Reducing gut microbiome-driven adipose tissue inflammation alleviates metabolic syndrome.

BACKGROUND: The gut microbiota contributes to macrophage-mediated inflammation in adipose tissue with consumption of an obesogenic diet, thus driving the development of metabolic syndrome. There is a need to identify and develop interventions that abrogate this condition. The hops-derived prenylated flavonoid xanthohumol (XN) and its semi-synthetic derivative tetrahydroxanthohumol (TXN) attenuate high-fat diet-induced obesity, hepatosteatosis, and metabolic syndrome in C57Bl/6J mice. This coincides with a decrease in pro-inflammatory gene expression in the gut and adipose tissue, together with alterations in the gut microbiota and bile acid composition. RESULTS: In this study, we integrated and interrogated multi-omics data from different organs with fecal 16S rRNA sequences and systemic metabolic phenotypic data using a Transkingdom Network Analysis. By incorporating cell type information from single-cell RNA-seq data, we discovered TXN attenuates macrophage inflammatory processes in adipose tissue. TXN treatment also reduced levels of inflammation-inducing microbes, such as Oscillibacter valericigenes, that lead to adverse metabolic phenotypes. Furthermore, in vitro validation in macrophage cell lines and in vivo mouse supplementation showed addition of O. valericigenes supernatant induced the expression of metabolic macrophage signature genes that are downregulated by TXN in vivo. CONCLUSIONS: Our findings establish an important mechanism by which TXN mitigates adverse phenotypic outcomes of diet-induced obesity and metabolic syndrome. TXN primarily reduces the abundance of pro-inflammatory gut microbes that can otherwise promote macrophage-associated inflammation in white adipose tissue. Video Abstract.

Interstitial incisional hernia of greater omentum: An incidental finding during routine cadaveric dissection.

Omental herniation, located between the rectus abdominis muscle and the anterior blade of the rectus sheath, can be triggered after a transverse suprapubic incision. It causes the development of an incisional interstitial hernia (IIH), which is an extremely rare and poorly understood condition. Based on this information, our work presents the first anatomical description of incisional interstitial hernia found during routine dissection at the Human Anatomy Laboratory of the Federal University of Ceará in a formalized female corpse.

Comparative in vitro metabolism of benzo[a]pyrene by recombinant zebrafish CYP1A and liver microsomes from beta-naphthoflavone-treated rainbow trout.

The zebrafish (Danio rerio) is a sensitive non-mammalian model used for studying polycyclic aromatic hydrocarbon (PAH)-induced chemical carcinogenesis. The susceptibility of zebrafish to PAH-induced carcinogenesis may be related to the ability of the zebrafish P450s to bioactivate these procarcinogens. As a part of our overall effort to identify the various P450 enzymes that are involved in the activation and detoxification of PAHs in zebrafish, therefore, we have examined the ability of recombinant zebrafish CYP1A (zCYP1A) expressed in yeast to metabolize BaP in vitro. Comparison studies also were conducted with liver microsomes from beta-naphthoflavone (BNF)-treated rainbow trout (Oncorhynchus mykiss). Results demonstrated that the trout liver microsomes were almost twice as active as zCYP1A in oxidizing BaP, with Vmax values of 1.7 and 0.94 nmol/min/nmol P450 for trout and zebrafish preparations, respectively. Like trout CYP1A1, cDNA-expressed zCYP1A was found to oxidize BaP to phenols, quinones and diols (BaP-7,8-diol and BaP-9,10-diol) in the presence of exogenous human microsomal epoxide hydrolase (hEH). BaP-7,8-diol is the precursor of the ultimate carcinogen, BaP-7,8-diol-9,10-epoxide (BaPDE). The ability of zCYP1A to bioactivate BaP was confirmed by the formation of DNA adducts when calf thymus DNA was added to the incubation mixture. BaP-DNA binding was enhanced by the addition of hEH to the incubation mixture. HPLC analysis of the [33P]-postlabeled DNA adducts showed the formation of at least four adducts mediated by both zCYP1A and trout liver microsomes, and one of these adducts co-migrated with BaPDE-dG in HPLC analysis. The addition of hEH to the incubation mixture decreased the formation of BaPDE-dG by zCYP1A and by trout liver microsomes while increasing the formation of an unidentified DNA adduct in the case of zCYP1A. zCYP1A also mediated the binding of BaP to protein, providing further evidence that this enzyme is capable of oxidizing BaP to reactive metabolites that bind to macromolecules. It thus appears that zCYP1A may play an important role in BaP-induced carcinogenesis in the zebrafish model by catalyzing the sequential formation of the ultimate diol epoxide carcinogenic metabolite of BaP.

