The effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on estrogen metabolism in MCF-7 breast cancer cells: Evidence for induction of a novel 17β-estradiol 4-hydroxylase

Rates of microsomal 17β-estradiol (E₂) hydroxylation at the C-2, -4, -6, and -15 positions are each induced greater than 10-fold by treating MCF-7 breast cancer cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The TCDD-induced activities at the C-2, -6 and -15 positions have been attributed to cytochrome P450 1A1 (CYP1A1); however, the low K_m 4-hydroxylase induced by TCDD appears to be a distinct enzyme. We report here that antibodies to cytochrome P450-EF (mouse CYP1B1) selectivity inhibited the C-4 hydroxylation of E₂ catalyzed by microsomes from TCDD-treated MCF-7 cells. Western blots probed with anti-CYP1B antibodies showed the induction of a 52 kDa microsomal protein in response to treatment with TCDD in MCF-7 cells. Western blots of microsomes from HepG2 cells did not show the TCDD-induced 52 kDa protein, and microsomes from TCDD-treated HepG2 cells did not catalyze a low K_m hydroxylation of E₂ at C-4. Cellular metabolism experiments also showed induction of both the C-2 and -4 hydroxylation pathways in TCDD-treated MCF-7 cells as evidenced by elevated 2- and 4-methoxyestradiol (MeOE₂) formation. In contrast, TCDD-treated HepG2 cells showed 2-MeOE₂ formation predominantly over 4-MeOE₂. Northern blots of RNA isolated from untreated and TCDD-treated cells, when probed with the human CYP1B1 cDNA, showed induction of a 5.2 kb RNA in MCF-7 cells but not in HepG2 cells in response to treatment with TCDD. These results provide additional evidence for the induction by TCDD of a novel E₂ 4-hydroxylase in MCF-7 cells but not in HepG2 cells and indicate possible endocrine regulatory roles for the newly discovered group of enzymes of the CYP1B subfamily.     

NADPH- and hydroperoxide-supported 17beta-estradiol hydroxylation catalyzed by a variant form (432L, 453S) of human cytochrome P450 1B1

Human cytochrome P450 1B1 (CYP1B1) catalyzes the hydroxylation of 17beta-estradiol (E(2)) at C-4, with a lesser activity at C-2. The E(2) 4-hydroxylase activity of human CYP1B1 was first observed in studies of MCF-7 breast cancer cells. Sequencing of polymerase chain reaction products revealed that CYP1B1 expressed in MCF-7 cells was not the previously characterized enzyme but a polymorphic form with leucine substituted for valine at position 432 and serine substituted for asparagine at position 453. To investigate the NADPH- and organic hydroperoxide-supported E(2) hydroxylase activities of the 432L, 453S form of human CYP1B1, the MCF-7 CYP1B1 cDNA was cloned and the enzyme was expressed in Sf9 insect cells. In microsomal assays supplemented with human NADPH:cytochrome P450 oxidoreductase, the expressed 432L, 453S form catalyzed NADPH-supported E(2) hydroxylation with a similar preference for 4-hydroxylation as the 432V, 453N form, with maximal rates of 1.97 and 0.37 nmol (min)(-1)(nmol cytochrome P450)(-1) for 4- and 2-hydroxylation, respectively. Cumeme hydroperoxide efficiently supported E(2) hydroxylation by both the 432V, 453N and 432L, 453S forms at several-fold higher rates than the NADPH-supported activities and with a lesser preference for E(2) 4- versus 2-hydroxylation (2:1). The hydroperoxide-supported activities of both forms were potently inhibited by the CYP1B1 inhibitor, 3,3',4, 4',5,5'-hexachlorobiphenyl. These results indicate that the 432V, 453N and 432L, 453S forms of CYP1B1 have similar catalytic properties for E(2) hydroxylation, and that human CYP1B1 is very efficient in catalyzing the hydroperoxide-dependent formation of catecholestrogens.     

