James R. Fouts

A rapid method for assaying the metabolism of 7-ethoxyresorufin by microsomal subcellular fractions

Abstract The formation of resorufin from 7-ethoxyresorufin by microsomal mixed-function oxidase may be assayed rapidly by precipitating the protein in incubation mixtures with methanol and measuring the fluorescence of resorufin in the supernatant after centrifugation. Conditions are described for minimizing the effects of the instability of resorufin and of inhibition by the product. Higher activity and linear plots of microsomal protein concentration versus activity are obtained by including bovine serum albumin or ethylenediamine-tetraacetate in the incubation mixture.

Hepatic and extrahepatic metabolism, in vitro, of an epoxide (8-14C-styrene oxide) in the rabbit

Abstract Two epoxide-metabolizing enzymes, glutathione- S -epoxide transferase and epoxide hydrase, were studied in subcellular fractions of rabbit liver, lungs, intestinal mucosa and kidney. Glutathione- S -epoxide transferase in soluble fraction was assayed by a new specific radiochemical method using styrene oxide (−8- 14 C) as substrate, and some properties of this enzyme are described. Liver had the highest specific activity of each enzyme, but there was no correlation between the relative specific activities of glutathione- S -epoxide transferase and epoxide hydrase in the extrahepatic organs. Feeding rabbits a purified diet did not alter the specific activities of epoxide hydrase or glutathione- S -epoxide transferase in liver and intestine. Rat and guinea pig had much higher specific activities of glutathione- S -epoxide transferase than rabbit in liver and kidney, and slightly higher specific activities in lung and intestine. Species did not differ markedly in epoxide hydrase activities when the same organ (liver, lung, intestine or kidney) was compared in rabbits, rats and guinea pigs, except that rat intestine had much lower epoxide hydrase activity than intestine from rabbit or guinea pig.

Isolation and identification of clara cells from rabbit lung

SummaryA procedure has been developed for the isolation of nonciliated bronchiolar epithelial cells (Clara cells) from rabbit lung. Following pulmonary lavage to eliminate macrosphages, cells (5% Clara cells) were released by digestion with 0.1% Protease I in HEPES-buffered balanced salt solution containing 0.5 mM ethylene glycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid instilled through the trachea. These cells were then separated on the basis of size using the Beckman JE-6 elutriator rotor. The fourth fraction collected from the elutriator contained about 30% Clara cells. This fraction was then layered on a two-polymer aqueous phase system consisting of 5% dextran T500 (DT) and 3.8% polyethylene glycol 6000 (PEG) in sodium phosphate buffer. A cell fraction was obtained from the PEG phase, which included approximately 70% Clara cells. These cells were found to be greater than 90% viable by trypan blue dye exclusion.Identification of isolated Clara cells was confirmed by light microscopic observation of nitro blue tetrazolium staining and by ultrastructural characteristics as observed by electron microscopy.

Xenobiotic metabolism by alveolar type II cells isolated from rabbit lung

Abstract A procedure for the isolation of alveolar type II cells from rabbit lung was developed. Following pulmonary lavage to minimize macrophage contamination, viable cells (30% type II cells) were released by digestion with 0.1% Protease type I (Sigma) in 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid (HEPES)-buffered balanced salt solution containing 0.5 mM ethyleneglycolbis (amino-ethylether) tetra-acetate (EGTA) instilled via the trachea. Type II cells were enriched to 50–60 per cent purity by centrifugal elutriation. Density gradient centrifugation in metrizamide was used to increase the purity of the type II cells from the elutriator fraction to 80 per cent. Whole, freshly isolated alveolar type II cells metabolized 7-ethoxycoumarin at a rate of 30 pmoles umbelliferone formed per mg protein/min. However, only traces of coumarin hydroxylase activity were detected which could be accounted for by 1–2% Clara cell contamination in the type II cell fraction. NADPH-cytochrome c (cyt c) reductase activity in the sonicated type II cell fraction was 44 nmoles cyt c reduced per mg protein/min compared to 25 for lung homogenate. Benzo[a]pyrene hydroxylase and N,N-dimethylanilineN-oxidase activities were also demonstrated in the type II cell fraction to be 13 pmoles 3-OH benzo[a]pyrene · (mg protein)−1 · min−1 and 0.8 nmole DMA N-oxide · (mg protein)−1 · min−1 respectively. Microsomes prepared from the isolated type II cell fraction contained 74 pmoles cytochrome P-450/mg protein and 90 pmoles cytochrome b5/mg protein.

