Expression Of Flavin-containing Monooxygenase Research Articles

Mice, rats, and guinea pigs differ in FMOs expression and tissue concentration of TMAO, a gut bacteria-derived biomarker of cardiovascular and metabolic diseases.

Increased plasma trimethylamine oxide (TMAO) is observed in cardiovascular and metabolic diseases, originating from the gut microbiota product, trimethylamine (TMA), via flavin-containing monooxygenases (FMOs)-dependent oxidation. Numerous studies have investigated the association between plasma TMAO and various pathologies, yet limited knowledge exists regarding tissue concentrations of TMAO, TMAO precursors, and interspecies variability. Chromatography coupled with mass spectrometry was employed to evaluate tissue concentrations of TMAO and its precursors in adult male mice, rats, and guinea pigs. FMO mRNA and protein levels were assessed through PCR and Western blot, respectively. Plasma TMAO levels were similar among the studied species. However, significant differences in tissue concentrations of TMAO were observed between mice, rats, and guinea pigs. The rat renal medulla exhibited the highest TMAO concentration, while the lowest was found in the mouse liver. Mice demonstrated significantly higher plasma TMA concentrations compared to rats and guinea pigs, with the highest TMA concentration found in the mouse renal medulla and the lowest in the rat lungs. FMO5 exhibited the highest expression in mouse liver, while FMO3 was highly expressed in rats. Guinea pigs displayed low expression of FMOs in this tissue. Despite similar plasma TMAO levels, mice, rats, and guinea pigs exhibited significant differences in tissue concentrations of TMA, TMAO, and FMO expression. These interspecies variations should be considered in the design and interpretation of experimental studies. Furthermore, these findings may suggest a diverse importance of the TMAO pathway in the physiology of the evaluated species.

FMO family may serve as novel marker and potential therapeutic target for the peritoneal metastasis in gastric cancer.

To explore the relationship between flavin-containing monooxygenases (FMOs) and peritoneal metastasis (PM) in gastric cancer (GC). TIMER 2.0 was used to perform pan-cancer analysis and assess the correlation between the expression of FMOs and cancers. A dataset from The Cancer Genome Atlas (TCGA) was used to analyze the correlation between FMOs and clinicopathological features of GC. PM is well established as the most common mode of metastasis in GC. To further analyze the correlation between FMOs and PM of GC, a dataset was obtained from the National Center for Biotechnology Information Gene Expression Omnibus (GEO) database. The results were validated by immunohistochemistry. The relationship between FMOs and PM of GC was explored, and a novel PM risk signature was constructed by least absolute shrinkage and selection operator (LASSO) regression analysis. The regression model's validity was tested by multisampling. A nomogram was established based on the model for predicting PM in GC patients. The mechanism of FMOs in GC patients presenting with PM was assessed by conducting Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses in TCGA and GEO datasets. Finally, the potential relationship between FMOs and immunotherapy was analyzed. The pan-cancer analysis in TCGA and GEO datasets showed that FMO1 was upregulated, while FMO2 and FMO4 were downregulated in GC. Moreover, FMO1 and FMO2 correlated positively with the T and N stage of GC in the TCGA dataset. FMO1 and FMO2 expression was a risk factor for GC (hazard ratio: 1.112 and 1.185). The overexpression of FMO1 was significantly correlated with worse disease-free-survival (DFS) and overall survival (OS). However, no relationship was found between FMO2 expression in GC and DFS and OS. PM was highly prevalent among GC patients and typically associated with a worse prognosis. FMO1 was highly expressed in GC with PM. FMO1 and FMO2 were positively correlated with PM in GC. We identified a 12-gene panel for predicting the PM risk signature by LASSO (Area Under Curve (AUC) = 0.948, 95%CI: 0.896-1.000). A 10-gene panel for PM prediction was identified (AUC = 0.932, 95%CI: 0.874-0.990), comprising FMO1 and FMO2. To establish a model for clinical application, a 7-gene panel was established (AUC = 0.927, 95% CI: 0.877-0.977) and successfully validated by multisampling. (AUC = 0.892, 95% CI: 0.878-0.906). GO and KEGG analyses suggest that FMO1 and FMO2 regulate the extracellular matrix and cell adhesion. FMO1 and FMO2 were positively correlated with the immune score of GC, and their expression was associated with the infiltration of immune cells. PM in GC is strongly correlated with FMOs. Overall, FMO1 and FMO2 have huge prospects for application as novel diagnostic and therapeutic targets.

Proatherogenic gut-microbiome metabolite trimethylamine elicits a gut-liver circuit to regulate TMAO metabolism and atherosclerosis via mediation of CREBH

Abstract Introduction In humans, circulating metabolite Trimethylamine N-oxide (TMAO) is closely associated with higher risk of cardiovascular disease. Trimethylamine (TMA), a precursor of TMAO, is produced by gut microbiome using dietary components, i.e., choline and carnitine, as substrates. The gut-derived TMA is then transferred to the liver where it is further oxidized to TMAO by the flavin-containing monooxygenases (FMOs). The ER-resident transcription factor c-AMP responsive element binding protein H (CREBH/CREB3L3) is exclusively expressed in the liver and intestine. Perturbation of CREBH activity contributes to the development of hyperlipidemia and cardiovascular disease. Therefore, the Purpose of this study is to investigate the regulatory effect of a gut bacterium, Akkermansia muciniphila (A. muciniphila), on TMA and TMAO metabolism and the role of CREBH in this process. Methods Two groups of wild type (WT) and CREBH knockout (CREBH-KO) mice were inoculated with 200 μL of A. muciniphila (2×108 cfu/0.2 mL) in PBS or the vehicle (PBS) alone as control every other day through oral gavage for 2 weeks. Plasmas, liver and intestinal tissues were collected for metabolomics analysis, immunoblotting analysis and q-RT-PCR. Results Metabolomics analysis of the plasmas from the experimental mice revealed that increased colonization of A. muciniphila in the gut significantly reduced circulating TMA in the WT mice but not in CREBH-KO mice (P<0.05), suggesting that depletion of CREBH altered the microenvironment of gut microbiome which affected the metabolism of TMA by gut bacteria. In the livers, A. muciniphila treatment markedly reduced mRNA expression of FMO1 and FMO3 (P<0.05), which subsequently inhibited the enzymatic conversion of TMA to TMAO in hepatocytes. Immunoblotting analysis further revealed that LDL receptor was upregulated whereas ER stress markers, GRP94 and JNK1/2, were downregulated in the A. muciniphila treated KO mice, indicating an acceleration in lipoprotein (VLDL remnant) clearance from the circulation and the improvement of metabolic inflammation. In vitro, incubation of mouse hepatocytes AML12 with TMA (600 mM) for 12 hours stimulated expression of FMOs to facilitate the conversion of TMA to TMAO and induced lipotoxicity. Conclusion CREBH mediates the crosstalk between gut microbiome and liver metabolic system that regulates TMA and TMAO metabolism, which contributes to the induction of metabolic inflammation and atherogenesis. This novel finding may lend support to the therapeutic strategy of atherosclerosis. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): British Heart Foundation

Functional assessment of rat pulmonary flavin-containing monooxygenase activity

The expression of flavin-containing monooxygenase (FMO) varies extensively between human and commonly used preclinical species such as rat and mouse. The aim of this study was to investigate the pulmonary FMO activity in rat using benzydamine. Furthermore, the contribution of rat lung to the clearance of benzydamine was investigated using an in vivo pulmonary extraction model.Benzydamine N-oxygenation was observed in lung microsomes and lung slices. Thermal inactivation of FMO and CYP inhibition suggested that rat pulmonary N-oxygenation is predominantly FMO mediated while any contribution from CYPs is negligible.The predicted lung clearance (CLlung) estimated from microsomes and slices was 16 ± 0.6 and 2.1 ± 0.3 mL/min/kg, respectively. The results from in vivo pulmonary extraction indicated no pulmonary extraction following intravenous and intra-arterial dosing to rats. Interestingly, the predicted CLlung using rat lung microsomes corresponded to approximately 35% of rat CLliver suggesting that the lung makes a smaller contribution to the whole body clearance of benzydamine.Although benzydamine clearance in rat appears to be predominantly mediated by hepatic metabolism, the data suggest that the lung may also make a smaller contribution to its whole body clearance.