Radical intermediates in the catalytic oxidation of hydrocarbons by bacterial and human cytochrome P450 enzymes

Cytochromes P450cam and P450BM3 oxidize alpha- and beta-thujone into multiple products, including 7-hydroxy-alpha-(or beta-)thujone, 7,8-dehydro-alpha-(or beta-)thujone, 4-hydroxy-alpha-(or beta-)thujone, 2-hydroxy-alpha-(or beta-)thujone, 5-hydroxy-5-isopropyl-2-methyl-2-cyclohexen-1-one, 4,10-dehydrothujone, and carvacrol. Quantitative analysis of the 4-hydroxylated isomers and the ring-opened product indicates that the hydroxylation proceeds via a radical mechanism with a radical recombination rate ranging from 0.7 +/- 0.3 x 10(10) s(-1) to 12.5 +/- 3 x 10(10) s(-1) for the trapping of the carbon radical by the iron-bound hydroxyl radical equivalent. 7-[2H]-alpha-Thujone has been synthesized and used to amplify C-4 hydroxylation in situations where uninformative C-7 hydroxylation is the dominant reaction. The involvement of a carbon radical intermediate is confirmed by the observation of inversion of stereochemistry of the methyl-substituted C-4 carbon during the hydroxylation. With an L244A mutation that slightly increases the P450(cam) active-site volume, this inversion is observed in up to 40% of the C-4 hydroxylated products. The oxidation of alpha-thujone by human CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 occurs with up to 80% C-4 methyl inversion, in agreement with a dominant radical hydroxylation mechanism. Three minor desaturation products are produced, with at least one of them via a cationic pathway. The cation involved is proposed to form by electron abstraction from a radical intermediate. The absence of a solvent deuterium isotope effect on product distribution in the P450cam reaction precludes a significant role for the P450 ferric hydroperoxide intermediate in substrate hydroxylation. The results indicate that carbon hydroxylation is catalyzed exclusively by a P450 ferryl species via radical intermediates whose detailed properties are substrate- and enzyme-dependent.     

Androgen hydroxylation catalysed by a cell line (SD1) that stably expresses rat hepatic cytochrome P-450 PB-4 (IIB1).

Androgen hydroxylation catalysed by Chinese hamster fibroblast SD1 cells, which stably express cytochrome P-450 form PB-4, the rat P450IIB1 gene product, was assessed and compared to that catalysed by purified cytochrome P-450 PB-4 isolated from rat liver. SD1 cell homogenates catalysed the NADPH-dependent hydroxylation of androstenedione and testosterone with a regioselectivity very similar to that purified by P-450 PB-4 (16 beta-hydroxylation/16 alpha-hydroxylation = 6.0-6.8 for androstenedione; 16 beta/16 alpha = 0.9 for testosterone). Homogenates prepared from the parental cell line V79, which does not express detectable levels of P-450 PB-4 or any other cytochrome P-450, exhibited no androgen 16 beta- or 16 alpha-hydroxylase activity. The hydroxylase activities catalysed by the SD1 cell homogenate were selectively and quantitatively inhibited (greater than 90%) by a monoclonal antibody to P-450 PB-4 at a level of antibody (40 pmol of antibody binding sites/mg of SD1 homogenate) that closely corresponds to the P-450 PB-4 content of the cells (48 pmol of PB-4/mg of SD1 homogenate). Fractionation of cell homogenates into cytosol and microsomes revealed that the P-450 PB-4-mediated activities are associated with the membrane fraction. Although the P-450 PB-4-specific content of the SD1 microsomes was 15% of that present in phenobarbital-induced rat liver microsomes, the P-450 PB-4-dependent androstenedione 16 beta-hydroxylase activity of the SD1 membrane fraction was only 2-3% of that present in the liver microsomes. This activity could be stimulated several-fold, however, by supplementation of SD1 microsomes with purified rat NADPH P-450 reductase. These studies establish that a single P-450 gene product (IIB1) can account for the hydroxylation of androgen substrates at multiple sites, and suggest that SD1 cells can be used to assess the catalytic specificity of P-450 PB-4 with other substrates as well.     

