ABSTRACT
Enhanced c-erbB-2/neu expression has been linked with a poor prognosis in human bladder cancer. Previous reports have shown that a point mutation at nucleotide T2012 in the coding region of the transmembrane domain of the rat gene is sufficient to confer transformation potential on this gene. We examined the comparative levels of p185neu as well as the sequence around the hotspot (T2012) of the neu gene of rat bladder cells transformed by 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) or established in culture from N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT)-induced rat bladder tumors. We concluded that increased p185neu expression did not correlate significantly with tumorigenicity. No alterations in nucleotide sequences of the neu gene were observed in either in vitro model.
Subject(s)
ErbB Receptors/genetics , Proto-Oncogene Proteins/genetics , Urinary Bladder Neoplasms/genetics , Animals , Base Sequence , Blotting, Southern , Carcinogens , Cell Transformation, Neoplastic , Cells, Cultured , FANFT/analogs & derivatives , Gene Expression , Molecular Sequence Data , Rats , Receptor, ErbB-2 , Urinary Bladder/metabolism , Urinary Bladder Neoplasms/chemically inducedABSTRACT
Enhanced c-erbB-2/neu expression has been linked with a poor prognosis in human bladder cancer. Previous reports have shown that a point mutation at nucleotide T2012 in the coding region of the transmembrane domain of the rat gene is sufficient to confer transformation potential on this gene. We examined the comparative levels of p185neu as well as the sequence around the hotspot (T2012) of the neu gene of rat bladder cells transformed by 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) or established in culture from N-[-4-(-5-nitro-2-furyl)-2- thiazolyl]formamide (FANFT)-induced rat bladder tumors. We concluded that increased p185neu expression did not correlate significantly with tumorigenicity. No alterations in nucleotide sequences of the neu gene were observed in either in vitro model.
Subject(s)
Carcinoma, Transitional Cell/chemically induced , Carcinoma, Transitional Cell/genetics , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Neoplastic/genetics , ErbB Receptors/genetics , ErbB Receptors/physiology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/physiology , Urinary Bladder Neoplasms/chemically induced , Urinary Bladder Neoplasms/genetics , 3T3 Cells , Animals , Base Sequence , Carcinoma, Transitional Cell/metabolism , Cells, Cultured , Epithelium/drug effects , Epithelium/pathology , Epithelium/physiology , ErbB Receptors/analysis , FANFT/analogs & derivatives , Gene Expression , Immunohistochemistry , Mice , Molecular Sequence Data , Mutation , Proto-Oncogene Proteins/analysis , Rats , Rats, Inbred F344 , Receptor, ErbB-2 , Urinary Bladder/drug effects , Urinary Bladder/pathology , Urinary Bladder/physiology , Urinary Bladder Neoplasms/metabolismABSTRACT
Phenylbutazone, a nonsteroidal anti-inflammatory drug, elicits therapeutic as well as toxic effects by unknown pathways. Phenylbutazone was shown to form a conjugate with the heterocyclic amine bladder carcinogen 2-amino-4-(5-nitro-2-furyl)-thiazole (ANFT). To understand further the reactivity of these compounds, this study was conducted to identify the conjugate formed and determine the mechanism of conjugate formation. Both prostaglandin H synthase and horseradish peroxidase catalyzed conjugate formation. This conjugate was identified by 1H-NMR to be 4-[2-amino-4-(5-nitro-2-furyl)-5-thiazolyl]-4-butyl-1,2-diphenyl-3,5- pyrazolidinedione. Phenylbutazone-mediated oxygen uptake was inhibited by ANFT (0.1 mM) and the spin traps 5,5-dimethyl-1-pyrroline-N-oxide (200 mM) and tert-nitrosobutane (4 mM). By contrast, phenol (0.005 to 0.25 mM) and aminopyrine (0.4 mM) stimulated oxygen uptake. None of these agents mediated oxygen uptake in the absence of phenylbutazone. Conjugate formation was significantly increased by phenol (0.005-0.25 mM) and aminopyrine (0.4 mM), as well as in the absence of oxygen. Conjugate formation was inhibited by 5,5-dimethyl-1-pyrroline-N-oxide (200 mM), tert-nitrosobutane (4 mM), ascorbic acid (2 mM), and 95% oxygen. Horseradish peroxidase initiated conjugate formation at much lower concentrations than it metabolized ANFT. The stoichiometric relationship between phenylbutazone and ANFT, with respect to conjugate formation, was complex. With the concentration of ANFT fixed at 0.05 mM, phenylbutazone exhibited saturation kinetics with a Km of 0.2 mM. In contrast, saturation kinetics were not observed with ANFT.Km values for ANFT varied with the concentration of phenylbutazone used.