FMO3 Expression Research Articles

222-LB: Diabetes Induces Liver Inflammation and FGF21 through C-Jun NH2-Terminal Kinase Pathways

Fibroblast growth factor 21 (FGF-21) is a hormone that regulates important metabolic pathways. Serum fibroblast FGF-21 levels were significantly higher in obesity and type 2 diabetes mellitus (T2DM). Previously, we have found that the JNK pathway played a critical role in diabetes-induced Kupffer cell activation, and pJNK1 inhibition decreases Kupffer cell activation induced by diabetes. We aim to study the involvement c-Jun NH2-terminal kinase pathways in the regulatory mechanisms of liver inflammation and FGF-21 in diabetes. Type 1 diabetic Akita mice (C57BL/6J-Ins2Akita) and type 2 diabetic Leprdb/db mice (C57BL/6J- Leprdb/db) were used. Leprdb/db-JNK1-/- and Ins2Akita-JNK1-/- mice were produced and used to study the role of pJNK in the regulatory mechanisms of diabetes on liver inflammation and FGF-21. Diabetic mice demonstrated increased ICAM, iNOS, interleukin 1 beta (IL-1β), FGF-21, and FMO3 expression of liver; and tumor necrosis factor alpha (TNF-α), and iNOS expression in Kupffer cells; and plasma FGF-21 levels. Ins2Akita-JNK1-/- and Leprdb/db-JNK1-/- mice demonstrated decreased ICAM, iNOS, IL-1β, and FMO3 expression of liver; and ICAM, iNOS, and FMO3 expression in aorta, tumor necrosis factor alpha (TNF-α), and iNOS expression in Kupffer cells, plasma IL-6 and TNF-α levels; dipeptidyl peptidase (DPP-4) activity. JNK inhibitor, SP600125, injection into adipose tissue of Leprdb/db mice significantly decreased ICAM, iNOS, IL-1β, and FMO3 expression of liver and serum FGF-21 levels. Altogether, our data suggest that diabetes induces liver inflammation, FGF-21, and insulin resistance through c-Jun NH2-terminal kinase. Disclosure L. Chen: None. Funding National Science Council (MOST 107-2314-B-010-043-MY3)

Supplementation of endogenous Ahr ligands reverses insulin resistance and associated inflammation in an insulin-dependent diabetic mouse model

Aryl-hydrocarbon receptor (Ahr) plays an important role in the regulation of intestinal homeostasis.Diabetes is characterized by vascular complications and intestinal dysfunction. We aimed at understanding the relationship between intestinal defense impairment and inflammation in diabetes and effects of Ahr ligands on diabetes-induced insulin resistance, endovascular inflammation, and intercellular adhesion molecule (ICAM) and flavin mono-oxygenase (FMO3) expression. Effects of Ahr ligands, such as tryptophan (Trp) and indole-3-carbinol (I3C) on intestinal barrier and inflammation of Ins2Akita mice were examined. Myeloid differentiation primary response 88 (MYD88) is the adaptor for inflammatory signaling pathways. Ins2Akita-MyD88-/- mice were used to study the role of MyD88. Ins2Akita mice demonstrated decreased Ahr and regenerating islet-derived 3-β (Reg3β) expression, and increased Klebsiella pneumoniae translocation. Ins2Akita mice demonstrated increased inducible nitric oxide synthase (iNOS) expression of intestine; ICAM, iNOS, interleukin 1 beta (IL-1β), and FMO3 expression of liver; and ICAM, iNOS, and FMO3 expression in aorta. Trp and I3C decreased diabetes-induced translocation and increased Ahr and Reg3β expression of intestine. Ahr ligands reduced diabetes-induced ICAM and FMO3 expression in liver and aorta; IL-6, tumor necrosis factor alpha (TNF-α), and iNOS expression in Kupffer cells; plasma IL-6 and TNF-α levels; dipeptidyl peptidase (DPP4) activity; and insulin insensitivity. Ins2Akita-MyD88-/- mice demonstrated decreased expression of p-NF-κB of liver and ICAM of aorta compared with Ins2Akita mice. Altogether, our data suggest that diabetes induces ICAM and FMO3 expression through the decrease in intestinal defense and MyD88. Ahr ligands reverse diabetes-induced intestinal defense impairment, insulin insensitivity, FMO3/ICAM expression, and systemic inflammation.

FMO1 gene expression independently predicts favorable recurrence-free survival of classical papillary thyroid cancer.

