Interaction of cytochrome P450 3A4 and UDP-glucuronosyltransferase 2B7: evidence for protein-protein association and possible involvement of CYP3A4 J-helix in the interaction.

We have reported that the protein-protein interaction between UDP-glucuronosyltransferase (UGT) 2B7 and cytochrome P450 3A4 (CYP3A4) alters UGT2B7 function. However, the domain(s) involved in the interaction are largely unknown. To address this issue, we examined in more detail the CYP3A4-UGT2B7 association by means of immunoprecipitation, overlay assay, and cross-linking involving 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide.

Induction of the hepatic cytochrome P450 2B subfamily by xenobiotics: research history, evolutionary aspect, relation to tumorigenesis, and mechanism.

The cytochrome P450 belonging to the CYP2B subfamily has long been of great interest because it can be induced by xenobiotics. While a well known diagnostic ligand-receptor theory explains the induction of the CYP1A subfamily, the mechanism by which xenobiotics induce the CYP2B subfamily is not fully understood. Although the constitutive androstane receptor (CAR) undoubtedly plays a crucial role in the induction, many questions regarding the mechanism of CAR activation by xenobiotics have not yet been answered.

Inhibition of UDP-glucuronosyltransferase 2b7-catalyzed morphine glucuronidation by ketoconazole: dual mechanisms involving a novel noncompetitive mode.

Glucuronidation of morphine in humans is predominantly catalyzed by UDP-glucuronosyltransferase 2B7 (UGT2B7). Since our recent research suggested that cytochrome P450s (P450s) interact with UGT2B7 to affect its function [Takeda S et al. (2005) Mol Pharmacol 67:665-672], P450 inhibitors are expected to modulate UGT2B7-catalyzed activity.

Modulation of UDP-glucuronosyltransferase 2B7 function by cytochrome P450s in vitro: differential effects of CYP1A2, CYP2C9 and CYP3A4.

Effects of cytochrome P450 isoforms, CYP1A2, CYP2C9 and CYP3A4, on the catalytic activity of UDP-glucuronosyltransferase 2B7 (UGT2B7) expressed in COS cell microsomal membranes were investigated using morphine as a substrate. When detergent-untreated COS cell microsomes were used as the enzyme source, the activity of morphine-3-glucuronide formation by UGT2B7 was reduced by addition of purified CYP1A2 and CYP2C9 in a concentration-dependent manner. The effect of CYP1A2 was greater than that of CYP2C9.

[Possible candidates for the compound which is expected to attenuate dioxin toxicity].

This review deals with the three candidate compounds which may combat with dioxins' toxicity. Geranylgeranylacetone (GGA), an antiulcer drug, counteracts suppression of body weight gain and lethality produced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in C57BL/6J mice. Similarly, curcumin, a food ingredient anticipates the TCDD's toxicity on body weight gain.

Suppression of carbonic anhydrase III mRNA level by an aryl hydrocarbon receptor ligand in primary cultured hepatocytes of rat.

The effect of an aryl hydrocarbon receptor (AhR) ligand on the carbonic anhydrase III (CAIII) mRNA level was studied using primary cultured hepatocytes of rats. CAIII gene which is highly suppressible by dioxins in vivo, was also suppressible in primary cultured hepatocytes of rats by an AhR ligand, 3-methylchlanthrene (3MC). The suppression of CAIII by 3MC was observed in a dose-dependent fashion.

Modulation of UDP-glucuronosyltransferase function by cytochrome P450: evidence for the alteration of UGT2B7-catalyzed glucuronidation of morphine by CYP3A4.

Modulation of UDP-glucuronosyltransferase 2B7 (UGT2B7)-catalyzed morphine glucuronidation by cytochrome P450 (P450) was studied. The effects of P450 isozymes on the kinetic parameters of UGT2B7-catalyzed glucuronidation of the morphine 3-hydroxyl group were examined by simultaneous expression of UGT2B7 and either CYP3A4, -1A2, or -2C9 in COS-1 cells. Although coexpression of CYP3A4 with UGT2B7 had little effect on Vmax, the Km was increased by about 9.

Functional protein-protein interaction of drug metabolizing enzymes.

Cytochrome P450 (P450, CYP), a major class of enzymes involved in Phase I drug metabolism, is expressed in the cellular endoplasmic reticulum together with other enzymes, such as microsomal epoxide hydrolase (mEH) and UDP-glucuronosyltransferase (UGT). In many cases, the metabolite produced by P450 is sequentially metabolized by other enzymes to increase its water solubility. It would be reasonable to assume that the metabolite produced by P450 is directly transferred to the other enzymes participating in its subsequent metabolism via protein-protein interactions for rapid metabolism.

Enhancement of acetaminophen cytotoxicity in selenium-binding protein-overexpressed COS-1 cells.

The role of selenium-binding protein (SeBP), which has a high ability to associate with acetaminophen (AAP), on the cytotoxicity of AAP was studied. To clarify this issue, we examined the cytotoxic effect of AAP using COS cells stably expressing SeBP. Expression of SeBP enhanced the susceptibility of the cells to AAP-induced cytotoxicity.

