Isoform distinct time-, dose-, and castration-dependent alterations in flavin-containing monooxygenase expression in mouse liver after 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment.
ABSTRACT: Flavin-containing monooxygenase (FMO) expression in male mouse liver is altered after 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure or castration. Because TCDD is slowly eliminated from the body, we examined hepatic Fmo mRNA alterations for up to 32 days following 10 or 64 microg/kg TCDD exposure by oral gavage in male C57BL/6J mice. Fmo2 mRNA was significantly induced at 1, 4, and 8 days whereas Fmo3 mRNA was also induced at 32 days relative to controls. Fmo3 mRNA levels exhibited a dose-dependent increase at 4, 8, and 32 days after exposure; Fmo1, Fmo4, and Fmo5 mRNA did not exhibit clear trends. Because castration alone also increased Fmo2, Fmo3, and Fmo4 mRNA we examined the combined effects of castration and TCDD treatment on FMO expression. A greater than additive effect was observed with Fmo2 and Fmo3 mRNA expression. Fmo2 mRNA exhibited a 3-5-fold increase after castration or 10 microg/kg TCDD exposure by oral gavage, whereas an approximately 20-fold increase was observed between the sham-castrated control and castrated TCDD-treated mice. Similarly, treatment with 10 microg/kg TCDD alone increased Fmo3 mRNA 130- and 180-fold in the sham-castrated and castrated mice compared to their controls respectively, whereas, Fmo3 mRNA increased approximately 1900-fold between the sham control and castrated TCDD-treated mice. An increase in hepatic Fmo3 protein in TCDD-treated mice was observed by immunoblotting and assaying methionine S-oxidase activity. Collectively, these results provide evidence for isoform distinct time-, dose-, and castration-dependent effects of TCDD on FMO expression and suggest cross-talk between TCDD and testosterone signal transduction pathways.
Project description:Flavin-containing monooxygenases (FMOs) are important in detoxication but generally are considered not to be inducible by xenobiotics. Our recent microarray studies revealed induction of FMO2 and FMO3 mRNAs by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in liver of mice with wild-type aryl hydrocarbon receptor (AHR) but not in Ahr-null mice. The aim of the present study was to delineate mechanisms of FMO regulation. In adult male mice, basal FMO3 mRNA is low but was induced 6-fold at 4 h and 6000-fold at 24 h. The ED50 was approximately 1 microg/kg for FMO2 and FMO3, similar to that for the classic AHR-regulated gene, Cyp1a1. In adult female mice basal FMO3 mRNA is high and was not induced at 4 h but was elevated 8-fold at 24 h. FMO5 mRNA was significantly down-regulated by TCDD in both male and female adult mice. Juvenile mice show no sex difference in response to TCDD; FMO3 was induced 4 to 6-fold by TCDD in both sexes. Chromatin immunoprecipitation demonstrated recruitment of AHR and aryl hydrocarbon nuclear translocator proteins to Fmo3 regulatory regions, suggesting that induction by TCDD is a primary AHR-mediated event. Although FMO2 and FMO3 mRNAs were highly induced by TCDD in adult males, overall FMO catalytic activity increased only modestly. In contrast to the striking up-regulation of FMO2 and FMO3 in mouse liver, TCDD has little effect on FMO mRNA in rat liver. However, FMO2 and FMO3 mRNAs were highly induced in transgenic mice that express wild-type rat AHR, indicating that lack of induction in rat is not due to an incompetent AHR in this species.