Aim:To examine the expression profile of FMO1 in papillary thyroid cancer (PTC) and its prognostic value in recurrence-free survival (RFS). Methods:A retrospective analysis was performed using data from the Cancer Genome Atlas and Human Protein Atlas. Results: The most frequent variants of PTC had decreased FMO1 expression compared with their respective adjacent normal tissues. However, even under the best cut-off model, high FMO1 expression was only significantly associated with better RFS in classical PTC (p<0.001), but not in other two variants. High FMO1 expression independently predicted favorable RFS (hazard ratio: 0.202; 95%CI: 0.084-0.487;p<0.001) in classical PTC. Conclusion: High FMO1 expression might serve as a biomarker that independently predicts favorable RFS in classical PTC patients.

Effects of fenbendazole and triclabendazole on the expression of cytochrome P450 1A and flavin-monooxygenase isozymes in bovine precision-cut liver slices

Combinations of the anthelmintics fenbendazole (FBZ) and triclabendazole (TCBZ) have shown enhanced efficacy against the liver fluke Fasciola hepatica. This study aimed to measuring the constitutive expression of CYP1A1, CYP1A2, FMO1 and FMO3, thought to be involved in the metabolism of those compounds, by using an absolute quantitative real time (RT)-PCR approach in bovine precision-cut liver slices (PCLS). It also aimed to characterize the effects of FBZ and TCBZ (alone and in combination) on the expression and activity of the aforementioned isozymes.Both FMO1 and FMO3 were equally represented in control PCLS, whereas CYP1A2 was expressed more than CYP1A1 (P<0.05). PCLS cultured in the presence of beta naphthoflavone (β-NF; CYP1A inducer) had higher mRNA levels of CYP1A1, CYP1A2, FMO1 and FMO3 (P<0.05). No clear-cut evidence of transcriptional effects of the anthelmintics were recorded. After incubation of PCLS with FBZ, there was a significant increase (P<0.05) vs. controls and TBCZ was observed for CYP1A1. PCLS treated with FBZ showed a higher (P<0.05) expression of CYP1A2 compared to controls, TCBZ alone, and the combination FBZ+TCBZ. The gene expression profiles of FMO1 and FMO3 were not affected by the presence of the anthelmintics; the only exception was an upregulation of FMO3 by TCBZ alone. The observed transcriptional effects of the xenobiotics were not mirrored by increased enzyme activities using prototypical substrates of the isozymes under study. Although further confirmatory studies are needed, these results suggest that PCLS represent an alternative in vitro tool for studies on the expression, regulation and function of relevant xenobiotic-metabolizing enzymes in cattle.

Berberine treatment reduces atherosclerosis by mediating gut microbiota in apoE-/- mice

BackgroundBerberine (BBR) has long been used for treating bacterial diarrhea due to its antimicrobial effect and is currently used to treat obesity, diabetes, hyperlipemia and atherosclerosis. Given the poor oral bioavailability of BBR, the mechanisms through which BBR mediates metabolic disorders are not well understood. The present study was designed to explore the role of BBR-induced gut microbiota modulation in the development of atherosclerosis. MethodsMale apoE-/- mice were fed a high-fat diet (HFD) with or without the intragastric administration of BBR. Because mice are coprophagic and can transfer their gut microbiota to each other, we cohoused BBR-treated HFD-mice with non-BBR-treated HFD-fed mice. ResultsAfter 12 weeks of HFD feeding, compared with non-BBR-treated HFD-fed mice, BBR-treated HFD-fed mice exhibited a significant reduction in both atherosclerosis development and inflammatory cytokine expression. In addition, cohousing BBR-treated HFD-fed mice with non-BBR-treated HFD-fed mice decreased atherosclerosis development and inflammatory cytokine expression. The denaturing gradient gel electrophoresis and principal component analyses showed that the gut microbial profiles of BBR-treated HFD-fed mice were significantly different from those of HFD-fed mice but were similar to those of cohoused mice. The abundances ofFirmicutes and Verrucomicrobia in cohoused and BBR-treated mice were different from those in HFD-fed and normal chow-fed mice. Moreover, BBR reduced hepatic FMO3 expression and serum trimethylamine N-oxide levels. ConclusionThe antiatherosclerotic effect of BBR is related to alterations in gut microbiota compositions, indicating the potential therapeutic value of pharmacological approaches that may modulate the gut microbiota in treating atherosclerosis.