Consecutive emamectin benzoate and deltamethrin treatments affect the expressions and activities of detoxification enzymes in the rainbow trout (Oncorhynchus mykiss)

Rainbow trout (Oncorhynchus mykiss) subjected to three consecutive, alternating treatments with emamectin benzoate (EMB) and deltamethrin (DM) during outbreaks of Caligus rogercresseyi in a farm located in southern Chile (Hornopiren, Chiloé), were studied to determine the effects of these treatments on the protein and enzymatic activity levels of cytochrome P450 1A (CYP1A), flavin-containing monooxygenase (FMO) and glutathione S-transferase (GST) in different tissues. Consecutive and alternating EMB/DM treatments resulted in a 10-fold increase and 3-fold decrease of CYP1A protein levels in the intestine and gills, respectively. Notably, CYP1A activity levels decreased in most of the analyzed tissues. FMO protein and activity levels markedly increased in the kidney and the intestine. GST was up-regulated in all tissues, either as protein or enzyme activity. When comparing consecutive EMB/DM treatments against previous studies of EMB treatment alone, CYP1A activity levels were similarly diminished, except in muscle. Likewise, FMO activity levels were increased in most of the analyzed tissues, particularly in the muscle, kidney, and intestine. The increases observed for GST were essentially unchanged between consecutive EMB/DM and EMB only treatments. These results indicate that consecutive EMB/DM treatments in rainbow trout induce the expression and activity of FMO and GST enzymes and decrease CYP1A activity. These altered activities of detoxification enzymes could generate imbalances in metabolic processes, synthesis, degradation of hormones and complications associated with drug interactions. It is especially important when analyzing possible effects of consecutive antiparasitic treatments on withholding periods and salmon farming yields.

Developmental expression and regulation of flavin-containing monooxygenase by the unfolded protein response in Japanese medaka (Oryzias latipes)

Flavin-containing monooxygenases (FMOs) play a key role in xenobiotic metabolism, are regulated by environmental conditions, and are differentially regulated during mammalian development. Japanese medaka (Oryzias latipes) are a common model organism for toxicological studies. The goal of the current research was to characterize developmental expression and regulation of FMOs in Japanese medaka embryos to better understand the role of FMOs in this model species. Five putative medaka fmos were characterized from the medaka genome through the National Center for Biotechnology Information (NCBI) database by protein motifs and alignments, then identified as fmo4, fmo5A, fmo5B, fmo5C and fmo5D for the current study. Fmo gene expression was analyzed at 1dpf, 3dpf, 6dpf and 9dpf and distinct developmental patterns of expression were observed. Fmo4 and fmo5D increased 3-fold during mid organogenesis (6dpf), while fmo5B and fmo5C decreased significantly in early organogenesis (3dpf) and fmo5A was unaltered. Promoter analysis was performed for transcription factor binding sites and indicated regulation by developmental factors and a role for the unfolded protein response in fmo modulation. Fmo regulation by the UPR was assessed with treatments of 1μg/ml, 2μg/ml, and 4μg/ml Tunicamycin (Tm), and 2mM and 4mM dithiothreitol (DTT), well-known inducers of endoplasmic reticulum stress, for 24h from 5–6dpf. High concentrations to Tm induced fmo4 and fmo5A up to two-fold, while DTT significantly decreased expression of fmo5A, fmo5B, and fmo5C. Results suggest that medaka fmos are variably regulated by the UPR during organogenesis with variable developmental expression, and suggesting potential stage-dependent activation or detoxification of xenobiotics.