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Carcinogens/metabolism , FANFT/analogs & derivatives , Phenylbutazone/metabolism , Biotransformation , Carcinogens/pharmacokinetics , Chromatography, High Pressure Liquid , FANFT/metabolism , FANFT/pharmacokinetics , Horseradish Peroxidase/metabolism , Kinetics , Peroxides/metabolism , Protein BindingABSTRACT
1. Oxidative metabolism of the bladder carcinogens FANFT/ANFT was examined in vitro in guinea pig (resistant species) relative to rat (susceptible species). 2. The total rate of ANFT hepatic metabolism by guinea pig (soluble metabolites plus protein bound, 354 pmol/min per mg protein) was approx. 4 times that in rat. 3. The total rate of FANFT metabolism was similar in both species and approx. one-quarter that for ANFT in guinea pig. In rat, the rate of total metabolism of FANFT and ANFT was similar. 4. Cytochrome P450 inhibitors, 2,4-dichloro-6-phenylphenoxyethylamine, 7,8-benzoflavone, and n-octylamine largely inhibited metabolism in guinea pig, but had little effect in rat. 5. H.p.l.c. analysis of ANFT metabolites indicated distinctly different products in guinea pig compared to rat. 7,8-Benzoflavone decreased metabolite formation by 80% in guinea pig, but only 30% in rat. 6. Flavin-dependent monooxygenases may participate in metabolism of these carcinogens in rat, but not guinea pig. 7. Because ANFT is thought to be a more proximate carcinogen than FANFT, the increased rate of ANFT metabolism and the formation of different products in guinea pig compared to rat may partially explain the resistance of guinea pig to FANFT-induced bladder cancer.
Subject(s)
Carcinogens/metabolism , FANFT/analogs & derivatives , FANFT/metabolism , Liver/metabolism , NADP/metabolism , Animals , DNA/metabolism , Enzyme Inhibitors/pharmacology , Guinea Pigs , Kidney/metabolism , Liver/enzymology , Male , Microsomes, Liver/enzymology , Organ Specificity , Oxidation-Reduction , Protein Binding/drug effects , Rats , Rats, Inbred F344 , Urinary Bladder Neoplasms/chemically inducedABSTRACT
The formation of thioether conjugates is an important pathway for inactivation of certain carcinogens. This study assessed the mechanism by which the bladder carcinogen 2-amino-4-(5-nitro-2-furyl)-thiazole (ANFT) forms a glutathione conjugate (ANFT-SG). Peroxidatic metabolism of ANFT, in the presence of glutathione, results in ANFT-SG formation. Both prostaglandin H synthase and horseradish peroxidase can catalyze this reaction. Metabolism of the reducing co-substrates ANFT, phenol, and aminopyrine elicit increases in oxidized glutathione (GSSG). ANFT-SG formation is potentiated by phenol and aminopyrine. tert-Nitrosobutane (tNB), a thiyl radical trap, prevented increases in both GSSG and ANFT-SG. Increasing concentrations of ANFT elicited corresponding increases in both GSSG and ANFT-SG. Peroxidatic metabolism of ANFT in the presence of glutathione, but not in the absence of glutathione, resulted in oxygen uptake. The formation of GSSG and oxygen uptake are consistent with the presence of thiyl radicals during ANFT metabolism. 5,5-Dimethyl-1-pyrroline N-oxide, a thiyl radical trap, was not as effective as tNB in inhibiting the formation of ANFT-SG and GSSG. Ascorbic acid, a reducing cosubstrate and antioxidant, was very effective in preventing ANFT-SG and GSSG formation, while the strong nucleophile methionine was ineffective. To clarify effects of different test agents, their effects on aminopyrine cation radical formation were assessed. Results are consistent with ANFT reacting with thiyl radicals to form ANFT-SG. ANFT appears to be a thiyl radical trap. Peroxidatic metabolism of ANFT probably results in the formation of a cation radical rather than a carbon-centered radical.
Subject(s)
Carcinogens/chemistry , FANFT/analogs & derivatives , Sulfides/chemistry , Urinary Bladder Neoplasms/chemically induced , Carcinogens/toxicity , FANFT/chemistry , FANFT/toxicity , Free Radicals , Glutathione/metabolism , Horseradish Peroxidase/metabolism , Oxygen/metabolism , Prostaglandin-Endoperoxide Synthases/metabolismABSTRACT
N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) administration to rats followed by sodium saccharin results in transitional cell carcinomas of the bladder, of which 24% harbor an activated H-ras gene. Since 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) is the mutagenic and carcinogenic metabolite of FANFT in vivo, we wished to examine ras activation in in vitro ANFT-transformed rat bladder epithelial cells as well as four cell lines established in culture from in vivo FANFT-induced rat bladder tumors. Screening by Western blotting revealed no enhanced levels of p21ras in ANFT-transformed cells nor in cells established in culture from FANFT-induced rat bladder carcinomas. Further investigations using immunohistochemical staining with a different pan-reactive p21 monoclonal antibody (Cetus Corporation) specific for this method, however, showed two groups of cells from FANFT-induced rat bladder tumors had enhanced immunoreactivity. Apart from this, p21ras expression of most of the cells groups varied little from the controls. We examined the reported hot spots (exons 1 and 2) of each of the ras genes (H-, K- and N-ras) by direct sequencing of amplified DNA. No mutations were present. We conclude, therefore, that ANFT transformation of primary rat bladder epithelial cells in vitro may not in this case be mediated by ras activation, although this is difficult to determine since others have observed that optimal culture conditions can select for certain populations of cells without ras activation.
Subject(s)
Carcinogens/toxicity , Cell Transformation, Neoplastic , FANFT/analogs & derivatives , FANFT/toxicity , Genes, ras , Urinary Bladder Neoplasms/genetics , Animals , Base Sequence , Blotting, Western , Cell Transformation, Neoplastic/genetics , Cells, Cultured , DNA , Epithelial Cells , Immunohistochemistry , Molecular Sequence Data , Oligonucleotides , Polymerase Chain Reaction , Rats , Urinary Bladder/cytology , Urinary Bladder Neoplasms/chemically inducedABSTRACT
To establish a rat urinary bladder carcinogenesis model in vitro, primary rat bladder epithelial cells were grown in media containing 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT), the water-soluble metabolite of N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide (FANFT), for 4 weeks followed by long-term (4-7 months) exposure to control medium, sodium saccharin (NaS), or urea. Another set of cultures were exposed to ANFT, NaS and urea simultaneously. Several phenotypic changes were observed in the chemically exposed cell cultures, namely differences in cell morphology, increased growth rate and the ability to grow on plastic instead of rat-tail collagen support. All of the chemically exposed cultures were anchorage independent except one of those treated with NaS. The ANFT-treated cells followed by control medium or urea and cells treated with ANFT, NaS and urea were tumorigenic when transplanted to nude mice, whereas NaS or ANFT followed by NaS treatment were not. The tumors were carcinomas and their epithelial differentiation was verified by strong positive staining for cytokeratin. These studies demonstrate the urothelial transforming capability of ANFT in cell culture without the necessity for a long exposure to a secondary chemical.
Subject(s)
Carcinogens/toxicity , Cell Transformation, Neoplastic/chemically induced , FANFT/analogs & derivatives , Urinary Bladder Neoplasms/chemically induced , Urinary Bladder/drug effects , Animals , Cells, Cultured , Drug Synergism , Epithelial Cells , Epithelium/drug effects , FANFT/toxicity , Female , Male , Mice , Mice, Inbred Strains , Mice, Nude , Neoplasm Transplantation , Rats , Rats, Inbred F344 , Saccharin/toxicity , Urea/toxicity , Urinary Bladder/cytologyABSTRACT
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.
Subject(s)
Carcinogens/metabolism , FANFT/analogs & derivatives , FANFT/metabolism , Urinary Bladder Neoplasms/chemically induced , Animals , Carbon Radioisotopes , Disease Susceptibility , FANFT/pharmacokinetics , Glucuronosyltransferase/metabolism , Guinea Pigs , Male , Mass Spectrometry , Rats , Rats, Inbred F344 , Species Specificity , Tissue DistributionABSTRACT
The mechanism of activation of the bladder carcinogen 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT) was investigated by comparison with benzidine. In comparison with benzidine, ANFT has a higher electrochemical potential (approximately 700 mV) and is less effective as a reducing co-substrate for either prostaglandin H synthase (PHS) or horseradish peroxidase. Activation was monitored by measuring binding to protein (BSA) and DNA. ANFT binding to protein was reduced by indomethacin, a fatty acid cyclooxygenase inhibitor; phenol and aminopyrine, competitive reducing co-substrates; ascorbic acid, an antioxidant; and glutathione, thioether conjugate formation. These results are consistent with those previously reported for benzidine and demonstrate a peroxide co-substrate requirement, interaction of peroxidase with amine, formation of reactive intermediates and inactivation of reactive intermediates. 5,5-Dimethyl-1-pyrroline N-oxide (DMPO), a radical trap, also reduced ANFT binding to protein. Similar results were observed whether activation by PHS or horseradish peroxidase was investigated. Peroxidative activation of ANFT and benzidine to bind DNA was inhibited by these test agents in a manner similar to that observed with protein except that DMPO did not reduce binding. In addition, 2-methyl-2-nitrosopropane and methyl viologen, which are radical traps, and methionine and p-nitrobenzyl-pyridine, which are strong nucleophiles, did not reduce ANFT or benzidine binding to DNA. These agents also did not prevent binding of benzidinediimine, the two-electron product of benzidine oxidation, to polydeoxyguanosine. Glutathione inhibited diimine binding by forming a conjugate. Results demonstrate that activation of ANFT to bind protein and DNA is similar to benzidine. Peroxidative activation of benzidine occurs by both one- and two-electron oxidation. A similar mechanism would explain ANFT binding to protein (one electron) and DNA (two electron).
Subject(s)
Benzidines/metabolism , FANFT/analogs & derivatives , Alkenes/metabolism , DNA/metabolism , Electrochemistry , FANFT/metabolism , Horseradish Peroxidase/metabolism , Oxidation-Reduction , Peroxides/metabolism , Prostaglandin-Endoperoxide Synthases/metabolismABSTRACT
An in vivo urinary metabolite of the bladder carcinogen 2-amino-4-(5-nitro-2-furyl) thiazole was isolated from guinea pig urine and was identified by direct analysis using thermospray mass spectrometry/high-performance liquid chromatography as 1-(2-amino-4-(5-nitro-2-furyl)-2-thiazolyl)-1-deoxy-beta-D-glucopyran uronic acid. The structure of this metabolite was also established by chemical synthesis. Both positive and negative ion thermospray mass spectrometry of the conjugate showed fragment ions resulting from cleavage across the pyran ring of the glucuronic acid comprising of aglycone moiety. These characteristic fragment ions may be diagnostic for identification of N-glucuronides from O-glucuronides.
Subject(s)
Carcinogens , FANFT/analogs & derivatives , Glucuronates/urine , Urinary Bladder Neoplasms/chemically induced , Animals , FANFT/chemistry , FANFT/metabolism , Gas Chromatography-Mass Spectrometry/methods , Glucuronates/chemistry , Guinea Pigs , Male , Rats , Rats, Inbred F344ABSTRACT
Aromatic and heterocyclic amines are environmental chemicals which can cause bladder cancer in man. Because these chemicals cause carcinomas at a site distal to their portals of entry, metabolic processes are involved in initiation of their carcinogenic effects. N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) and its deformylated analogue, ANFT, were used as model compounds to assess metabolism. Electrochemical properties of ANFT made liquid chromatography with electrochemical detection a specific and sensitive method for analysis. Peroxidatic metabolism of ANFT by prostaglandin H synthase (PHS) in the presence of N-acetylcysteine resulted in the formation of 2-amino-4-(5-nitro-2-furyl)-5-(N-acetylcystein-S-yl)thiazole (ANFT-MA). This thioether product has an oxidation potential significantly lower than ANFT. Rat urinary excretion of ANFT-MA was significantly decreased with peroxidase inhibitors, 6-n-propyl-2-thiouracil and methimazole. Inhibitors did not alter excretion of ANFT or prostaglandin E2, a PHS product of arachidonic acid metabolism. 1H and 13C-NMR were selected to explore potential structural differences between ANFT and FANFT which might explain preferential PHS metabolism of ANFT. Evidence for a "zwitterion" configuration for ANFT but not FANFT was observed. ANFT in the "zwitterion" configuration would be a better reducing co-substrate. Chemical synthesis and GC-MS fragmentation patterns identified 3-(2,3-dihydro-1-methyl-2-pyrrolyl)pyridine as a peroxidatic product of nicotine metabolism. This peroxidatic product was found in urine from a cigarette smoker in an amount approximately 6% that observed for continine. Thus, a potential rôle for peroxidative metabolism was demonstrated in man.