Mixed-function oxidases and the alveolar macrophage

Abstract Biphenyl 4-hydroxylase, benzpyrene hydroxylase and D-(+)-benzphetamine N -demethylase activities in subcellular fractions of the alveolar macrophage were investigated and found to be extremely low. Cytochrome P-450 was absent from the microsomal fraction but both NADPH-cytochrome c reductase and NADH-cytochrome c reductase activities were present. A b-type cytochrome, which was probably cytochrome b 5 , was detected in the microsomal fraction. Glutathione S -aryltransferase and UDP-glucuronyl transferase activities were absent from the soluble and microsomal fractions respectively. These results indicate that alveolar macrophages may not play a significant role in the detoxication of foreign compounds by the lung.

Amdxidation and demethylation of N,N-dimethylaniline by rabbit liver and lung microsomes effects of age and metals

Lung N-oxidase enzyme activity was about three times higher than liver N-oxidase at the pH optimum, about pH 8.9, whereas the activities were nearly the same at more physiological ranges of pH. The lung N-oxidase was also stimulated about 2-fold by 100 mM Mg2+ and by 0.1 mM Hg2+, whereas liver N-oxidase activity was inhibited by these concentrations of ions. The difference in response of liver and lung enzymes to Mg2+ and Hg2+ was not altered by preparing the microsomes in the presence of 50 mM ethylenediamine tetraacetic acid (EDTA) in 0.1 M Tris (hydroxymethyl) amino methane (Tris) buffer or 50 mM EDTA in 0.1 M KPO4 buffer, both at pH 7.6, indicating that the differences are probably not due to the presence of endogenous metals. The difference between the liver and lung N-oxidase systems may be due to the tissue environment rather than to the enzyme itself since mercury stimulation of lung N-oxidation began to disappear upon partial purification of the N-oxidase enzymes. In contrast to the effects of Hg2+ and Mg2+, 1 mM Ni2+ enhanced liver N-oxidase activity about 30% and 5 mM Ni2+ stimulated lung enzyme activity about 30% whereas concentrations above 10 mM were inhibitory to both N-oxidases. Both liver and lung demethylase activities were inhibited by these concentrations of Mg2+, Hg2+ and Ni2+. Various suifhydryl reagents were also tested for their effects on these enzymes. The mercurials, para-chloromercurybenzoate (pCMB) and phenylmercuryacetate (PMA) at concentrations of 0.1 mM had the same effect as HgCl2 inhibiting both demethylases and liver N-oxidase, but stimulating lung N-oxidase activity. However, 0.1 mM to 1 mMN-ethylmaleimide (NEM) and iodoacetamide had little if any effect on either liver or lung N-oxidase. It was also shown that Hg2+ effects on N-oxidase activity could be overcome by dilution. Changes in N,N-dimethyl aniline (DMA) metabolism with age were followed in rabbits from 4 days old to adult. There was a steady increase in lung demethylase activity and N-oxidase activity in the liver and lung to adult levels. However, the liver demethylase had a sharp increase in activity between 2 weeks and 1 month much like that seen with benzphetamine demethylase in rabbit liver. Activities of N-demethylase in liver and lung, and N-oxidr.se in liver from new-born rabbits were from 10 to 20 % of adult levels. However, in lung, N-oxidase activities in the newborn were about 50 % of adult levels. Microsomal N-oxidation in lungs from 2-day-old rabbits was stimulated by 0.1 mM mercury just as in the adult.