Plant polyphenols and multidrug resistance: effects of dietary flavonoids on drug transporters in Caco-2 and MDCKII-MDR1 cell transport models.

The hypothesis tested was that specific flavonoids such as epicatechin gallate, epigallocatechin gallate, genistein, genistin, naringenin, naringin, quercetin and xanthohumol will modulate cellular uptake and permeability (P(e)) of multidrug-resistant substrates, cyclosporin A (CSA) and digoxin, across Caco-2 and MDCKII-MDR1 cell transport models. (3)H-CSA/(3)H-digoxin transport and uptake experiments were performed with and without co-exposure of the flavonoids. Aglycone flavonoids reduced the P(e) of CSA to a greater extent than glycosylated flavonoids with 30 microM xanthohumol producing the greatest effect (7.2 x 10(-6) to 6.6 x 10(-7) and 17.9 x 10(-6) to 4.02 x 10(-6) cm s(-1) in Caco-2 and MDCKII-MDR1 cells, respectively); while no measurable effects were seen with digoxin. Xanthohumol significantly demonstrated (1) saturable efflux, (2) increased uptake of (3)H-digoxin and (3) decreased uptake of (3)H-CSA in the Caco-2 cells. The transport data suggests that xanthohumol effects transport of CSA in a manner that is distinct from the digoxin efflux pathway and suggests that intestinal transport of these MDR1 substrates is more complex than previously reported.

CYP2K6 from zebrafish (Danio rerio): cloning, mapping, developmental/tissue expression, and aflatoxin B1 activation by baculovirus expressed enzyme.

A full-length zebrafish (Danio rerio) cytochrome P450 (CYP) 2K6 cDNA, was obtained (GenBank accession No. AF283813) through polymerase chain reaction cloning using degenerated primers based on a consensus CYP2 sequence and the heme-binding domain. This first CYP2K family member cloned from zebrafish had 1861 bp which contained 27 bp of 5'-untranslated region (5'-UTR), an open reading frame (ORF) of 1518 bp, and a 300 bp 3'-UTR with a poly A tail. The deduced 506 amino acid sequence of CYP2K6 had 63%, 62% and 59% identity with rainbow trout CYP2K1, CYP2K4 and CYP2K3, respectively; and 45%, 42%, and 42% identity with rabbit CYP2C1, human CYP2C19 and mouse CYP2C39, respectively. CYP2K6 mapped to 107.49cR on LG3 using the LN54 radiation hybrid panel. Its mRNA was detected at 5 days post-fertilization and in the adult liver and ovary among nine tissues examined. The ORF, including the 27 bp of the 5'-UTR, was cloned into pFastBac donor vector and then transferred into the baculovirus genome (bacmid DNA) in DH10Bac competent cells. The recombinant bacmid DNA was used to infect Spodoptera frugiperda insect cells to express the CYP2K6 protein (Bv-2K6). As its ortholog, rainbow trout Bv-2K1 [Yang, Y.H., Miranda, C.L., Henderson, M.C., Wang-Buhler, J.-L., Buhler, D.R., 2000. Heterologous expression of CYP2K1 and identification of the expressed protein (Bv-2K1) as lauric acid (omega-1)-hydroxylase and aflatoxin B1 exo-epoxidase. Drug Metab. Disp. 28,1279-83.], Bv-2K6 also catalyzed the conversion of aflatoxin B1 (AFB1) to its exo-8,9-epoxide as assessed by the trapping of a glutathione (GSH) adduct in the presence of a specific mouse alpha class glutathione S-transferase. The identity of the AFB1-GSH adduct was verified by liquid chromatography-mass spectrometry (LC-MS) and mass spectrometry-mass spectrometry (MS-MS) analysis. Although rainbow trout Bv-2K1 was capable of oxidizing lauric acid, zebrafish Bv-2K6 protein showed no activity against this substrate.