Subject(s)
Carcinogens/metabolism , FANFT/analogs & derivatives , FANFT/metabolism , Urinary Bladder Neoplasms/chemically induced , Animals , Chromatography, High Pressure Liquid , Electrochemistry , Prostaglandin-Endoperoxide Synthases/pharmacology , RatsABSTRACT
The electron ionization mass spectra of 2-amino-4-(5-nitro-2-furyl)thiazole metabolites obtained from microsomal incubations and chemical syntheses were studied. The identities of the metabolites were established by chemical ionization, high resolution, and metastable measurements. The compounds studied showed multiple modes of cleavage, skeletal rearrangements and hydrogen back-transfer.
Subject(s)
Carcinogens/analysis , FANFT/analysis , Thiazoles/analysis , Animals , Carcinogens/metabolism , Chromatography, High Pressure Liquid , FANFT/analogs & derivatives , FANFT/metabolism , Gas Chromatography-Mass Spectrometry , In Vitro Techniques , Male , Mass Spectrometry , Microsomes/metabolism , Oxidation-Reduction , SheepABSTRACT
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.
Subject(s)
Carcinogens/metabolism , FANFT/metabolism , Methimazole/pharmacology , Peroxidases/antagonists & inhibitors , Propylthiouracil/pharmacology , Thiazoles/metabolism , Acetylcysteine/metabolism , Animals , FANFT/analogs & derivatives , Glutathione/metabolism , Male , Prostaglandin-Endoperoxide Synthases/physiology , Rats , Rats, Inbred F344 , Urinary Bladder Neoplasms/chemically inducedABSTRACT
Renal metabolic/excretory coupling is the enhancement of urinary excretion dependent upon renal metabolism. The nitrofurothiazoles, N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) and 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT), are model compounds used to study metabolic/excretory coupling. FANFT is deformylated to ANFT by renal deformylase enhancing ANFT excretion. In the rat, ANFT excretion after oral FANFT administration was 100-fold greater than ANFT excretion when ANFT was administered. FANFT and ANFT uptake into purified proximal tubules achieved equilibrium within 60 s and was demonstrated in nonviable tubules. FANFT partitioned into oil better than ANFT. Albumin inhibited FANFT and ANFT uptake into oil and decreased tubular uptake by 65%. Tubular FANFT uptake was threefold or greater than that of ANFT uptake with or without albumin. Renal deformylase was predominantly cytosolic and yielded apparent Km and Vmax of 6.7 microM and 6.1 nmol ANFT.min-1.mg protein-1, respectively. Deformylase activity was abolished by boiling, was specific for N-formylated compound, and was not altered by dinitrophenol treatment. Renal metabolic/excretory coupling for FANFT/ANFT combines energy-independent uptake with metabolism (deformylation), resulting in enhanced urinary ANFT excretion.
Subject(s)
Kidney/metabolism , Animals , Biological Availability , Chromatography, High Pressure Liquid , FANFT/analogs & derivatives , FANFT/pharmacokinetics , Kidney Tubules, Proximal/metabolism , Metabolic Clearance Rate , Rabbits , Rats , Rats, Inbred Strains , p-Aminohippuric Acid/metabolismABSTRACT
Epidemiological studies have demonstrated an association between urinary tract infection and the development of bladder cancer. The present study aimed at evaluating the influence of urinary tract infection in male Sprague-Dawley rats exposed to a sub-carcinogenic dose of N-[4-(nitro-2-furyl)-thiazolyl]formamide (FANFT). A single injection of Escherichia coli into the bladder resulted in a persistent upper urinary tract infection in a high percentage of the rats. Thirty-two percent of the rats exposed to FANFT and E. coli infection developed urinary tract tumors, all but one occurring in the renal pelvis. Urinary tract tumors were not found in rats treated with FANFT or E. coli alone. The present results support that inflammation resulting from infection is actively involved in urinary tract tumorigenesis and may support the epidemiological studies showing an association between infection and human urinary tract cancer. The formation of dimethylnitrosamine or other nitroso compounds from nitrates in the urine or increased cell proliferation due to chronic inflammation or both may be important pathogenetic factors in tumor development.
