A microsomal fraction, prepared from mouse skin, catalyzed the hydroxylation of benzpyrene and aniline and the deethylation of 7-ethoxycoumarin. Contamination of the preparation by cytochrome oxidase and cytochrome P-420 was determined by spectral analysis. The enzyme activities studied in mouse skin (Swiss-Webster CD-1) did not respond to topical application of 3-MC. Twenty-four hours after topical application of TCDD to mice, microsomes from skin had 50% greater benzpyrene hydroxylase and 7-ethoxycoumarin deethylase activity, and 4- to 8-fold greater activity of these enzymes was seen after 72 hr. Increases in cytochrome P-450 content of skin microsomes could be demonstrated 24 and 72 hr after topical TCDD treatment of mice. Cholate treatment (solubilization) of skin microsomes, followed by centrifugation, removed the contaminating cytochrome oxidase. Quantitative and qualitative analyses of cytochrome P-450 difference spectra were made from the solubilized preparations.

Aniline Hydroxylase↗

PubMedDate not supplied

Reconstitution of the rabbit pulmonary microsomal mixed-function oxidase system from solubilized components.

Lung microsomal cytochrome P-450 was solubilized and purified 2-fold. NADPH-cytochrome c reductase (EC 1.6.2.3) was solubilized and purified 4-6 fold by three methods with use of sonication and detergent digestion followed by either DEAE-cellulose chromatography or ammonium sulfate fractionation. Benzphetamine N-demethylase and 7-ethoxycoumarin deethylase activities were reconstituted when NADPH-cytochrome c reductase and cytochrome P-450 fractions were combined. Reductase fractions prepared by sodium cholate digestion of microsomes were highly active in supporting the hydroxylation activity in the reconstituted systems, whereas those prepared with sodium deoxycholate were not. About twice as much NADPH-cytochrome c reductase was required for saturation of the 7-ethoxycoumarin deethylase activity as for saturation of the benzphetamine N-demethylase activity. A heat-stable lipid fraction was necessary for maximum hydroxylation activity. NADH did not support benzphetamine N-demethylation in the reconstituted system or increase the rate of reaction when added with NADPH.

Animals↗

PubMedDate not supplied

Effect of pregnancy or treatment with cetain steroids on N,N-dimethylaniline demethylation and N-oxidation by rabbit liver or lung microsomes.

Rates of lung microsomal dimethylaniline (DMA) demethylation and N-oxidation as well as cytochrome P-450 concentrations from 20- and 28-day pregnant rabbits were observed to be as much as 2 times the values measured in lung microsomes from adult female nonpregnant rabbits. These increases were not seen in lung microsomes from 10-day pregnant rabbits, and only small increases were observed in lung microsomes from 15-day pregnant rabbits. In contrast, no differences were seen in hepatic microsomal DMA demethylase or N-oxidase activities at any stage of pregnancy. An attempt was made to correlate high plasma concentrations of steroids during pregnancy with the elevated enzyme activities observed in lung microsomes. Adult nonpregnant rabbits were treated with a variety of steroids, the concentrations of which have been shown to increase in plasma during late pregnancy. No effect on lung or liver microsomal DMA metabolism was observed when rabbits were treated with testosterone propionate. However, after animal pretreatment with the glucocorticoids, dexamethasone and hydrocortisone, there was a 40-60% increase in DMA demethylase and N-oxidase activities in liver and lung microsomes from adult male and nonpregnant female rabbits. Following pretreatment of adult female nonpregnant rabbits with the mineralocorticoid, deoxycorticosterone, an increase of 50% in rats of microsomal DMA metabolism in lung but not in liver was observed.

Aniline Compounds↗

PubMedDate not supplied

Species differences in benzene hydroxylation to phenol by pulmonary and hepatic microsomes.

The metabolism of benzene to phenol by microsomal preparations from lung and liver has been compared in hamsters, rats, and rabbits. There were wide differences in the apparent Vmax of benzene hydroxylation among the various species and tissues and smaller differences in the apparent KM values for benzene hydroxylase. Benzene can inhibit its own metabolism in vitro when present in high concentrations. Phenol was the only metabolite of benzene identified, under the conditions of the assay, in incubation mixtures containing microsomes from lung or liver of any of the three animal species. When incubated with microsomes under the conditions used to measure benzene metabolism, phenol was further metabolized in liver but not in lung preparations. Phenol metabolism was almost completely inhibited when 11.2 mM benzene was included in the incubation mixture containing hepatic microsomes. The variation in rates of benzene hydroxylation by microsomal preparations from lungs or livers of the three animal species was similar to the variation in rates of benzypyrene hydroxylation in the same preparations.

Aniline Hydroxylase↗

PubMedDate not supplied

Biotransformation of xenobiotics in individual rabbit hepatocytes. Application of microspectrofluorometry.

We have investigated several methods for determining rates of xenobiotic biotransformation in individual rabbit hepatocytes by microspectrofluorometry. Experiments designed to measure monooxygenase activity by following fluorescent product formation (i.e. 7-ethoxycoumarin deethylase or oxidation of the fluorescein derivatives ethoxyfluorescein ethyl ester and 5- and 6-ethoxycarbonyl ethoxyfluorescein ethyl ester) demonstrated that the fluorescent products were released from cells to the surrounding media. Thus, metabolically active cells probably had underestimated activities, and inactive cells could accumulate fluorescent product and appear metabolically active. We also utilized benzo(a)pyrene (BP) as a substrate in our system. By selecting appropriate wavelengths (370 nm excitation, 407 nm emission), it was possible to selectively monitor BP disappearance in single cells. In hepatocytes from rabbits, disappearance of fluorescence was observed after a 10-min incubation at 37 degrees C with 1-40 microM BP followed by a wash to remove extracellular substrate. The decrease in fluorescence followed apparent first-order kinetics. Measurement of extracellular fluorescence indicated that the substrate did not leave the hepatocytes. Furthermore, in both control and beta-naphthoflavone-treated rabbits, the observed fluorescence changes in the cells were inhibited by the presence of equimolar concentrations of ellipticine or alpha-naphthoflavone, inhibitors of BP metabolism. Storage of hepatocytes from untreated animals on ice for 3.5 hr did not affect rate constant values in cells with a normal appearance, whereas those lacking refringent edges or having numerous cytoplasmic blebs demonstrated greatly reduced (up to 88% loss) activities compared with normal, freshly isolated cells. Potential applications of this system (e.g. to other cell types) are discussed.

Animals↗