Influence of prenylated and non-prenylated flavonoids on liver microsomal lipid peroxidation and oxidative injury in rat hepatocytes.

Prenylated chalcones from hops and beer were compared with non-prenylated flavonoids [chalconaringenin (CN), naringenin (NG), genistein (GS) and quercetin (QC)] for their ability to inhibit lipid peroxidation in rat liver microsomes. Chalcones with prenyl- or geranyl-groups (5 and 25 microM) were more effective inhibitors of microsomal lipid peroxidation than CN, NG or GS induced by Fe(2+)/ascorbate. Prenylated chalcones were effective inhibitors of microsomal lipid peroxidation induced by Fe(3+)-ADP/NADPH and by tert-butyl hydroperoxide (TBH) but to a lesser extent compared to the Fe(2+)/ascorbate system. An increase of prenyl substituents decreased antioxidant activity in the lipid peroxidation systems. Certain flavonoids behaved as prooxidants in the iron-dependent lipid peroxidation systems. For example, at 5 microM, NG enhanced iron/ascorbate-induced lipid peroxidation whereas CN, diprenylxanthohumol and tetrahydroxanthohumol enhanced Fe(3+)-ADP/NADPH-induced lipid peroxidation. None of the flavonoids (25 microM), except QC, inhibited NADPH cytochrome P450-reductase activity of rat liver microsomes, suggesting that the mechanism of inhibition of lipid peroxidation induced by Fe(3+)-ADP/NADPH is not due to inhibition of the reductase enzyme. Chalcones exhibiting antioxidant activity against TBH-induced lipid peroxidation such as xanthohumol and 5'-prenylxanthohumol, and NG, with no antioxidant property at 5 microM concentration protected cultured rat hepatocytes from TBH toxicity. Other antioxidants (desmethylxanthohumol and CN) in the TBH system were not cytoprotective. These results demonstrate the importance of prenyl groups in the antioxidant activity of hop chalcones in the various in vitro systems of lipid peroxidation. Furthermore, the antioxidant activity of the flavonoids has little or no bearing on their ability to protect rat hepatocytes from the toxic effects of TBH.

Effects of 17beta-estradiol and testosterone on hepatic mRNA/protein levels and catalytic activities of CYP2M1, CYP2K1, and CYP3A27 in rainbow trout (Oncorhynchus mykiss).

There is growing concern that exposure to chemicals in the environment can disrupt the endocrine systems of wildlife and humans, causing reproductive problems or other adverse effects. The expression of many cytochrome P450s (CYPs) is under hormonal control, hence, levels of these enzymes can be affected by exposure to endocrine-disrupting chemicals. Previous research has reported that treatment of fish and other animals with the estrogenic and androgenic hormones 17beta-estradiol (E2) and testosterone (T) alters the P450 content or enzyme activities in the treated animals. However, the results of many of these studies are either incomplete or in disagreement and in most cases the effect on specific P450 forms has not been determined. Therefore, to better understand the effects of gonadal hormones on the expression of P450s and their associated enzyme activities, it was of interest to undertake a comprehensive investigation of the transcriptional and translational expression of three constitutive hepatic P450s in the rainbow trout (Oncorhynchus mykiss) following hormone exposure. Accordingly, juvenile trout were injected intraperitoneally with propylene glycol vehicle and the most active estrogenic and androgenic hormones E2 (3 mg/kg) or T (3 mg/kg) on days 1, 4, 7, 13, and 15 and euthanized on day 19. After treatment with E2, hepatic microsomes showed significantly lower levels (percentage of control) in total P450 contents (52%), lauric acid hydroxylase (32%), and 6beta-progesterone hydroxylase activities (27%), [(3)H]aflatoxin-DNA binding (31%), and the protein levels of individual cytochrome P450s (CYPs) LMC1 (CYP2M1), LMC2, (CYP2K1), and LMC5 (CYP3A27) (average for three isoforms a reduction to 29% of control values) with only minor differences between sexes. Treatment with T had either no effect or resulted in small increases in total P450 in males (42%), in lauric acid hydroxylase in females (24%), and in 6beta-progesterone hydroxylase activity in males (21%). Biological variabilities among fish were high and a polymorphic or new LMC2-like form was detected at about 52 kDa in some liver microsomal samples after exposure of fish to either hormone. Female liver RNAs were analyzed through Northern blots and an average decrease of 94% in CYP2 M1, CYP2K1, and CYP3A27 mRNA levels occurred in the E2-treated trout. In livers from T-treated trout, the changes of mRNA levels of CYP2M1 and CYP3A27 were negligible, but CYP2K1 mRNA level decreased by about 60%. Additional CYP2K1 cDNA hybridizable mRNAs were seen in some fish as faint bands at about 2.8 kb for both hormone treatments. Results of this study, therefore, indicated that E2 down-regulated while T produced small but variable effects on the hepatic mRNA/protein levels of CYP2K1, CYP2M1, and CYP3A27 in juvenile rainbow trout. This study, therefore, suggests that exposure of fish and other wildlife to environmental endocrine disruptors, especially estrogen mimics, can adversely affect a number of physiological processes through mechanisms involving altered levels of expression of specific P450 isozymes.

