Effect of age on the conversion of 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 by kidney of rat.

The decreased absorption of calcium by the small intestine of the adult may reflect changes in vitamin D metabolism with age. The purpose of this study was to compare the capacity of young (1.5 mo of age) and adult (12 mo of age) vitamin D-deficient rats to convert 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D, the physiologically active form of vitamin D. Young rats responded to an oral dose of 25-hydroxyvitamin D3 with significantly increased intestinal absorption of calcium and a three-fold increase in the intestinal content of vitamin D-stimulated calcium-binding protein. Adult rats showed no significant increase in these parameters. The conversion of 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 was measured in the whole animal by administering a dose of tritiated 25-hydroxyvitamin D3 and determining the appearance of tritiated metabolites in plasma and small intestine. In the adult rat, only 2.1 +/- 0.6% of the plasma radioactivity was in the form of 1,25-dihydroxyvitamin D3 after 24 h compared with 20.8 +/- 3.0% in the young. The conversion of tritiated 25-hydroxyvitamin D3 to its products was also measured directly in isolated slices of renal cortex. 1,25-Dihydroxyvitamin D3 production by adult renal slices was found to be less than one-tenth that of slices from the young. These results indicate that there is a marked decrease in the capacity of the vitamin D-deficient adult rat to convert 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3. This is probably due to the decreased capacity of the adult kidney to 1-hydroxylate 25-hydroxyvitamin D3. These studies also demonstrate the usefulness of renal slices in measuring changes in the renal conversion of 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 in the mammal.

Role of renal metabolism and excretion in 5-nitrofuran-induced uroepithelial cancer in the rat.

5-Nitrofurans have been used in the study of chemical carcinogenesis. There is substantial evidence that N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide (FANFT) is deformylated to 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) in the process of FANFT-induced bladder cancer. Paradoxically, ANFT is less potent as a uroepithelial carcinogen than FANFT when fed to rats. Feeding aspirin with FANFT to rats decreases the incidence of bladder cancer. Isolated kidneys were perfused with 5-nitrofurans to determine renal clearances and whether aspirin acts to decrease urinary excretion of the carcinogen. In FANFT-perfused kidneys, FANFT was deformylated to ANFT and excreted (1.06 +/- 0.22 nmol/min) at a rate eightfold higher than excretion of FANFT. In kidneys perfused with equimolar ANFT, excretion of ANFT was 0.25 +/- 0.05 nmol/min, which suggests a coupling of renal deformylation of FANFT to excretion of ANFT in FANFT-perfused kidneys. Neither aspirin nor probenecid altered the urinary excretion or half-life of FANFT or ANFT. In rats fed 0.2% FANFT as part of their diet, coadministration of aspirin (0.5%) increased urinary excretion of ANFT during a 12-wk feeding study, which suggests decreased tissue binding or metabolism of ANFT. Kidney perfusion with acetylated ANFT (NFTA), a much less potent uroepithelial carcinogen, resulted in no ANFT excretion or accumulation, which indicates the specificity of renal deformylase. Renal deformylase activity was found in broken cell preparations of rat and human kidney. These data describe a unique renal metabolic/excretory coupling for these compounds that appears to explain the differential carcinogenic potential of the 5-nitrofurans tested. These results are consistent with the hypothesis that aspirin decreases activation of ANFT by inhibiting prostaglandin H synthase.

Modulation of the cyclic AMP content of rat renal inner medulla by oxygen: possible role of local prostaglandins.