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.

Isolation and Culture of Mouse Hepatocytes: Gender-Specific Gene Expression Responses to Chemical Treatments.

In this chapter, the isolation of primary mouse hepatocytes and their response to chemical treatment are described. We show that it is important to consider, in the experimental design, the sex of the animals to be used. We demonstrate this by measuring the effect of sex hormones or xenobiotics on the expression of flavin-containing monooxygenase 5 in cultures of primary hepatocytes isolated from male and female mice.

Exploring nicotinamide cofactor promiscuity in NAD(P)H-dependent flavin containing monooxygenases (FMOs) using natural variation within the phosphate binding loop. Structure and activity of FMOs from Cellvibrio sp. BR and Pseudomonas stutzeri NF13

Flavin-containing monooxygenases (FMOs) catalyse asymmetric oxidation reactions that have potential for preparative organic synthesis, but most use the more expensive, phosphorylated nicotinamide cofactor NADPH to reduce FAD to FADH2 prior to formation of the (hydro)peroxy intermediate required for substrate oxygenation. A comparison of the structures of NADPH-dependent FMO from Methylophaga aminisulfidivorans (mFMO) and SMFMO from Stenotrophomonas maltophilia, which is able to use both NADPH and NADH, suggested that the promiscuity of the latter enzyme may be due in part to the substitution of an Arg–Thr couple in the NADPH phosphate recognition site in mFMO, for a Gln–His couple in SMFMO (Jensen et al., 2012, Chembiochem, 13, 872–878). Natural variation within the phosphate binding region, and its influence on nicotinamide cofactor promiscuity, was explored through the cloning, expression, characterisation and structural studies of FMOs from Cellvibrio sp. BR (CFMO) and Pseudomonas stutzeri NF13 (PSFMO), which possess Thr–Ser and Gln–Glu in the putative phosphate recognition positions, respectively. CFMO and PSFMO displayed 5- and 1.5-fold greater activity, respectively, than SMFMO for the reduction of FAD with NADH, and were also cofactor promiscuous, displaying a ratio of activity with NADH:NADPH of 1.7:1 and 1:1.3, respectively. The structures of CFMO and PSFMO revealed the context of the phosphate binding loop in each case, and also clarified the structure of the mobile helix–loop–helix motif that appears to shield the FAD-binding pocket from bulk solvent in this class of FMOs, a feature that was absent from the structure of SMFMO.

Regulation of FMO and PON Detoxication Systems in ALS Human Tissues

Amyotrophic lateral sclerosis (ALS) is an adult-onset, progressive, and fatal neurodegenerative disease with unknown etiology. Recent evidence suggests an association between the exposure to toxic environmental factors and sporadic ALS. The flavin-containing monooxygenases (FMOs) and paraoxonase (PONs) genes encode enzymes involved in xenobiotic detoxication and are associated with ALS. FMO and PON gene expression has been examined in the human central nervous system including human brain subregions defined as the spinal cord, medulla, and cerebral cortex and in the peripheral tissues (lymphocytes, fibroblasts) in ALS patients and normal control subjects. FMO expression was generally higher in tissues from ALS subjects than in control tissues, with the largest increases in FMO expression detected in the spinal cord. In peripheral tissues, the FMO mRNA level was found to be lower compared with FMO expression in brain tissue, and no differences were detected between ALS patients and the control tissue. FMO and PON gene expression was low in peripheral tissues. In contrast to FMO5 expression, the PON2 gene was down-regulated in ALS patients compared to the controls. Because FMO and PON are involved in the detoxication processes and their functional activity to bioactivate chemicals to toxins has been documented, the data herein suggest that environmental toxin exposure may play a role in a subset of individuals who contract ALS by altering FMO and PON gene expression. Although the precise pathogenic link is presently unknown, these findings suggest a role at FMO and PON genes in the development of ALS.