Radical rebound mechanism in cytochrome P-450-catalyzed hydroxylation of the multifaceted radical clocks alpha- and beta-thujone

Alpha-thujone (1alpha) and beta-thujone (1beta) were used to investigate the mechanism of hydrocarbon hydroxylation by cytochromes P-450(cam) (CYP101) and P-450(BM3) (CYP102). The thujones are hydroxylated by these enzymes at various positions, but oxidation at C-4 gives rise to both rearranged and unrearranged hydroxylation products. Rearranged products result from the formation of a radical intermediate that can undergo either inversion of stereochemistry or ring opening of the adjacent cyclopropane ring. Both of these rearrangements, as well as a C-4 desaturation reaction, are observed. The ring opening clock gives oxygen rebound rates that range from 0.2 x 10(10) to 2.8 x 10(10) s(-1) for the different substrate and enzyme combinations. The C-4 inversion reaction provides independent confirmation of a radical intermediate. The phenol product expected if a C-4 cationic rather than radical intermediate is formed is not detected. The results are consistent with a two-state process and provide support for a radical rebound but not a hydroperoxide insertion mechanism for cytochrome P-450 hydroxylation.     

Monoclonal antibody-directed characterization of rat hepatic P450 catalyzing the omega-1 and omega-2 hydroxylation of prostaglandins

Previous studies have demonstrated that methylcholanthrene (MC) treatment of rats increases 10-fold the omega-2 hydroxylation of prostaglandin E2 (PGE2) by liver microsomes (K. A. Holm, R. J. Engell, and D. Kupfer (1985) Arch. Biochem. Biophys. 237, 477-489). The current study identifies the cytochrome P450 form, which catalyzes a major portion of the omega-2 hydroxylation of prostaglandins in liver microsomes of MC-treated rats (MC-microsomes) and examines whether the same enzyme catalyzes this reaction in microsomes from untreated rats (control microsomes). Three monoclonal antibodies (MAbs), MC 1-7-1, 1-31-2, and 1-36-1, raised against the major liver P450 from MC-treated rats were used. MAb 1-7-1 binds P450(57K) and P450(56K) (P450c and P450d, respectively); MAb 1-31-2 binds primarily P450(57K); and 1-36-1 binds solely P450(57k). MAb 1-7-1 inhibited omega-2 and omega-1 PGE2 hydroxylations in MC-microsomes by 70 and 45%, respectively. By contrast, MAb 1-31-2 and 1-36-1 were not inhibitory. MAb 1-7-1 did not inhibit PGE2 omega-2 hydroxylation in control or in microsomes from phenobarbital-treated rats (PB-microsomes). Since MAb 1-7-1 binds to both P450c and P450d, and 1-31-2 and 1-36-1 bind to P450c but are not inhibitory, these findings did not permit the determination of whether in MC microsomes a single isozyme (P450c or P450d) or both isozymes catalyze the omega-2 hydroxylation. This question was partially resolved by the observation that immunoaffinity-isolated P450c, supplemented with purified NADPH-P450 reductase, catalyzes effectively the omega-2 hydroxylation and to a lesser extent the omega-1 hydroxylation. There was no activity in the absence of reductase. The P450 antibody complex exhibits characteristics similar to those of the omega-2 hydroxylating activity in intact MC-microsomes supported by H2O2, by demonstrating a much higher activity when H2O2 is used instead of reductase and NADPH. Furthermore, a reconstituted monooxygenase composed of rat liver reductase and P450c, purified by conventional means, hydroxylated PGE2 at the omega-2 and omega-1 sites at a ratio of 2.8, similar to that obtained with the P450-antibody complex. These findings demonstrate that a major portion of the omega-2 hydroxylation of PGs in MC-microsomes is catalyzed by P450c; however, the possibility that some omega-2 hydroxylating activity is due to P450d was not ruled out.(ABSTRACT TRUNCATED AT 400 WORDS)     

A proteolytically sensitive region common to several rat liver cytochromes P450: effect of cleavage on substrate binding.