Abstract P145: The Bacterial Metabolite Trimethylamine (TMA), but Not Trimethylamine N-Oxide (TMAO), Causes Vascular Contraction

Circulating levels of TMAO are elevated in humans with cardiometabolic diseases. The gut microbiota-initiated TMA/flavin-containing monooxygenase 3 (FMO3)/TMAO pathway has been identified as a potential modulator of host cardiometabolic phenotypes. Like TMA/TMAO, FMO3 expression is positively correlated with body mass index and high-fat diet-induced obesity We hypothesized that TMA/TMAO stimulate vascular contraction which could be modified by perivascular adipose tissue (PVAT) expression of FMO3. Our model was the isolated thoracic aorta with (+) or without (-) PVAT, in presence (+) or in absence (+) of endothelium (E) of male Sprague Dawley rat to perform tissue bath studies to measure isometric tone using a large range of TMA/TMAO concentrations: 1 nM-10 -1 M. Immunohistochemistry (IHC) studies were done to identify the presence of FMO3 in aorta. TMA and TMAO, at any concentration, did not relax half maximally contracted tissues when compared to vehicle control. TMA and TMAO elicited arterial contraction independent of PVAT or E status. Interestingly, contractions stimulated by TMA were significantly higher (% PE max 123.7±15.6%) than by TMAO (% PE max 38.3±15.5%). IHC studies revealed FMO3 in aortic PVAT, but the FMO3 inhibitor methimazole (100 μM) did not affect aortic contraction stimulated by TMA (+PVAT). The L type Ca2+ channel antagonist nifedipine (100 nM), reduced the TMA-induced contraction (% PE max: +PVAT: 73±9%; -PVAT: 63±6.7%), compared to vehicle (+PVAT: 149.8±18%; -PVAT: 111.8±9%). These results support that TMA can induce arterial contraction with higher potency and efficacy compared to TMAO, independent of the conversion to TMAO. The contractile mechanism is ≈ 50% dependent of Ca 2+ influx through L-type VOC. Considering the high concentration needed to achieve the contraction it is unlikely that increasing vascular tone is a mechanism that could contribute to CVD but dependence of contraction on Ca 2+ channel activation suggests specific activity of TMA in the vessel wall that warrants further study.

Decreased Kidney Function Is Associated with Enhanced Hepatic Flavin Monooxygenase Activity and Increased Circulating Trimethylamine N-Oxide Concentrations in Mice.

Circulating trimethylamine N-oxide (TMAO) predicts poor cardiovascular outcomes in patients with chronic kidney disease (CKD). Accumulation of serum TMAO has been observed in CKD patients; however, the mechanisms contributing to this finding have been inadequately explored. The purpose of this study was to investigate the mechanisms responsible for TMAO accumulation in the setting of decreased kidney function using a CKD mouse model. Mice were fed a diet supplemented with 0.2% adenine to induce CKD, which resulted in increased serum TMAO concentrations (females: CKD 29.4 ± 32.1 μM vs. non-CKD 6.9 ± 6.1 μM, P < 0.05; males: CKD 18.5 ± 13.1 μM vs. non-CKD 1.0 ± 0.5 μM, P < 0.001). As anticipated, accumulation of circulating TMAO was accompanied by a decrease in renal clearance (females: CKD 5.2 ± 3.8 μl/min vs. non-CKD 90.4 ± 78.1 μl/min, P < 0.01; males: CKD 10.4 ± 8.1 μl/min vs. non-CKD 260.4 ± 134.5 μl/min; P < 0.001) and fractional excretion of TMAO. Additionally, CKD animals exhibited an increase in hepatic flavin monooxygenase (FMO)-mediated formation of TMAO (females: CKD 125920 ± 2181 pmol/mg per 60 minutes vs. non-CKD 110299 ± 4196 pmol/mg per 60 minutes, P < 0.001; males: CKD 131286 ± 2776 pmol/mg per 60 minutes vs. non-CKD 74269 ± 1558 pmol/mg per 60 minutes, P < 0.001), which likely resulted from increased FMO3 expression in CKD mice. The current study provides evidence that both decreased renal clearance and increased hepatic production of TMAO may contribute to increments in serum TMAO in the setting of CKD. Hepatic FMO activity may represent a novel therapeutic target for lowering circulating TMAO in CKD patients.

Development of a Physiologically Based Pharmacokinetic Model of Ethionamide in the Pediatric Population by Integrating Flavin-Containing Monooxygenase 3 Maturational Changes Over Time.