Flavin-Containing Monooxygenase mRNA Levels are Up-Regulated in ALS Brain Areas in SOD1-Mutant Mice

Flavin-containing monooxygenases (FMOs) are a family of microsomal enzymes involved in the oxygenation of a variety of nucleophilic heteroatom-containing xenobiotics. Recent results have pointed to a relation between Amyotrophic Lateral Sclerosis (ALS) and FMO genes. ALS is an adult-onset, progressive, and fatal neurodegenerative disease. We have compared FMO mRNA expression in the control mouse strain C57BL/6J and in a SOD1-mutated (G93A) ALS mouse model. Fmo expression was examined in total brain, and in subregions including cerebellum, cerebral hemisphere, brainstem, and spinal cord of control and SOD1-mutated mice. We have also considered expression in male and female mice because FMO regulation is gender-related. Real-Time TaqMan PCR was used for FMO expression analysis. Normalization was done using hypoxanthine-guanine phosphoribosyl transferase (Hprt) as a control housekeeping gene. Fmo genes, except Fmo3, were detectably expressed in the central nervous system of both control and ALS model mice. FMO expression was generally greater in the ALS mouse model than in control mice, with the highest increase in Fmo1 expression in spinal cord and brainstem. In addition, we showed greater Fmo expression in males than in female mice in the ALS model. The expression of Fmo1 mRNA correlated with Sod1 mRNA expression in pathologic brain areas. We hypothesize that alteration of FMO gene expression is a consequence of the pathological environment linked to oxidative stress related to mutated SOD1.

The potential of knockout mouse lines in defining the role of flavin-containing monooxygenases in drug metabolism

Importance of the field: Flavin-containing monooxygenases (FMOs) metabolize a number of therapeutic drugs. However, their role in drug metabolism has been overlooked compared with that of the cytochromes P450 (CYPs). Genetic variants of FMOs are expected to influence drug response and/or contribute to adverse drug reactions.Areas covered in the review: We review tissue-specific expression of FMOs and genetic variation that may influence drug metabolism. We discuss how the use of mouse lines in which Fmo genes have been deleted can demonstrate the role an FMO plays in the metabolism of a drug, particularly if the drug is subject to metabolism by other enzymes, for example, CYPs, or undergoes retro-reduction. We cite seminal papers and review articles to give the reader an appreciation of the FMO field.What the reader will gain: Insights into the problems associated with determining the contribution of an FMO to the metabolism of a drug and how an Fmo-knockout mouse line has revealed the role of FMO1 in the metabolism of imipramine in vivo.Take home message: The use of an Fmo-knockout mouse line demonstrates a more important role for FMOs in multi-pathway drug metabolism than has previously been appreciated.

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

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 μg/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 μg/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 μg/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.

Effect of cortisol and urea on flavin monooxygenase activity and expression in rainbow trout, Oncorhynchus mykiss

Expression of flavin-containing monooxygenase(s) (FMO) correlates with salinity exposure in certain species of euryhaline fish, such as the rainbow trout, Oncorhynchus mykiss. The mechanism(s) by which salinity regulates FMO is unclear. Adult rainbow trout were infused through the dorsal aorta with either cortisol or urea. At 500 ng/ml, cortisol caused a significant increase in FMO-catalyzed thiourea oxidase activity in gill and liver microsomes. FMO1 expression, however, was significantly increased by the high cortisol dose only in gill microsomes. The levels of TMAO and urea were not altered by cortisol. In the liver, urea infusion caused an increase in hepatic FMO activity. FMO expression and activity correlated with elevated tissue urea levels, but TMAO concentrations were not related. These results indicate that FMO expression and activity may be partially controlled by the osmoregulatory/stress hormone, cortisol, and concentrations of the organic osmolyte, urea, in the rainbow trout.

Effect of 17beta-estradiol and testosterone on the expression of flavin-containing monooxygenase and the toxicity of aldicarb to Japanese medaka, Oryzias latipes.