The lower O2 tension and more active anerobic metabolism that pertain in the inner medulla (IM) of kidney relative to cortex (C) are well recognized, but there is no evidence that O2 availability constitutes a limiting or regulatory factor in IM metabolism or function. In the present in vitro study, we examined the effects of O2 on adenosine 3',5'-monophosphate (cAMP) metabolism in slices of rat renal C and IM. After a 20-min incubation of slices in Krebs Ringer bicarbonate buffer with 95% O2 + 5% CO2 serving as the gas phase, the cAMP content of IM was 6-10 fold higher than that of C in either the presence or absence of 2 mM 1-methyl-3-isobutylxanthine in the incubation media. In slices of IM incubated for 20 min with 1-methyl-3-isobutylxanthine, cAMP was 22.5+/-SE 2.48 pmol/mg wet weight at 95% O2 and 4.37 without O2. Oxygenation of O2-deprived IM increased cAMP twofold in 2 min, an effect fully expressed in 5 min (fivefold increase). Further, cAMP of IM rose progressively and significantly over a range of atmospheric O2 content from 0 to 50% conditions which should reproduce and encompass O2 tensions that pertain in tissues in vivo. By contrast, basal cAMP content of C varied less than twofold in the presence of 95% versus no O2, implying that O2 modulation of cAMP was specific for IM. Indomethacin and meclofenamate, structurally distinct inhibitors of prostaglandin synthesis, both significantly decreased basal cAMP accumulation in oxygenated slices of IM but not of C. Meclofenamate also reduced basal adenylate cyclase activity determined in homogenates prepared from slices of IM which had been incubated at 95% O2. In slices of IM previously exposed to indomethacin or meclofenamate at 95% O2, a maximally effective concentration of exogenous prostaglandin E1 restored cAMP and adenylate cyclase activity to levels which approximated those observed at 95% O2 in the absence of an inhibitor of prostaglandin synthesis. These results suggest that O2 enhancement of cAMP content in IM may be mediated at least in part by local prostaglandins.

Prostaglandin hydroperoxidase-mediated 2-amino-4-(5-nitro-2-furyl) [14C]thiazole metabolism and nucleic acid binding.

Prostaglandin hydroperoxide-mediated metabolism and binding of 2-amino-4-(5-nitro-2-furyl) [14C]thiazole ([14C]ANFT) metabolite to nucleic acids and proteins were investigated with rabbit bladder transitional epithelial and solubilized ram seminal vesicle microsomes. Metabolism was assessed by spectrophotometric and radiochemical techniques. Substrate and inhibitor studies are consistent with both metabolism and binding of [14C]ANFT occurring by the prostaglandin hydroperoxidase activity of prostaglandin endoperoxide synthetase. The ratio of the rates of [14C]ANFT product formation is approximately 3:7:10 (organic soluble:non-trichloroacetic acid precipitable: trichloroacetic acid precipitable) over a wide range of arachidonic acid concentrations. Approximately 2 and 1% of the total [14C]ANFT metabolized binds to transfer RNA and DNA, respectively. The metabolite isolated from the organic phase had a chromatographic profile and ultraviolet spectra different from authentic ANFT. If transfer RNA or DNA is added at the end of a 5-min incubation, no binding to nucleic acids was observed. The demonstration of prostaglandin hydroperoxidase-mediated covalent binding to nucleic acids is consistent with the involvement of this enzyme in 5-nitrofuran-induced bladder carcinogenesis.

Metabolism of N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide by prostaglandin endoperoxide synthetase.

Cooxidative metabolism of the urinary bladder carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) was examined using solubilized and particulate microsomal preparations from the rabbit renal inner medulla and the ram seminal vesicle. Metabolism was measured by the rate of decrease in absorbance at 400 nm. In these soluble and particulate preparations, FANFT metabolism was observed only in the presence of specific fatty acids. These fatty acids are substrates for prostaglandin endoperoxide synthetase. Structurally dissimilar inhibitors of prostaglandin endoperoxide synthetase such as indomethacin, aspirin, 5,8,11,14-eicosatetraynoic acid, ethoxyquin, and meclofenamic acid specifically inhibited FANFT metabolism. Other inhibitor and substrate specificity studies suggest that FANFT was not metabolized by nitroreductase, xanthine oxidase, lipoxygenase, lipid peroxidation, or mixed-function oxidases. In addition, the lack of detectable 2-amino-4-(5-nitro-2-furyl)thiazole formation suggests that arylformamidase was not participating in FANFT metabolism measured in these experiments. The data indicate that prostaglandin endoperoxide synthetase can mediate FANFT metabolism by a cooxidative process.

Peroxidase metabolism of the urinary bladder carcinogen 2-amino-4-(5-nitro-2-furyl)thiazole.

Metabolism of 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) by a variety of different peroxidases was examined. Metabolism of ANFT was measured by the binding of radiolabeled substrates to protein and DNA. Prostaglandin hydroperoxidase but not horseradish peroxidase, lactoperoxidase, or chloroperoxidase metabolically activated ANFT. All four peroxidases catalyzed the binding of benzidine to protein and DNA. With peroxide substrates, peroxidase-catalyzed binding of both carcinogens was observed with or without molecular oxygen. Arachidonic acid-dependent binding of ANFT and benzidine by prostaglandin endoperoxide synthetase was inhibited by anaerobic conditions and aspirin. Chloroperoxidase activation of benzidine was also inhibited by aspirin. Vitamin E inhibited activation of both carcinogens by all enzymes examined. Prostaglandin hydroperoxidase-catalyzed binding of benzidine to protein was inhibited by the 5-nitrofurans ANFT and 3-hydroxymethyl-1-(([3-(5-nitro-2-furyl)allydidene] amino))hydantoin and acetaminophen, while only acetaminophen inhibited horseradish peroxidase-catalyzed binding. These results indicate that different peroxidases may exhibit specificity with respect to their activation of carcinogens. Only prostaglandin hydroperoxidase activated the 5-nitrofuran ANFT, while a number of peroxidases activated the aromatic amine benzidine.

Transport of the renal carcinogen 3-hydroxymethyl-1-([3-(5-nitro-2-furyl)allydidene]amino) hydantoin by renal cortex and cooxidative metabolism by prostaglandin endoperoxide synthetase.

Transport of the renal carcinogen 3-hydroxymethyl-1-([3-(5-nitro-2-furyl)-allydidene]amino) hydantoin (HMN) by the renal cortex and metabolism by the kidney was evaluated. Organic acid and base transport by renal cortical slices was determined using [131I]Hippuran and [14C]tetraethylammonium, respectively. HMN caused a dose-dependent reversible inhibition of [131]Hippuran accumulation but did not alter [14C]tetraethylammonium uptake. By contrast, benzidine inhibited organic base but not acid transport. The decrease in absorbance at 405 nm was used as an index of microsomal metabolism of HMN. Reduced nicotinamide adenine dinucleotide phosphate-dependent metabolism of HMN was not observed with either cortical or medullary microsomes. However, there was prostaglandin endoperoxide synthetase-mediated metabolism of HMN. Specific substrate, cofactor, and inhibitor studies suggest that metabolism occurs by the prostaglandin hydroperoxidase activity of prostaglandin endoperoxide synthetase. At least one product of HMN metabolism was characterized and shown to be different from HMN by its high-pressure liquid chromatographic and ultraviolet spectral properties. The renal mixed-function oxidases system, lipid peroxidation, nitroreduction, and lipoxygenase did not seem to be involved in HMN metabolism. These results are consistent with the hypothesis that the kidney is a site for cooxidative metabolism of chemicals which elicit carcinogenic and nephrotoxic effects in the kidney. Facilitated transport of HMN into renal tissue by the organic acid transport system may explain the greater potential for HMN to elicit renal carcinogenesis compared to other tissues.

Benzidine binding to nucleic acids mediated by the peroxidative activity of prostaglandin endoperoxide synthetase.

The cooxidative metabolism of the urinary bladder carcinogen benzidine was examined using renal inner medullary microsomes. The products of [14C]benzidine metabolism were recovered in the aqueous but not in the organic soluble fraction of reacting mixtures. The reactive metabolites formed during cooxidative metabolism of benzidine bound to DNA and transfer RNA. Cooxidative metabolism of benzidine and subsequent binding to nucleic acids was dependent upon specific fatty acid substrates and was blocked by inhibitors of prostaglandin endoperoxide synthetase. The ratio of the rates of benzidine product formation was approximately 10:3:1 (trichloroacetic acid precipitable:non-trichloroacetic acid precipitable:transfer RNA bound) over a wide range of arachidonic acid concentrations. Cumene hydroperoxide also initiated cooxidative metabolism of benzidine but was less effective than was arachidonic acid. In contrast to arachidonic acid, cumene hydroperoxide-mediated metabolism of benzidine and fuaiacol peroxidase activity was not blocked by indomethacin. Using electron paramagnetic resonance, radicals were detected after addition of arachidonic acid or cumene hydroperoxide to the microsomal preparation. Radical production was completely quenched by addition of benzidine or guaiacol. These results demonstrate that the peroxidative activity of renal medullary prostaglandin endoperoxide synthetase mediates benzidine metabolism and subsequent binding to nucleic acids.