Currently, ethionamide is the most frequently prescribed second-line antituberculosis drug in children. After extensive metabolism by flavin-containing monooxygenase (FMO) isoform 3 in the liver, the drug may exert cytotoxic effects. The comparison of children in different age groups revealed a significant age-related increase in ethionamide elimination in vivo. However, to date, the exact mechanism underlying this dynamic increase in ethionamide elimination has not been elucidated. We hypothesized that the age-dependent changes in ethionamide elimination were predominantly a result of the progressive increases in the expression and metabolic capacity of FMO3 during childhood. To test this hypothesis, a full physiologically based pharmacokinetic (PBPK) model of ethionamide was established and validated in adults through incorporation of comprehensive metabolism and transporter profiles, then expanded to the pediatric population through integration of FMO3 maturational changes over time. Thus, a good prediction PBPK model was validated successfully both in adults and children and applied to demonstrate the critical contribution of FMO3 in the mechanistic elimination pathway of ethionamide. In addition, a significant correlation between clearance and age was observed in children by accounting for ethionamide maturation, which could offer a mechanistic understanding of the age-associated changes in ethionamide elimination. In conclusion, this study underlines the benefits of in vitro-in vivo extrapolation and a quantitative PBPK approach for the investigation of transporter-enzyme interplay in ethionamide disposition and the demonstration of FMO3 ontogeny in children.

Lactobacillus plantarum reverse diabetes-induced Fmo3 and ICAM expression in mice through enteric dysbiosis-related c-Jun NH2-terminal kinase pathways.

Diabetes mellitus (DM) is characterized by increased fatality associated with the atherogenetic process. Circulating trimethylamine-N-oxide (TMAO) levels are closely associated with atherosclerosis. The flavin mono-oxygenase family (Fmo) members oxidize trimethylamine (TMA) to TMAO. The effect and the regulatory mechanism of intestinal microflora on diabetes-induced Fmo3 and intercellular adhesion molecule (ICAM) expression were examined in streptozotocin-induced diabetic mice (STZDM) and Akita mice (C57BL/6J-Ins2Akita). STZDM-JNK1-/- and Ins2Akita-JNK1-/- mice were produced and used to study the role of pJNK in the regulatory mechanisms. Diabetic mice exhibited decreased Lactobacilli growth and reactive oxygen species (ROS) production in the intestinal mucosa; increased levels of pJNK and iNOS proteins in the intestinal mucosa; increased levels of serum nitrate, IL-1β, and TNF-α expression in Kupffer cells; increased Fmo3 expression in the liver; and increased ICAM expression in the aorta. Reversal of diabetes-induced enteric dysbiosis by prebiotic (FOS) or probiotic (dead L. plantarum) treatment decreased diabetes-induced pJNK and iNOS expression in the intestine, Fmo3 expression in the liver, IL-1β expression in Kupffer cells, and ICAM expression in the aorta and liver. Ins2Akita-JNK1-/- and STZDM-JNK1-/- mice demonstrated decreased levels of serum NO, IL-1β expression in Kupffer cells, Fmo3 expression in the liver, and ICAM expression in the aorta. GF mice cohoused with DM mice demonstrated an increase in ICAM expression in the liver. In conclusion, diabetes induced the expression of both Fmo3 and ICAM expression and possible vascular impairment through enteric dysbiosis. Diabetes-induced Fmo3 and ICAM expression could be reversed by pJNK inhibition or by correcting enteric dysbiosis.

Pharmacogenetic profiling and metabolic activity of human embryonic stem cell derived hepatocytes: focus on CYP450-mediated oxidation

&lt;p class="ADMETkeywordsheading"&gt;Primary human hepatocytes are commonly used to evaluate liver drug metabolism and toxicity. Pluripotent stem cell derived hepatocytes (SCDHs) have the potential to overcome access and function-related limitations associated with primary hepatocytes. SCDHs may also provide an improved system for evaluating genotype-phenotype relationships, e.g. cytochrome P450 (CYP) gene polymorphisms and their impact on drug metabolism and toxicity. However, in order for SCDHs to become routinely used in preclinical drug metabolism and toxicity screening, they must demonstrate reproducible activity of drug metabolism proteins, particularly the oxidative CYP enzymes. We characterized the differentiation status of SCDHs, with emphasis on the expression and functional capacity of drug-metabolizing enzymes, and genetically profiled the commonly used WiCell® hESC lines, focusing on CYP2D6 in our analysis. An assay of enzymatic function in SCDHs using a CYP cocktail assay designed to measure 8 different isozymes revealed only minimal activity for CYP3A, below that observed in primary hepatocytes. With regard to CYP2D6 gene copy number variation, we found the H1 line has only one gene copy, which also harbors the CYP2D6*41 splicing defect, predictive of a CYP2D6 poor/intermediate metabolizer. We identified no CYP2D6 gene duplications, indicating no representative ultra-rapid metabolizer. The H7 and H14 lines are heterozygous for the non-functional CYP2D6*4 variant resulting in a predicted intermediate metabolizer phenotype. In addition, we compared the penetrance of the CYP2D6*41 splicing defect in SCDHs and liver tissue via reverse-transcription PCR assay. We found incomplete penetrance of the CYP2D6*41 allele in liver tissue and variable penetrance in SCDHs. Based on gene expression profiling, SCDHs most closely resemble fetal hepatocytes, especially with regards to AFP, CYP3A7 and FMO1 expression. Finally, these studies indicate a low degree of genetic diversity of pharmacogenetically-relevant genes in the WiCell® hESC lines.&lt;/p&gt;

