Microsomal Mixed-function Oxidase Research Articles

Methoxol's Impact on the Hepatic Microsomal Mixed Function Oxidase System Induced by Phenobarbital and Pyrene in Rats

Insecticides like methomyl (Lannate) are commonly used to keep pests at bay in vineyards. The mixed-function oxidase system undergoes dramatic change after being exposed to methomyl. This research set out to determine if methomyl inhibits phenobarbital-induced CYP 2B1, 2B2, and 3A, as well as benzo[a]pyrene-induced CYP 2D6 (CYP 1A1). There were a total of eight groups, each including six adult male rats. Calcium precipitation was used to separate the microsomes. Extinction coefficients were used to quantify the amounts of the electron transport components cytochrome P450, cytochrome B5, and cytochrome c-reductase. Drug metabolizing enzymes were tested for their efficacy. Mixed function oxidase was significantly elevated in rats when exposed to inducers like phenobarbital and benzo[a]pyrene. Inducer-(Phenobarbital, benzop [a] pyrene) pretreated rats given 4mg/kg methomyl had significantly lower levels of electron transport components and drug-metabolizing enzyme activity compared to inducer treatment alone. The enzymes involved in mixed-function oxidation were likewise affected by methomyl's influence on the induction of phenobarbital. The levels of electron transport components and drug metabolizing enzymes in methomyl-pretreated rats treated with benzo[a]pyrene were much lower than in rats treated with benzo[a]pyrene alone. These findings show that phenobarbital and benzo[a]pyrene increased cytochrome P450 isoform susceptibility to methomyl (CYP 2B1, 2B2, 3A; and CYP 1A1) and the induction pattern of some inducers with respect to CYP 450 isoforms are both modified by methomyl

Role of Cytochrome P450 and Oxidative Stress in Alcohol-Induced Liver Injury

Alcohol-induced liver injury is a significant global health problem. The mechanisms by which alcohol causes cell injury are still not clear. Many of the hepatic toxic effects of ethanol have been linked to its metabolism in the liver and shown to correlate with cytochrome P450 2E1 (CYP2E1) levels, lipid peroxidation, and elevated formation of reactive oxygen species (ROS). CYP2E1 is induced under a variety of pathophysiological conditions, by drugs, and by alcohol. The goal of this review is to describe and characterize the liver cytochrome P450 microsomal mixed function oxidase system including the generation of ROS. The review then discusses biochemical and toxicological properties of CYP2E1 and its contribution to ethanol-elevated oxidant stress and liver pathology.

Lethal and sublethal effects of the chitin synthesis inhibitor chlorfluazuron on Bradysia odoriphaga Yang and Zhang (Diptera: Sciaridae)

Bradysia odoriphaga Yang and Zhang is the primary insect pest that affects Chinese chive in northern China. Nevertheless, very few studies have been conducted on the use of chitin synthesis inhibitors (CSIs) for the control of B. odoriphaga. Here, lethal and sublethal effects of the CSI chlorfluazuron on B. odoriphaga were studied to explore the use for integrated pest management (IPM) of B. odoriphaga. A contact and ingestion toxicity bioassay showed that chlorfluazuron was more active against B. odoriphaga than three other CSIs, with a 72h LC50 of 0.1593mg/L. Treatment with the LC50 dose of chlorfluazuron decreased both the intrinsic and finite rates of increase of B. odoriphaga, in addition to reproduction rate, survival rate, and fecundity, and the mean generation time, total preovipositional period and larval development duration were shortened, compared with those of the control and LC10 groups. The mean generation time, total preovipositional period and larval development duration were all also markedly decreased by treatment with chlorfluazuron at the LC10. Furthermore, chlorfluazuron inhibited the feeding of the final instar larvae for a short period. Glutathione S-transferase and microsomal mixed function oxidase activities increased after exposure to the chemical. These results showed that chlorfluazuron at the sublethal LC50 treatment inhibited B. odoriphaga population growth, whereas the danger of causing rapid population growth by using a lower sublethal concentration was demonstrated with the sublethal LC10 treatment. Therefore, chlorfluazuron should be used with caution in an IPM program for B. odoriphaga.