Eicosanoid synthesis by cultured human urothelial cells: potential role in bladder cancer.

Prostaglandin (PG) H synthase and eicosanoid products of arachidonic acid metabolism have been implicated in several steps in the carcinogenic process. This study assessed these parameters using primary cultures of human urothelial cells. To determine the possible presence of permeability barriers to agonist stimulation, incubations were performed with adherent cells in the presence or absence of thioglycolate pretreatment or with cell suspensions. No evidence for permeability barriers was observed. With adherent cells in the absence of thioglycolate, radioimmunoassayable PGE2 was stimulated by epinephrine less than 12-O-tetradecanoylphorbol-13-acetate = thrombin less than bradykinin = A23187 much less than arachidonic acid. Tumor promoters but not non-tumor promoters stimulated PGE2 synthesis. 1-Oleoyl-2-acetylglycerol which like 12-O-tetradecanoylphorbol-13-acetate activates protein kinase C also increased PGE2 synthesis. Cells prelabeled with [14C]arachidonic acid were exposed to agonists and the profile of eicosanoids synthesized was assessed by high performance liquid chromatography. With bradykinin, the pattern of eicosanoids synthesized was 6-keto-PGE1 alpha (12% of total 14C label), thromboxane B2 (0.4%), PGF2 alpha (1.7%), PGE2 (18%), PGD2 (1%), leukotrienes (0.4 to 1%), 12-hydroxy-5,8,10-heptadecatrienoic acid (3%), 15-hydroxy-5,8,11,13-eicosatetraenoic acid (4%), 12-hydroxy-5,8,10,14-eicosatetraenoic acid (0%) and 5-hydroxy-5,8,12,14-eicosatetraenoic acid (2%). Thus, human urothelial cells contain both prostaglandin H synthase and lipoxygenase pathways with the former being more prominent. These pathways may participate in urinary bladder carcinogenesis.

Renal reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase-mediated metabolism of the carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide.

N-[4-(5-Nitro-2-furyl)-2-thiazolyl]acetamide (NFTA) metabolism was examined in vitro using microsomes prepared from rat liver and renal cortex and from rabbit liver and renal cortex and outer and inner medulla. NFTA nitroreduction was observed with each tissue. Three mol of NADPH were used per mol of NFTA reduced. Substrate and inhibitor specificity suggested that the microsomal nitroreduction was due to NADPH:cytochrome c reductase. Metabolite(s) formed bound to protein, RNA, DNA, and synthetic polyribonucleotides. Maximum covalent binding was seen with polyguanylic acid. A guanosine-NFTA adduct was isolated. Binding was inhibited by sulfhydryl compounds and vitamin E. The [14C]NFTA:glutathione or [3H]glutathione:NFTA conjugates obtained from microsomal incubations showed identical chromatographic properties as the product obtained by the reaction of synthetic N-hydroxy-NFTA with [3H]glutathione. Structures of synthetic N-hydroxy-NFTA and the microsomal reduction product 1-[4-(2-acetylaminothiazolyl)]-3-cyano-1-propanone were established by mass spectrometry. The latter reduction product did not bind macromolecules. These results suggest that renal NADPH:cytochrome c reductase reduces NFTA to an N-hydroxy-NFTA intermediate that binds nucleophilic sites on macromolecules.

Effect of peroxidase inhibitors on an in vivo metabolite of the urinary bladder carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide in rats.

Peroxidase metabolism of 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) was evaluated in vitro and in vivo. In vitro metabolism of ANFT was characteristic of the hydroperoxidase activity of prostaglandin H synthase. The peroxidase inhibitors, 6-n-propyl-2-thiouracil and methimazole, significantly reduced ANFT binding to trichloroacetic acid precipitable material and glutathione conjugate formation. Isolated perfused kidneys rapidly converted the glutathione conjugate to its corresponding mercapturic acid (ANFT-MA). With both radiochemical and electrochemical techniques, ANFT-MA was identified in the urine of rats given N-[14C]-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide, the carcinogenic N-formyl analogue of ANFT. ANFT was the major urinary metabolite with N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide not detected. A 30-min pretreatment with 6-n-propyl-2-thiouracil and methimazole significantly reduced urinary excretion of ANFT-MA in rats given N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (150 mg/kg) from 14.8 +/- 2.1 (SE) to 7.9 +/- 0.8 and 6.2 +/- 1.1 nmol/18 h, respectively. Peroxidase inhibitor pretreatment did not alter the excretion of ANFT or prostaglandin E2. These results provide further in vitro and in vivo support for the involvement of peroxidases, i.e., the hydroperoxidase activity of prostaglandin H synthase, in ANFT metabolism.

Metabolic activation of carcinogenic aromatic amines by dog bladder and kidney prostaglandin H synthase.

Microsomal enzyme preparations from dog liver, kidney, and bladder were used to assess the prostaglandin H synthase-catalyzed activation of carcinogenic aromatic amines to bind covalently to proteins and nucleic acids. Benzidine, a urinary bladder carcinogen, bound to protein of bladder transitional epithelial and renal inner and outer medullary microsomes and was dependent upon addition of arachidonic acid, but not upon reduced nicotinamide adenine dinucleotide phosphate. Bladder transitional epithelial microsomes also activated o-dianisidine, 4-aminobiphenyl, and 2-naphthylamine to bind to protein and transfer RNA and benzidine and O-dianisidine to bind DNA. Cosubstrate and inhibitor specificities were consistent with activation by prostaglandin H synthase. Binding of benzidine to protein was not observed with either hepatic or renal cortical microsomes upon addition of arachidonic acid or reduced nicotinamide adenine dinucleotide phosphate. Prostaglandin H synthase and mixed-function oxidase-catalyzed bindings of 2-naphthylamine to protein and to transfer RNA were compared using liver and bladder microsomes. Only mixed-function oxidase-catalyzed binding was observed in liver, and only prostaglandin H synthase-catalyzed binding was observed in bladder. The rate of binding catalyzed by bladder microsomes was considerably greater than that catalyzed by hepatic microsomes. In addition, the bladder content of prostaglandin H synthase activity was approximately 10 times that of kidney inner medullary, a tissue reported to have a relatively high content of this enzyme in other species. These results are consistent with involvement of bladder transitional epithelial prostaglandin H synthase in the genesis of primary aromatic amine-induced bladder cancer.

Inhibition by aspirin of N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide initiation and sodium saccharin promotion of urinary bladder carcinogenesis in male F344 rats.

N-[4-(5-Nitro-2-furyl)-2-thiazolyl]formamide (FANFT) is metabolically activated by several enzyme systems, including prostaglandin H synthase. Aspirin is an inhibitor of prostaglandin H synthase and has been shown to inhibit FANFT-induced bladder carcinogenesis when coadministered in the diet. To further evaluate the effects of aspirin on bladder carcinogenesis in the rat, we have coadministered aspirin with FANFT during the initiation phase and with sodium saccharin during the promotion phase of carcinogenesis. FANFT was administered in the diet at a level of 0.2% for 6 weeks as the initiator and sodium saccharin was administered in the diet at a level of 5% for 61 weeks as promoting stimulus. Aspirin was administered at a level of 0.5% with FANFT or with sodium saccharin, and appropriate control groups were included. Weanling male Fischer 344 rats were utilized and the chemicals were added to Agway Prolab 3000 rat chow. A 1-week interval was included between the FANFT and sodium saccharin administration during which the rats received either aspirin containing diet or control chow, depending on the treatment regimen of the group. Thirty rats were included in each group at the beginning of the experiment, except for the control group which contained 40. Rats given FANFT followed by saccharin had a bladder carcinoma incidence of 83%. Rats given aspirin with FANFT but not with saccharin had a carcinoma incidence of 20% and the rats fed aspirin with the saccharin but not with the FANFT had an incidence of 28%. FANFT followed by control diet resulted in a bladder carcinoma incidence of 10%, as was true for the rats given FANFT plus aspirin followed by control diet. However, the hyperplastic effects induced in the bladder epithelium by saccharin without prior FANFT administration were inhibited by coadministration with aspirin. These results indicate that aspirin inhibits both FANFT initiation and sodium saccharin promotion of bladder carcinogenesis, but the mechanisms involved would most probably be different for each.