Simultaneous expression of guinea pig UDP-glucuronosyltransferase 2B21 (UGT2B21) and 2B22 in COS-7 cells enhances UGT2B21-catalyzed chloramphenicol glucuronidation.

Our previous study suggested that hetero-oligomer formation of guinea pig liver UDP-glucuronosyltransferases (UGTs) 2B21 and 2B22 enhances UGT2B21-catalyzed morphine-6-glucuronidation. In this work, further evidence for a functional hetero-oligomer between UGT2B21 and UGT2B22 was provided by studies of the glucuronidation of chloramphenicol with dual expression in COS-7 cells. UGT2B21 expressed in COS cells was capable of glucuronidating the 3-hydroxyl group of morphine, 4-hydroxybiphenyl, borneol, testosterone, androsterone, and estriol, whereas it had some effect on chloramphenicol.

Reduction of the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in mice using an antiulcer drug, geranylgeranylacetone.

The protective effect of geranylgeranylacetone (GGA), an antiulcer drug, against the acute toxicity and teratogenicity produced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was examined in C57BL/6J mice. When mice were co-treated, GGA reduced the loss of body weight gain and lethality produced by TCDD but hepatomegaly and thymic atrophy were not improved. Additionally, no protective effect of GGA was observed in the formation of cleft palate and hydronephrosis in mouse fetuses caused by maternal exposure to TCDD.

Comparison of genomic and cDNA sequences of guinea pig CYP2B18 and rat CYP2B2: absence of a phenobarbital-responsive enhancer module in the upstream region of the CYP2B18 gene.

Potential mechanisms were investigated whereby CYP2B18, a cytochrome P450 gene exhibiting high constitutive expression but only low levels of phenobarbital-inducibility in the guinea pig liver, may be differentially regulated versus the highly inducible rat CYP2B2 gene. To comparatively assess potential regulatory sequences associated with CYP2B18, a guinea pig genomic library was screened enabling isolation of the CYP2B18 gene. The genomic screening process resulted in the identification of at least four closely-related CYP2B18 genes, designated here as CYP2B18A-D.

Formation of highly analgesic morphine-6-glucuronide following physiologic concentration of morphine in human brain.

3H-Morphine at physiologic concentration was metabolized in vitro to its 3- and 6-glucuronides by human brain homogenate. Recombinant UGT2B7, one of the UDP-glucuronosyltransferase (UGT) isoforms, is able to glucuronidate the 3- and 6-hydroxy groups of morphine at nanomolar concentrations. These results suggest that endogenous morphine is converted to its 6-glucuronide, a more highly analgesic substance than the parent compound, to suppress effectively pain symptoms in humans.

Cytochrome P450 1A1 (CYP1A1) inhibitor alpha-naphthoflavone interferes with UDP-glucuronosyltransferase (UGT) activity in intact but not in permeabilized hepatic microsomes from 3-methylcholanthrene-treated rats: possible involvement of UGT-P450 interactions.

The effects of cytochrome P450 (P450, CYP) ligands and permeabilization of microsomes on 3-hydroxybenzo(a)pyrene [3-OH-B(a)P] glucuronidation mediated by rat hepatic microsomes were studied. While the UDP-glucuronosyltransferase (UGT) activity with non-permeabilized microsomes from 3-methylcholanthrene (MC)-treated rats was markedly reduced by alpha-naphthoflavone (NF), this inhibitor had hardly any effect when permeabilized microsomes were used in which the inhibitor was expected to have easy access to UGT. Kinetic analysis indicated that the inhibitory effect of alpha-NF is competitive.

Activation of microsomal epoxide hydrolase by interaction with cytochromes P450: kinetic analysis of the association and substrate-specific activation of epoxide hydrolase function.

The kinetics of the association between cytochrome P450 (P450) and microsomal epoxide hydrolase (mEH) was studied by means of resonant mirror based on the principle of surface plasmon resonance. The dissociation equilibrium constants (K(D)) for the affinity of P450 enzymes for mEH were estimated by resonant mirror using an optical biosensor cell covalently bound to rat mEH. Comparable K(D) values were obtained for CYP1A1 and 2B1, and these were greater by one order of magnitude than that for the CYP2C11.

Estrogen-dependent regulation of the expression of hepatic Cyp2b and 3a isoforms: assessment using aromatase-deficient mice.

The role of estrogen in the expression and induction of hepatic Cyp2b and Cyp3a isoforms was studied using mice [Ar (-/-) mice] lacking aromatase, a key enzyme for estrogen biosynthesis. The expression of P450s was determined by reverse transcription-polymerase chain reaction, immunoblotting, and measuring testosterone 6beta- and 16alpha-hydroxylase activity as markers. Basic expression of Cyp3a11 mRNA and protein was seen in both sexes of Ar (+/+) mice.

Dansyl chloride derivatization of methamphetamine: a method with advantages for screening and analysis of methamphetamine in urine.

The screening and quantitation of methamphetamine (MP) in urine using dansyl chloride (DNC) as the derivatization reagent were studied. Urinary MP derivatized with DNC could be detected by visual observation of the fluorescence in a solid-phase extraction column such as a Sep-Pak C18 cartridge to which the whole reaction solution was applied. The DNC-derivatized MP was eluted from the cartridge and then identified and quantitated by gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC).