Carcinogenesis by isomers of N-nitroso-3,5-dimethylpiperidine.

The cis- and trans-isomers of N-nitroso-3,5-dimethylpiperidine were administered separately in drinking water solution to groups of 20 female F344 rats for 50 weeks. The concentrations of the solutions were 0.72 m M for the cis-isomer and 0.14 mM for the trans-isomer. In both groups the animals died with tumors of the upper gastrointestinal (GI) tract, mainly carcinomas of the esophagus, at about the same time. A third group of animals was given a mixture of the two isomers in the ratio of 5 cis:1 trans, and these animals died more rapidly with the same upper GI tumors. The trans-isomer appeared to be a more potent carcinogen than the cis-isomer. The 3,5-dimethyl derivative is a less potent carcinogen than nitrosopiperidine.

Esophageal carcinogenesis in F344 rats by nitrosomethylethylamines substituted in the ethyl group.

The carcinogenic activity in male F344 rats of five nitrosomethylalkylamines related to nitrosomethylethylamine has been examined. All rats were tested by administration of controlled doses of the compounds in the drinking water. Nitrosomethylbenzylamine was the most potent carcinogen of the five, causing death of most animals with tumors of the upper gastrointestinal tract (mainly the esophagus) within 6 months after a total dose of 0.2 mmol. The remaining compounds, all of which can be considered true derivatives of nitrosomethylethylamine, were less potent than nitrosomethylbenzylamine but also induced a high incidence of tumors of the esophagus. Nitrosomethyl-2-phenylethylamine and nitrosomethylneopentylamine were of comparable potency, while nitrosomethyltrifluoroethylamine was considerably less potent, as measured by the time to death with esophageal tumors. Deuterium-labeled nitrosomethylethylamine, in which deuterium replaces hydrogen on the beta-carbon atom of nitrosomethylethylamine, induced a high incidence of esophageal tumors as well as liver tumors after administration of identical doses. Reactions on the beta-carbon atom of nitrosomethylethylamine might be important as well as oxidation at the alpha-carbon atoms in determining carcinogenic effects.

Carcinogenesis by derivatives of 1-nitroso-3,5-dimethylpiperazine in rats.

Four mononitrosopiperazines were administered to groups of 20 female Fischer 344 rats to compare their effectiveness as carcinogens. The four, 1-nitroso-3,5-dimethylpiperazine, its 4-acetyl derivative, its 4-benzoyl derivative, and 1-nitroso-3,4,5-trimethylpiperazine, were given as 0.7 mM solutions in drinking water, 100 ml to each rat per week. The length of treatment varied from 26 weeks for nitrosotrimethylpiperazine to 50 weeks for 1-nitroso-3,5-dimethyl-4-benzoylpiperazine. Dimethyl- and trimethylnitrosopiperazine gave rise to virtually 100% incidence of undifferentiated lymphomas of the thymus and leukemias within 30 weeks (in contrast to the non-C-methylated analogs which are noncarcinogenic or only weakly so). Acetyldimethylnitrosopiperazine was also a potent carcinogen, all of the rats treated with it dying within 30 weeks with tumors of the esophagus. In contrast, benzoyldimethylnitrosopiperazine was weakly carcinogenic, inducing only a small number of tumors of the forestomach and reducing the normal life span of the rats very little.

Enzymatic sulfation of steroids. II. The sulfation of corticosterone by the glucocorticoid sulfotransferases of rat liver cytosol.

A radioisotopic assay for the cytoplasmic corticosterone sulfotransferase activity of rat liver was developed. The steroid inhibits the enzyme reaction. For reliable results, a complex assay method, using three different corticosterone concentrations, each studied with several different amounts of enzyme, was necessary. This "mosaic" assay compensates for observed biological, gonadal and seasonal enzyme fluctuations. Cytosols from female rats contain 6--9-times the enzyme activity found in males. The sulfation product with both sexes is corticosterone-21-sulfate. The effects of castration and of androgen administration on hepatic cortisol and corticosterone sulfation were compared in female rats. Ovariectomy resulted in 20--32% and 25--35% decreases of hepatic corticosterone and cortisol sulfotransferase activity, respectively. Androgen administration caused 37--55% and 40--60% decreases of sulfation of the two steroids. The data suggest the equivalence of hepatic cortisol and corticosterone sulfotransferases. Fractionation of cytosols from female rats, on DEAE-Sephadex A-50 columns, resolved three peaks of corticosterone sulfotransferase activity which eluted concurrently with the hepatic cortisol sulfotransferases I, II and III. They appear to be the same enzymes. Cytosol from males contained cortisosterone sulfotransferase activity due mostly to sulfotransferase III. Sulfotransferases I and II appear to have higher turnover numbers for hepatic cortisol than for corticosterone. The reverse is true for sulfotransferase III.