Mechanism of 12-O-tetradecanoylphorbol-13-acetate enhanced metabolism of arachidonic acid in dog urothelial cells.

The mechanism of 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced arachidonic acid metabolism was investigated in dog urothelial cells. Primary cultures of dog urothelial cells were grown to confluency and evaluated in the presence or absence of overnight prelabeling with [3H]arachidonic acid. High-performance liquid chromatography analysis of media from TPA stimulated cells indicated that prostaglandin E2 (PGE2) was the major eicosanoid produced. Lipoxygenase products were not detected. Control cell media contained only arachidonic acid. Effects of selected inhibitors on TPA and exogenous arachidonic acid mediated increases in radioimmunoassayable PGE2 were assessed. Prostaglandin H synthase inhibitors (indomethacin and aspirin) prevented both TPA and arachidonic acid increases in PGE2. By contrast, inhibitors of phospholipases (quinacrine, W-7, and trifluoropromazine), protein synthesis (cycloheximide), and protein kinase C (staurosporine) prevented TPA but not arachidonic acid increases in PGE2. The latter agents also reduced TPA mediated increases in the release of total radioactivity from cells labeled with [3H]arachidonic acid. However, aspirin reduced the amount of 3H-prostaglandins formed with TPA. A calcium requirement was demonstrated when increases in radioimmunoassayable PGE2 elicited by TPA and the calcium ionophore A23187 were reduced with calcium depleted media. When epidermal growth factor in combination with either TPA or bradykinin was used, at least additive effects were observed with respect to release of [3H]arachidonic acid, 3H-prostaglandins, and radioimmunoassayable PGE2. These experiments suggest that separate pathways may be involved in enhanced arachidonic acid metabolism demonstrated with different agonists. For TPA, increased arachidonic acid release occurs by a calcium dependent process involving phospholipase(s), protein synthesis, and protein kinase C.

Metabolism and disposition of bladder carcinogens in rat and guinea pig: possible mechanism of guinea pig resistance to bladder cancer.

The metabolism and disposition of N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) and 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) were studied in rat and guinea pig. Rat is susceptible whereas guinea pig is resistant to FANFT-induced bladder cancer. Rats and guinea pigs were p.o. administered either 2-[14C]ANFT or 2-[14C]FANFT (100 mg/kg), and 18-h urine and feces were collected. Tissue distribution of radiolabel was determined. In both species, the highest concentrations of radioactivity expressed as nmol/g tissue were observed in the urine and intestines. Urinary metabolites were separated by high-performance liquid chromatography and radioactivity determined by radioanalytical detection. FANFT was not detected in urine from either species under any experimental condition. More ANFT was observed in urine following FANFT than ANFT administration. This deformylation-dependent excretion of FANFT was demonstrated in both species and has been previously described as renal metabolic/excretory coupling. Less ANFT, the carcinogen more proximate than FANFT, is excreted in guinea pigs compared with rats. A unique ANFT metabolite was identified in guinea pig but not rat urine. This metabolite represented 80 and 18% of radioactivity recovered in guinea pig urine following ANFT and FANFT administration, respectively. A metabolite produced by guinea pig liver and kidney microsomes in the presence of uridine-5'-diphosphoglucuronic acid coeluted with this unique metabolite. The urinary metabolite was characterized using hydrolytic enzymes, acid hydrolysis, and mass spectrometry and identified as an ANFT-N-glucuronide. A unique UDP-glucuronosyl-transferase appears to be responsible, at least in part, for the reduced amount of free ANFT excreted by guinea pigs compared with rats. Reduced levels of urinary ANFT observed in guinea pigs may partially explain the resistance of this species to FANFT-induced bladder cancer.

