Enzymes mixed-function oxidases

Most phase one reactions are catalyzed by the drug-metabolizing enzymes (mixed function oxidases, oxygenases) located in the endoplasmic reticulum of liver and, to a lesser extent, in intestine, kidney, and lung. These enzymes have been the subject of intensive research (G7, G8, LI). 

Not only are enzyme activities inhibited, but they can also be induced by a toxicant agent. Quantitative measures exist for a broad variety of these enzymes. Mixed-function oxidases are perhaps the best studied, with approximately 100 now identified from a variety of organisms. Activity can be measured or the synthesis of new mixed-function oxidases may be identified using antibody techniques. DNA repair enzymes can also be measured and their induction is an indication of DNA damage and associated genotoxic effects. 

The biochemical mechanism of bacterial luminescence has been studied in detail and reviewed by several authors (Hastings and Nealson, 1977 Ziegler and Baldwin, 1981 Lee et al., 1991 Baldwin and Ziegler, 1992 Tu and Mager, 1995). Bacterial luciferase catalyzes the oxidation of a long-chain aldehyde and FMNH2 with molecular oxygen, thus the enzyme can be viewed as a mixed function oxidase. The main steps of the luciferase-catalyzed luminescence are shown in Fig. 2.1. Many details of this scheme have been experimentally confirmed. 

Cytochrome P450 (CYP) mono-oxygenases, also called mixed function oxidases, are versatile hemoprotein enzymes that catalyze the cleavage of molecular oxygen to incoiporate one oxygen atom into a substrate molecule and one atom into water [1]. The general stoichiometry of the reaction is as follows (S-H, substrate) ... 

Acetaminophen, which depletes hepatic glutathione, does not potentiate the toxicity of methyl parathion in mice. A possible mechanism of action may be competition between acetaminophen and methyl parathion for mixed function oxidases and subsequent prevention of activation of methyl parathion to methyl paraoxon (Costa and Murphy 1984). Diethyl maleate, an agent that depletes cytosolic glutathione and is not an enzyme inducer, potentiates toxicity of methyl parathion in mice (Mirer et al. 1977). 

These include the mitochondrial respiratory chain, key enzymes in fatty acid and amino acid oxidation, and the citric acid cycle. Reoxidation of the reduced flavin in oxygenases and mixed-function oxidases proceeds by way of formation of the flavin radical and flavin hydroperoxide, with the intermediate generation of superoxide and perhydroxyl radicals and hydrogen peroxide. Because of this, flavin oxidases make a significant contribution to the total oxidant stress of the body. 

Oxidation is intimately linked to the activation of polycyclic aromatic hydrocarbons (PAH) to carcinogens (1-3). Oxidation of PAH in animals and man is enzyme-catalyzed and is a response to the introduction of foreign compounds into the cellular environment. The most intensively studied enzyme of PAH oxidation is cytochrome P-450, which is a mixed-function oxidase that receives its electrons from NADPH via a one or two component electron transport chain (10. Some forms of this enzyme play a major role in systemic metabolism of PAH (4 ). However, there are numerous examples of carcinogens that require metabolic activation, including PAH, that induce cancer in tissues with low mixed-function oxidase activity ( 5). In order to comprehensively evaluate the metabolic activation of PAH, one must consider all cellular pathways for their oxidative activation. 

Tyrosine hydroxylase is the rate-limiting enzyme for the biosynthesis of catecholamines. Tyrosine hydroxylase (TH) is found in all cells that synthesize catecholamines and is a mixed-function oxidase that uses molecular oxygen and tyrosine as its substrates and biopterin as its cofactor [1], TH is a homotetramer, each subunit of which has a molecular weight of approximately 60,000. It catalyzes the addition of a hydroxyl group to the meta position of tyrosine, thus forming 3,4-dihydroxy-L-phenylalanine (l-DOPA). 

Environmental agents that influence microsomal reactions will influence hexachloroethane toxicity. The production of tetrachloroethene as a metabolite is increased by agents like phenobarbital that induce certain cytochrome P-450 isozymes (Nastainczyk et al. 1982a Thompson et al. 1984). Exposure to food material or other xenobiotics that influence the availability of mixed function oxidase enzymes and/or cofactors will change the reaction rate and end products of hexachloroethane metabolism and thus influence its toxicity. 

Fabacher DL, Hodgson E. 1976. Induction of hepatic mixed-function oxidase enzymes in adult and neonatal mice by Kepone and mirex. Toxicol Appl Pharmacol 38 71-77. 

NB the %02 shown at the top appears as water at the end of the sequence. This is typical of mixed function oxidase enzymes. 

The desaturation process is particularly interesting as it provides an example of a microsomal (as opposed to mitochondrial) electron transport system. The enzymes responsible, fatty acyl-CoA desaturases, are examples of mixed function oxidases... 

The CYP enzymes active in phase I reactions are often oxidases or hydroxylases, sometimes called mixed function oxidase (MFO). An oxidase enzyme introduces into the substrate (i.e. the unwanted compound) both atoms of an oxygen molecule whilst... 

