Inhibition of human UGT2B7 gene expression in transgenic mice by the constitutive androstane receptor.

The xenobiotic receptors, constitutive androstane receptor (CAR), and pregnane X receptor (PXR) regulate and alter the metabolism of xenobiotic substrates. Among the 19 functional UDP-glucuronosyltransferases (UGTs) in humans, UGT2B7 is involved in the metabolism of many structurally diverse xenobiotics and plays an important role in the clearance and detoxification of many therapeutic drugs. To examine whether this gene is regulated by CAR and PXR in vivo, transgenic mice expressing the entire UGT2B7 gene (TgUGT2B7) were created. Gene expression profiles revealed that UGT2B7 is differentially expressed in liver, kidney, adipocytes, brain, and estrogen-sensitive tissues, such as ovary and uterus. Liver UGT2B7 expression levels were decreased when TgUGT2B7 mice were treated with the CAR ligand 1,4-b-s-[2-(3,5,-dichloropyridyloxy)] (TCPOBOP) but not the PXR ligand pregnenolone 16α-carbonitrile. Although TCPOBOP decreased the levels of UGT2B7 mRNA in TgUGT2B7 mice, it had no affect on Tg(UGT2B7)Car(-/-) mice, adding support for a CAR-dependent mechanism contributing toward UGT2B7 gene suppression. Expression of promoter constructs in HepG2 cells showed the CAR-dependent inhibition was linked to hepatocyte nuclear factor-4α (HNF4α)-mediated transactivation of the UGT2B7 promoter. The inhibitory effect of CAR on UGT2B7 gene expression was validated in chromatin immunoprecipitation assays in which TCPOBOP treatment blocked HNF4α binding to the UGT2B7 promoter. These results suggest that HNF4α plays an important role in the constitutive expression of hepatic UGT2B7, and CAR acts as a negative regulator by interfering with HNF4α binding activity.

The contribution of UDP-glucuronosyltransferase 1A9 on CYP1A2-mediated genotoxicity by aromatic and heterocyclic amines.

The importance of environmental and dietary arylamines, and heterocyclic amines in the etiology of human cancer is of growing interest. These pre-carcinogens are known to undergo bioactivation by cytochrome P450 (CYP)-directed oxidation, which then become substrates for the UDP-glucuronosyltransferases (UGTs). Thus, glucuronidation may contribute to the elimination of CYP-mediated reactive intermediate metabolites, preventing a toxic event. In this study, human UGTs were analyzed for their ability to modulate the mutagenic actions of N-hydroxy-arylamines formed by CYP1A2. Studies with recombinant human UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7 and UGT2B15 expressed in heterologous cell culture confirmed that UGT1A9 glucuronidated the mutagenic arylamines N-hydroxy-2-acetylaminofluorene (N-hydroxy-2AAF) and 2-hydroxyamino-1-methyl-6-phenylimidazo(4,5-b)pyridine (N-hydroxy-PhIP). To examine the mutagenic potential of these agents, a genotoxicity assay was employed using Salmonella typhimurium NM2009, a bacterial strain expressing the umuC SOS response gene fused to a beta-galactosidase reporter lacZ gene. DNA modification results in the induction of the umuC gene and subsequent enhancement of beta-galactosidase activity. Both N-hydroxy-2AAF and N-hydroxy-PhIP stimulated a dose-dependent increase in bacterial beta-galactosidase activity. In addition, the procarcinogens 2AAF and PhIP were efficiently bioactivated to bacterial mutagens when incubated with Escherichia coli membranes expressing CYP1A2 and NADPH reductase. CYP1A2 generated 2AAF- and PhIP-mediated DNA damage, but only the action of N-hydroxy-2AAF was blocked by expressed UGT1A9. These results indicate that UGT1A9 can control the outcome of a genotoxic response. The results also indicate that while a potential toxicant such as N-hydroxy-PhIP can serve as substrate for glucuronidation, its biological actions can exceed the capacity of the detoxification pathway to prevent the mutagenic episode.

Characterization of expressed full-length and truncated FMO2 from rhesus monkey.

