Craig B. Marcus

Expression of cytochrome P450 CYP1B1 in breast cancer

The expression of CYP1B1 has been identified in breast cancer using the reverse transcriptase‐polymerase chain reaction and immunoblotting. CYP1B1 mRNA was expressed in the majority of breast tumours and immunoblotting of breast tumours identified a single protein band of molecular weight 60 kDa corresponding to the predicted molecular weight of human CYP1B1. This is the first study to identify CYP1B1 expression in a tumour where it may represent a previously unknown pathway for the metabolism of oestradiol and chemotherapeutic drugs.

Role of the aryl hydrocarbon receptor in drug metabolism

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that regulates the transcription of certain key enzymes involved in the metabolism of xenobiotic substances including some drugs. The AhR can be activated by a wide range of classes of compounds (e.g. polycyclic aromatic hydrocarbons, benzimidazoles and flavonoids), and interacts with a number of other proteins, including nuclear hormone receptors such as the oestrogen and androgen receptors. Activation of the AhR antagonises the oestrogen receptor and can lead to modulation of its transcriptional activity; thus, activating the AhR may serve as a target for breast cancer therapy. Disruption of normal signalling by drug interactions with the AhR or downstream components of this pathway could result in adverse effects, such as the bioactivation of procarcinogens or the disruption of normal homeostasis. The cytochrome P450s CYP1A1, -1B1, -1A2 and -2S1 are regulated by the AhR, and they are all involved in the metabolism of endogenous substrates as well as xenobiotics. Polymorphisms in the AhR, or polymorphisms in enzymes regulated by the AhR, may cause variations in response to certain drugs in different individuals; this needs to be taken into consideration when administering drugs that interact with this pathway.

Evidence for an Aryl Hydrocarbon Receptor-Mediated Cytochrome P450 Autoregulatory Pathway

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor responsible for mediating the cellular response to the toxic compound 2,3,7,8,-tetrachlorodibenzo-p-dioxin. An essential role for the AhR in cellular biology has been established previously, but no high-affinity endogenous ligand has yet been identified. We have confirmed the presence of a putative endogenous ligand(s) in CV-1 cells through transient transfection with various cytochrome P450 isoforms. Expression of cytochromes P450 1A1, 1A2, or 1B1 reduced AhR-mediated luciferase reporter activity, whereas cytochrome P450 2E1 exhibited no significant effect. Studies with 2,4,3′,5′-tetramethoxystilbene, a potent and specific inhibitor of cytochrome P450 1B1, was able to partially block cytochrome P450 1B1-mediated reduction in reporter gene activity. These results provide evidence of the existence of a possible feedback mechanism in which AhR-regulated cytochromes P450 from the CYP1A and CYP1B families are able to metabolically alter putative endogenous ligand(s). Several experiments were performed to provide initial characterization of these putative endogenous ligands, including electrophoretic mobility shift assay analyses, which demonstrated that these ligands directly activate the AhR. Soluble extracts from various C57BL/6J and Ahr-null mouse tissues were also analyzed for the presence of AhR activators. Studies revealed that Ahr-null mouse lung tissue had a 4-fold increase in AhR-mediated reporter activity in cells. Quantitative polymerase chain reaction analysis revealed that lung tissue exhibits relatively high constitutive CYP1A1 mRNA levels. These results suggest that there is an autoregulatory feedback loop between the AhR and cytochrome P450 1A1 in mouse lung.

Immobilized artificial membrane chromatography : rapid purification of functional membrane proteins

A solid-phase membrane mimetic system, denoted as immobilized artificial membranes (IAM), has been developed and utilized as a novel high-performance liquid chromatography (HPLC) matrix for the first step in the rapid purification of functional membrane proteins. IAM phases consist of monolayers of amphiphilic membrane lipid molecules covalently bonded to a rigid silica particle. These monolayers of lipids have proved remarkably effective for the chromatography of biomolecules. Several cytochrome P450 isozymes, an extremely important family of hydrophobic membrane proteins with a labile heme catalytic center, have been partially purified in functional conformations from rat liver, kidney, and adrenal microsomes on IAM supports. Functionality of purified P450 and P450 reductase has been demonstrated by optical difference spectroscopy, by carbon monoxide binding, and by reconstitution of enzymatic activity in vitro. Other membrane proteins, including rat liver plasma membrane NADH oxidase and ferricyanide oxidoreductase have also been partially purified by IAM HPLC. The methods for purification of these proteins are described.