Photo-initiated crosslinking extends mapping of the protein–protein interface to membrane-embedded portions of cytochromes P450 2B4 and b5

Protein–protein interactions play a central role in the regulation of many biochemical processes (e.g. the system participating in enzyme catalysis). Therefore, a deeper understanding of protein–protein interactions may contribute to the elucidation of many biologically important mechanisms. For this purpose, it is necessary to establish the composition and stoichiometry of supramolecular complexes and to identify the crucial portions of the interacting molecules.This study is devoted to structure–functional relationships in the microsomal Mixed Function Oxidase (MFO) complex, which is responsible for biotransformation of many hydrophobic endogenous compounds and xenobiotics. In particular, the cytochrome b5 interaction with MFO terminal oxygenase cytochrome P-450 (P450) was studied. To create photolabile probes suitable for this purpose, we prepared cytochrome b5 which had a photolabile diazirine analog of methionine (pMet) incorporated into the protein sequence, employing recombinant expression in Escherichia coli. In addition to wild-type cytochrome b5, where three methionines (Met) are located at positions 96, 126, and 131, six mutants containing only one Met in the sequence were designed and expressed (see Table 1). In these mutants, a single Met was engineered into the catalytic domain (at positions 23, 41, or 46), into the linker between the protein domains (at position 96), or into the membrane region (at positions 126 or 131). These mutants should confirm or exclude these portions of cytochrome b5 which are involved in the interaction with P450.After UV irradiation, the pMet group(s) in the photolabile cytochrome b5 probe was(were) activated, producing covalent crosslinks with the interacting parts of P450 2B4 in the close vicinity. The covalent complexes were analyzed by the “bottom up” approach with high-accuracy mass spectrometry. The analysis provided an identification of the contacts in the supramolecular complex with low structural resolution. We found that all the above-mentioned cytochrome b5 Met residues can form intermolecular crosslinks and thus participate in the interaction. In addition, our results indicate the existence of at least two P450:cytochrome b5 complexes which differ in the orientation of individual proteins.The results demonstrate the advantages of the photo-initiated crosslinking technique which is able to map the protein–protein interfaces not only in the solvent exposed regions, but also in the membrane-embedded segments (compared to a typical crosslinking approach which generally only identifies crosslinks in solvent exposed regions).

Molecular mechanisms of the microsomal mixed function oxidases and biological and pathological implications

The cytochrome P450 mixed function oxidase enzymes play a major role in the metabolism of important endogenous substrates as well as in the biotransformation of xenobiotics. The liver P450 system is the most active in metabolism of exogenous substrates. This review briefly describes the liver P450 (CYP) mixed function oxidase system with respect to its enzymatic components and functions. Electron transfer by the NADPH-P450 oxidoreductase is required for reduction of the heme of P450, necessary for binding of molecular oxygen. Binding of substrates to P450 produce substrate binding spectra. The P450 catalytic cycle is complex and rate-limiting steps are not clear. Many types of chemical reactions can be catalyzed by P450 enzymes, making this family among the most diverse catalysts known. There are multiple forms of P450s arranged into families based on structural homology. The major drug metabolizing CYPs are discussed with respect to typical substrates, inducers and inhibitors and their polymorphic forms. The composition of CYPs in humans varies considerably among individuals because of sex and age differences, the influence of diet, liver disease, presence of potential inducers and/or inhibitors. Because of such factors and CYP polymorphisms, and overlapping drug specificity, there is a large variability in the content and composition of P450 enzymes among individuals. This can result in large variations in drug metabolism by humans and often can contribute to drug–drug interactions and adverse drug reactions. Because of many of the above factors, especially CYP polymorphisms, there has been much interest in personalized medicine especially with respect to which CYPs and which of their polymorphic forms are present in order to attempt to determine what drug therapy and what dosage would reflect the best therapeutic strategy in treating individual patients.

Effect of enrofloxacin on hepatic microsomal mixed function oxidases in camel

Administration of enrofloxacin at a dose of 5 mg/kg intramuscularly for 3 days has resulted in a significant decrease in aminopyrine-N-demathylase and aniline-4-hydroxylase in liver of camel compared to saline treated (controls). It is suggested that co-administration of drugs metabolised by phase-I reaction with enrofloxacin should be avoided in camels.