Sex‐dependent expression of cytoprotective elements in the kidney in response to a hepatotoxic dose of acetaminophen

Acetaminophen (APAP) overdose can cause liver and kidney necrosis. Male mice are known to be more susceptible to APAP hepatotoxicity than females, suggesting that gender‐related genotypic traits play a role in APAP toxicity. Gender‐related differences in susceptibility to APAP nephrotoxicity in mice are also well documented and attributed to differential renal expression of CYP2E1. Recent studies in our laboratory investigated the role of the transcription factor nuclear factor erythroid 2‐related factor 2 (Nrf2), the efflux transporter multidrug resistance‐associated protein 4 (Mrp4), and the drug metabolizing enzyme flavin‐containing mono‐oxygenase 3 (Fmo3) in sex‐dependent susceptibility to APAP‐induced hepatotoxicity in mice. However, the expression and potential role of Nrf2, Fmo3, and Mrp4 in the responsiveness of male and female mouse kidneys to toxic doses of APAP has not been investigated. In the current study, we examined gene expression and protein levels of Nrf2, Mrp4, and Fmo3 in the kidneys of APAP (400 mg/kg) and vehicle treated male and female mice. Plasma alanine aminotransferase (ALT) and blood urea nitrogen (BUN) levels were measured to assess liver and renal damage. Confirmation of tissue damage was made by histopathological analysis. Immunoblots and qRT‐PCR analysis revealed that basal renal Fmo3 gene and protein expression is significantly greater in male than female mice, while Mrp4 expression is higher in females. No differences in basal Nrf2 gene and protein expression were observed among genders. High plasma ALT values and histopathological analysis indicated the presence of hepatotoxicity in both males and females receiving APAP. Renal damage was not evident, as BUN values were within the normal range for both genders and the kidney histology was normal. In response to APAP treatment, no changes in renal expression of Mrp4 or Fmo3 were observed in either gender, consistent with the absence of renal pathology. However, renal Nrf2 gene and protein expression decreased significantly in female mice after APAP dosing. In conclusion, this study documents for the first time the difference in basal renal expression levels of Nrf2 and Fmo3 in male and female mice. Further studies will investigate the expression and regulation of these genes at doses of APAP resulting in renal oxidative stress and toxicity and their potential involvement in the sex‐dependent responses to APAP induced renal damage.Support or Funding InformationSupported by NSF and NIH

Flavin-Containing Monooxygenase 3 Reduces Endoplasmic Reticulum Stress in Lipid-Treated Hepatocytes.

Isoforms of flavin-containing monooxygenase (FMO) are involved in xenobiotic metabolism but have also been implicated in the regulation of glucose and lipid homeostasis and in the development of atherosclerosis. However, we have recently shown that improved insulin action is associated with increased FMO expression in livers of protein kinase C-deficient mice. Here, we investigated whether FMO3 expression affected insulin signaling, glucose metabolism, and endoplasmic reticulum (ER) stress in hepatocytes. HepG2 and IHH hepatocytes were transfected with FMO3 cDNA for overexpression, or small interfering RNA for knockdown. Cells were treated with palmitate to induce insulin resistance and insulin signaling, phosphoenolpyruvate carboxykinase (PEPCK) gene expression and ER stress markers were examined by immunoblotting and RT-PCR. Glycogen synthesis was measured using [(14)C]glucose. Palmitate treatment reduced insulin signaling at the level of Akt phosphorylation and glycogen synthesis, which were little affected by FMO3 overexpression. However, the fatty acid also increased the levels of several ER stress markers and activation of caspase 3, which were counteracted by FMO3 overexpression and exacerbated by FMO3 knockdown. Although FMO3 expression did not reverse lipid effects on protein thiol redox in hepatocytes, it did prevent up-regulation of the gluconeogenic enzyme PEPCK by pharmacological ER stress inducers or by palmitate. ER stress and PEPCK levels were also reduced in livers of fat-fed protein kinase Cδ-deficient mice. Our data indicate that FMO3 can contribute to the regulation of glucose metabolism in the liver by reducing lipid-induced ER stress and the expression of PEPCK, independently of insulin signal transduction.

