by the aryl hydrocarbon receptor (AhR) in primary cultures of rat mammary fibroblasts (RMF) and rat embryo fibroblasts (REF). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induced the 5.2-kilobase CYP1B1 mRNA in RMF (12-fold) and REF (14-fold) after a 6-hr treatment, with comparable increases in the microsomal protein. The synthetic glucocorticoid dexamethasone (DEX) suppresses TCDD-induced expression of CYP1B1 in RMF and REF. Suppression of CYP1B1 mRNA in RMF (maximal suppression, 70%) was observed when DEX was added 2 hr before TCDD, but was not observed with co-administration. The concentration dependence (EC50 approximately 10 nM) and reversal by the antagonist, RU486, implicates the glucocorticoid receptor. DEX inhibition of TCDD-induced CYP1B1 protein needed more extensive preincubation (>6 hr). TCDD induction of CYP1B1-luciferase constructs in RMF was mediated by a 265-base-pair upstream region (-810 to -1075), which was similarly suppressed (50-70%) by a 2-hr preincubation with 10(-7) M DEX via this enhancer region. Expression of the AhR is suppressed by DEX (70% after 12 hr), but not after the 2-hr period that was sufficient for suppression of transcription. The AhR nuclear translocator is not affected by this treatment. We conclude that glucocorticoid receptor rapidly suppresses activity of the AhR/AhR nuclear translocator complex in the CYP1B1 enhancer region, even though lacking glucocorticoid responsive element(s). DEX inhibits proliferation of RMF in this same concentration range (35%, EC50 approximately 5 nM), indicating additional effects on intracellular activity that may link to this suppression.

Characterization of CYP1B1 and CYP1A1 expression in human mammary epithelial cells: role of the aryl hydrocarbon receptor in polycyclic aromatic hydrocarbon metabolism.

CYP1B1 and CYP1A1 expression and metabolism of 7,12-dimethylbenz(a)anthracene (DMBA) have been characterized in early-passage human mammary epithelial cells (HMECs) isolated from reduction mammoplasty tissue of seven individual donors. The level of constitutive microsomal CYP1B1 protein expression was donor dependent (<0.01-1.4 pmol/mg microsomal protein). CYP1B1 expression was substantially induced by exposure of the cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to levels ranging from 2.3 to 16.6 pmol/mg among the seven donors. Extremely low, reproducible levels of constitutive CYP1A1 expression were detectable in three donors (0.03-0.16 pmol/mg microsomal protein). TCDD inductions were larger for CYP1A1, as compared to CYP1B1, demonstrating substantial variability in the induced levels among the donors (0.8-16.5 pmol/mg). Northern and reverse transcriptase PCR analyses corroborate the donor-dependent differences in protein expression, whereby CYP1B1 mRNA (5.2 kb) was constitutively expressed and was highly induced by TCDD (33-fold). The contributions of CYP1B1 and CYP1A1 to the metabolism of DMBA were analyzed using recombinant human CYP1B1 and CYP1A1, as references, in conjunction with antibody-specific inhibition analyses (anti-CYP1B1 and anti-CYP1A1). Constitutive microsomal activity exhibited a profile of regioselective DMBA metabolism that was characteristic of human CYP1B1 (increased proportions of 5,6- and 10,11-DMBA-dihydrodiols), which was inhibited by anti-CYP1B1 (84%) but not by anti-CYP1A1. TCDD-induced HMEC microsomal DMBA metabolism generated the 8,9-dihydrodiol of DMBA as the predominant metabolite, with a regioselectivity similar to that of recombinant human CYP1A1, which was subsequently inhibited by anti-CYP1A1 (79%). A CYP1B1 contribution was indicated by the regioselectivity of residual metabolism and by anti-CYP1B1 inhibition (25%). DMBA metabolism analyses of one of three donors expressing measurable basal expression of CYP1A1 confirmed DMBA metabolism levels equivalent to that from CYP1B1. The HMECs of all donors expressed similar, very high levels of the aryl hydrocarbon receptor and the aryl hydrocarbon nuclear translocator protein, suggesting that aryl hydrocarbon receptor and aryl hydrocarbon nuclear translocator protein expression are not responsible for differences in cytochrome P450 expression. This study indicates that CYP1B1 is an important activator of polycyclic aromatic hydrocarbons in the mammary gland when environmental chemical exposures minimally induce CYP1A1. Additionally, certain individuals express low levels of basal CYP1A1 in HMECs, representing a potential risk factor of mammary carcinogenesis through enhanced polycyclic aromatic hydrocarbon bioactivation.