Enzymatic sulfation of steroids. XVI. Very rapid effects of steroid hormones on hepatic cortisol sulfation in intact male rats.

Hepatic glucocorticoid sulfotransferase activity in male rats was elevated approximately 200, 100 or 60%, respectively, by administration of 0.20 mg estradiol, 1.0 mg testosterone or 12 mg progesterone daily for 2 days. Administration of 3.0 mg corticosterone daily, for 2 days, was without effect. All observed hormone effects were due to elevation of the concentration of sulfotransferase III, the glucocorticoid-preferring steroid sulfotransferase of rat liver. The response of the enzyme activity to the estrogen was blocked by puromycin or actinomycin D. The relationship between these studies and general endocrine control of sulfotransferase production is discussed.

Enzymatic sulfation of steroids. XV. Studies differentiating between rat liver androgen, estrogen, bile acid, glucocorticoid and phenol sulfotransferases.

Earlier reports left the number of enzymes that catalyzed phenol, androgen, estrogen, bile acid and glucocorticoid sulfation obscure. Here, we have utilized chromatographic, immunochemical and endocrinologic methods to compare and differentiate these enzymes in rat liver. Sulfotransferases I, II, and III--which sulfate glucocorticoids--were used in this comparison. We found that phenols were sulfated by phenol sulfotransferases 1 and 2, which were unrelated to the other enzymes studied here. Large amounts of phenol sulfotransferase 1 were found in both sexes. Large amounts of phenol sulfotransferase 2 were restricted to males. By contrast, the small amount of androgen sulfation found in both sexes appeared to be mediated by sulfotransferase II, which preferred 3 beta-hydroxysteroids, but also sulfated estrogens and glucocorticoids to lesser extents. The sulfation of estrogens presented a more complex picture. Most estradiol sulfotransferase activity in both sexes was due to an enzyme that sulfated estrone poorly, and did not sulfate the other steroids tested. This specific estradiol sulfotransferase was unrelated to the other sulfotransferases described here. Smaller amounts of estrogen sulfotransferase activity that sulfated estradiol and estrone equally well were present at concentrations dependent on the sex of test animals. This enzyme activity appeared to be due to sulfotransferases I, II and III. Most bile acid sulfotransferase activity eluted from DEAE-Sephadex A-50 columns with sulfotransferases I and II. However, data with males suggested that these enzymes were not responsible. Thus, phenols, androgens, estrogens and glucocorticoids were sulfated by six enzymes of differing substrate specificity: phenol sulfotransferases 1 and 2, specific estradiol sulfotransferase, and sulfotransferases I, II, and III. Unique bile acid sulfotransferases also appeared probable.

Enzymatic sulfation of steroids: II. The control of the hepatic cortisol sulfotransferase activity and of the individual hepatic steroid sulfotransferases of rats by gonads and gonadal hormones.

Ovariectomy has relatively little effect on hepatic cortisol sulfotransferase activity (HCSA) in female rats, diminishing it only 30%. On the other hand, castration more than doubles HCSA in males. HCSA is due to 3 steroid sulfotransferases, STI, STII, and STIII. Its dimunition in ovariectomized rats is due to decreased STI and STII. Castration of males results in elevation of STII. Thus, ovaries appear to stimulate STI and STII production and testes appear to inhibit production of STII and perhaps STI. Studies with testosterone and estradiol-17beta support a role for sex hormones as mediators of gonadal effects on HCSA, by stimulating or inhibiting production of the individual enzymes. Estradiol-17beta administration reverses the effect of ovariectomy on HCSA. Testosterone administration to intact or castrated females decreases HCSA by 60-70%, due to disappearance of all STI and most STII activity. Thus, androgen administration appears to suppress both STI and STII production. In intact males testosterone administration elevates HCSA 70-80% due to increased STIII. Estradiol-17beta administration to intact or castrated males elevates HCSA 9-10-fold. In intact animals this is due to elevated STI and STII but not STIII. In castrates all three enzymes are elevated by the estrogen.

Enzymatic sulfation of steroids: I. The enzymatic basis for the sex difference in cortisol sulfation by rat liver preparations.