Heterologous expression of CYP2K1 and identification of the expressed protein (BV-CYP2K1) as lauric acid (omega-1)-hydroxylase and aflatoxin B1 exo-epoxidase.

LMC2 is the most abundant constitutively expressed hepatic cytochrome P450 found in sexually immature rainbow trout (Onchorynchus mykiss) and is also the isozyme that activates the carcinogen aflatoxin B1 (AFB1). This P450 has been cloned, sequenced, and designated as CYP2K1. The present report describes the heterologous expression of enzymatically active CYP2K1 (BV-CYP2K1) in baculovirus Spodoptera frugiperda (Sf9) insect cells and its catalytic and immunoreactivity characterization in comparison with that of the previously purified LMC2 P450. Homogenates of Sf9 cells expressing the CYP2K1 enzyme and LMC2 both catalyzed the hydroxylation of lauric acid and the epoxidation of AFB1 in the presence of rat NADPH-cytochrome P450 reductase. Both LMC2 and BV-CYP2K1 catalyzed the oxidation of lauric acid primarily at the (omega-1) position plus small amounts at the (omega-2) position. Formation of AFB1 epoxide was shown indirectly by the appearance of an AFB1 epoxide-glutathione conjugate when P450 incubation mixtures contained AFB1, glutathione (GSH) together with mouse liver cytosol or purified rat GSH-transferase. When the AFB1 epoxide-GSH conjugate produced by BV-CYP2K1 and purified LMC2 was analyzed by HPLC using a chiral column, it had a retention time identical to that produced by CYP3A4, a human P450 known to form exclusively the AFB1 exo-epoxide. These results, therefore, confirm that the cDNA-expressed CYP2K1 protein is catalytically and immunologically identical to purified trout LMC2 and that these two enzymes produce primarily the highly carcinogenic stereoisomeric exo-epoxide form of AFB1.

Prenylflavonoids from hops inhibit the metabolic activation of the carcinogenic heterocyclic amine 2-amino-3-methylimidazo[4, 5-f]quinoline, mediated by cDNA-expressed human CYP1A2.

The heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) is a potential human carcinogen found in cooked food that requires initial metabolic activation by cytochrome P450s, primarily CYP1A2. The present study was conducted to examine whether recombinant human CYP1A2 expressed in insect cells mediates the metabolic activation of IQ and whether prenylflavonoids found in hops and beer would modulate the CYP1A2-mediated activation of IQ. The cDNA-expressed human CYP1A2 was found to strongly activate IQ as measured by the Ames Salmonella assay and by the covalent binding of IQ metabolites to calf thymus DNA and protein. Inhibition studies showed that the prenylchalcone xanthohumol and the prenylflavanones 8-prenylnaringenin and isoxanthohumol strongly inhibited the mutagenic activation of IQ mediated by cDNA-expressed human CYP1A2 in the Ames Salmonella assay. The three prenylflavonoids also markedly inhibited the human CYP1A2-mediated binding of IQ to metabolites that bind to DNA. The inhibition of the metabolic activation of IQ was paralleled by the inhibition of acetanilide 4-hydroxylase activity of human CYP1A2. Thus, xanthohumol, isoxanthohumol, and prenylflavanones 8-prenylnaringenin are potent inhibitors of the metabolic activation of IQ and may have the potential to act as chemopreventive agents against cancer induced by heterocyclic amines activated by CYP1A2.

