Teresa Gessner

A unified method for the assay of uridine diphosphoglucuronyl-transferase activities toward various aglycones using uridine diphospho[U-14C]glucuronic acid

Abstract A method is described for the assay of UDP-glucuronyltransferase activity for various aglycones using UDP-[U 14 -C]glucuronic acid. The radioactive glucuronide conjugates formed during the reactions were separated from unmetabolized UDP-[U- 14 C]glucuronic acid, and from other uncharacterized radioactive products, by thin-layer chromatography. The conjugates were identified by comparison of their R f values on thin-layer chromatography with those of several authentic glucuronides, and by hydrolysis with β-glucuronidase in the absence or presence of d -saccharic acid-1,4-lactone, an inhibitor of β-glucuronidase. The procedure is general for the determination of the enzymic activity with various aglycones. It has been utilized for eleven aglycones and is capable of extension to many others.

Glucuronidation of oxygenated benzo(a)pyrene derivatives by UDP-glucuronyltransferase of nuclear envelope

Abstract Several benzo(a)pyrene phenols, dihydrodiols, epoxides, quinones, and a diolepoxide were tested as possible substrates for UDP-glucuronyltransferase located in rat liver nuclear envelope fraction. Only phenolic derivatives of benzo(a)pyrene served as substrates for the nuclear membrane transferase, under the conditions tested. The specific activities observed in nuclear envelope preparations were greater than or equal to, those of the microsomal fraction indicating that a very effective detoxication mechanism for these phenols is present in the nuclear compartment.

Effects of superoxide generated in vitro on glucuronidation of benzo[a]pyrene metabolites by mouse liver microsomes

Glucuronidation of benzo[a]pyrene (B[a]P) metabolites, generated in situ by oxidative pathways, was studied using mouse liver uninduced microsomes. No coupling was evident between UDP-glucuronyltransferase and oxygenation of B[a]P. UDPGA protected microsomal macromolecules against binding of reactive B[a]P metabolites. Superoxide, and other reactive oxygen species decreased both the overall B[a]P metabolism and glucuronidation of some B[a]P metabolic products, and caused more extensive binding to macromolecules; UDPGA was less protective in this condition. Peroxidation of microsomes differentially affected glucuronidation of various metabolites of B[a]P, and of various model substrates, indicating that multiple glucuronyltransferases are involved in the conjugation of hydroxylated metabolites of B[a]P.

Action of xanthine-xanthine oxidase system on microsomal benzo(a)pyrene metabolism in vitro.

The effect of superoxide anion-radical and other reactive oxygen species on the metabolism of benzo(a)pyrene was studied with isolated mouse liver microsomes. Reactive oxygen species were generated in vitro by xanthine-xanthine oxidase plus Fe3+ X FeEDTA and benzo(a)pyrene metabolism was followed by reverse-phase high pressure liquid chromatography. The following results were obtained: The reactive oxygen species induced one-electron oxidation of benzo(a)pyrene and increased production of free epoxide as well as protein-binding intermediates. The reactive oxygen species triggered microsomal lipid peroxidation in the presence of Fe3+ X FeEDTA. As a result of microsomal lipid peroxidation a decreased activity of cytochrome P-450, epoxide hydrolase and UDP-glucuronyltransferase was found. It is suggested that active oxygen species changed the balance between bioactivation and conjugation of benzo(a)pyrene metabolites causing accumulation of the epoxide and protein-binding intermediates. The role of iron ions and chelates in this process is discussed.

Asbestos-catalyzed oxidation of benzo(a)pyrene by superoxide-peroxidized microsomes.

Asbestos and benzo(a)pyrene (B(a)P) are ubiquitous in our environment and both are recognized as causal factors for cancer in man and animals. In vitro studies showed a synergism in morphological transformation of mammalian cells treated with asbestos and B(a)P. It has been shown that asbestos can mediate lipid peroxidation and that iron cations might be involved in the catalytic activity of asbestos fibers. A previous study of B(a)P metabolism by microsomes showed that peroxidative conditions change the balance between activation and deactivation of B(a)P and demonstrated that catalytically active iron can play a role in this process. The present investigation examines the effect of asbestos on oxidation of B(a)P by superoxide - peroxidized microsomes in vitro.

Effects of inhibition of nadph: cytochrome p-450 reductase on benzo(a)pyrene metabolism in mouse liver microsomes

1. Effects of antioxidants (butylated hydroxytoluene and nor-dihydroguaiaretic acid), vitamin K-related quinones (vitamin K1 and coenzyme Q10) and inorganic copper (CuSO4), in concentrations inhibiting NADPH: cytochrome P-450 reductase, were re-examined on benzo(a)pyrene metabolism in mouse liver uninduced microsomes. 2. It was found that all these compounds decrease production of the two-electron oxygenation products of benzo(a)pyrene (monophenoles, diols) and the amounts of glucuronides in a manner parallel to their inhibitory potency against NADPH: cytochrome P-450 reductase. 3. No correlation was found between amounts of one-electron oxidation products of benzo(a)pyrene and inhibition of NADPH: cytochrome P-450 reductase. 4. Without added UDPGA the compounds studied decreased protein associated benzo(a)pyrene metabolites in parallel to the decreased overall metabolism of this polyaromatic hydrocarbon. 5. The mode of action of the studied compounds is discussed.

Inhibition of the redox cycling of vitamin K3 (menadione) in mouse liver microsomes.

1. Concentration-dependent effects of vitamin K1, coenzyme Q10, butylated hydroxytoluene, nor-dihydroguaiaretic acid and Fe-initiated lipid peroxidation on redox cycling of vitamin K3 were studied in mouse liver microsomes in vitro. 2. The antioxidants (butylated hydroxytoluene, nor-dihydroguaiaretic acid) caused apparent non-competitive inhibition of vitamin K3 redox cycling. 3. Vitamin K1 and coenzyme Q10 caused competitive inhibition of the redox cycling (Ki = 33 and 46 microM, respectively). 4. Fe-initiated microsomal lipid peroxidation caused irreversible decrease of one-electron reduction of vitamin K3. 5. The role of NADPH:cytochrome P-450 reductase along with mechanisms of these inhibitions are discussed.

Interaction between vitamin K3 and benzo(a)pyrene metabolism in uninduced microsomes

1. Relationship between quinone recycling, glucuronidation and benzo(a)pyrene (BaP) oxygenation was investigated in uninduced mouse liver microsomes--native and modified by Fe3+.FeEDTA and/or superoxide (O2-.)-initiated lipid peroxidation. 2. A functional coupling between glucuronidation of reduced quinones and BaP metabolism, not discernible during BaP metabolism by native uninduced microsomes, was demonstrable in the presence of a model quinone, vitamin K3 (menadione). 3. Menadione inhibited BaP oxygenation in microsomal preparations, by siphoning off electrons from cytochrome P-450, while addition of UDPGA reversed this effect by glucuronidation of menadiol. 4. Fe3+.FeEDTA and/or O2-.-initiated lipid peroxidation decreased, to different extent, the microsomal enzymatic activities involved in quinone metabolism. The most sensitive was quinone reductase activity, which was reduced by 77%. Under peroxidative conditions menadione was a less effective inhibitor of BaP metabolism. 5. The important role of the balance between quinone reductase and UDP-glucuronyltransferase activities in the coupling with BaP oxygenation is discussed. A mechanism by which vitamin K3 could exert a regulatory effect on BaP metabolism is proposed.