Polycyclic Aromatic Hydrocarbons (PAHs) Mediate Transcriptional Activation of the ATP Binding Cassette Transporter ABCB6 Gene via the Aryl Hydrocarbon Receptor (AhR)

Liver is endowed with a mechanism to induce hepatic cytochromes P450 (CYP450s) in response to therapeutic drugs and environmental contaminants, leading to increased detoxification and elimination of the xenobiotics. Each CYP450 is composed of an apoprotein moiety and a heme prosthetic group, which is required for CYP450 activity. Thus, under conditions of CYP450 induction, there is a coordinate increase in heme biosynthesis to compensate for the increased expression of CYP450s. ABCB6, a mitochondrial ATP binding cassette transporter, which regulates coproporphyrinogen transport from the cytoplasm into the mitochondria to complete heme biosynthesis, represents a previously unrecognized rate-limiting step in heme biosynthesis. However, it is not known if exposure to drugs and environmental contaminants induces ABCB6 expression, to assure an adequate and apparently coordinated supply of heme for the generation of functional cytochrome holoprotein. In the present study, we demonstrate that polycyclic aromatic hydrocarbons (PAHs), the widely distributed environmental toxicants shown to induce porphyrin accumulation causing hepatic porphyria, up-regulate ABCB6 expression in both mice and humans. Using siRNA technology and Abcb6 knock-out mice, we demonstrate that PAH-mediated increase in hepatic porphyrins is compromised in the absence of ABCB6. Moreover, in vivo studies in aryl hydrocarbon receptor (AhR) knock-out mice demonstrate that PAH induction of ABCB6 is mediated by AhR. Promoter activation studies combined with electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrate direct interactions between the AhR binding sites in the ABCB6 promoter and the AhR receptor, implicating drug activation mechanisms for ABCB6 similar to those found in inducible cytochrome P450s. These studies are the first to describe direct transcriptional activation of both mouse and human ABCB6 by xenobiotics.

Characteristics of cytochrome P450 and its application

Cytochrome P450(CYP450)is the most important family of enzymes in microsomal mixedfunction oxidase,widely distributed in vivo.CYP450 is involved in the metabolism of many exogenous compounds,the generation of endogenous substances,in particularly,affecting the occurrence and development of the tumors and their drug treatment,in the fields of medical and pharmaceutical research,cytochrome P450 has been very noticeable. Key words: Cytochrome P-450 enzyme system; Drug interactions; Neoplasms

Gender-related difference in the toxicity of 2-(2′-hydroxy-3′,5′-di-tert-butylphenyl)benzotriazole in rats: Relationship to the plasma concentration, in vitro hepatic metabolism, and effects on hepatic metabolizing enzyme activity

Previously, we showed that the toxic susceptibility of male rats to an ultraviolet absorber, 2-(2′-hydroxy- 3′,5′-di-tert-butylphenyl)benzotriazole (HDBB), was nearly 25 times higher than that of females. The present study aimed to clarify the mechanism of gender-related differences in HDBB toxicity. Male and female rats were given HDBB by gavage at 0.5, 2.5, or 12.5 mg/kg/day for 28 days, and plasma HDBB levels were measured at various time points by using liquid chromatography–tandem mass spectrometry. HDBB was rapidly absorbed and eliminated from the plasma in both sexes, and no sexual variations were found in the plasma levels. In the plasma, HDBB metabolites were not detected at any dose by the liquid chromatography–photodiode array detector. In an in vitro metabolic study using hepatic microsomes from male and female rats, HDBB was slightly metabolized, but no sexual differences were found in the residual HDBB ratio after a 60-minute incubation with an NADPH-generation system. Following 28-day HDBB administration, sexually different changes were found in cytochrome P450–dependent microsomal mixed-function oxidase activities in the liver. In males, 7-ethoxyresorufin O-deethylase activity decreased and lauric acid 12-hydroxylase activity increased at all doses. Decreases in aminopyrine N-demethylase activity and testosterone 2α- and 16α-hydroxylase activity were also found at 2.5 mg/kg and above in males. In females, the only significant change was increased lauric acid 12-hydroxylase activity at 12.5 mg/kg. These findings indicate that HDBB would have hepatic peroxisome proliferative activity, and the difference in susceptibility of male and female rats to this effect might lead to marked gender-related differences in HDBB toxicity.

