Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver.

Eight different forms of cytochrome P-450 (P-450) were purified to electrophoretic homogeneity by a common procedure from liver microsomes of rats treated with phenobarbital or beta-naphthoflavone. Antibodies were prepared to seven of these forms in rabbits. The eight P-450s were distinguished by spectral properties of the ferric, ferrous, and ferrous carbonyl forms, apparent monomeric molecular weights, peptide mapping, immunological reactivity as discerned by double-diffusion immunoprecipitin analysis and crossed immunoelectrophoresis, and catalytic activities toward the substrates acetanilide, aminopyrine, aniline, benzo[a]-pyrene, d-benzphetamine, N,N-dimethylnitrosamine, 7-ethoxycoumarin, 7-ethoxyresorufin, ethylmorphine, p-nitroanisole, testosterone, and (R)- and (S)-warfarin. Crossed sodium dodecyl sulfate-polyacrylamide gel immunoelectrophoresis was used to estimate the levels of each of the eight forms of P-450 present in the liver microsomes of untreated rats and rats treated with phenobarbital, 5,6-benzoflavone, pregnenolone-16 alpha-carbonitrile, isosafrole, or the polychlorinated biphenyl mixture Aroclor 1254. In each situation, the sum of the levels of these eight P-450s was at least as high as the spectrally determined P-450 content. The results clearly demonstrate that individual forms of P-450 can be induced by different compounds and that a single compound can lower the level of one form of P-450 while inducing one or more other forms of P-450. Catalytic activities toward each of the substrates observed with microsomal preparations are compared to rates predicted on the basis of the content of each of the eight P-450s. These studies provide a basis for further studies on the regulation of individual P-450s, the physical properties of the different P-450s, and the metabolic consequences of changes in the forms of P-450 in rat liver models.

Purification and characterization of liver microsomal cytochromes p-450: electrophoretic, spectral, catalytic, and immunochemical properties and inducibility of eight isozymes isolated from rats treated with phenobarbital or beta-naphthoflavone.

Molecular and cellular aspects of thiol-disulfide exchange.

DT-diaphorase as a quinone reductase: a cellular control device against semiquinone and superoxide radical formation.

Agriculture and fishing are the primary source of almost all our food, as well as of many other products, so remain vital activities even though their share in the economy is small and in continuous decline. Partly for this reason public policy in these areas is almost entirely integrated at European level in the Common Agricultural Policy (CAP) and Common Fisheries Policy (CFP). Forestry policies are also heavily affected by EU-level decision-making. As agriculture dominates in terms of direct contribution to GDP and numbers of people engaged in it, as well as accounting for the largest amount of public support expenditure, it is agriculture that will receive the greatest attention here.

Agriculture, Forestry and Fisheries

A reliable, sensitive, and convenient radioactive assay for benzpyrene monooxygenase.

Summary The epoxide hydrase assay developed by Oesch et al. (Biochim. Biophys. Acta, 227: 685–691, 1971) using [ 3 H]styrene oxide as substrate was modified in three ways for use with rat lung microsomes: the substrate was purified before use, the volume of the incubation mixture was scaled down 4-fold, and the incubation time was extended to 45 min (activity was found to be linear for at least 60 min). These modifications increased the sensitivity of the assay procedure 75- to 150-fold. The procedure was found to be linear with lung microsomal protein up to at least 1.8 mg protein per incubation mixture. This modified assay for epoxide hydrase was used to characterize the enzyme in rat lung. Its apparent V max is 0.5 nmole of styrene glycol formed per min per mg microsomal protein, and its apparent K m was 0.11 to 0.25 mm. The pH optimum is around 9.7. Upon subcellular fractionation of lung tissue, expoxide hydrase distributes in the same manner as a marker for the endoplasmic reticulum (reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase) and in a different way from markers for the nuclei, mitochondria, concentric lamellar organelles, lysosomes, Golgi membranes, plasma membrane, and soluble cytoplasm. The specific activity of epoxide hydrase in rough and smooth lung microsomes is about the same. Treatment i.p. of rats with methylcholanthrene (3 injections of 20 mg/kg), phenobarbital (5 daily injections of 80 mg/kg), or styrene oxide (5 daily injections of 40 mg/kg) did not induce lung microsomal epoxide hydrase activity. 1,1,1-Trichloropropene 2,3-oxide was shown to be an uncompetitive inhibitor, and cyclohexene oxide was a noncompetitive inhibitor of this enzyme. Ethanol and butanol activate the epoxide hydrase of lung microsomes at low concentrations and inhibit it at higher concentrations.

Characterization of Rat Lung Epoxide (Styrene Oxide) Hydrase with a Modified Radioactive Assay of Improved Sensitivity

Publisher Summary The purpose of this chapter is to provide information on the radioactive assay of aryl hydrocarbon monooxygenase and epoxide hydrase. Polycyclic hydrocarbons are found in cigarette smoke and in the waste products of our technology and may be a major cause of cancer in human beings. Assay of epoxide hydrase using styrene oxide is very simple; extraction with petroleum ether removes remaining substrate, while the product styrene glycol is retained in the aqueous incubation medium. The first step in the metabolism of these compounds by mammalian tissues is catalyzed by the NADPH-dependent, cytochrome P-450-1inked monooxygenase system localized on the endoplasmic reticulum, and the first product is an epoxide. A number of alternative assay procedures for epoxide hydrase are also reported, including procedures where an epoxide of a polycyclic hydrocarbon is used as substrate and the products are separated from remaining substrate by gas chromatography or by high-pressure liquid chromatography.

Maria S. Moron

Papers

Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver.

A reliable, sensitive, and convenient radioactive assay for benzpyrene monooxygenase.

Characterization of Rat Lung Epoxide (Styrene Oxide) Hydrase with a Modified Radioactive Assay of Improved Sensitivity

Radioactive assay of aryl hydrocarbon monooxygenase and epoxide hydrase.

The topology of epoxide hydratase and benzpyrene monooxygenase in the endoplasmic reticulum of rat liver.