Antioxidant and prooxidant actions of prenylated and nonprenylated chalcones and flavanones in vitro.

Prenylated flavonoids found in hops and beer, i.e., prenylchalcones and prenylflavanones, were examined for their ability to inhibit in vitro oxidation of human low-density lipoprotein (LDL). The oxidation of LDL was assessed by the formation of conjugated dienes and thiobarbituric acid-reactive substances (TBARS) and the loss of tryptophan fluorescence. At concentrations of 5 and 25 microM, all of the prenylchalcones tested inhibited the oxidation of LDL (50 microg protein/ml) induced by 2 microM copper sulfate. The prenylflavanones showed less antioxidant activity than the prenylchalcones, both at 5 and 25 microM. At 25 microM, the nonprenylated chalcone, chalconaringenin (CN), and the nonprenylated flavanone, naringenin (NG), exerted prooxidant effects on LDL oxidation, based on TBARS formation. Xanthohumol (XN), the major prenylchalcone in hops and beer, showed high antioxidant activity in inhibiting LDL oxidation, higher than alpha-tocopherol and the isoflavone genistein but lower than the flavonol quercetin. When combined, XN and alpha-tocopherol completely inhibited copper-mediated LDL oxidation. These findings suggest that prenylchalcones and prenylflavanones found in hops and beer protect human LDL from oxidation and that prenylation antagonizes the prooxidant effects of the chalcone, CN, and the flavanone, NG.

Isoform specificity of N-deacetyl ketoconazole by human and rabbit flavin-containing monooxygenases.

N-Deacetyl ketoconazole (DAK) is the major metabolite of orally administered ketoconazole. This major metabolite has been demonstrated to be further metabolized predominately by the flavin-containing monooxygenases (FMOs) to the secondary hydroxylamine, N-deacetyl-N-hydroxyketoconazole (N-hydroxy-DAK) by adult and postnatal rat hepatic microsomes. Our current investigation evaluated the FMO isoform specificity of DAK in a pyrophosphate buffer (pH 8.8) containing the glucose 6-phosphate NADPH-generating system. cDNA-expressed human FMOs (FMO1, FMO3, and FMO5) and cDNA-expressed rabbit FMOs (FMO1, FMO2, FMO3, and FMO5) were used to assess the metabolism of DAK to its subsequent FMO-mediated metabolites by HPLC analysis. Human and rabbit cDNA-expressed FMO3 resulted in extensive metabolism of DAK in 1 h (71.2 and 64.5%, respectively) to N-hydroxy-DAK (48.2 and 47.7%, respectively) and two other metabolites, metabolite 1 (11.7 and 7.8%, respectively) and metabolite 3 (10.5 and 10.0%, respectively). Previous studies suggest that metabolite 1 is the nitrone formed after successive FMO-mediated metabolism of N-hydroxy-DAK. Moreover, these studies display similar metabolic profiles seen with adult and postnatal rat hepatic microsomes. The human and rabbit FMO1 metabolized DAK predominately to the N-hydroxy-DAK in 1 h (36.2 and 25.3%, respectively) with minimal metabolism to the other metabolites (

In vitro inhibition of human P450 enzymes by prenylated flavonoids from hops, Humulus lupulus.