Regulation of cytochrome P4501B1 in cultured rat adrenocortical cells by cyclic adenosine 3',5'-monophosphate and 2,3,7,8- tetrachlorodibenzo-p-dioxin.

Cytochrome P4501B1 (CYP1B1), which is responsible for metabolism of 7,12-dimethylbenz[a]anthracene in the rat adrenal gland, is partially dependent on ACTH in vivo. The regulation of CYP1B1 and possible involvement in steroidogenesis have been characterized in cultured rat adrenocortical cells. Relatively high basal expression of CYP1B1 is maintained in vitro and is, therefore, independent of ACTH. CYP1B1 expression is elevated 4-fold in primary cultures of fasciculata cells after 24 h of ACTH treatment, as measured by selective 7,12-dimethylbenz[a]anthracene metabolism, immunoblot analysis, and parallel changes in the 5.2-kilobase CYP1B1 messenger RNA (mRNA). Corticosterone synthesis was stimulated about 40-fold in these cells after this ACTH treatment. Maximal stimulation of CYP1B1 protein and mRNA by ACTH has been duplicated in fasciculata cells by 8-bromo-cAMP and the adenylyl cyclase agonist, forskolin, indicating that cAMP mediates this induction. CYP1B1 is similarly stimulated by ACTH in rat adrenal glomerulosa cells, although constitutive expression of CYP1B1 is about 4-fold lower. Angiotensin II treatment of glomerulosa cells, which stimulated aldosterone synthesis 3-fold, had no effect on CYP1B1 activity or expression. Treatment of fasciculata and glomerulosa cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin resulted in small increases in CYP1B1 activity (1.8- and 2.5-fold, respectively), but much larger increases (5- and 6-fold, respectively) in CYP1B1 at the mRNA level 2,3,7,8-Tetrachlorodibenzo-p-dioxin had no effect in the presence of ACTH stimulation. CYP1B1 is not coordinately expressed with steroidogenic enzymes. CYP11A1 and CYP21 mRNAs are far more responsive to ACTH, in part because of lower basal expression. CYP1B1 exhibited a transient response to ACTH that peaked (9-fold) at 6 h before declining to about 4-fold at 36 h, the time when CYP21 mRNA was maximally stimulated. The complete inactivation of CYP1B1 activity in fasciculata cells by a mechanism-based inhibitor, 1-ethynylpyrene, did not affect corticosterone production, indicating that this protein does not have a direct physiological role in the steroidogenic response.

Identification of a rat adrenal cytochrome P450 active in polycyclic hydrocarbon metabolism as rat CYP1B1. Demonstration of a unique tissue-specific pattern of hormonal and aryl hydrocarbon receptor-linked regulation.