Liver cytosols from female rats contained 6-8 times as much cortisol sulfotransferase activity as those from males. The reaction product, with both sexes, appeared to be cortisol-21 sulfate. Liver cytosols from male and female rats showed different substrate preferences when tested with cortisol, estradiol-17beta, testosterone, and dehydroepiandrosterone, suggesting that they contained different steroid sulfotransferases. Fractionation of cytosols from female rats on DEAE Sephadex A-50 columns resolved 3 steroid sulfotransferases, or families of steroid sulfotransferases (STI, STII, and STIII). These enzymes exhibited different substrate preferences. STI and STIII had the greatest preferences for cortisol, although none of the enzymes was restricted to the glucocorticoid. Fractionation of cytosols from males resolved 2 sulfotransferases which eluted at salt concentrations identical to STII and STIII from females. Study of the development of cortisol sulfotransferase activity with age showed little enzyme activity in rats of either sex at 2 days after birth. Enzyme activity developed in parallel in both sexes until 30 days after birth. Then the sulfotransferase activity began to rise in females and to drop in males. By day 50-55 both sexes attained adult enzyme levels. STII was the major enzyme in all immature animals. STIII was also present, but STI was absent. In male rats STIII activity began to rise by day 30. Soon after, STII activity began to drop. By day 55 adult male patterns developed. STI was the major enzyme in females by day 30. In ensuing days all 3 enzyme levels rose, until by day 50 adult enzyme patterns and levels were attained. The data suggest that the ovaries stimulated production of all 3 sulfotransferases and that the testes suppressed production of STII (and perhaps STI). Preliminary studies with gonadectomized rats supported the suppressive role of the testes, but suggested that the ovaries were not the only factor controlling sulfotransferase production in female rats.

Decomposition reactions of (hydroxyalkyl) nitrosoureas and related compounds: possible relationship to carcinogenicity.

(Hydroxyalkyl)nitrosoureas and the related cyclic carbamates N-nitrosooxazolidones are potent carcinogens. The decompositions of four such compounds, 1-nitroso-1-(2-hydroxyethyl)urea (I), 3-nitrosooxazolid-2-one (II), 1-nitroso-1-(2-hydroxypropyl)urea (III), and 5-methyl-3-nitrosooxazolid-2-one (IV), in aqueous buffers at physiological pH were studied to determine if any obvious differences in decomposition pathways could account for the variety of tumors obtained from these four compounds. The products predicted by the literature mechanisms for nitrosourea and nitrosooxazolidone decompositions (which were derived from experiments at pH 10-12) were indeed the products formed, including glycols, active carbonyl compounds, epoxides, and, from the oxazolidones, cyclic carbonates. Furthermore, it was shown that in pH 6.4-7.4 buffer epoxides were stable reaction products. However, in the presence of hepatocytes, most of the epoxide was converted to glycol. The analytical methods developed were then applied to the analysis of the decomposition products of some related dialkylnitrosoureas, and similar results were obtained. The formation of chemically reactive secondary products and the possible relevance of these results to carcinogenesis studies are discussed.

Enzymatic sulfation of steroids--XX. Effects of ten drugs on the hepatic glucocorticoid sulfotransferase activity of rats in vitro and in vivo.

The effects of ten drugs on hepatic glucocorticoid sulfotransferase activity (HGSTA) were examined in male rats. The enzyme activity per 100 g body weight was elevated 152, 94.9, 140, 140, 73.1, 63.9, 76.9, and 140% after administration of daily i.p. doses of 111 mg spironolactone/kg (6-10 days), 66.7 mg WIN-24540/kg (6-10 days), 150 mg metyrapone/kg (19-31 days), 33.3 mg pentachlorophenol/kg (9-16 days), 16.5 mg aspirin/kg (10-16 days), 90.5 mg alloxan/kg (23.27 days), 104 mg aminoglutethimide/kg (12-20 days), and 16.8 mg propranolol/kg (21-27 days). Shorter experimental periods or lower drug doses caused smaller effects on HGSTA. Most notably, spironolactone (111 mg/kg) and WIN-24540 (66.7 mg/kg) caused 50-75% elevation of HGSTA in 2 days. Effects of WIN-24540, aspirin and pentachlorophenol were due mostly to elevation of hepatic levels of sulfotransferase III (STIII), the glucocorticoid-preferring sulfotransferase of rat liver. Effects of the other test drugs were due to elevation of hepatic levels of sulfotransferases I and II (STI and STII), which much prefer dehydroepiandrosterone as substrate, but also catalyze glucocorticoid sulfation. Enzyme inhibition studies showed that the test drugs interacted with the HGSTA in vitro in a fashion that appeared to be related to the in vivo effects already described. None of the drugs interacted exclusively with STI, STII or STIII in vitro. However, some differences of the strengths of individual drug-sulfotransferase interactions were observed. The drug effects are discussed in relation to drug and glucocorticoid actions.

Carcinogenesis in F-344 rats induced by nitrosohydroxyalkyl-chloroethylureas.