Limited proteolysis of rat liver microsomes was used to probe the topography and structure of cytochrome P450 bound to the endoplasmic reticulum. Three cytochromes P450 from two families were examined. Monoclonal antibodies to cytochrome P450 forms 1A1, 2B1, and 2E1 were used to immunopurify these proteolyzed cytochromes P450 from microsomes from rats treated with 3-methylcholanthrene, phenobarbital, and acetone, respectively. Electrophoretic and immunoblot analysis of tryptic fragments revealed a highly sensitive cleavage site in all three cytochromes P450. N-Terminal sequencing was performed on the fragments after transfer onto poly(vinylidene difluoride) membranes and showed that this preferential cleavage site is at amino acid position 298 of P450 1A1, position 277 of P450 2B1, and position 278 of P450 2E1. Multiple sequence alignment revealed that these positions are at the amino terminal of a highly conserved region of these cytochromes P450. The important functional role implied by primary sequence conservation along with the proteolytic sensitivity at its amino terminal suggests that this region is a protein domain. Comparison with the known structure of the bacterial cytochrome P450cam predicts that this proteolytically sensitive site is within an interhelical turn region connected to the distal helix that partially encompasses the heme-containing active site. Substrate binding to the cleaved cytochromes P450 was examined in order to determine whether the newly added conformational freedom near the cleavage site functionally altered these cytochromes P450. Cleavage of P450 2B1 abolished benzphetamine binding, which indicates that the cleavage site contains an important structural determinant for binding this substrate. However, cleavage did not affect benzo[a]pyrene binding to P450 1A1.     

Partial purification of cytochrome P450 from rat brain and demonstration of estradiol hydroxylation

Cytochrome P450 was partially purified from brain microsomes of untreated rats. A difference spectrum of the dithionite-reduced CO-complex of the purified P450 showed essentially the hemeprotein absorbing exclusively at 449 nm. The purified brain P450 was able to catalyze estradiol (E2) hydroxylation leading to the formation of 6 alpha- and 6 beta-hydroxy(OH)E2, 4-OHE2, estrone, 6-oxoE2, 2-OHE2, 15 alpha-OHE2 and estriol. These results demonstrate that rat brain P450 is active in estradiol hydroxylation.     

The sexually differentiated metabolism of [6,7-3H]17 beta-oestradiol in rats: male-specific 15 alpha- and male-selective 16 alpha-hydroxylation and female-selective catechol formation.

The oxygenated-metabolite profiles of exogenous 17 beta-oestradiol (E2) in adult male and female Wistar rats have been characterized and major sex-dependent biotransformations observed which correlate with the regioselectivities of known sexually differentiated hepatic P450. [6,7-3H]E2 (27 micrograms/kg) was given i.v. The metabolites of E2 were rapidly and extensively excreted in bile (46 and 78% of the dose over 1 and 6 h, respectively). Female rats metabolized E2 by one major pathway: oxidation to oestrone (E1) followed by C-2 hydroxylation and O-methylation; the principal aglycones (0-1 h bile collections) were E1 (14%), 2-hydroxyE1 (2-OHE1) (42%) and 2-methoxyE1 (24%). Male rats metabolized E2 principally by two parallel composite pathways of E1 hydroxylation which yielded a complex mixture of mono- and di-oxygenated compounds: 15 alpha-OHE1 (33%), 2,15 alpha-diOHE1 (7%), and 2-methoxy-15 alpha OHE1 (14%); 16 alpha-OHE1 (13%), 2,16 alpha-diOHE1 (4%) and 2-methoxy-16 alpha-OHE1 (2%). 15 alpha-Hydroxylation was unique to males. The balance of aromatic and alkyl hydroxylation in males was dose-dependent: at 3 mg/kg, 15 alpha-hydroxylation was decreased approx. 50% in favour of 2-hydroxylation whilst 16 alpha-hydroxylation was largely unaffected. The male-specific 15 alpha-hydroxylation and male-predominant 16 alpha-hydroxylation of E1 derived from E2 in vivo may be ascribable to the male-specific isoforms P450IIC13 and P450IIC11, respectively.     