Quantification of Flavin-containing Monooxygenases 1, 3, and 5 in Human Liver Microsomes by UPLC-MRM-Based Targeted Quantitative Proteomics and Its Application to the Study of Ontogeny

Flavin-containing monooxygenases (FMOs) have a significant role in the metabolism of small molecule pharmaceuticals. Among the five human FMOs, FMO1, FMO3, and FMO5 are the most relevant to hepatic drug metabolism. Although age-dependent hepatic protein expression, based on immunoquantification, has been reported previously for FMO1 and FMO3, there is very little information on hepatic FMO5 protein expression. To overcome the limitations of immunoquantification, an ultra-performance liquid chromatography (UPLC)-multiple reaction monitoring (MRM)-based targeted quantitative proteomic method was developed and optimized for the quantification of FMO1, FMO3, and FMO5 in human liver microsomes (HLM). A post-in silico product ion screening process was incorporated to verify LC-MRM detection of potential signature peptides before their synthesis. The developed method was validated by correlating marker substrate activity and protein expression in a panel of adult individual donor HLM (age 39-67 years). The mean (range) protein expression of FMO3 and FMO5 was 46 (26-65) pmol/mg HLM protein and 27 (11.5-49) pmol/mg HLM protein, respectively. To demonstrate quantification of FMO1, a panel of fetal individual donor HLM (gestational age 14-20 weeks) was analyzed. The mean (range) FMO1 protein expression was 7.0 (4.9-9.7) pmol/mg HLM protein. Furthermore, the ontogenetic protein expression of FMO5 was evaluated in fetal, pediatric, and adult HLM. The quantification of FMO proteins also was compared using two different calibration standards, recombinant proteins versus synthetic signature peptides, to assess the ratio between holoprotein versus total protein. In conclusion, a UPLC-MRM-based targeted quantitative proteomic method has been developed for the quantification of FMO enzymes in HLM.

Induction of Flavin-Containing Monooxygenase in Mice by Oral Administration of Phellinus baumii (Agaricomycetes) Extract.

Phellinus baumii is a yellow mushroom long used in alternative medicine in Korea and other central Asian countries. To identify genes affected by a single or 7-day oral administration of a water extract of Ph. Baumii, mouse liver tissue was analyzed using microarrays. The results showed that 8 and 23 genes were upregulated and 3 and 11 genes downregulated more than 3-fold by single and multiple oral administrations of 100 mg/kg PBE, respectively. Among the upregulated genes, the expression of 3 flavin-containing monooxygenase (Fmo) family genes, Fmo2-4, was upregulated in a concentration-dependent manner. The microarray analysis also showed that single and multiple administrations of PBE increased Fmo3 expression in the mouse liver by 5.1- and 17.6-fold, respectively. To validate the Fmo expression microarray data, polymerase chain reaction was used to confirm the induction of Fmo subclass genes. Mice were orally administered Ph. Baumii extract (PBE), Ph. Baumii water, or Ph. Baumii β-glucan fraction (PBG) for 7 days, and induction of the expression of the Fmo subclasses in the liver, lung, and kidney was investigated. Fmo2, Fmo3, and Fmo4 expression was induced by both PBE and PBG in the lung, liver, and kidney, respectively. However, no induction of Fmo1 and Fmo5 was detected. To investigate the metabolic acceleration of xenobiotic by PBE, carbendazim was orally administered to mice and its clearance from the blood analyzed. High-performance liquid chromatography analysis showed accelerated clearance of serum carbendazim by oral administration of PBE for 7 days, as evidenced by the reduced peak plasma concentration, time to reach the peak plasma concentration, and area under the curve values. Moreover, PBE increased the carbendazim clearance rate at the higher concentration. These data indicate that oral administration of PBE resulted in modulation of gene expression: PBE was responsible for the induction of Fmo2, Fmo3, and Fmo4 expression. PBE also accelerated the metabolic clearance of carbendazim in vivo and so could be applied to the detoxification of xenobiotics such as drugs, pesticides, and nicotine.