Previous studies in our laboratory indicated gender differences in salinity-enhanced acute toxicity of aldicarb in Japanese medaka with females being more susceptible. In the current study, the effects of the sex steroids, 17beta estradiol (E2) and testosterone (T) on aldicarb toxicity was examined. Adult Japanese medaka were separated by sex and exposed to 100 microg/l E2 or T for 6 days followed by exposure to the 96-h LC50 (0.5 mg/l) of aldicarb. The toxicity of aldicarb to adult males was significantly lowered by E2 and T whereby the mortality percentage was reduced to 23.3 +/- 5.8% and 3.3 +/- 5.8%, respectively, compared to the fish not receiving steroids (46.7 +/- 5.8% mortality). In females, T caused significant reduction in aldicarb toxicity to 16.7 +/- 5.8%, while E2 significantly enhanced the toxicity to 96.7 +/- 5.8% mortality. Since the flavin-containing monooxygenase (FMO) enzyme system had been shown to play a critical role in aldicarb toxicity, the effect of E2 and T on FMO expression was examined. Gill FMO activity showed a direct correlation with the overall toxicity of aldicarb in both male and female medaka. Expression of FMO1-like protein was significantly reduced by T in male livers and gills, and T did not affect the expression of FMOs in female tissues. In contrast, E2 significantly reduced FMO1-like protein expression in male gills and female livers, as well as FMO3 expression in both male and female livers, but significantly increased gill FMO1 expression in females. Since aldicarb acts by inhibiting the enzyme cholinesterase (ChE), the effect of sex hormones on the activity of this enzyme was also examined. In both male and female medaka, T counteracted the inhibitory effect of aldicarb on muscle ChE. In male fish, E2 had similar effects but did not seem to counteract the ChE inhibition in females. In conclusion, E2 and T modulation of aldicarb toxicity in Japanese medaka seems to be mediated via alteration of gill FMO and ChE actitivies.

Effect of salinity on flavin-containing monooxygenase expression and activity in rainbow trout (Oncorhynchus mykiss).

In order to obtain more information about the physiological role(s) of flavin-containing monooxygenases (FMOs) in euryhaline teleost fishes, two experimental series were performed using adult and juvenile rainbow trout (Oncorhynchus mykiss). Cannulated adult trout were exposed to freshwater or 21% seawater for 48 h, whereas juvenile trout were acclimated to one of four different salinities: freshwater, 7%, 14%, or 21% during a 2-week period. FMO expression and activity were determined in red blood cells (RBC), liver, gill, kidney, gut, heart and brain. Furthermore, the content of trimethylamine oxide (TMAO; an FMO metabolite and an osmolyte) as well as urea were determined in various tissues. FMO expression and activity increased significantly and in a salinity dependent manner in osmoregulatory organs (gills, kidney and gut) in both juveniles and adult trout and, furthermore, in RBC in adults. No significant changes were observed in liver or heart. Urea content increased significantly and in a salinity dependent manner in all tissues, whereas TMAO was accumulated primarily in muscle tissue. Salinity dependent adjustment of FMO expression and activity primarily in osmoregulatory organs as well as regulation of TMAO content in muscle is consistent with previous studies showing an association of FMO with osmoregulation in euryhaline teleosts. However, the lack of a parallel increase of TMAO with urea in other tissues of fish at high salinity indicates other mechanisms of protection from intracellular urea may exist in non-muscular tissues.

Effect of urea and low temperature on the expression and activity of flavin-containing monooxygenase in the liver and gill of rainbow trout (Oncorhynchus mykiss)