Aspirin inhibition of N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide-induced lesions of the urinary bladder correlated with inhibition of metabolism by bladder prostaglandin endoperoxide synthetase.

The effects of aspirin on N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide (FANFT) -induced urinary bladder lesions, endogenous bladder prostaglandin E2 synthesis, and the metabolism of FANFT by bladder epithelial microsomes were examined. Rats were fed 0.5% aspirin and/or a diet containing 0.1% or 0.2% FANFT. Bladder lesions were observed with light and scanning electron microscopy, and the prostaglandin E2 content of rat bladder was measured by radioimmunoassay. Metabolism of FANFT was measured by decreased absorbance at 400 nm. Aspirin inhibited the appearance of hyperplastic lesions induced by feeding 0.1% or 0.2% FANFT for 6 or 12 weeks. Aspirin reduced bladder prostaglandin E2 content at 1, 2, 6, and 13 weeks compared to corresponding control values. Rat and rabbit microsomal metabolism of FANFT were dependent upon specific fatty acid substrate and prevented by specific inhibitors (including aspirin) of prostaglandin endoperoxide synthetase. Other inhibitor and substrate specificity studies suggest that FANFT was not metabolized by xanthine oxidase, lipoxygenase, lipid peroxidation, or mixed-function oxidases. These results suggest that the metabolism of FANFT by prostaglandin endoperoxide synthetase may be involved in the metabolic activation of FANFT necessary for the induction of bladder cancer in rats.

Human N-acetylation of benzidine: role of NAT1 and NAT2.

These studies were designed to assess metabolism of benzidine and N-acetylbenzidine by N-acetyltransferase (NAT) NAT1 and NAT2. Metabolism was assessed using human recombinant NAT1 and NAT2 and human liver slices. For benzidine and N-acetylbenzidine, Km and Vmax values were higher for NAT1 than for NAT2. The clearance ratios (NAT1/NAT2) for benzidine and N-acetylbenzidine were 54 and 535, respectively, suggesting that N-acetylbenzidine is a preferred substrate for NAT1. The much higher NAT1 and NAT2 Km values for N-acetylbenzidine (1380 +/- 90 and 471 +/- 23 microM, respectively) compared to benzidine (254 +/- 38 and 33.3 +/- 1.5 microM, respectively) appear to favor benzidine metabolism over N-acetylbenzidine for low exposures. Determination of these kinetic parameters over a 20-fold range of acetyl-CoA concentrations demonstrated that NAT1 and NAT2 catalyzed N-acetylation of benzidine by a binary ping-pong mechanism. In vitro enzymatic data were correlated to intact liver tissue metabolism using human liver slices. Samples incubated with either [3H]benzidine or [3H]N-acetylbenzidine had a similar ratio of N-acetylated benzidines (N-acetylbenzidine + N',N'-diacetylbenzidine/ benzidine) and produced amounts of N-acetylbenzidine > benzidine > N,N'-diacetylbenzidine. With [3H]benzidine, p-aminobenzoic acid, a NAT1-specific substrate, increased the amount of benzidine and decreased the amount of N-acetylbenzidine produced, resulting in a decreased ratio of acetylated products. This is consistent with benzidine being a NAT1 substrate. N-Acetylation of benzidine or N-acetylbenzidine by human liver slices did not correlate with the NAT2 genotype. However, a higher average acetylation ratio was observed in human liver slices possessing the NAT1*10 compared to the NAT1*4 allele. Thus, a combination of human recombinant NAT and liver slice experiments has demonstrated that benzidine and N-acetylbenzidine are both preferred substrates for NAT1. These results also suggest that NAT1 may exhibit a polymorphic expression in human liver.

Formation of adenosine 3',5'-monophosphate from adenosine in mouse thymocytes.