Preferential inducibility of CYP1A1 and CYP1A2 by TCDD: differential regulation in primary human hepatocytes versus transformed human cells

Cytochrome P4501A1 (CYP1A1) induction, a marker of aryl hydrocarbon (Ah) receptor activation, has been associated with carcinogenicity of the environmental agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Consistently, we show that TCDD treatment led to induction of CYP1A1 in responsive human cancer cell lines including HepG2, LS174T, and MCF-7, as determined by Western blotting and CYP1A form-selective R-warfarin 6- and 8-hydroxylation. TCDD, however, preferably induced CYP1A2, not CYP1A1, in primary human hepatocytes. Such CYP1A form-preferred induction at the protein level was apparently uncorrelated with non-preferred mRNA induction in any cells studied. Moreover, while both genes were up-regulated by TCDD in primary hepatocytes and HepG2 cells, the induction of CYP1A1 and CYP1A2 at the mRNA level was distinguishable, indicated by the marked differences in activation kinetics and the response to the protein synthesis inhibitors, anisomycin and cycloheximide. Furthermore, formation of total benzo(a)pyrene (BaP)-DNA adducts was not altered following BaP exposure in TCDD-treated primary hepatocytes, whereas significantly elevated, in a CYP1A1-dependent manner, in the treated HepG2 cells. Taken together, our findings, demonstrating the complexities of TCDD-associated human Ah receptor function and differential regulations of CYP 1A enzymes, suggest clearly the need for caution when extrapolating data obtained in cell-based models.     

Activation and irreversible binding of regiospecifically labeled catechol estrogen by rat liver microsomes: evidence for differential cytochrome P-450 catalyzed oxidations

Estradiol and 2-hydroxyestradiol labeled with 3H at different positions in rings A or B were incubated with male rat liver microsomes, and their oxidative transformation was followed by the transfer of 3H into 3H2O. 14C-labeled estrogen or catechol estrogen was used to determine the fraction that becomes bound covalently to microsomal protein. The further metabolism of 2-hydroxyestradiol involves activation of the steroid at C-4 and, to a much lesser extent at C-1, by a cytochrome P-450 mediated reaction as indicated by the effects of NADPH, spermine, SKF-525A, and CO in the microsomal system. Glutathione promoted the loss of 3H from C-4 of either estradiol or 2-hydroxyestradiol but had less effect on this reaction at C-1 and inhibited it at C-6,7. It also abolished the irreversible binding of 14C-labeled estradiol and 2-hydroxyestradiol to microsomal protein. NADPH was needed specifically for glutathione to exert its effect both on the transfer of 3H into 3H2O and on the formation of water-soluble products from catechol estrogen by rat liver microsomes. It could not be replaced by NADP, NAD, or NADH. Ascorbic acid inhibited these enzymatic reactions but did not affect significantly the initial 2-hydroxylation of estradiol. Evidence is also provided for the further hydroxylation of 2-hydroxyestradiol at C-6 (or C-7). These results indicate that cytochrome P-450 activates catechol estrogens by an electron abstraction process.     