1. Several unique flavonoid compounds have recently been isolated from hops, Humulus lupulus, and their presence has been detected in beer. Their chemical structures are similar to other plant-derived compounds, many present in the human diet, that have been shown to have cancer chemopreventive properties due, in part, to inhibition of cytochrome P450 enzymes that activate carcinogens. Additionally, preliminary studies have shown these flavonoids (at 100 microM) to be inhibitory of P450-mediated activation reactions in a variety of in vitro systems. Thus, the in vitro effects of these phytochemicals on cDNA-expressed human CYP1A1, CYP1B1, CYP1A2, CYP3A4 and CYP2E1 were currently examined by the use of diagnostic substrates and the carcinogen AFB1. 2. At 10 microM, the prenylated chalcone, xanthohumol (XN), almost completely inhibited the 7-ethoxyresorufin O-deethylase (EROD) activity of CYP1A1. At the same concentration, other hop flavonoids decreased the EROD activity by 90.8-27.0%. 3. At 10 microM, XN completely eliminated CYP1B1 EROD activity, whereas the other hop flavonoids showed varying degrees of inhibitory action ranging from 99.3 to 1.8%. 4. In contrast, the most effective inhibitors of CYP1A2 acetanilide 4-hydroxylase activity were the two prenylated flavonoids, 8-prenylnaringenin (8PN) and isoxanthohumol (IX), which produced > 90% inhibition when added at concentrations of 10 microM. 5. CYP1A2 metabolism of the carcinogen AFB1 was also inhibited by IX and 8PN as shown by decreased appearance of dihydrodiols and AFM1 as analysed by hplc. IX and 8PN also decreased covalent binding of radiolabelled AFB1 to microsomal protein in a concomitant manner. 6. XN, IX and 8PN, however, were poor inhibitors of CYP2E1 and CYP3A4 as measured by their effect on chorzoxazone hydroxylase and nifedipine oxidase activities respectively. 7. These results suggest that the hop flavonoids are potent and selective inhibitors of human cytochrome P450 and warrant further in vivo investigations.

Prenylated chalcones and flavanones as inducers of quinone reductase in mouse Hepa 1c1c7 cells.

The objective of this study was to determine if prenylchalcones (open C-ring flavonoids) and prenylflavanones from hops and beer are inducers of quinone reductase (QR) in the mouse hepatoma Hepa 1c1c7 cell line. All the prenylchalcones and prenylflavanones tested were found to induce QR but not CYP1A1 in this cell line. In contrast, the synthetic chalcone, chalconaringenin, and the flavanone, naringenin, with no prenyl or geranyl groups, were ineffective in inducing QR. The hop chalcones, xanthohumol and dehydrocycloxanthohumol hydrate, also induced QR in the Ah-receptor-defective mutant cell line, Hepa 1c1c7 bp(r)c1. Thus, the prenylflavonoids represent a new class of monofunctional inducers of QR.

Antiproliferative and cytotoxic effects of prenylated flavonoids from hops (Humulus lupulus) in human cancer cell lines.

Six flavonoids [xanthohumol (XN), 2',4',6',4-tetrahydroxy-3'-prenylchalcone (TP); 2',4',6',4-tetrahydroxy-3'-geranylchalcone (TG); dehydrocycloxanthohumol (DX); dehydrocycloxanthohumol hydrate (DH); and isoxanthohumol (IX)] from hops (Humulus lupulus) were tested for their antiproliferative activity in human breast cancer (MCF-7), colon cancer (HT-29) and ovarian cancer (A-2780) cells in vitro. XN, DX and IX caused a dose-dependent (0.1 to 100 microM) decrease in growth of all cancer cells. After a 2-day treatment, the concentrations at which the growth of MCF-7 cells was inhibited by 50% (IC50) were 13.3, 15.7 and 15.3 microM for XN, DX and IX, respectively. After a 4-day treatment, the IC50 for XN, DX and IX were 3.47, 6.87 and 4.69 microM, respectively. HT-29 cells were more resistant than MCF-7 cells to these flavonoids. In A-2780 cells, XN was highly antiproliferative with IC50 values of 0.52 and 5.2 microM after 2 and 4 days of exposure, respectively. At 100 microM, all the hop flavonoids were cytotoxic in the three cell lines. Growth inhibition of XN- and IX-treated MCF-7 cells was confirmed by cell counting. XN and IX inhibited DNA synthesis in MCF-7 cells. As antiproliferative agents, XN (chalcone) and IX (flavanone isomer of XN) may have potential chemopreventive activity against breast and ovarian cancer in humans.

Intervention based on monthly monitoring decreases hemodialysis access thrombosis.