The effect of adenosine on the mouse thymocyte adenylate cyclase-adenosine 3':5'-monophosphate (cyclic AMP) system was examined. Adenosine, like prostaglandin E1, can cause 5-fold or greater increases in thymocyte cyclic AMP content in the presence but not in the absence of certain cyclic phosphodiesterase inhibitors. Two non-methylxanthine inhibitors potentiated the prostaglandin E1 and adenosine responses, while methylxanthines selectively inhibited the adenosine response. Adenosine increased cyclic AMP content significantly within 1 min and was maximal by 10 to 20 min with approx. 2 and 10 muM adenosine being minimal and half-maximal effective doses, respectively. Combinations of prostaglandin E1, isoproterenol and adenosine were near additive and not synergistic. Of the adenosine analogues tested, only 2-chloro- and 2-fluoroadenosine significantly increased cyclic AMP. Thymocytes prelabeled with [14C]adenine exhibited dramatic increases in cyclic [14C]AMP 10 min after addition of adenosine or prostaglandin E1 which corresponded to simultaneously determined increases in total cyclic AMP. Using [14C]adenosine, the percent of total cyclic AMP increase due to adenosine was only 16%. Adenosine was also shown to elicit a 40% increase in particulate thymocyte adenylate cyclase activity. Therefore, the increased content of cyclic AMP seen in mouse thymocytes after incubation with adenosine was due primarily to stimulation of adenylate cyclase and only partially to conversion of adenosine to cyclic AMP. The increased cellular content of cyclic AMP may be, in part, responsible for various immunosuppressive effects of adenosine.

Properties of cholera toxin- and NaF-stimulated adenylate cyclase from mouse thymocytes.

Kinetic parameters of mouse thymocyte adenylate cyclase activity were determined. NaF and cholera toxin stimulated adenylate cyclase. Stimulation by either agent did not change the pH or Mg2+ optima relative to control (unstimulated cyclase). The Km value for ATP of adenylate cyclase stimulated by NaF was significantly reduced from control. By contrast, cholera toxin treatment did not change the Km relative to control. Adenylate cyclase, when stimulated by NaF, had an optimum for Mn2+ alone, or Mn2+ in combination with Mg2+, at least twice that of control. In contrast, cyclase activity prepared from cells treated with cholera toxin remained unchanged with regard to these divalent cations when compared to control. Addition of NaF to adenylate cyclase prepared from cells treated with cholera toxin resulted in a significant reduction (30%) in activity suggesting that both NaF and cholera toxin were acting on the same cyclase. NaF inhibition of cholera toxin-stimulated activity was shown to be a direct interaction of fluoride on the stimulated cyclase enzyme. This inhibition appeared to be immediate and independent on pH, Mg2+ or ATP concentrations. Although NaF inhibition was lost when Mn2+ was present in the reaction mixture, the activity expressed by addition of NaF to cyclase prepared from cholera toxin-treated cells was much less than by addition of NaF to control. As observed with cholera toxin stimulation alone, activity expressed by the inhibited enzyme (cholera toxin treated + NaF) exhibited a Km for ATP and an optimum for Mn2+ alone or in combination with Mg2+ similar to control.

6-Ketoprostaglandin E1 stimulation of rat and rabbit renal adenylate cyclase-cyclic AMP systems.

6-Ketoprostaglandin E1 effects on rat and rabbit renal adenylate cyclase-cyclic AMP systems were examined. Adenylate cyclase activity was assessed in the 1000 X g fractions prepared from different areas of kidney. 6-Ketoprostaglandin E1 caused a dose-dependent increase in rat cortical and medullary adenylate cyclase activity with 8 x 10(-6) M being the lowest effective concentration. Combinations of maximal stimulatory concentrations of 6-ketoprostaglandin E1 and prostaglandin I2 caused stimulation similar to that seen with either agent alone. In contrast, the combination of either prostaglandin with parathyroid hormone (cortex) or antidiuretic hormone (medulla) resulted in enzyme activity significantly greater than with either agent alone. Similar results were observed in the rabbit. In addition, rabbit cortical and medullary slice cyclic AMP content was increased by 6-ketoprostaglandin E1. Maximal stimulatory effects of 6-ketoprostaglandin E1 on adenylate cyclase activity and cyclic AMP content were similar to prostaglandin I2. Therefore, the similarity in physiologic actions of 6-ketoprostaglandin E1 and prostaglandin I2 may be due to the stimulation of adenylate cyclase by both agents. These prostaglandins and the polypeptide hormones appear to activate different renal adenylate cyclase-cyclic AMP systems.