Little is known about the physiological role of flavin containing monooxygenases (FMOs) in teleost fish. Recent studies have indicated induction during saltwater adaptation in several telost species including rainbow trout. A physiological product of FMOs, trimethylamine N-oxide (TMAO) and urea have also been shown to increase in rainbow trout muscle during saltwater adapatation. TMAO counteracts the denaturing effects of urea. In order to evaluate urea as a possible inducer of FMO expression and activity, adult rainbow trout were infused for 48 h with urea/saline solutions at a loading rate of 8 mmol urea/kg/day. To determine whether low temperature had any effect on FMO expression and activity, one group of animals was infused with urea and exposed to low temperature (2–3 °C) for 48 h. Gill FMO-catalyzed thiourea oxygenase activity was significantly induced by low temperature, with twice the activity observed at low temperature (1.116±0.356 nmol/min/mg) compared to urea infusion at 10 °C (0.585±0.282 nmol/min/mg). Low temperature without urea treatment caused a 50% increase in gill FMO activity. In the liver, urea infusion caused an increase in liver FMO activity (from 0.144±0.053 nmol/min/mg to 0.464±0.237 nmol/min/mg), but was unaffected by co-exposure to low temperature (0.523±219 nmol/min/mg). FMO expression and activity correlated with elevated tissue urea levels, but TMAO concentrations were not related. The interactions between urea, temperature and the tissue-specific induction of FMOs indicate FMOs may contribute to other physiological and cellular processes besides osmoregulation.

Random expression of branchial flavin-containing monooxygenase (FMO) activity in the Atlantic flounder ( Platichthys flesus)

In an attempt to better understand the regulatory basis explaining relationships between FMO and osmoregulation, sexually immature Atlantic flounder were treated for 18 days with abdominal implants containing the osmoregulatory modulators cortisol (CO) and 17 beta-estradiol (E2). NaKATPase mRNA expression was significantly repressed in E2 animals, and tended to be elevated in CO-treated animals. FMO mRNA expression was extremely variable with a slight reduction in E2-treated animals, but no significant differences were observed between any treatment groups. TMA oxygenase activity was unchanged by CO or E2 treatment, and detected in only 40% of the animals (8 out of 20). To assess individual variability, 11 male and nine female flounder were examined for branchial FMO expression and activity. Although FMO mRNA was observed in all animals, FMO activity was only observed in 63% (7 out of 11) of males and 33% of females (3 out of 9). No significant correlations were observed between FMO mRNA expression, FMO activity, serum CO, or E2. Results of this study indicate variable expression of FMO mRNA and sporadic deletion of catalytic activity in Atlantic flounder.

Occurrence of flavin-containing monooxygenases in non-mammalian eukaryotic organisms

Flavin-containing monooxygenases (FMOs) have been identified in various organisms from bacteria to humans. However, because of the importance of these enzymes in the biotransformation of xenobiotics, the majority of studies have focused almost entirely upon the mammalian forms of the enzyme. Consequently, this review is an attempt to document the occurrence of FMO expression (mRNA, proteins, activities) in non-mammalian species in an attempt to provide insight about its putative physiological and toxicological roles. Activity indicative of FMO has been observed in numerous invertebrate species but corresponding proteins or transcripts have not been identified. There is a significant gap of information pertaining to insects, echinoderms, avian, reptilian and amphibian species. Significant homology of structure and function is observed in lower vertebrates. Evidence is provided primarily from studies with piscine forms of the enzyme suggesting a possible osmoregulatory role of FMOs, especially in euryhaline species of fish.

Expression of branchial flavin-containing monooxygenase is directly correlated with salinity-induced aldicarb toxicity in the euryhaline fish ( Oryzias latipes)

Earlier studies in our laboratory have demonstrated a reduction of flavin-containing monooxygenase (FMO) activity when salt-water adapted euryhaline fish were transferred to water of less salinity. Since FMOs have been shown to be responsible for the bioactivation of the carbamate insecticide, aldicarb, to a more potent cholinesterase inhibitor, it would be predicted that euryhaline fish exposed to aldicarb at higher salinity should have greater levels of FMO and, hence, be more susceptible to aldicarb toxicity. It was not surprising, therefore, that mature female Japanese medaka ( Oryzias latipes) were significantly more susceptible to aldicarb toxicity at higher salinity. A direct correlation was observed between salinity and FMO expression, but salinity had no significant effect on the uptake of aldicarb. These data suggest that FMOs are upregulated by salinity and may be responsible for the enhanced toxicity of aldicarb at higher salinities.