Antibodies against a novel adrenocorticotropic hormone-inducible cytochrome P450 (P450RAP), responsible for polycyclic aromatic hydrocarbon metabolism in rat adrenal microsomes (Otto, S., Bhattacharyya, K.K., and Jefcoate, C.R. (1992) Endocrinology 131, 3067-3076), identified a cDNA clone encoding a partial cytochrome P450 sequence from a rat adrenal cDNA library. Rescreening a second cDNA library yielded several clones up to 5.0 kilobases (kb) encoding a 1629-base pair open reading frame. The deduced amino acid sequence (543 residues) matched completely with five peptides cleaved from P450RAP. The amino acid sequence of P450RAP is 92% identical to a 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible CYP1B1, cloned from mouse C3H10T1/2 (10T1/2) embryo fibroblast cells (Savas, U., Bhattacharyya, K. K., Christou, M., Alexander, D.L., and Jefcoate, C. R. (1994) J. Biol. Chem. 269, 14905-14911), which shows nearly the same characteristics in polycyclic aromatic hydrocarbon metabolism. The available 5'- and 3'-noncoding regions show, respectively, 93 and 83% sequence identity. We conclude that P450RAP protein is encoded by a rat CYP1B1 gene orthologous to the mouse CYP1B1 gene. Alignment of rat CYP1B1 amino acid sequences with rat CYP1A1 (39% identical) indicated eight regions of high identity for each (60-78%), interspersed by extensive regions of less than 30% similarity. The CYP1B1 cDNAs hybridize a 5.2-kb mRNA in rat adrenals, consistent with the length of the longest clones and the mRNA recognized in 10T1/2 cells. CYP1B1 mRNA was elevated by a 2-day adrenocorticotropic hormone treatment but much less than CYP11A1 (cytochrome P450 side chain cleavage) mRNA (2-fold versus 4-fold). The lower levels of the 5.2-kb mRNA in other steroidogenic cells (ovary) was consistent with the amount of immunodetectable CYP1B1 protein and, unlike the adrenal, expression in the ovary was stimulated 5-fold by beta-naphthoflavone, an aryl hydrocarbon receptor agonist, in parallel with CYP1A1 induction. In several other tissues (liver > lung > uterus >> kidney), CYP1B1 mRNA and protein were constitutively undetectable but highly induced by beta-naphthoflavone, although at much lower levels than CYP1A1. Rat CYP1B1, therefore, exhibits regulation through hormonal signaling and the aryl hydrocarbon receptor in a cell-specific manner.

The induction of five rat hepatic P450 cytochromes by phenobarbital and similarly acting compounds is regulated by a sexually dimorphic, dietary-dependent endocrine factor that is highly strain specific.

The phenobarbital (PB)-mediated expression of five forms of cytochrome P450 (CYP2A1, CYP2B1, CYP2B2, CYP2C6, and CYP3A1) and epoxide hydrolase has been examined in male and female rats from three inbred strains [Fischer (F344), Wistar Furth (WF), and Wistar Kyoto (WK)]. Evidence is presented that the regulation of induction of each protein involves a very similar endocrine control process. Each induction shows the same marked strain differences that are observable to a much greater extent in females than in males. The differences are largely removed by hypophysectomy and are each greatly enhanced by a shift in diet (standard [Teklad (W) 8604] to defined [Teklad AIN-76A]). The induction of each gene in female rats, as measured by specific immunoblots, follows the same strain selectivity (F344 >> WK > WF). These differences were similarly demonstrated in isoform-specific metabolism and comparable variations in the levels of the specific P450 mRNA suggest that these differences reflect alterations in gene transcription. For CYP3A1, CYP2B1, and CYP2B2, the differences between the F344 and WF strains approach 10-fold when using the defined diet, while the differences decreased approximately 3-fold with the standard chow, in parallel with much more effective induction of total P450. The same trend was also observed for the induction of CYP2A1, CYP2C6, and epoxide hydrolase, but the differences were less because of higher constitutive levels which were insensitive to these effects and smaller induction factors. These strain differences were not observed for CYP1A2, which is unresponsive to PB. Similar sex- and strain-selectivity for each P450 gene occurs for D-limonene, a structurally and chemically dissimilar PB-type inducer. Each of these measurements indicates a suppression of expression in female WF rats relative to a set of similar levels in male WF rats and F344 rats of both sexes. Hypophysectomy relieves the suppression through selective stimulation of induced levels in female WF rats. Many of the same differences between the F344 and WF strains can be observed in the very low basal expression of these PB-inducible genes. Thus, we have identified a gender-selective, pituitary-mediated polymorphism that probably affects basal regulatory factors.