Subject(s)
Escherichia coli Infections/pathology , FANFT , Thiazoles , Urinary Tract Infections/pathology , Urologic Neoplasms/chemically induced , Animals , Cocarcinogenesis , Disease Susceptibility , FANFT/analogs & derivatives , Hyperplasia/pathology , Inflammation/pathology , Kidney Pelvis/pathology , Male , Rats , Rats, Inbred StrainsABSTRACT
Nitrofurothiazoles such as 2-amino-4-(5-nitro-2-furyl) thiazole (ANFT) and N-formylated ANFT (FANFT) are model compounds used in the study of chemical carcinogenesis. FANFT is a more potent uroepithelial carcinogen than ANFT but previous studies have shown extensive deformylation of FANFT to ANFT in vivo and ANFT to be the putative proximate carcinogen. To investigate this paradox, disposition of radiolabeled FANFT and ANFT was determined in rats prepared for clearance experiments. After 2 hr, 27.3 +/- 2.1% of recovered ANFT was excreted in urine compared to 44.2 +/- 1.8% of FANFT (P less than .001). In addition, approximately 20% of both FANFT and ANFT were excreted in bile after 2 hr. The disposition of nonradiolabeled FANFT and ANFT was also determined. The urinary excretion rate for ANFT with i.v. ANFT administration was 0.9 +/- 0.1 nmol/min. Following i.v. FANFT administration, the urinary excretion rate for ANFT was 49.7 +/- 8.6 nmol/min (P less than .001). The elimination half-lives were 23 +/- 3 and less than 5 min, for ANFT and FANFT, respectively. The differences in renal handling of ANFT and FANFT could not be accounted for by differences in protein binding. Large differences were found in urinary metabolite excretion between FANFT and ANFT administration. These results demonstrate deformylation dependent excretion (renal metabolic/excretory coupling) exists for FANFT resulting in much higher concentrations of ANFT reaching the urinary tract than when ANFT only is administered. Biliary excretion accounts for significant early clearance of both ANFT and FANFT. Renal metabolic/excretory coupling may explain the difference in uroepithelial carcinogenicity between FANFT and ANFT.
Subject(s)
FANFT/metabolism , Thiazoles/metabolism , Urinary Bladder Neoplasms/chemically induced , Animals , FANFT/analogs & derivatives , Glomerular Filtration Rate , Metabolic Clearance Rate , Protein Binding , Rats , Rats, Inbred Strains , Serum Albumin/metabolism , Tissue DistributionABSTRACT
The chemical synthesis of 4-(5-amino-2-furyl)thiazole (AFT) and its formation during the in vitro reductive metabolism of 4-(5-nitro-2-furyl)thiazole (NFT) by rat liver tissues on anaerobic incubation with NADPH were examined. AFT was synthesized by catalytic reduction of NFT with 5% palladium on activated carbon. Purified AFT, a pale yellow powder, melted at 105 degrees C and had an extinction coefficient of 16.3 mM-1 cm-1 at 297 nm in methanol. The proton n.m.r. spectrum, i.r. and mass spectra were consistent with the assigned structure. Analysis of the ethyl acetate extract, following incubation of NFT with rat liver tissue preparations, revealed a metabolite whose chromatographic and mass spectral characteristics were the same as those obtained with synthetic AFT, thus establishing the structural identity of the metabolite as AFT. These data show that AFT is formed on reduction and could act as a precursor for the formation of 1-(4-thiazolyl)-3-cyano-1-propanone as postulated earlier.
Subject(s)
FANFT/metabolism , Thiazoles/analysis , Thiazoles/metabolism , Animals , Chromatography, High Pressure Liquid , FANFT/analogs & derivatives , Magnetic Resonance Spectroscopy , Mass Spectrometry , Microsomes, Liver/metabolism , NADP/metabolism , RatsABSTRACT
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.