Microsomal MFO system

Benzo(a)pyrene is converted by the microsomal MFO system of mammals (18) and trout (25) to a number of oxidized products. 

One of our more interesting observations is illustrated in Table III. The administration of DBA to winter flounder increased hepatic microsomal AHH and 7-ethoxyresorufin deethylase activities as expected, and AHH activity was strongly inhibited in the DBA-treated flounder by 10" M a-naphthoflavone as we have previously reported for both little skate (4) and sheepshead (9). However, the presence of high AHH and 7-ethoxyresorufin deethylase activities in one control flounder, and the inhibition of AHH activity by a-naphthoflavone in this animal suggested that the hepatic microsomal MFO system of this fish was already induced. 

The endoplasmic reticulum contains high amounts of lipids, especially phospholipids, rich in polyunsaturated fatty acids. Lipids may influence the detoxication process by affecting the cytochrome P-450 system because phosphatidylcholine is an essential component of the hepatic microsomal MFO system. A high-fat diet may favor more oxidation to occur, as it may contribute to more incorporation of membrane material. 

Crustaceans also exhibit a range in ability to biotransform PAHs. A study with the blue crab (Callinectes sapidus) found that this species was able to extensively metabolize PAHs (naphthalenes, fluorene, and benzo-[a]pyrene) and produce high concentrations of metabolites (Lee et al. 1976). Conversely, Burns (1976) found that another crab (Uca pugnax) possessed a very weak, uninducible microsomal MFO system for metabolizing xenobi-otic hydrocarbons. It was noted that this species was very sensitive to oil pollution because of its limited ability to metabolize hydrocarbons. The larval spot shrimp (Pandalus platyceros) only weakly metabolized naphthalene with 9%-21% of the total " C-naphthalene in tissues present as metabolites (Sanborn and Malins 1977). Contrary to this, fairly extensive biotransformation of naphthalene was demonstrated for the copepod Calanus helgolandicus by Corner et al. (1976) and Harris et al. (1977). 

MFO). Similar MFO microsomal redox systems operate for the metabolic detoxification of drugs and some waste products (Section 6.4). 

In general, our studies with cytochrome P-450-dependent metabolism have emphasized the similarity of the hepatic MFO system in marine fish to that found in mammals. Thus, in the little skate (Raja erinaoea), a marine elasmobranch, enzyme activity is localized in the microsomal fraction, requires NADPH and molecular oxygen for maximum activity, and can be inhibited with CO (1, 2). Moreover, when hepatic microsomes from the little skate were solubilized and separated into cytochrome P-450, NADPH-cytochrome P-450 reductase, and lipid fractions, all three fractions were required for maximal MFO activity in the reconstituted system (3). We have also found, as have others, that the administration of polycyclic hydrocarbons (3-methylcholanthrene, 1,2,3,4-dibenzanthracene [DBA]), 2,3,7,8-tetrachlorodibenzo-p-dioxin... 

When 4C-benzo(a)pyrene (100 nmol) was incubated with the reconstituted MFO system, the reaction components were increased 10-fold (maintaining the original incubation volume, and substrate and NADPH concentrations). Metabolites were extracted and analyzed by HPLC as described for the microsomal incubations. 

Several properties of hepatic microsomal AHH activity were compared in control and DBA-pretreated male little skates as shown in Table I. Following treatment there was an approximately 35-fold increase in specific enzyme activity, as quantitated by fluorescence of the phenolic metabolites formed (3, 21). The pH optimum, which was fairly broad, and the concentration of benzo(a)-pyrene (0.06 mM) that had to be added to the incubation mixture to achieve maximum enzyme activity were the same for both control and induced skate hepatic microsomes. The shorter periods observed for linearity of product formation with microsomes from the induced skates is thought to be related to the much higher AHH activity present, and may be due to substrate depletion or the formation of products which are inhibitory (i.e., compete with the MFO system as they are substrates themselves). A similar explanation may be relevant for the loss of linear product formation at lower microsomal protein concentrations in the induced animals. 

For foreign compounds, the majority of oxidation reactions are catalyzed by monooxygenase enzymes, which are part of the mixed function oxidase (MFO) system and are found in the SER (and also known as microsomal enzymes). Other enzymes involved in the oxidation of xenobiotics are found in other organelles such as the mitochondria and the cytosol. Thus, amine oxidases located in the mitochondria, xanthine oxidase, alcohol dehydrogenase in the cytosol, the prostaglandin synthetase system, and various other peroxidases may all be involved in the oxidation of foreign compounds. 

Microsomal flavin-containing monooxygenases. As well as the cytochromes P-450 MFO system, there is also a system, which uses FAD. This flavin-containing monooxygenase or FMO enzyme system is found particularly in the microsomal fraction of the liver, and the monomer has a molecular weight of around 65,000. Each monomer has one molecule of FAD associated with it. The enzyme may accept electrons from either NADPH or NADH although the former is the preferred cofactor. It also requires molecular oxygen, and the overall reaction is as written for cytochromes P-450 ... 