The two isomeric N-nitroso derivatives of 1-chloroethyl-3-(2-hydroxyethyl)-urea and of 1-chloroethyl-3-(2-hydroxypropyl)-urea were prepared and isolated. They were given by gavage in ethyl acetate/corn oil to groups of 20 male and female F-344 rats. The two nitroso-1-chloroethyl compounds were nephrotoxic and most animals died within 20 weeks; no neoplasms were seen in any of these animals. Nitroso-1-hydroxyethyl-3-chloroethylurea was given at 2 concentrations, 21 and 10.5 mg/ml; in both groups almost all animals died with neoplasms related to the treatment. These included hepatocellular and cholangiocellular neoplasms of the liver; many of the former metastasized. Many rats also had tubular cell neoplasms in the kidney. Nitroso-1-(2-hydroxypropyl)-3-chloroethylurea was a less potent carcinogen at equimolar doses, inducing fewer liver neoplasms than the nitrosohydroxyethyl analog and only few kidney neoplasms. Both of these carcinogens were less effective in female rats than in males, although the females, which were smaller, received a higher dose per unit body weight. The spectrum of neoplasms induced by the nitrosohydroxyalkyl-chloroethylureas was quite different from that induced by equimolar doses of each corresponding nitrosohydroxyalkylurea, neither of which induced neoplasms of the liver, although they were potent inducers of neoplasms in other organs.

The same enzymes catalyze sulfation of minoxidil, minoxidil analogs and catecholamines.

Rat liver sulfation of minoxidil and minoxidil analogs is described and the enzymes responsible are compared with those that sulfate catecholamines. Our study of minoxidil sulfation showed male-dominant sex dimorphism of the enzyme activity due to two enzymes that coelute with dopamine sulfotransferases. The most abundant isoform, in our routine assay, is minoxidil sulfotransferase 2 (P2). Sulfation ability by this enzyme parallels minoxidil analog antihypertensive ability. Minoxidil, analog and dopamine sulfotransferases were not separable by several different chromatographic methods, supporting their identity. The minoxidil sulfotransferase activity dropped in hypophysectomized males, due mostly to diminished levels of minoxidil sulfotransferase 2/dopamine sulfotransferase II, which appears to be aryl sulfotransferase IV. Its relationship to the minoxidil sulfotransferase reported by the Falany group is not clear. Here, we describe the exploration of rat liver sulfation of antihypertensive minoxidil (2,4-diamino-6-piperidinopyrimidine-3-oxide), and several minoxidil analogs. Minoxidil sulfation was first reported in rat and human liver cytosol by Johnson's group at Upjohn [1,2]. Our interest in the sulfation process arose because direct action of minoxidil sulfate had been implicated both in vivo [3,4] and in vitro [5] in blood pressure control processes.

Perfluoro-N-decanoic acid effects on enzymes of fatty acid metabolism.

In vitro perfluorodecanoate (PFDA) effects on Pseudomonas acyl-CoA synthetase, Candida acyl-CoA oxidase and pigeon muscle carnitine acetyltransferase were examined. Synthetase made little PFDA-CoA from PFDA. It used palmitate, oleate, laurate and decanoate more extensively. PFDA inhibited acyl-CoA formation from these acids. Palmitoyl-CoA formation was affected most. That of decanoyl-CoA was affected least. Inhibitions appeared to be competitive. Acyl-CoA oxidase test substrates were palmitoyl-CoA, lauroyl-CoA and decanoyl-CoA. Oxidase preferred C-10 and C-12 acyl-CoAs. PFDA inhibited oxidation of C-10 and C-12 acyl-CoAs more than that of palmitoyl-CoA. Inhibitions with C-16 and C-10 acyl-CoAs were competitive, KIs 593 +/- 150 and 76 +/- 6.0 microM. Acetyl-CoA was the best acetyltransferase substrate. C-2 to C-8 transfer from acyl-CoAs was inhibited similarly by PFDA. Inhibitions of C-2 and C-8 transfer were competitive and non-competitive, respectively, KIs 111 +/- 15 and 76 +/- 28 microM.

Decomposition N-nitrosohydroxyalkylureas and N-nitrosooxazolidones in aqueous buffer.

The decomposition of N-nitroso-2-hydroxyethylurea, N-nitrosooxazolidone, N-nitroso-2-hydroxypropylurea and N-nitroso-5-methyloxazolidone in pH 7.4 phosphate buffer was studied. The aldehydes and ketones formed in the decompositions were monitored through the formation of dinitrophenylhydrazine derivatives, which were then analysed by high-performance liquid chromatography. N-Nitroso-2-hydroxyethylurea gave a 25% yield of acetaldehyde dinitrophenylhydrazone, while N-nitrosooxazolidone gave only 2%. The major product from the latter is 1,3-dioxolane-2-one (ethylene carbonate); ethylene glycol is a major product from both. The other two compounds studied gave similar products, but in much lower yields.