Table II summarizes the results together with the detailed experimental conditions. As is evident, metabolic activities were detectable in these 3 aquatic species, but the rate was far lower as compared with mammalian hepatic enzume preparations, and the oxidative activities in snail were particularly low although the possibility was not ruled out of the presence of inhibitors of mixed-function oxidases in the fractions. The O-demethylation reaction proceeds extremely slowly in the enzyme preparation of aquatic animals, at less than one hundredth that of mammals.

Enzymes of the hepatic microsomes of most marine organisms, with the notable exception of certain molluscs, metabolize xeno-biotic substrates however, as much as 600-fold variations in enzyme activities have been noted between different species of marine teleosts (40). The hepatic enzyme activities of aquatic species are generally lower, with most substrates tested, than the hepatic enzymes of mammals (40). The mixed function oxidase enzymes in marine organisms are inducible by hydrocarbons, such as 3-methylcholanthrene or benzo[a]pyrene. Moreover, it is known... 

Mixed-function Oxidase Induction.-- Indirect evidence of environmental induction of detoxifying enzymes in the R fish has been observed as an increase in the acute toxicity of parathion and a simultaneous decrease in NADPH-dependent parathion... 

Hepatic mixed-function oxidase activities demonstrated seasonal trends, with higher specific activities in the cold weather months in both populations with few differences in enzyme activities or cytochrome levels between the two populations. Metabolism of aldrin, dieldrin and DDT was similar between the two populations. R fish have larger relative liver size and, therefore, a greater potential for xenobiotic metabolism. However, biotransformation appears to be of minor importance in chlorinated alicyclic insecticide resistance in mosquitofish barriers to penetration appear to be of greater importance and an implied target site insensitivity appears to be the most important factor in resistance. 

Addison, R.F., Zinck, M.E. and Willis, D.E. Mixed function oxidase enzymes in trout (Salvelinus fontinalis) liver Absence of induction following feeding of p,p -DDT or p,p -DDE. Comp. Biochem. Physiol. (1977) 57C 39-43. 

Dent, J.G. Characteristics of cytochrome P-450 and mixed function oxidase enzymes following treatment with PBB s. Environ. Health. Perspect. (1978) 23 301-308. 

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). 

Exposure of various invertebrate species to high concentrations of petroleum did not induce mixed function oxidase activity. Enzyme activity was stimulated, however, in a number of fish tissues by petroleum. Different permutations can be addressed as to the significance of basal or induced levels of mixed function oxidases and hydrocarbon toxicity. AHH may have a physiological role in enhancing hydrocarbon clearance but may also increase the mutagenic-carcinogenic potential of hydrocarbons. Both of these concepts have been demonstrated in studies with fish (29,30). Induced AHH levels may permit a more rapid oxidative transformation with concomitant "disappearance" of parent hydrocarbons, but potentially toxic metabolites could be retained in tissues for longer periods (31). It is likely that at the enzymic level the... 

Considerable interest has developed concerning the nature of the mixed function oxidase system in fish and the role that this system may play in the development of toxic responses in these animals. Studies have shown that components of the mixed function oxidase system are present in relatively high concentrations in fish liver (4, 5, 6) and that the enzyme systems in this organ are capable of many of the biotransformation reactions already described for the mammalian liver (7, 8, 9). The presence of this complement of enzymes in the livers of many fishes suggests that this organ too may be particularly sensitive to insult from sub lethal concentrations of many waterborne toxicants. For this reason, methods to evaluate liver function in fish may be particularly useful in identifying the sublethal effects of certain classes of toxicants. 

An effect of ozone on lung microsomes has been suggested by morpholine studies that indicated alterations in the endoplasmic reticulum, Biochemical evidence of an effect on microsomal enzymes was originally obtained in the studies of Palmer et who demonstrated that ozone exposure (0.75-10 ppm for 3 h) resulted in a decrease in activity of Syrian hamster lung benzopyrene hydroxylase, a mixed-function oxidase that depends on cytochrome P-450. No changes in hepatic activities of this enzyme were observed, and the results were similar in animals in which high activities of benzopyrene hydroxylase had been induced. The maximal effect was not observed until a few days after the single ozone exposure. Palmer et also reported a decrease in rabbit tracheobronchial mucosal benzopyrene hydroxylase activity after exposure to similar ozone concentrations. 

N-dealkylation results from an alkyl substitution on an aromatic molecule, which is one of the first places where microorganisms initiate catabolic transformation of atrazine, a xenobiotic molecule (Fig. 15.2). It is a typical example of a reaction leading to transformation of pesticides like phenyl ureas, acylanihdes, carbamates, s-tri-azines, and dinitranilines. The enzyme mediating the reaction is a mixed-function oxidase, requiring a reduced nicotinamide nucleotide as an H donor. 

Kraft bleach mill effluent produced by oxygen delignification or 100% chlorine dioxide Ereshwater fish Induction of mixed function oxidase (MEO) enzymes following exposure to 4% and 12% effluent in artificial streams Bankey et al., 1995 [28]... 

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