Effects of synthetic and naturally occurring flavonoids on benzo[a]pyrene metabolism by hepatic microsomes prepared from rats treated with cytochrome P-450 inducers

Activity-directed fractionation of Trifolium pratense resulted in isolation of the isoflavone biochanin A, a potent inhibitor of metabolic activation of the carcinogen benzo[a]pyrene (B[a]P) in cells in culture. To determine the structural features required for maximal inhibition of cytochrome P-450 mediated metabolism of B[a]P, the inhibitory potencies of 23 flavonoids on metabolism of B[a]P to water-soluble derivatives were examined in liver S-9 homogenate from rats induced with Aroclor 1254. Flavones were much more efficient inhibitors than their corresponding isoflavone or flavanone analogs. Most flavonols were as effective inhibitors as their flavone analogs with the exception of kaempferide. Flavones with two hydroxyl or two methoxyl groups at positions 5 and 7 were the most active. Although all eight flavonoids tested effectively inhibited B[a]P metabolism by beta-naphthoflavone-induced microsomes, none were very effective inhibitors of B[a]P metabolism by phenobarbitol-induced microsomes, and only three were effective inhibitors of B[a]P metabolism by microsomes from non-induced rats. These results indicate that flavones or flavonols that contain free 5- and 7-hydroxyls are potent inhibitors of P-450 induced by beta-naphthoflavone (P-450IA1 and/or P-450IA2) and may potentially be useful as chemopreventive agents against hydrocarbon-induced carcinogenesis.

Heat Shock Protein 90 Inhibitors Suppress Aryl Hydrocarbon Receptor–Mediated Activation of CYP1A1 and CYP1B1 Transcription and DNA Adduct Formation

The aryl hydrocarbon receptor (AhR), a client protein of heat shock protein 90 (HSP90), plays a significant role in polycyclic aromatic hydrocarbon (PAH)–induced carcinogenesis. Tobacco smoke, a source of PAHs, activates the AhR, leading to enhanced transcription of CYP1A1 and CYP1B1, which encode proteins that convert PAHs to genotoxic metabolites. The main objectives of this study were to determine whether HSP90 inhibitors suppress PAH-mediated induction of CYP1A1 and CYP1B1 or block benzo(a)pyrene [B(a)P]–induced formation of DNA adducts. Treatment of cell lines derived from oral leukoplakia (MSK-Leuk1) or esophageal squamous cell carcinoma (KYSE450) with a saline extract of tobacco smoke, B(a)P, or dioxin induced CYP1A1 and CYP1B1 transcription, resulting in enhanced levels of message and protein. Inhibitors of HSP90 [17-allylamino-17-demethoxygeldanamycin (17-AAG); celastrol] suppressed these inductive effects of PAHs. Treatment with 17-AAG and celastrol also caused a rapid and marked decrease in amounts of AhR protein without modulating levels of HSP90. The formation of B(a)P-induced DNA adducts in MSK-Leuk1 cells was inhibited by 17-AAG, celastrol, and α-naphthoflavone, a known AhR antagonist. The reduction in B(a)P-induced DNA adducts was due, at least in part, to reduced metabolic activation of B(a)P. Collectively, these results suggest that 17-AAG and celastrol, inhibitors of HSP90, suppress the activation of AhR-dependent gene expression, leading, in turn, to reduced formation of B(a)P-induced DNA adducts. Inhibitors of HSP90 may have a role in chemoprevention in addition to cancer therapy.

Selective induction of cytochrome P450 isozymes in rat liver by 4-n-alkyl-methylenedioxybenzenes

To examine the structural requirements of cytochrome P450 induction by 4-n-alkyl-substituted methylenedioxybenzenes (MDBs), rats were treated in vivo with a series of MDBs that differed in alkyl carbon side-chain length (0, 1, 2, 3, 4, 5, 6, or 8). Expression patterns of specific P450 isozymes were evaluated with Western and Northern blotting, enzymatic assays, and solution hybridization assays. As determined by carbon monoxide difference spectroscopy, maximal hepatic induction of total P450 content occurred when rats were treated with MDB derivatives with alkyl chain lengths of five or six carbons. However, maximum induction of the specific P450s--P450IA1, P450IIB1, and P450IIB2--occurred with n-hexyl-MDB. In contrast to effects observed with phenobarbital, treatment with MDBs resulted in higher levels of P450IIB2 than of P450IIB1 in rat hepatic microsomes. Western blot quantitation of MDB-induced hepatic P450IIB1 and P450IIB2 apoenzymes did not correlate to measured levels of the corresponding P450 mRNAs. In fact, P450IIB1 and P450IIB2 apoenzyme levels were consistently lower than expected based on Northern blot and solution hybridization measures of the respective mRNAs. These data suggest that the n-alkyl-MDBs effect increases in levels of hepatic P450 in a complex manner, producing accumulation of P450 mRNAs concomitant with alterations in processes regulating steady-state levels of P450 apoenzyme.