Effect of biliary obstruction and internal biliary drainage on hepatic cytochrome P450 isozymes in rats

To investigate the total cytochrome P450 (CYP) content, microsomal mixed-function oxidase (MFO) activity, and expression of mRNAs for various CYP isozymes in a simple rat model of reversible obstructive jaundice. Obstructive jaundice was created in male rats by causing bile duct obstruction with polyester tape. In another group of rats, bile duct obstruction was followed by internal biliary drainage after releasing the tape. The expression of various CYP isozyme mRNAs was semi-quantitatively assessed by competitive RT-PCR. The total CYP content and microsomal MFO activity showed a significant decrease after biliary obstruction, but returned to respective control levels after biliary drainage. A marked reduction in the expression of CYP1A2, 2B1/2, 2C11, 2E1, 3A1, and 3A2 mRNA was detected during biliary obstruction, while expression increased significantly toward the control level after biliary drainage. Although expression of CYP4A1 mRNA showed no reduction during biliary obstruction, it still increased significantly after biliary drainage. These results suggest that not only obstructive jaundice, but also the subsequent internal biliary drainage may affect regulatory medications of the synthesis of individual CYP isozymes differently.

Recalling P446. P4501A1 (CYP1A1) Opting for Clinical Application

This review goes back to spectral studies [see ]. The findings of apparent absolute spectra of two interconvertible forms of microsomal mixed function oxidases are looked back on to recall whether their impact sustained scrutiny or are rather remembered as of sentimental value only.The second part summarizes studies on the clinical relevance of CYP1A1 with special reference to our investigations. The impact of genetic variability of CYP1A1 on cancer susceptibility, differential effects of polyphenols and hyperforin on toxification and detoxification pathways of benzo[a]pyrene, and differential metabolite patterns of 17ß-estradiol, estrone, and eicosapentaenoic acid are presented.

Testing the ecotoxicology of vegetable versus mineral based lubricating oils: 2. Induction of mixed function oxidase enzymes in barramundi, Lates calcarifer, a tropical fish species

An increasing number of vegetable-based oils are being developed as environmentally friendly alternatives to petroleum products. However, toxicity towards key tropical marine species has not been investigated. In this study we used laboratory-based biomarker induction experiments to compare the relative stress of a vegetable-based lubricating oil for marine 2-stroke engines with its mineral oil-based counterpart on tropical fish. The sub-lethal stress of 2-stoke outboard lubricating oils towards the fish Lates calcarifer (barramundi) was examined using liver microsomal mixed function oxidase (MFO) induction assays. This study is the first investigation into the use of this key commercial species in tropical North Queensland, Australia in stress assessment of potential hydrocarbon pollution using ethoxyresorufin O-deethylase (EROD) induction. Our results indicated that barramundi provide a wide range of inducible rates of EROD activity in response to relevant organic stressors. The vegetable- and mineral-based lubricants induced significant EROD activity at 1.0 mg kg−1 and there was no significant difference between the two oil treatments at that concentration. At increasing concentrations of 2 and 3 mg kg−1, the mineral-based lubricant resulted in slightly higher EROD activity than the vegetable-based lubricant. The EROD activity of control and treated barramundi are found to be within ranges for other species from temperate and tropical environments. These results indicate that vegetable-based lubricants may be less stressful to barramundi than their mineral counterparts at concentrations of lubricant ⩾2 mg kg−1. There is great potential for this species to be used in the biomonitoring of waterways around tropical North Queensland and SE Asia.

Effect of the Degree of Hydrogenation of Fish Oil on the Enzymatic Activity and on the Fatty Acid Composition of Hepatic Microsomes from Young and Aged Rats