Flavin-containing monooxygenase (FMO) metabolizes a wide variety of nitrogen, sulfur, and phosphorous-containing xenobiotics. FMO2 is highly expressed in the lung of most mammals examined, but the protein has only recently been detected in humans, presumably due to a premature stop codon at AA472 in most individuals. In this study, full-length (mFMO2-535) and 3'-truncated (mFMO2-471) monkey FMO2 protein, produced by cDNA-mediated baculovirus expression, were characterized and compared with baculovirus-expressed rabbit FMO2 (rFMO2-535). Although baculovirus-expressed mFMO2-535 had properties similar to FMO in monkey lung microsomes and had catalytic properties similar to rFMO2-535, the expressed proteins differed in a number of properties in S-oxidation assays. Both enzymes had the same pH optima (pH 9.5); however, mFMO2-535 quickly lost activity at higher pH values whereas rFMO2-535 retained the majority of its activity. Also, mFMO2-535 was significantly less stable at elevated temperatures and in the presence of cholic acid but had greater activity in the presence of magnesium. mFMO2-535 had higher apparent K(m) and V(max)/K(m) values than rFMO2-535 did in N-oxygenation assays. mFMO2-471 was correctly targeted to the membrane fraction, but N- and S-oxygenation was not detected. Since the AA sequence identity of mFMO2 and human FMO2 is 97%, our results with mFMO2-535 suggest that individuals carrying the allele encoding full-length FMO2 are likely to have in vivo FMO2 activity. Such activity could result in marked differences in the metabolism, efficacy, and/or toxicity of drugs and xenobiotics for which lung is a portal of entry or target organ.

Ethnic differences in human flavin-containing monooxygenase 2 (FMO2) polymorphisms: detection of expressed protein in African-Americans.

The flavin-containing monooxygenases (FMOs) are a family of xenobiotic-metabolizing enzymes that are expressed in a species- and tissue-specific manner. FMO2 expression has been observed in pulmonary tissue from several species, but not human. Two human FMO2 point mutations have been reported: a cytosine to thymidine transition at position 1414 resulting in a premature stop codon and a thymidine insertion at position 1589 resulting in a frameshift. To define the frequency of these sequence variations and explore their significance, unrelated African-American, Caucasian, and Korean individuals were genotyped. In the African-American population tested (n = 180), the 1414C allele occurred at a 13% frequency; however, all of the tested Caucasians (n = 52) and Koreans (n = 100) were homozygous for the 1414T allele. The T1589 allele occurred at frequencies of 6.9 and 13.0% in African-Americans (n = 175) and Caucasians (n = 23), respectively, and appears to segregate with the 1414T allele. Thus, it would have no further impact on FMO2 activity. Western blot analysis of pulmonary microsomes failed to detect immunoreactive protein in 1414T homozygotes. A heterozygotic individual did exhibit a single band of the expected size, but no detectable FMO activity in the corresponding lung microsomes. Sequence analysis, however, was consistent with the 1414C allele encoding an active FMO2 enzyme. FMO2 mRNA expression was observed in most individuals, but failed to correlate with genotype or protein expression. In summary, functional FMO2 is expressed in only a small percentage of the overall population. However, in certain ethnic groups, active pulmonary FMO2 enzyme will be present in a significant number of individuals.

Flavin-containing monooxygenase isoform 2: developmental expression in fetal and neonatal rabbit lung.

Mammalian flavin-containing monooxygenase functions in the oxygenation of numerous xenobiotics containing a soft nucleophile, usually a nitrogen or sulfur. A total of five distinct flavin monooxygenase (FMO) isoforms are expressed in mammals. Individual isoforms are expressed in a sex-, age-, and tissue-specific fashion. In this study, we document the early developmental appearance of the major isoform in rabbit lung, FMO2. FMO2 catalytic activity as well as protein and mRNA are not only present in fetal and neonatal lung but, in some instances, approach levels found in the adult. The expression pattern of FMO2 is similar to that of the two major constitutive cytochromes P450 found in rabbit lung, 2B4 and 4B1. The early developmental appearance of these monooxygenases indicate an important role in the protection of the fetus and neonate against toxic insult from foreign chemicals.

Pulmonary flavin-containing monooxygenase (FMO) in rhesus macaque: expression of FMO2 protein, mRNA and analysis of the cDNA.

Pulmonary microsomes from Rhesus macaque express a flavin-containing monooxygenase (FMO) resembling the FMO2 ortholog from rabbit with respect to immunochemical cross-reactivity and expression in lung, but not liver. A full-length cDNA was cloned following screening of a Rhesus macaque lung cDNA library. The nucleotide sequence contained an open reading frame encoding 535 amino acids with 85 and 84% identity to FMO2 from rabbit and guinea pig, respectively, and an identical location of the putative FAD- and NADP-binding sites. Northern blots of monkey lung mRNA revealed multiple size FMO2 transcripts. These mRNA transcripts are expressed in lung, but not in liver or kidney.