Project description:The significance of active versus inactive flavin-containing monooxygenase 2 (FMO2) for human drug and xenobiotic metabolism and sensitivity is unknown, but the underlying ethnic polymorphism is well documented. We used quantitative real-time PCR to measure message levels of Fmo1, Fmo2, Fmo3 and Fmo5 in lung and liver from eight strains of 8 week old female mice to determine if a strain could be identified that predominately expressed Fmo2 in lung, recapitulating the human FMO expression profile and being the ideal strain for Fmo2 knockout studies. We also characterized enzyme activity of baculovirus expressed mouse Fmo1, Fmo2 and Fmo3 to identify a substrate or incubation conditions capable of discriminating Fmo2 from Fmo mixtures. Fmo transcript expression patterns were similar for all strains. In lung, 59% of total FMO message was Fmo2, but Fmo1 levels were also high, averaging 34%, whereas Fmo3 and Fmo5 levels were 2 and 5%, respectively. In liver, Fmo1, Fmo2, Fmo3 and Fmo5 contributed 16, 1, 7 and 76% respectively, of detected message. Peak activity varied by isoform and was pH- and substrate-dependent. Fmo3 oxidation of methyl p-tolyl sulfide was negligible at pH 9.5, but Fmo3 oxidation of methimazole was comparable to Fmo1 and Fmo2. Heating microsomes at 50 degrees C for 10min eliminated most Fmo1 and Fmo3 activity, while 94% of Fmo2 activity remained. Measurement of activity in heated and unheated lung and liver microsomes verified relative transcript abundance. Our results show that dual Fmo1/2 knockouts will be required to model the human lung FMO profile.
Project description:Flavin-containing monooxygenases often are thought not to be inducible but we recently demonstrated aryl hydrocarbon receptor (AHR)-dependent induction of FMO mRNAs in mouse liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Celius et al., Drug Metab Dispos 36:2499, 2008). We now evaluated FMO induction by other AHR ligands and xenobiotic chemicals in vivo and in mouse Hepa1c1c7 hepatoma cells (Hepa-1). In mouse liver, 3-methylcholanthrene (3MC) induced FMO3 mRNA 8-fold. In Hepa-1 cells, 3MC and benzo[a]pyrene (BaP) induced FMO3 mRNA >30-fold. Induction by 3MC and BaP was AHR dependent but, surprisingly, the potent AHR agonist, TCDD, did not induce FMO3 mRNA in Hepa-1 cells nor did chromatin immunoprecipitation assays detect recruitment of AHR or ARNT to Fmo3 regulatory elements after exposure to 3MC in liver or in Hepa-1 cells. However, in Hepa-1, 3MC and BaP (but not TCDD) caused recruitment of p53 protein to a p53 response element in the 5'-flanking region of the Fmo3 gene. We tested the possibility that FMO3 induction in Hepa-1 cells might be mediated by Nrf2/anti-oxidant response pathways, but agents known to activate Nrf2 or to induce oxidative stress did not affect FMO3 mRNA levels. The protein synthesis inhibitor, cycloheximide (which causes "superinduction" of CYP1A1 mRNA in TCDD-treated cells), by itself caused dramatic upregulation (>300-fold) of FMO3 mRNA in Hepa-1 suggesting that cycloheximide prevents synthesis of a labile protein that suppresses FMO3 expression. Although FMO3 mRNA is highly induced by 3MC or TCDD in mouse liver and in Hepa-1 cells, FMO protein levels and FMO catalytic function showed only modest elevation.
Project description:The objective of the study was to investigate the regulation of hepatic flavin-containing monooxygenases (Fmo) Fmo1, Fmo3, Fmo4, and Fmo5 in three different mouse models of inflammation, including treatment with Citrobacter rodentium, lipopolysaccharide (LPS), and dextran sulfate sodium (DSS). Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to evaluate the steady-state mRNA levels for the various Fmo isoforms in these mouse models of inflammation during different treatment time courses. Fmo3 mRNA was most significantly down-regulated in C. rodentium-treated female mice. Fmo1, Fmo3, and Fmo5 mRNAs were also found to be down-regulated in LPS models of inflammation. The significant down-regulation of hepatic FMO3 protein during C. rodentium treatment was confirmed with Western blot analysis of liver microsomes from treated animals. Toll-like receptor (TLR) 4 is known to be responsible for LPS signaling in association with several proteins. To investigate whether TLR4 was responsible for regulation of Fmo genes in both LPS and C. rodentium animal models, Fmo mRNA levels in female wild-type (C3H/HeOuJ) and TLR4 mutant (C3H/HeJ) mice were compared in both inflammatory models by real-time RT-PCR. The results showed that Fmo3 down-regulation during C. rodentium infection is independent of TLR4. Whereas TLR4 is likely to play only a partial role in Fmo1 gene regulation in LPS-treated animals, our results show that the down-regulation of Fmo3 and Fmo5 in this model is TLR4-dependent. Unlike cytochrome P450 regulation measured in the same mouse strains, Fmo3 expression was largely refractory to down-regulation in the DSS model of inflammatory colitis.