By modifying the degree of hydrogenation of dietary fat, it is possible to modify the fatty acid composition and the biochemical activity of cellular tissues. The age can be another variable influencing these modifications. The effect of isocaloric diets containing oils with different degrees of hydrogenation: fish oil (FO, 0.3% trans), partially hydrogenated fish oil (PHFO, 29% trans), or highly hydrogenated fish oil (HHFO, 2.3% trans), in the fatty acid composition (cis and trans isomers) of hepatic microsomes from young (70-day-old) and aged (18-month-old) rats, in the microsomal cytochrome P-450 (C-450) content, and in the aminopyrine N-demethylase (AND), aniline hydroxylase (AH), NADPH cytochrome P-450 reductase (NCR), UDP-glucuronyl transferase (UGT), and GSH-S transferase (GST) enzymatic activities were studied. Fatty acid composition and n–6/n–3 ratio of microsomal membranes was modified to a higher extent in young rats. C-450 content and AND activity were reduced when the degree of hydrogenation of dietary fat was increased in the young and the aged rats. AH activity was higher after the PHFO diet in the young rats only. NCR activity was reduced in the young animals when the hydrogenation of the fat was increased. However, in aged rats the enzyme exhibited a higher activity after the PHFO and HHFO diet. UGT and GST activities where not affected by the level of hydrogenation of the dietary fat in both the young and the aged rats. However, UGT activity was higher in the young rats, while GST activity was higher in the aged animals. We conclude that hydrogenation of dietary fat can modify the fatty acid composition of hepatic microsomes, young animals being more sensitive to these changes than aged animals. These effects were also reflected in the amount and/or the activity of some molecular components of the hepatic microsomal mixed-function oxidase enzyme system. Microsomal trans fatty acid composition is not affecting the activity of the enzymes, the age of the animals being the most important factor

Activated transformations of organophosphorus insecticides in the case of non-AChE inhibitory oxons.

Many organophosphorus (OP) compounds are of the thiono form and in insects or animals are converted by microsomal mixed function oxidases (MFO) into the oxon forms which inhibit acetylcholinesterase (AChE) and give toxic activity. However, certain S-alkyl phosphorothiolates (RS-P(O) <) such as methamidophos, profenophos and prothiophos oxon are strongly insecticidal, but very poor inhibitors of AChE in vitro. Their oxons are converted further to the S-oxides, which either inhibit AChE or decompose, depending on the alkyl substituents on the sulfur atom. It is also inferred in the case of prothiophos oxon that its S-oxide not only inhibits AChE but also conjugates with glutathione (GSH) by the action of glutathione S-transferase (GST), and the conjugate inhibits AChE. Certain phosphoramidates (R2N-P(O) <) such as isofenphos oxon, schradan and propetamphos oxon are weak AChE inhibitors, but strongly insecticidal. It is well known that isofenphos oxon is converted into the stable N-desalkyl form (H2N-P(O) <) by oxidative dealkylation to inhibit AChE. The authors have studied activation of phosphoramidates using 2,4-dichlorophenyl methyl N-alkylphosphoramidates as model compounds using various approaches including computational chemistry, and these studies indicated that the O-aminophosphate structure (R2N-O-P(O) <) is an activated form.

Effects of dietary organic chromium on gilthead seabream (Sparus aurata L.) performances and liver microsomal metabolism

The effect of yeast and chromium yeast on gilthead seabream (Sparus auratus L) performance, carcass indices and body composition was studied. Whether supplementation affected liver microsomal mixed function oxidases using either multibioprobes (testosterone) or highly specific substrates to cytochrome P450 (CYP) isoforms was also investigated. Seabream juveniles (35‐37 g initial weight) were allocated into 12 800 L tanks of 50 fish each for 87 days and fed pelleted experimental diets, i.e. control, yeast supplemented (1.6%) and chromium yeast supplemented at both low (800 p.p.b.) and high chromium level (53 810 p.p.b.). At the end of the experiment, growth, feed conversion ratio, thermal-unit growth coefficient, carcass yield, hepatosomatic index, and carcass and fillet proximate compositions were similar among treatments and only condition factor was statistically different. Organic chromium at both doses affected CYP-catalysed drug reactions slightly, as shown by the modest effect on the regio- and stereoselective hydroxylations of testosterone, as well as the metabolism of the selected probes. Overall, we found that chromium yeast did not change performance substantially, nor carcass indices, carcass and fillet chemical compositions, or hepatic xenobiotic metabolizing enzymes of gilthead seabream.

Induction of hepatic microsomal drug-metabolizing enzymes by inhibitors of 5-lipoxygenase (5-LO): studies in rats and 5-LO knockout mice.