We randomized 103 patients (68 arteriovenous [AV] fistulas, 35 polytetrafluoroethylene [PTFE] grafts; mean follow-up 197 days) to monthly measurement of access flow (QAT), monthly measurement of static venous pressure (VPS), or no monthly monitoring (control patients) to determine whether access thrombosis would decrease. Patients with access flow <750 cc/min or with static venous pressure > or =0.5 were referred for angiography and angioplasty of stenotic lesions > or =50%. Six of sixty-two (9.7%) of monthly monitored patients (MM) developed access thrombosis vs. 9 of 41 (22%) of control patients (p<0.05). Fewer MM patients developed thrombosis in AV fistulas (2.4% [2 of 42] vs. 15.4% [4 of 26] control patients; p<0.05). Monthly monitored patients had fewer thrombotic episodes than control patients (19 vs. 125 per 100 patient-years; p<0.01). Thrombosis rates were lowest in patients receiving monthly access flow measurement (5.9 [QAT] vs. 30.3 per 100 patient-years [VPS]; p<0.05). In conclusion, intervention based on monthly access flow measurement or static venous pressure decreased hemodialysis access thrombosis. Measurement of access flow tended to result in lower thrombosis rates than after static venous pressure. We believe that monthly access flow measurement will ensure the lowest incidence of thrombosis and decrease the cost of access maintenance.

Species differences in the hepatic microsomal enzyme metabolism of the pyrrolizidine alkaloids.

Species differences in pyrrolic metabolites and senecionine (SN) N-oxide formation among eight animal species (sheep, cattle, gerbils, rabbits, hamsters, Japanese quail, chickens, rats) varying in susceptibility to pyrrolizidine alkaloid (PA) intoxication were measured in vitro by hepatic microsomal incubations. The results suggested that there is not a strong correlation between the production of pyrrolic metabolites and susceptibility of animals to PA toxicity. The rate of PA activation in hamsters, a resistant species, measured by formation of (+/-)6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) far exceeded the rate of SN N-oxide formation (detoxification) (DHP/N-oxide = 2.29). In contrast, SN N-oxide was the major metabolite in sheep, another resistant species, with much lower production of DHP (DHP/N-oxide = 0.26). The roles of cytochrome P450s and flavin-containing monooxygenases (FMO) in bioactivation and detoxification of pyrrolizidine alkaloids (PA) were studied in vitro using sheep and hamster hepatic microsomes. Chemical and immunochemical inhibition data suggested that the conversion of SN to DHP is catalyzed mainly by cytochrome P450s (68-82%), whereas the formation of SN N-oxide is carried out largely by FMO (55-71%). There also appeared to be a high rate of glutathione-DHP conjugation in hamster (63%) and sheep (79%) liver microsomal incubation mixtures. Therefore, low rates of pyrrole metabolite production coupled with glutathione conjugation in sheep may explain the resistance of sheep to SN, whereas the high rate of GSH-DHP conjugation may be one of the factors contributing to the resistance of hamsters to intoxication by this PA.

Cloning, sequencing, and tissue expression of CYP3A27, a new member of the CYP3A subfamily from embryonic and adult rainbow trout livers.

Screening of lambdagt11 and lambdagt22A cDNA libraries of livers from adult females and embryos of rainbow trout (Oncorhynchus mykiss), respectively, using rabbit anti-rainbow trout cytochrome P450 LMC5 polyclonal antibodies showed that there were identical cDNAs of 1802-bp nucleotides with open reading frames coding for proteins containing 518 amino acids (59,206 Da, pI = 6.39). The cDNA was assigned CYP3A27 by the P450 Nomenclature Committee to represent the first CYP3A subfamily member reported for aquatic species. The deduced N-terminal sequence of CYP3A27 was in agreement with 8 of the first 12 confirmed amino acid residues from Edman degradation of LMC5, a P450 previously isolated from juvenile trout liver. In similarity comparisons between species by positional alignment, the deduced amino acid sequence of rainbow trout CYP3A27 was 56.4% identical with dog CYP3A12, 56.0% with monkey CYP3A8, 54.9% with human CYP3A4, 54.7% with rat CYP3A9, and 54.2% with sheep CYP3A24. Marked differences in sex, age, and tissue expression of CYP3A27 in rainbow trout were observed at the mRNA level as shown by Northern blots. The major extrahepatic expression site for CYP3A27 was upper small intestine. Females expressed considerably more CYP3A27 mRNA than male in the fish examined. Southern blot analysis of restriction enzyme-digested rainbow trout genomic DNA demonstrated that multiple CYP3A27-related genes exist in rainbow trout.

