Oxidation of 7-ethoxycoumarin and conjugation of umbelliferone by intact, viable epidermal cells from the hairless mouse.

Intact, viable (greater than 80%) epidermal cells were isolated from the hairless mouse. These cells metabolized 7-ethoxycoumarin (7-EC) to umbelliferone ( UMB ) (3 pmol/min/10(6) cells) and UMB to the sulfate and glucuronide conjugates (1 pmol/min/10(6) cells). The rate of oxidation in intact cells compared well with that in disrupted cells with added NADPH, but conjugation proceeded more rapidly in disrupted cells with added cofactors, due to a combination of "activation" of the UDP-glucuronosyltransferase, and to a limitation of activity by the concentration of UDP-glucuronic acid in the intact cells. Pretreatment of the animals with 5,6-benzoflavone resulted in a 5-fold increase in the rate of oxidation, and a 2-fold increase in both the rate of conjugation and the intracellular concentration of UDP-glucuronic acid. UDP-glucuronic acid concentration in isolated cells increased during incubation with glucose, and was regenerated to a steady-state concentration on incubation of cells with UMB . Pretreatment of animals with 5,6-benzoflavone decreased the percentage of metabolite conjugated (from 30% to 15%), whereas adding an inhibitor of oxidation, ellipticine, to cells isolated from pretreated animals, increased the percentage of metabolite conjugated (from 15% to 40%). Sulfation of UMB was almost undetectable, except at very low concentrations (less than 10 nM) of substrate. Thus, glucuronidation of UMB in epidermal cells may be limited by UDP-glucuronic acid availability; sulfation in the epidermis may contribute little to the conjugation of UMB ; and greater than 70% of the products of 7-EC oxidation in the skin may remain unconjugated.

The mitochondrial metabolism of 1,2-disubstituted hydrazines, procarbazine and 1,2-dimethylhydrazine.

Sonication of isolated rat hepatocytes caused a pronounced decrease in the metabolism of biphenyl due to dilution of the cytosolic pool of NADPH, but did not greatly reduce the rate of procarbazine oxidation to its azo derivative. This result suggested the existence of two enzyme systems which can oxidize 1,2-disubstituted hydrazines: an NADPH-dependent (cytochrome P-450) and an NADPH-independent hydrazine oxidase. Upon assaying the various cell fractions of the hepatocyte, it was noted that the NADPH-independent hydrazine oxidase activity was localized in the mitochondria. The reaction was not linked to mitochondrial electron transport, but preincubation of isolated mitochondria with N,N-dimethylpropargylamine markedly inhibited both monoamine oxidase activity (benzylamine and kynuramine deamination) and procarbazine oxidation. During a 15-fold purification of the enzyme, benzylamine oxidase and procarbazine oxidase activity copurified, demonstrating that the rat liver mitochondrial monoamine oxidase can convert procarbazine to its respective azo derivative. 1,2-Dimethyl- and monomethylhydrazine were also metabolized by monoamine oxidase. Procarbazine did not inactivate monoamine oxidase like other hydrazines; this most likely reflects the fact that the oxidation product of the 1,2-disubstituted hydrazines is a stable azo derivative. However, procarbazine is a competitive substrate for the enzyme and may exert some of its toxic neurological effects by altering the metabolism of biogenic amines.(ABSTRACT TRUNCATED AT 250 WORDS)

Properties of an Escherichia coli mutant deficient in phosphoenolpyruvate carboxylase catalytic activity.

A mutant Escherichia coli (Ppcc-) which was unable to grow on glucose as a sole carbon source was isolated. This mutant had very low levels of phosphoenolpyruvate carboxylase activity (approximately 5% of the wild type). Goat immunoglobulin G prepared against wild-type phosphoenolypyruvate carboxylase cross-reacted with the Ppcc- enzyme. The amount of enzyme protein in the mutant cells was similar to that found in wild-type cells, but it had greatly diminished specific activity. The catalytically less active mutant enzyme retained the ability to interact with fructose 1,6-bisphosphate, but did not exhibit stabilization of the tetrameric form by aspartate. The pI of the mutant protein was lower (4.9) than that of the wild-type protein (5.1). After electrophoresis and immunoblotting of the partially purified protein, several immunostaining bands were seen in addition to the main enzyme band. A novel method for showing that these bands represented proteolytic fragments of phosphoenolpyruvate carboxylase was developed.

Foreign compound metabolism by isolated skin cells from the hairless mouse.

A method for isolating mouse skin cells by enzymatic digestion with trypsin was developed. Cell populations of 33% viability could be further separated by metrizamide and Percoll gradient centrifugations into three fractions enriched in different cell types. in one fraction 80% of the cells were sebaceous, in the second fraction 50% of the cells were basal and the third fraction consisted predominantly of differentiated keratinocytes. Different cell types were characterized by electron microscopy, light microscopy, staining and enzyme activities. Measurement of benzo(a)pyrene hydroxylase, 7-ethoxycoumarin O-deethylase, UDP-glucuronosyltransferase and GSH-S-transferase activities in different cell types from control mice and mice topically treated with beta-naphthoflavone showed that different cell populations metabolized foreign compounds at different rates. The sebaceous cells were the most active xenobiotic-metabolizing cells. beta-Naphthoflavone increased relative enzyme activities of the original cell population and basal cell-enriched fraction more than that of the already highly active sebaceous cell population.

7-ethoxycoumarin O-deethylation activity in viable basal and differentiated keratinocytes isolated from the skin of the hairless mouse.

Epidermal cells in suspension were prepared from the skin of hairless mice by digestion of skin strips with pronase. The viability of cells in such suspensions was routinely greater than 75%. Fractions enriched in different cell types were prepared from the original cell suspensions using metrizamide gradients and elutriation techniques. These fractions were studied histologically and enzymically. The cells of greater size both were more differentiated and had higher xenobiotic metabolizing enzyme activities. Also increasing in parallel with cell size were parameters such as protein to DNA ratios. Lowest in all respects were basal cells (size, enzyme activities, protein/DNA ratios, etc.). The present techniques seem superior to previously described methods for isolating skin cells for study of xenobiotic metabolisms and possible distribution of these metabolisms in different cell types.