The effect of 5-lipoxygenase (5-LO) inhibitors on the hepatic microsomal mixed-function oxidase (MFO) system of rodents was investigated. After establishing the relative in vitro and in vivo potencies of the 3 test compounds, male Crl:CD (SD) BR rats received CJ-11,802 (0, 10, 50, or 200 mg/kg/day), zileuton (0, 10, 60, or 300 mg/kg/day) or ZD2138 (0 or 200 mg/kg/day) once daily by oral gavage for 14 (zileuton and ZD2138) or 30 (CJ-11,802) consecutive days. Controls were given an equivalent volume of 0.5% methylcellulose vehicle. At necropsy, all livers were weighed, and sections from representative animals (control and highest dose for each compound) were utilized to prepare hepatic microsomal fractions, which were assayed for cytochrome P-450 (CYP) content and the activities of cytochrome c reductase (CRed), para-nitroanisole O-demethylase (p-NOD), ethoxyresorufin O-deethylase (EROD), and pentoxyresorufin O-dealkylase (PROD). A dose-related increase in liver weight occurred in rats given CJ-11,802 and zileuton, while animals administered ZD2138 were unaffected. Rats given CJ-11,802 (200 mg/kg/day) and zileuton (300 mg/kg/day) had increases in CYP, EROD, PROD, CRed and p-NOD compared to corresponding controls, while only the latter two activities were elevated in animals administered ZD2138. To determine if induction of the hepatic microsomal MFO system was related to 5-LO inhibition, male DBA wild-type and 5-LO knockout mice were administered either CJ-11,802 (200 mg/kg/day) or vehicle by oral gavage for 14 consecutive days. At necropsy, liver weight, CYP content, and CRed activity were measured and all were increased similarly in the treated wild-type and knockout mice compared to corresponding controls, indicating that induction was not related to inhibiting 5-LO.

Chemopreventive Effects of Certain Nutritive Acids on Aflatoxin-induced Cytotoxicity

Many investigators have evaluated the effects of several plant products in modulating in vitro metabolism of aflatoxin B 1 (AFB 1 ) by microsomal mixed function oxidase (MFO), thereby reducing its toxicity. However, the importance of these studies is of limited significance as MFO is not present in all cells. Our earlier study revealed a concentrationdependent increase in the rate of haemolysis, indicating aflatoxin-induced cytotoxicity on red blood cells. It could be due to the direct action of aflatoxin on the plasma membrane, as MFO required for bioactivation of AFB 1 is absent in red blood cells. The present study was designed to evaluate the efficacy of some micronutrients, namely lactic, citric and folic acid, in ameliorating aflatoxin-induced cytotoxicity on red blood cells in vitro .

Cytochrome P450 2E1 metabolically activates propargyl alcohol: propiolaldehyde-induced hepatocyte cytotoxicity

Pargyline, an antihypertensive agent and monoamine oxidase inhibitor, induces hepatic GSH depletion and hepatotoxicity in vivo in rats [E.G. De Master, H.W. Sumner, E. Kaplan, F. N. Shirota, H.T. Nagasawa, Toxicol. Appl. Pharmacol. 65 (1982) 390–401]. Propargyl alcohol (2-propyn-1-ol), because of its structural similarity to allyl alcohol, was thought to be activated by alcohol dehydrogenase. However, it is a poor substrate compared to allyl alcohol and it was therefore proposed that propargyl alcohol-induced liver injury involved metabolic activation by catalase/H 2O 2 [E.G. De Master, T. Dahlseid, B. Redfern, Chem. Res. Toxicol. 7 (1994) 414–419]. In the following we showed that; (1) propargyl alcohol-induced cytotoxicity was markedly enhanced in CYP 2E1-induced hepatocytes and prevented by various CYP 2E1 inhibitors but was only slightly affected when alcohol dehydrogenase was inhibited with methylpyrazole/DMSO or when catalase was inactivated with azide or aminotriazole, (2) hepatocyte GSH depletion preceded cytotoxicity and was inhibited by cytochrome P450 inhibitors but not by catalase/alcohol dehydrogenase inhibitors. GSH conjugate formation during propargyl alcohol metabolism by microsomal mixed function oxidase in the presence of GSH was also prevented by anti-rat CYP 2E1 or CYP 2E1 inhibitors, (3) cytotoxicity was prevented when lipid peroxidation was inhibited with antioxidants, desferoxamine (ferric chelator) or dithiothreitol. Propargyl alcohol-induced cytotoxicity and reactive oxygen species formation were markedly increased in GSH-depleted hepatocytes. All of this evidence suggests that propargyl alcohol-induced cytotoxicity involves metabolic activation by CYP 2E1 to form propiolaldehyde that causes hepatocyte lysis as a result of GSH depletion and lipid peroxidation.