The lack of effects of pretreatment with phenobarbital and chlorpromazine on the acute toxicity of benzene in rats

Abstract Female CD rats were injected ip daily for 3 days with either phenobarbital (75 mg/kg) or chlorpromazine (15 mg/kg). On the fourth morning the animals were either subjected to a 4-hr inhalation exposure of benzene or given an ip injection of 50% ( v v ) of benzene and mineral oil. Animals were injected with doses of 1, 2, 3 and 4 g benzene/kg body weight. In the inhalation studies, animals were exposed to 6 levels of benzene ranging from 11,500 to 15,500 ppm. The LD50 for animals injected with benzene and the LC50 for animals inhaling benzene were calculated for control groups and those pretreated with either phenobarbital or chlorpromazine. Neither the LD50 or the LC50 were affected by any of the treatment protocols. In order to determine that the pretreatment was stimulating benzene metabolism, a method for measuring benzene metabolism has been developed using [14C]benzene. These studies have shown that phenobarbital and 3-MC do induce benzene metabolism in the liver, that chlorpromazine slightly induces benzene metabolism in the lung, and that pretreatment by these compounds does not affect the acute inhalation toxicity or the ip toxicity of benzene.

A comparative study of two procedures used in the determination of hepatic microsomal aniline hydroxylation

Abstract The hepatic microsomal parahydroxylation of aniline is measured by the determination of its product p -aminophenol (pAP). The pAP formed in incubation mixtures is assayed either after ether extraction or after trichloroacetic acid (TCA) precipitation. The TCA-precipitation method is simpler and less time-consuming than the ether-extraction method, but it has been found that the apparent recovery of pAP was lower in TCA supernatants than with the ether-extraction method. In the present work, it was found that the recovery of pAP by the TCA-precipitation method depended on the nature of NADPH-generating system used in the incubation mixture. When “soluble fraction” was used as a source of glucose-6-phosphate dehydrogenase for the reduction of NADP to NADPH, the recovery of pAP using the TCA-precipitation method was less than that with the ether-extraction method. But when “soluble fraction” was replaced by yeast glucose-6-phosphate dehydrogenase or by chemically prepared NADPH, the recovery of pAP was approximately equal with both the methods. Lowered recovery of pAP when added to the soluble fraction or its dialyzate might be due to the presence of sulfhydryl reacting groups. The addition of mercuric chloride to the soluble fraction dialyzate resulted in almost “full” recovery of pAP by the TCA-precipitation method, i.e., the apparent loss of pAP was prevented.

Cytochrome P-450 monooxygenase, epoxide hydrolase and flavin monooxygenase activities in Clara cells and alveolar type II cells isolated from rabbit.

The activities of several enzymes which metabolize xenobiotics were measured and compared in freshly isolated rabbit Clara cells (50–70% purity) and alveolar type II cells (80–95% purity) or microsomal preparations from the isolated cell fractions. The presence of 1 mM nicotinamide in protease and cell isolation buffers increased significantly 7-ethoxycoumarin (7-EC) deethylase and epoxide hydrolase activities in the isolated Clara and type II cells. Isolated Clara cell fractions metabolized 7-EC to umbelliferone at a rate of 241 ± 27 pmoles/mg prot/min (mean ± S.E., N=5), while the 7-EC deethylation rate in type II cells was 111 ± 15 pmoles/mg prot/min. Coumarin hydroxylation activity, however, was more than ten times greater in the Clara cells than in the type II cells on a per mg cellular protein basis. N-oxidation of N,N-dimethylaniline, catalyzed by a flavin monooxygenase, was about 2 times as great in microsomes of Clara cells as in microsomes of type II cells. Epoxide hydrolase activity with benzo(a)pyrene 4,5-oxide as substrate was about 10 times higher in Clara cells than in type II cells. Because of the greater cellular, structural and functional heterogeneity in lung, differential distribution of enzymes responsible for xenobiotic metabolism in this tissue may contribute to cell selective chemical toxicity and carcinogenesis.

Xenobiotic Metabolizing Enzymes in Marine Fish

In recent years, the interest of scientists from several disciplines has focused on the fate of the many xenobiotics, including pesticides, which are introduced into our environment. Many of these foreign chemicals eventually enter the oceans and can be ingested by marine species. Using in vivo and in vitro techniques, we have studied xenobiotic metabolism in a number of marine fish and crustacea. The pathways studied in vitro include oxidative metabolism by the cytochrome P-450-dependent mixed-function oxidases and metabolism of some products of oxidation, namely epoxides and arene oxides. A large number of pesticide molecules undergo metabolism by these reactions, which have been extensively studied in mammalian species. We also followed the uptake, distribution, and metabolism of a polychlorinated biphenyl, 2,4,5,2’,5’-pentachlorobiphenyl, and of the herbicides, 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid, after administration to selected marine species.