Project description:Flavin-containing monooxygenase (FMO) oxygenates drugs/xenobiotics containing a soft nucleophile through a C4a hydroperoxy-FAD intermediate. Human FMOs 1, 2 and 3, expressed in Sf9 insect microsomes, released 30-50% of O? consumed as H?O? upon addition of NADPH. Addition of substrate had little effect on H?O? production. Two common FMO2 (the major isoform in the lung) genetic polymorphisms, S195L and N413K, were examined for generation of H?O?. FMO2 S195L exhibited higher "leakage", producing much greater amounts of H?O?, than ancestral FMO2 (FMO2.1) or the N413K variant. S195L was distinct in that H?O? generation was much higher in the absence of substrate. Addition of superoxide dismutase did not impact H?O? release. Catalase did not reduce levels of H?O? with either FMO2.1 or FMO3 but inhibited H?O? generated by FMO2 allelic variants N413K and S195L. These data are consistent with FMO molecular models. S195L resides in the GxGxSG/A NADP(+) binding motif, in which serine is highly conserved (76/89 known FMOs). We hypothesize that FMO, especially allelic variants such as FMO2 S195L, may enhance the toxicity of xenobiotics such as thioureas/thiocarbamides both by generation of sulfenic and sulfinic acid metabolites and enhanced release of reactive oxygen species (ROS) in the form of H?O?.
Project description:Flavin-containing monooxygenase (FMO) oxygenates drugs and xenobiotics containing a "soft-nucleophile", usually nitrogen or sulfur. FMO, like cytochrome P450 (CYP), is a monooxygenase, utilizing the reducing equivalents of NADPH to reduce 1 atom of molecular oxygen to water, while the other atom is used to oxidize the substrate. FMO and CYP also exhibit similar tissue and cellular location, molecular weight, substrate specificity, and exist as multiple enzymes under developmental control. The human FMO functional gene family is much smaller (5 families each with a single member) than CYP. FMO does not require a reductase to transfer electrons from NADPH and the catalytic cycle of the 2 monooxygenases is strikingly different. Another distinction is the lack of induction of FMOs by xenobiotics. In general, CYP is the major contributor to oxidative xenobiotic metabolism. However, FMO activity may be of significance in a number of cases and should not be overlooked. FMO and CYP have overlapping substrate specificities, but often yield distinct metabolites with potentially significant toxicological/pharmacological consequences. The physiological function(s) of FMO are poorly understood. Three of the 5 expressed human FMO genes, FMO1, FMO2 and FMO3, exhibit genetic polymorphisms. The most studied of these is FMO3 (adult human liver) in which mutant alleles contribute to the disease known as trimethylaminuria. The consequences of these FMO genetic polymorphisms in drug metabolism and human health are areas of research requiring further exploration.
Project description:We have previously reported the cloning of cDNAs for a flavin-containing mono-oxygenase (FMO) of man, designated FMO1 [Dolphin, Shephard, Povey, Palmer, Ziegler, Ayesh, Smith & Phillips (1991) J. Biol. Chem. 266, 12379-12385], that is the orthologue of pig and rabbit hepatic FMOs. We now describe the isolation and characterization of cDNA clones for a second human FMO, which we have designated FMO2. The polypeptide encoded by the cDNAs is 558 amino acid residues long, has a calculated M(r) of 63337, and contains putative FAD- and NADP-binding sites that align exactly with those described in other mammalian FMOs. Human FMO2 has 51-53% primary sequence identity with human FMO1, rabbit pulmonary FMO and rabbit liver FMO form 2, and thus represents a fourth, distinct, member of the mammalian FMO family. The corresponding mRNA is present in low abundance in adult human liver. Southern blot hybridization with single-exon probes demonstrated that human FMO2 and FMO1 are the products of single genes. The gene encoding FMO2 (designated FMO2) was mapped, by the polymerase chain reaction, to human chromosome 1, the same chromosome on which FMO1 is located.