Purification and characterization of cholesterol 7 alpha-hydroxylase from rat liver microsomes

Cholesterol 7 alpha-hydroxylase (cholesterol, NADPH: oxygen oxidoreductase, 7 alpha-hydroxylating, EC 1.14.13.17) was purified from liver microsomes of cholestryramine-fed male rats by using high-performance ion-exchange chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 52,000), and its dithionite-reduced CO complex exhibited an absorption maximum at 450 nm. The specific content of the enzyme was 9 nmol of cytochrome P-450/mg of protein. Upon reconstitution with NADPH-cytochrome P-450 reductase, the enzyme showed a high activity of cholesterol 7 alpha-hydroxylation with the turnover number of 50 min-1 at 37 degrees C. The reaction was inhibited neither by aminoglutethimide nor by metyrapone, but inhibited markedly by iodoacetamide and disulfiram. The reaction was also inhibited significantly by CO. The enzyme catalyzed hydroxylation of cholesterol with strict regio- and stereoselectivity and was inert toward other sterols which are intermediates in the conversion of cholesterol to bile acids, i.e. 7 alpha-hydroxy-4-cholesten-3-one (12 alpha-hydroxylation), 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol (25-hydroxylation), and taurodeoxycholate (7 alpha-hydroxylation). Unlike other cytochromes P-450 isolated from rat liver microsomes, the enzyme showed no activity toward testosterone and xenobiotics such as 7-ethoxycoumarin and benzo[a] pyrene. The NH2-terminal amino acid sequence of the enzyme was Met-Phe-Glu-Val(Ile)-Ser-Leu-, which was distinct from those of any other cytochromes P-450 of rat liver microsomes hitherto reported. These results indicate that the enzyme is a novel species of cytochrome P-450 so far not isolated from liver microsomes.     

Enhancement of 2- and 16 alpha-estradiol hydroxylation in MCF-7 human breast cancer cells by 2,3,7,8-tetrachlorodibenzo-P-dioxin

2,3,7,8-Tetrachlorodibenzo-p-dioxin exhibits antiestrogenic activity and induces cytochromes P-450 in estrogen-dependent MCF-7 human breast-cancer cells. To determine whether induction of 2- or 16 alpha-hydroxylation of 17 beta-estradiol has a role in this antiestrogenic activity, MCF-7 cells which were exposed to this xenobiotic for 72 hrs were incubated with either [2-3H] or [16 alpha-3H] 17 beta-estradiol and the extent of tritiated H2O formation, indicative of site-specific hydroxylation, was determined. 2,3,7,8-Tetrachlorodibenzo-p-dioxin-treated MCF-7 cultures showed an 8-fold increase in 2-hydroxylation and a 2-fold increase in 16 alpha-hydroxylation. These results support the suggestion that increased hydroxylation of 17 beta-estradiol may have a role in the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in MCF-7 cells.     

Mitochondrial P450-dependent arachidonic acid metabolism by TCDD-induced hepatic CYP1A5; conversion of EETs to DHETs by mitochondrial soluble epoxide hydrolase

Several P450 enzymes localized in the endoplasmic reticulum and thought to be involved primarily in xenobiotic metabolism, including mouse and rat CYP1A1 and mouse CYP1A2, have also been found to translocate to mitochondria. We report here that the environmental toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces enzymatically active CYP1A4/1A5, the avian orthologs of mammalian CYP1A1/1A2, in chick embryo liver mitochondria as well as in microsomes. P450 proteins and activity levels (CYP1A4-dependent 7-ethoxyresorufin-O-deethylase and CYP1A5-dependent arachidonic acid epoxygenation) in mitochondria were 23-40% of those in microsomes. DHET formation by mitochondria was twice that of microsomes and was attributable to a mitochondrial soluble epoxide hydrolase as confirmed by Western blotting with antiEPHX2, conversion by mitochondria of pure 11,12 and 14,15-EET to the corresponding DHETs and inhibition of DHET formation by the soluble epoxide hydrolase inhibitor, 12(-3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA). TCDD also suppressed formation of mitochondrial and microsomal 20-HETE. The findings newly identify mitochondria as a site of P450-dependent arachidonic acid metabolism and as a potential target for TCDD effects. They also demonstrate that mitochondria contain soluble epoxide hydrolase and underscore a role for CYP1A in endobiotic metabolism.     