Membrane stabilising effects of natural polyphenols and flavonoids from Sempervivum tectorum on hepatic microsomal mixed-function oxidase system in hyperlipidemic rats

The extensive role of the microsomal mixed-function oxidase (MFO) system in the oxidation of endo-and xenobiotics, in the detoxication, in the generation of reactive free radicals and in the decomposition of the end products of lipid peroxides is well documented in the literature. Steatotic liver is a very frequent damage with different etiology. Drug metabolising reactions are suppressed in fatty liver, in which pathologically increased production of reactive oxygen intermediates may lead to the peroxidation of microsomal membrane lipids and to the change of membrane bound enzyme activities because of overwhelmed protective mechanisms. The subnormal activity of the MFO system may diminish the non specific resistance of the organism. Therefore we have studied the effects of natural flavonoids and polyphenolic compounds on the mixed-function oxidases. Antioxidant, O 2 − and OH scavenger properties of Sempervivum tectorum extract (STF1) were proved by EPR spectroscopic and chemiluminometric techniques. Potential bioactive constituents were determined by chromatography (HPLC, TLC) and spectrometric (UV, UV–VIS) methods. In the present study we reflect on the membrane stabilising, antioxidant and lipid metabolism modifying effects of this extract. It was established that activities of NAD(P)H reductase and content of cytochrome P450 were normalised in liver microsomes of hyperlipidemic rats, if the animals were treated with STF1 (2 g/bwkg for 9 days in drinking water parallel with fat-rich diet feeding). Fatty acid composition, examined by HRGLC analysis, was changed beneficially. NADPH induced lipid peroxidation was also decreased in microsomes in in vivo and in vitro experiments. At the same time the STF1 had no significant influence on MFO system in normolipidemic animals and on cytochrome b5 concentration of microsome fractions of hyperlipidemic rats.

Biochemical and molecular studies in mild flavin monooxygenase 3 deficiency.

Flavin-containing monooxygenases (FMOs) are membrane-associated microsomal mixed-function oxidases that are required for the oxidation and detoxification of many nitrogen-, sulphur- and phosphorus-containing substances and are present in nearly all secretory cell types (Dolphin et al 1997; Treacy et al 1998). At least five different isoforms (1 to 5) have been identified in mammalian tissues (Lawton et al 1994). In humans, FMO3 is the major catalytic isoform expressed in the liver (Lomri et al 1993), although it is also present in the kidney and probably in other tissues. Substrates include endogenous amines (e.g. adrenaline), tyramine, nicotine, drugs (e.g. tricyclic antidepressants, ranitidine), as well as trimethylamine (TMA), a volatile malodourous substance that is present in fish or may be produced by the action of gut bacteria on choline, lecithin or carnitine (Lin et al 1996; Ziegler 1993). It has been shown that a deficiency of FMO3 is the cause of the recessive disorder trimethylaminuria (TMA-uria) or fish-odour syndrome' (Dolphin et al 1997; Treacy et al 1998). Individuals affected by this disease have a reduced capacity to oxidize free TMA to its odourless, nonvolatile metabolite TMA N-oxide (TMAO) and display a strong, fish-like odour due to the excretion of large amounts of TMA in urine, sweat and breath (Ayesh et al 1993). The diagnosis is made biochemically on the basis of elevated urinary free TMA; under physiological conditions, >95% of the total TMA (= free TMA + TMAO) is excreted as TMAO. In classical fish-odour syndrome, the TMAO/total TMA ratio is strongly decreased. The FMO3 gene is located on chromosome 1q23-25 and contains one noncoding and eight coding exons. A small number of severe, disease-causing mutations and several polymorphic variants in the gene have been reported (Akerman et al 1999; Dolphin et al 1997; Treacy et al 1998). We have recently found that a frequent variant allele carrying two amino acid polymorphisms, c.472G > A and c.923A > G [E308G; E158K], is associated with reduced N-oxidation capacity after oral trimethylamine challenge (Zschocke et al 1999). Patients previously reported to suffer from 'transient' trimethylaminuria (Mayatepek and Kohlmuller 1998) were compound heterozygous for the variant allele and a severe FMO3 mutation, corresponding to mild enzyme deficiency. Here we present the results of molecular analyses and biochemical studies under physiological conditions in patients with mild FMO3 deficiency and report the first symptomatic patient who is homozygous for the variant allele.