DNA double-strand breaks induce the expression of flavin-containing monooxygenase and reduce root meristem size in Arabidopsis thaliana.

Plants use various mechanisms to cope with environmental stresses, which often threaten genome integrity. In Arabidopsis, DNA double-strand breaks (DSBs) reduce root meristem size in a SOG1-dependent manner. SOG1 is a key transcription factor controlling the response to DNA damage. However, the underlying mechanism remains largely unknown. In this study, we found that treatment with the DSB inducer zeocin increased the accumulation of H2O2 in root tips. Chromatin immunoprecipitation analysis showed that SOG1 directly binds to the promoter of FMO1, which encodes a flavin-containing monooxygenase and is associated with the production of reactive oxygen species (ROS), H2O2 in particular. Indeed, zeocin induced the expression of FMO1 in a SOG1-dependent manner, and neither the sog1 nor the fmo1 knockout mutant exhibited higher H2O2 accumulation in root tips. Consequently, both sog1 and fmo1 could tolerate exposure to zeocin, in terms of root growth and the maintenance of the meristem size. However, transgenic plants over-expressing FMO1 also accumulated H2O2 in response to zeocin exposure, suggesting that other ROS-synthesis genes are also involved in the regulation of ROS production. We conclude that SOG1-mediated regulation of ROS homeostasis plays a key role in the reduction of root meristem size under DNA stress conditions.

Tolerance to Acetaminophen Hepatotoxicity in the Mouse Model of Autoprotection Is Associated with Induction of Flavin-Containing Monooxygenase-3 (FMO3) in Hepatocytes

Acetaminophen (APAP) pretreatment with a hepatotoxic dose (400 mg/kg) in mice results in resistance to a second, higher dose (600 mg/kg) of APAP (APAP autoprotection). Recent microarray work by our group showed a drastic induction of liver flavin containing monooxygenase-3 (Fmo3) mRNA expression in our mouse model of APAP autoprotection. The role of liver Fmo3, which detoxifies xenobiotics, in APAP autoprotection is unknown. The purpose of this study was to characterize the gene regulation and protein expression of liver Fmo3 during APAP hepatotoxicity. The functional consequences of Fmo3 induction were also investigated. Plasma and livers were collected from male C57BL/6J mice over a period of 72 h following a single dose of APAP (400 mg/kg) to measure Fmo3 mRNA and protein expression. Although Fmo3 mRNA levels increased significantly following APAP treatment, protein expression changed marginally. In contrast, both Fmo3 mRNA and protein expression were significantly higher in APAP autoprotected livers. Unlike male C57BL/6J mice, female mice have ∼80-times higher constitutive Fmo3 mRNA levels and are highly resistant to APAP hepatotoxicity. Coadministration of APAP with the FMO inhibitor methimazole rendered female mice susceptible to APAP hepatotoxicity, with no changes in susceptibility detected in male mice. Furthermore, a human hepatocyte cell line (HC-04) clone over-expressing human FMO3 showed enhanced resistance to APAP cytotoxicity. Taken together, these findings establish for the first time induction of Fmo3 protein expression and function by xenobiotic treatment. Our results also indicate that Fmo3 expression and function plays a role in protecting the liver from APAP-induced toxicity. Although the mechanism(s) of this protection remains to be elucidated, this work describes a novel protective function for this enzyme.

Drug oxygenation activities mediated by liver microsomal flavin-containing monooxygenases 1 and 3 in humans, monkeys, rats, and minipigs

Liver microsomal flavin-containing monooxygenases (FMO, EC 1.14.13.8) 1 and 3 were functionally characterized in terms of expression levels and molecular catalytic capacities in human, cynomolgus monkey, rat, and minipig livers. Liver microsomal FMO3 in humans and monkeys and FMO1 and FMO3 in rats and minipigs could be determined immunochemically with commercially available anti-human FMO3 peptide antibodies or rat FMO1 peptide antibodies. With respect to FMO-dependent N-oxygenation of benzydamine and tozasertib and S-oxygenation of methimazole and sulindac sulfide activities, rat and minipig liver microsomes had high maximum velocity values (Vmax) and high catalytic efficiency (Vmax/Km, Michaelis constant) compared with those for human or monkey liver microsomes. Apparent Km values for recombinantly expressed rat FMO3-mediated N- and S-oxygenations were approximately 10–100-fold those of rat FMO1, although these enzymes had similar Vmax values. The mean catalytic efficiencies (Vmax/Km, 1.4 and 0.4min−1μM−1, respectively) of recombinant human and monkey FMO3 were higher than those of FMO1, whereas Vmax/Km values for rat and minipig FMO3 were low compared with those of FMO1. Minipig liver microsomal FMO1 efficiently catalyzed N- and S-oxygenation reactions; in addition, the minipig liver microsomal FMO1 concentration was higher than the levels in rats, humans, and monkeys. These results suggest that liver microsomal FMO1 could contribute to the relatively high FMO-mediated drug N- and S-oxygenation activities in rat and minipig liver microsomes and that lower expression of FMO1 in human and monkey livers could be a determinant factor for species differences in liver drug N- and S-oxygenation activities between experimental animals and humans.