Cytochrome P-450 isozymes from the marine teleost Stenotomus chrysops: their roles in steroid hydroxylation and the influence of cytochrome b5

Two new cytochrome P-450 forms were purified from liver microsomes of the marine fish Stenotomus chrysops (scup). Cytochrome P-450A (Mr = 52.5K) had a CO-ligated, reduced difference spectrum lambda max at 447.5 nm, and reconstituted modest benzo[a]pyrene hydroxylase activity (0.16 nmol/min/nmol P-450) and ethoxycoumarin O-deethylase activity (0.42 nmol/min/nmol P-450). Cytochrome P-450A reconstituted under optimal conditions catalyzed hydroxylation of testosterone almost exclusively at the 6 beta position (0.8 nmol/min/nmol P-450) and also catalyzed 2-hydroxylation of estradiol. Cytochrome P-450A is active toward steroid substrates and we propose that it is a major contributor to microsomal testosterone 6 beta-hydroxylase activity. Cytochrome P-450A had a requirement for conspecific (scup) NADPH-cytochrome P-450 reductase and all reconstituted activities examined were stimulated by the addition of purified scup cytochrome b5. Cytochrome P-450B (Mr = 45.9K) had a CO-ligated, reduced difference spectrum lambda max at 449.5 nm and displayed low rates of reconstituted catalytic activities. However, cytochrome P-450B oxidized testosterone at several different sites including the 15 alpha position (0.07 nmol/min/nmol P-450). Both cytochromes P-450A and P-450B were distinct from the major benzo[a]pyrene hydroxylating form, cytochrome P-450E, by the criteria of spectroscopic properties, substrate profiles, minimum molecular weights on NaDodSO4-polyacrylamide gels, peptide mapping and lack of cross-reaction with antibody raised against cytochrome P-450E. Cytochrome P-450E shares epitopes with rat cytochrome P-450c indicating it is the equivalent enzyme, but possible homology between scup cytochromes P-450A or P-450B and known P-450 isozymes in other vertebrate groups is uncertain, although functional analogs exist.     

12-O-tetradecanoylphorbol-13-acetate upregulates the Ah receptor and differentially alters CYP1B1 and CYP1A1 expression in MCF-7 breast cancer cells

Elevated expression of cytochrome P450 1B1 (CYP1B1) and estradiol 4-hydroxylation have been reported to be biomarkers of tumorigenesis in humans. The aromatic hydrocarbon receptor (AhR) regulates expression of human cytochrome P450 1A1 (CYP1A1) and CYP1B1, 17β-estradiol (E₂) 2- and 4-hydroxylases, respectively. There is also evidence that expression of estrogen receptor α (ERα) potentiates CYP1A1 inducibility in breast cancer cells. To characterize these relationships further, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), which downregulates ERα, and the high-affinity AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on the expression of AhR, ERα, CYP1A1, and CYP1B1 in MCF-7 human breast cancer cells. Treatment with TPA, which suppressed ERα mRNA levels, caused a greater than fourfold elevation of AhR mRNA and protein levels, whereas treatment with TCDD caused a decrease in AhR protein but no change in ERα or AhR mRNA levels. In MCF-7 cells treated with TPA prior to treatment with TCDD, the AhR mRNA level was elevated, the ERα mRNA level remained suppressed, and the ratio of CYP1B1 to CYP1A1 mRNA was increased compared with treatment with TCDD alone. A corresponding increase in the ratio of the rates of 4- to 2-hydroxylation pathways of E₂ metabolism was also observed in response to pretreatment with TPA prior to the addition of TCDD. These results demonstrate differential regulation of the human CYP1A1 and CYP1B1 genes and provide a cellular model to investigate further the mechanisms that may be involved in the elevated expression of CYP1B1 in tumorigenesis. J. Cell. Biochem. 70:289–296, 1998. © 1998 Wiley-Liss, Inc.