Cytochrome P450 purification and immunological detection in an insecticide resistant strain of German cockroach (Blattella germanica, L.).

A German cockroach strain, Munsyana (MA) had 80-fold resistance to the pyrethroid insecticide cypermethrin, 4.5-fold greater total cytochrome P450 content and 2.5-fold greater cytochrome P450-mediated N-demethylation of 4-chloro-N-methylaniline compared to the susceptible Johnson Wax (JWax) strain. Immobilized artificial membrane high performance liquid chromatography (IAM-HPLC) of microsomal proteins from the MA strain enriched cytochrome P450 greater than 70-fold. Following purification, a single protein band of M(r) = 49,000 (P450 MA), was detected by silver-staining SDS PAGE gels. Antiserum to the purified protein from the MA strain (anti-P450 MA) was produced in mice. Anti-P450 MA inhibited cytochrome P450-mediated N-demethylation by 4-fold in both MA and JWax strains. In Western blots of microsomal proteins, anti-P450 MA differentiated single MA and JWax individuals by recognizing and M(r) 49,000 protein band in only the MA strain. In JWax cockroaches, the M(r) 49,000 band was only detectable in Western analysis following induction with pentamethylbenzene (PMB). PMB induction also increases N-demethylation 2.6 and 8.0-fold in the MA and JWax strains, respectively. These results are consistent with the hypothesis that insecticide resistance in the MA strain is due to over-expression of a cytochrome P450.

Immobilized Artificial Membrane Chromatography: Surface Chemistry and Applications

Immobilized Artificial Membranes are solid surfaces containing phospholipids immobilized on silica particles at surface densities similar to the ligand density of reversed phase Chromatographic surfaces. Chromatographic and non-chromatographic applications of Immobilized Artificial Membrane surfaces are reviewed and compared to the Chromatographie and non-chromatographic applications of reversed phase columns. The methodology for synthesizing Immobilized Artificial Membranes and the stability of Immobilized Artificial Membranes are also described. Several examples are presented regarding the ability of Immobilized Artificial Membrane surfaces to model biological processes. Examples include predicting the transport of solutes across human skin, predicting the transport of amino acids across the blood brain barrier, and predicting the binding of solutes to liposome membranes. In addition, the purification of several membrane proteins, including cytochrome P450 from rat adrenals and rat livers, NADH oxidase, and rabbit intestinal phospholipid binding protein, are discussed.

Inhibition of benzo[a]pyrene metabolism by insulin, FITC-insulin and an FITC-insulin-antibody conjugate in the human hepatoma cell line HepG2.

Benzo[a]pyrene (BaP) can be metabolically activated to an ultimate carcinogen, (+)-anti-BaP-7,8-dihydrodiol-9,10-epoxide [(+)-anti-BaPDE] by cells in culture. This activation involves oxidation by specific isoforms of cytochrome P450s such as CYP1A1. The human hepatoma cell line, HepG2, was used to examine the effect of inhibition of CYP1A1 activity by anti CYP1A1 specific antibodies on BaP metabolism. Metabolism of BaP to water-soluble metabolites by HepG2 cells in culture was 50% lower in fluorescein isothiocyanate (FITC)-insulin-CYP1A1-antibody-conjugate-treated cells than in control cells. However, FITC-insulin (lacking anti CYP1A1 conjugates) or insulin alone also decreased BaP metabolism by 50%. This insulin-induced inhibition of BaP metabolism was observed for cultures treated with a concentration range of FITC-insulin from 50-1000 nM. FITC-conjugated gamma-globulin showed no significant binding to HepG2 cells by fluorescence microscopy, however, FITC-insulin-antibody conjugates bound extensively, suggesting that FITC-insulin conjugates still retain the ability to bind insulin receptors. These results demonstrate that free insulin, FITC-insulin or FITC-insulin conjugated to antibodies are effective inhibitors of BaP metabolism in cells in culture.