Project description:Flavin-containing monooxygenases (FMO) catalyze the metabolism of nucleophilic heteroatom-containing drugs and xenobiotics, including nicotine. Rare mutations in FMO3 are responsible for defective N-oxidation of dietary trimethylamine leading to trimethylaminuria, and common genetic variation in FMO3 has been linked to interindividual variability in metabolic function that may be substrate specific.A genetic model of CYP2A6 function is used as a covariate to reveal functional polymorphism in FMO3 that indirectly influences the ratio of deuterated nicotine metabolized to cotinine following oral administration. The association is tested between FMO3 haplotype and cigarette consumption in a set of nicotine-dependent smokers.FMO3 haplotype, based on all common coding variants in Europeans, significantly predicts nicotine metabolism and accounts for ?2% of variance in the apparent percent of nicotine metabolized to cotinine. The metabolic ratio is not associated with FMO2 haplotype or an FMO1 expression quantitative trait locus. Cross-validation demonstrates calculated FMO3 haplotype parameters to be robust and significantly improve the predictive nicotine metabolism model over CYP2A6 genotype alone. Functional classes of FMO3 haplotypes, as determined by their influence on nicotine metabolism to cotinine, are also significantly associated with cigarettes per day in nicotine-dependent European Americans (n=1025, P=0.04), and significantly interact (P=0.016) with CYP2A6 genotype to predict cigarettes per day.These findings suggest that common polymorphisms in FMO3 influence nicotine clearance and that these genetic variants in turn influence cigarette consumption.
Project description:Conventional biochemical and molecular techniques identified previously several genes whose expression is regulated by the aryl hydrocarbon receptor (AHR). We sought to map the complete spectrum of AHR-dependent genes in male adult liver using expression arrays to contrast mRNA profiles in Ahr-null mice (Ahrâ/â) with those in mice with wild-type AHR (Ahr+/+). Transcript profiles were determined both in untreated mice and in mice treated 19 h earlier with 1000 Âµg/kg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Expression of 456 ProbeSets was significantly altered by TCDD in an AHR-dependent manner, including members of the classic AHRE-I gene battery, such as Cyp1a1, Cyp1a2, Cyp1b1, and Nqo1. In the absence of exogenous ligand, AHR status alone affected expression of 392 ProbeSets, suggesting that the AHR has multiple functions in normal physiology. In Ahrâ/â mice, only 32 ProbeSets exhibited responses to TCDD, indicating that the AHR is required for virtually all transcriptional responses to dioxin exposure in liver. The flavin-containing monooxygenases, Fmo2 and Fmo3, considered previously to be uninducible, were highly induced by TCDD in an AHR-dependent manner. The estrogen receptor alpha as well as two estrogen-receptor-related genes (alpha and gamma) exhibit AHR-dependent expression, thereby extending cross-talk opportunities between the intensively studied AHR and estrogen receptor pathways. p53 binding sites are over-represented in genes down-regulated by TCDD, suggesting that TCDD inhibits p53 transcriptional activity. Overall, our study identifies a wide range of genes that depend on the AHR, either for constitutive expression or for response to TCDD. Experiment Overall Design: Mice bearing wild-type or genetically deleted AHRs were treated with corn oil vehicle or TCDD, and their livers analyzed by expression arrays.
Project description:The most appropriate time to introduce androgen deprivation therapy for prostate cancer remains controversial. Our aim was to evaluate the effects of early versus delayed surgical castration on prostate cancer progression and survival in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. TRAMP mice were randomly divided into three groups: the early castration group (on which castration was performed at the age of 4 weeks), the delayed castration group (on which castration was performed when abdominal tumours could be palpated), and the sham-castrated group. Mice were monitored daily throughout their lives until cancer-related death or the development of an obviously moribund appearance, at which time the individual mouse was killed. Androgen receptor expression in prostate tumours was also evaluated. The results shows that the average lifespan in early castration, delayed castration and sham-castrated groups were 54.1 weeks, 59.9 weeks and 39.1 weeks, respectively. Both early castration and delayed castration conferred a statistically significant survival advantage when compared with the sham-castrated group (P<0.001). However, the difference in lifespan between the early castration group and the delayed castration group was not statistically significant (P=0.85). The increase in lifespan in the TRAMP mice that received either early or delayed castration correlated with lower G/B value (genitourinary tract weight/body weight) at death than the sham-castrated mice. In conclusion, early and delayed castrations in TRAMP mice prolonged survival to a similar extent. This finding may provide a guide for clinical practice in prostate cancer therapy.