Abstract 18108: Decreased Fmo3 Expression is Associated With Increased Atherosclerosis and Impaired Liver Function in Apolipoprotein E Null Mice

Recent studies demonstrated a strong positive association between blood trimethylamine-N-oxide (TMAO) level and risk for cardiovascular disease. Dietary choline is converted to trimethylamine (TMA) by the gut bacteria, and TMA is then converted to TMAO in the liver, mainly by flavin containing monooxygenase 3 (Fmo3). To examine how FMO3 expression influences atherogenesis, we administered two different FMO3 or control antisense oligonucleotdies (ASOs) to apolipoprotein E (apoE) null mice for 15 weeks. During the last 12 weeks of the ASO treatment, the mice received a chow diet containing 1% choline to enhance TMA production. Hepatic FMO3 mRNA levels were decreased by 80% in mice that received either FMO3 ASO #1 or #2 versus those that received the control ASO. As expected, circulating TMA and TMAO levels were significantly increased and decreased, respectively, in the mice that received the two FMO3 ASOs versus the controls. Other observed changes in the FMO3 ASO-treated mice included: decreased plasma triglyceride, HDL, and glucose levels. Significantly increased VLDL/IDL/LDL cholesterol was observed only in FMO3 ASO #2-treated mice versus the controls. FMO3 ASO treatment increased circulating total bile acid levels by more than 2-fold. Compared to controls, serum markers of hepatic inflammation and necrosis (ALT, AST) were significantly increased in FMO3 ASO treated mice. Histological examination revealed obvious hepatic steatosis and increased inflammatory cell infiltration, suggesting that FMO3 abrogation may be a reasonable model for non-alcoholic steatohepatitis (NASH). We also found a significant increase in quantitative triglyceride and cholesterol content of these livers. Transcriptional profiling by microarrays showed increased hepatic expression of inflammatory genes in mice that received FMO3 ASOs versus the controls. In addition, a significant increase in spleen weight in these mice suggests increased systemic inflammation. Finally, FMO3 knockdown increased atherosclerosis by more than 50% when compared to controls (p &lt; 0.01). This work demonstrates that blocking FMO3 activity has a major impact on liver physiology and atherosclerosis. Our study suggests a protective role for FMO3 in atherosclerosis and liver function.

The Emerging Relevance of the Gut Microbiome in Cardiometabolic Health

The Emerging Relevance of the Gut Microbiome in Cardiometabolic Health

Farnesoid X receptor directly regulates xenobiotic detoxification genes in the long-lived Little mice

Activation of xenobiotic metabolism pathways has been linked to lifespan extension in different models of aging. However, the mechanisms underlying activation of xenobiotic genes remain largely unknown. Here we showed that although farnesoid X receptor (FXR, Nr1h4) mRNA levels do not change significantly, FXR protein levels are elevated in the livers of the long-lived Little mice, leading to increased DNA binding activity of FXR. Hepatic FXR expression is sex-dependent in wild-type mice but not in Little mice, implying that up-regulation of FXR might be dependent on the reduction of growth hormone in Little mice. Growth hormone treatment decreased hepatic expression of FXR and xenobiotic genes Abcb1a, Fmo3 and Gsta2 in both wild-type and Little mice, suggesting an association between FXR and xenobiotic gene expression. We found that Abcb1a is transactivated by FXR via direct binding of FXR/retinoid X receptor α (RXRα) heterodimer to a response element at the proximal promoter. FXR also positively controls Fmo3 and Gsta2 expression through direct interaction with the response elements in these genes. Our study demonstrates that xenobiotic genes are direct transcriptional targets of FXR and suggests that FXR signaling may play a critical role in the lifespan extension observed in Little mice.