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We determined UDP-glucuronyltransferase (UDP-GT) activities of hepatic and mammary gland microsomes of female rats with p-nitrophenol and the ring- and N-hydroxylated metabolites of N-2-fluorenylacetamide (2-FAA) and the effects of hepatic inducers of UDP-GT's on these glucuronidations. Pre-treatment of non-lactating (NL) and lactating (L) rats with beta-naphthoflavone (beta-NF) significantly increased glucuronidations, of p-nitrophenol, the phenolic metabolites of 2-FAA, especially of 5-hydroxy-2-FAA, and also of N-hydroxy-2-FAA by hepatic microsomes. Pre-treatment of L rats with beta-NF or 3-methyl-cholanthrene (3-MC) significantly increased glucuronidations of these compounds by mammary gland microsomes suggesting that both liver and mammary gland of L rats possess similar UDP-GT activities. In NL rats, UDP-GT activities of mammary microsomes toward phenols were greater than in L rats, and except for that of 5- and 7-hydroxy-2-FAA, were not inducible with beta-NF. The data obtained with L rats, the greater magnitude of stimulation of the hepatic UDP-GT of NL rats by beta-NF than by phenobarbital, and the lack of effect of the latter on UDP-GT of mammary microsomes suggested that the phenolic metabolites of 2-FAA and N-hydroxy-2-FAA share chiefly the characteristics of substrates for group 1 UDP-GT activities (i.e., those inducible with beta-NF or 3-MC). Neither inducer increased glucuronidation of 9-hydroxy-2-FAA, a relatively poor substrate for UDP-GT of mammary or hepatic microsomes. In contrast to hepatic microsomes which formed considerable amounts of the glucuronide of N-hydroxy-2-FAA, mammary gland microsomes glucuronidated this substrate to a minor extent only. This suggested that glucuronide of N-hydroxy-2-FAA may play a role in systemic, but not in local mammary tumorigenesis by N-hydroxy-2-FAA.

The kinetic mechanism of SS isozyme of horse liver alcohol dehydrogenase is shown by initial velocity and product inhibition studies to be asymmetrical, being random for ethanol oxidation and compulsory ordered for acetaldehyde reduction. Enzyme isomerization seems to account for the asymmetry in the mechanism. In its interaction with NADH, the SS isozyme resembles classical alcohol dehydrogenase; consequently, the maximal velocity in the direction from ethanol to acetaldehyde appears to be determined by the rate of NADH dissociation. In the direction from acetaldehyde to ethanol, the enzyme isomerization step appears to limit the maximal velocity.

A method for the assay of glucuronidation of C- and N-hydroxylated metabolites of the carcinogen N-2-fluorenylacetamide is described. The method employs UDP-[U-14C ))glucuronic acid and Baker C18 extraction columns for separation of the glucuronides from their aglycones and from the glucuronic acid. The 14C-labeled glucuronides, generated by rat liver microsomes, are eluted from the columns with 30% (v/v) methanol after prewashing the columns and elution of the radioactivity of 14C-glucuronic acid with 1 mM ammonium acetate, pH 6.9. The radioactivity of the eluates is measured by scintillation counting. The method is modified for assays of glucuronidation of alpha-naphthol and p-nitrophenol in that 1 mM phosphoric acid is used instead of 1 mM ammonium acetate, and the method is potentially adaptable to other aglycones. By monitoring radioactivity or uv absorbance of the column eluates, it is shown that all aglycones, except p-nitrophenol, are retained on the columns during elution of their glucuronides with 30% (v/v) methanol and are eluted only when absolute methanol is used. The identity of the glucuronides is shown by their response to hydrolysis by beta-glucuronidase in the presence and absence of D-saccharic acid-1,4-lactone and, in some instances, by chromatographic and spectral analyses of the released aglycones.

The most cathodal (on starch-gel electrophoresis), steroid-active band of horse liver alcohol dehydrogenase, whose catalytic properties were shown to be dependent on the livers used as a starting material [Pietruszko (1974) Biochem. Biophys. Res. Commun. 60, 687-694], has been prepared from A-type and S-type horse livers by identical methods. Results presented here show that different isoenzymes are present in these preparations.

Metabolic or synthetic N-hydroxylation of N-arylamides yields N-arylacylhydroxamic acids considerably more carcinogenic for the rat mammary gland than the parent amides both by systemic and topical administration. The size of the aryl moiety, the position of the nitrogen relative to the aryl moiety and the type of acyl group are determinants of the carcinogenic potency of N-arylacylhydroxamic acids. Induction of mammary tumors required ovarian hormones. Receptors for estrogen, androgen and progesterone were shown in the N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA)-induced mammary carcinoma. This tumor involved epithelial and stromal components of the mammary gland that were separated in culture and produced tumors of their respective origin in the isologous host. Both mammary epithelial cells and fibroblasts are capable of metabolism of carcinogens. The enzymes potentially involved in metabolic activation of N-arylamides and N-arylacylhydroxamic acids in the mammary gland include: a cytochrome P-450(P(1)-450) system, UDP-glucuronyltransferase, N,O-acyltransferases and peroxidases. Mammary microsomes in which cytochrome P(1)-450 was induced generated small amounts of N-OH-2-FAA from 2-FAA. N-OH-2-FAA and its carcinogenic isomer, N-OH-3-FAA, were oxidized by cytochrome c/H(2)O(2) to the nitroxyl free radicals which dismutated to the respective acetate esters and nitrosofluorenes. The addition of unsaturated lipid to either the free radicals or to the nitrosofluorenes gave electron spin resonance signals characteristic of immobilized radicals. It is proposed that interactions of carcinogens with lipids and with DNA play a role in mammary tumorigenesis.