The roles of CYP3A and CYP2B isoforms in hepatic bioactivation and detoxification of the pyrrolizidine alkaloid senecionine in sheep and hamsters.

The roles of cytochrome CYP3A and CYP2B isozymes in the bioactivation and detoxification of the pyrrolizidine alkaloid (PA) senecionine (SN) have been investigated in vitro with sheep and hamster hepatic microsomes. Our results show that the rate of SN activation measured by (+/-)-6, 7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) formation greatly exceeded the rate of SN N-oxide formation (detoxification) in hamsters. In contrast, SN N-oxide, a detoxification product, was the major metabolite in sheep with much lower DHP production. Immunoinhibition studies with anti-sheep CYP3A and CYP2B antibodies show that members of CYP3A subfamily play the major role in the conversion of PA to pyrrolic metabolites in both species (over 90% in sheep; 68% in hamster). These enzymes also contribute 38.8 and 41. 3% of SN N-oxidation in sheep and hamsters, respectively. In contrast, CYP2B isoforms have a limited capacity toward DHP formation in both species (47% in sheep; 32% in hamster), while these enzymes catalyzed only 24.6 and 35.4% SN N-oxidation in sheep and hamster, respectively. Using triacetyloleandomycin (TAO) and gestodene, two highly selective chemical inhibitors of CYP3A isoforms, our data show that 90% of DHP formation was inhibited by either inhibitor in sheep. Gestodene appeared to be more efficient than TAO in the inhibition of DHP production in hamsters. Testosterone 6beta-hydroxylase activity, a functional marker of CYP3A, was significantly inhibited by TAO and gestodene in sheep liver microsomes and by gestodene (100 microM) in hamster liver microsomes. These results suggest that CYP3A isozymes have important roles in bioactivation and detoxification of PA in both species, whereas CYP2B subfamily members are less efficient in biotransformation of PA.

CYP2M1: cloning, sequencing, and expression of a new cytochrome P450 from rainbow trout liver with fatty acid (omega-6)-hydroxylation activity.

A cDNA clone was isolated from a female rainbow trout liver lambda g tau 11 library using polyclonal antibodies raised against rainbow trout cytochrome P450 LMC1. This 2149-nucleotide clone contained an open reading frame encoding a protein of 499 amino acids with a calculated M(r) of 56,850 Da. On the basis of cytochrome P450 (P450) amino acid sequence comparisons, this rainbow trout P450 was assigned by the P450 Nomenclature Committee to a new P450 subfamily designated as CYP2M1. Northern blot results suggest that the expression of CYP2M1 at the transcriptional level was generally sex, tissue, and age specific. By use of a full-length CYP2M1 cDNA probe, it was observed that this cDNA hybridized strongly to a single 2.2-kb transcript in juvenile female rainbow trout trunk kidney and in liver from juvenile and sexually mature trout from both sexes. Negligible amounts of mRNA hybridizable to CYP2M1 cDNA were found in the juvenile and sexually mature male trunk kidney. cDNA-directed expression in COS-7 cells and of recombinant baculovirus in insect cells produced a protein that was reactive with rabbit anti-trout P450 LMC1 polyclonal antibody and exhibited the unique (omega-6)-hydroxylation toward lauric acid previously observed with rainbow trout P450 LMC1.

Expression of CYP2M1, CYP2K1, and CYP3A27 in brain, blood, small intestine, and other tissues of rainbow trout.

Expression of five constitutive forms of cytochrome P450 [(LMC1 (CYP2M1), LMC2 (CYP2K1), LMC3, LMC4, and LMC5 (CYP3A27)] in selected tissues from sexually immature 2-year old female and male rainbow trout (Oncorhynchus mykiss) were examined at the translational level by Western blot using polyclonal antibodies raised in rabbits against those purified trout hepatic P450s. Tissues examined were from brain, liver, muscle, blood, head kidney, trunk kidney, upper intestine, stomach, heart, and gonad (ovary or testis). The results showed that the liver was the major organ for expression of all the trout P450s studied. Trunk kidney was the secondary expression site except for LMC5. Selective translational expression of these P450 isoforms or similar proteins was observed for LCM1 and LMC5 in brain; for LMC2 and LMC5 in female upper intestine; and for LMC2 in blood plasma of the fish studied under the experimental and sampling conditions.