The confusion on the mechanism of the comutagenesis and mutagenesis of these pyrolysis products, especially pertaining to the enhancement and inhibition effects of Harman and Norharman,centers around the problem of the lack of certain fixed variables in the experimentation, particularly, the availability of the purified enzymes involved in the metabolic activation, which constitute the cytochrome P-450 mixed function oxidase system. We, therefore, undertake this problem to elucidate the mechanism of microsomal metabolism of these oyrolysis products with the purified mixed function oxidase(MFO) system. 

Microsomal Metabolism A typical reaction of microsomal metabolism or metabolism by the purified and reconstituted MFO system contained 1 ml of aqueous so1ution 0.075 vol. of drug in methanol,... 

The alternative explanation must involve the MFO system. The possible mechanism for the inhibitory effect or enhancement effect of Norharman upon covalent DNA binding and mutagenicity must be the results of the net balancing of substrate inhibition and membrane fluidization of the microsomal membrane or of the lipid vesicles in the reconstituted MFO system. A schematic pathway of the metabolism of these tryptophan pyrolysis products is postulated as shown in Figure 8. 

Foreign compouttds are usually strongly electrophilic before they encounter the microsomal mixed Atnctfon oxidase (MFO) system. 

The biotransformation systems involved in insecticide metabolism have been studied in the R and S populations to determine any differences which might be potential contributory factors to or results of insecticide resistance. In addition, the possibility of mixed-function oxidase induction has been investigated. Specifically, the studies have encompassed a seasonal study of microsomal mixed-function oxidase (mfo) components, and studies of aldrin, dieldrin and DDT metabolism. 

The marine environment acts as a sink for a large proportion of polyaromatic hydrocarbons (PAH) and these compounds have become a major area of interest in aquatic toxicology. Mixed function oxidases (MFO) are a class of microsomal enzymes involved in oxidative transformation, the primary biochemical process in hydrocarbon detoxification as well as mutagen-carcinogen activation (1,2). The reactions carried out by these enzymes are mediated by multiple forms of cytochrome P-450 which controls the substrate specificity of the system (3). One class of MFO, the aromatic hydrocarbon hydroxylases (AHH), has received considerable attention in relation to their role in hydrocarbon hydroxylation. AHH are found in various species of fish (4) and although limited data is available it appears that these enzymes may be present in a variety of aquatic animals (5,6,7,8). 

We employed various substrates to check for MFO in two bivalve species, a salt water mussel (Mytilus edulis) and a fresh water clam (Anodonta sp). Cytochrome P-450 was also studied. Organisms were exposed to 100 PPM Venezuelan crude in a stagnant system for up to one month. Enzyme assays were carried out with digestive gland 9000 g homogenates (17) and cytochrome P-450 analysis, with microsomes (21). The hydrocarbon substrates investigated included 1I+C-labelled benzo(a)pyrene, fluorene, anthracene, and naphthalene. The method used for separation of BP metabolites by thin layer radiochromatography has been described (7). The metabolite detection method for the other aromatic hydrocarbons was essentially the same except methylene chloride was used as metabolite extractant as well as TLC developer. Besides the hydrocarbon substrates, we also checked for other MFO reactions, N-dealkylase with C-imipramine (22) and 0-dealkylase with ethoxycoumarin (15). 

The renal cytochrome P-450 enzyme system is involved in oxidative reactions in which an atom of molecular oxygen is inserted in an organic molecule. The flavoprotein NADPH-cytochrome P-450 reductase is an essential component of the mixed-function oxidase systems (MFO). Microsomal membranes appear to be particularly subject to attack by reactive oxygen radicals due to their high content of unsaturated fatty acids and the presence of the cytochrome P-450 system [40]. Cephaloridine-induced peroxidation of membrane lipids is decreased by the cytochrome P-450 inhibitor cobalt chloride [31], suggesting a role for a cytochrome P-450 reductase in the P-lactam-induced generation of reactive oxygen species and subsequent peroxidation products. 

The principal biotransformation of toxicants is catalyzed by the microsomal mixed function oxidase system (MFO). A deficiency of essential fatty acids generally depresses MFO activities. This is also true with protein deficiency. The decreased MFO has different effects on the toxicity of chemicals. For example, hexobarbital and aminopyrine are detoxified by these enzymes and are thus more toxic to rats and mice with these nutrient deficiencies. On the other hand, the toxicity of aflatoxin is lower in such animals because of their depressed bioactivation of this toxicant. MFO activities are decreased in animals fed high levels of sugar. 

Many tissues in the human body, including liver, lung, colon, and lymphocytes, have the abihty to metabolize BaP. The most important enzymes that mediate the initial oxidation of PAHs in the body are cytochrome P450 mixed function oxidases (MFOs), which are found in microsomes, nuclei, and mitochondria of cells. MFO enzyme systems function together with NADPH-cytochrome P450 reductase and require phosphatidylcholine for activity. 

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