Enhancement of N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide-induced carcinogenesis by urinary tract infection in rats

Epidemiological studies suggest that urinary tract infection is an important risk factor in the development of bladder cancer. Chronic urinary tract infection in rats is associated with urothelial hyperplasia and papillomatosis. In the Sprague-Dawley strain, exposure to the 5-nitrofuran, N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT), is associated in particular with the development of renal pelvic tumors. The present study was designed to evaluate whether chronic urinary tract infection could enhance tumor development in FANFT-induced urinary tract carcinogenesis. One hundred forty-four female Sprague-Dawley rats were divided into the following groups. Group 1 received 0.2% FANFT in the diet for 7 wk followed by control diet. Group 2 received 0.2% FANFT in the diet for 7 wk followed by control diet. One wk after completion of FANFT administration, the suspension of 0.5 ml of Escherichia coli (06K13H1) was injected into the bladder through the urethra. Group 3 received 0.2% FANFT in the diet for 7 wk followed by control diet. One wk after completion of FANFT administration, a suspension of heat-killed E. coli (06K13H1) was injected into the bladder through the urethra. Group 4 received a suspension of 0.5 ml of E. coli (06K13H1) through the urethra and received control diet throughout the experiment. Group 5 was fed control diet only. The experiment continued for 104 wk. A significantly higher number of urinary tract tumors, particularly of the renal pelvis, was recorded in Group 2 compared to Groups 1, 3, 4, and 5. The majority of the rats in Groups 2 and 4 had morphological signs of urinary tract infections, particularly pyelitis and/or pyelonephritis. Thus, a single injection of E. coli (06K13H1) into the bladder results in an enhancement of FANFT-induced urinary tract carcinogenesis in the Sprague-Dawley rat, especially for renal pelvic tumors. 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 the tumor development.     

Long term dose response study of N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide-induced urinary bladder carcinogenesis

The effect of dose was evaluated for the bladder carcinogenicity of N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide (FANFT). The chemical was fed to male weanling F344 rats for 30 weeks followed by 74 weeks of control diet. The incidences of bladder carcinoma were 100, 100, 87, 0, 0 and 0% for doses of 0.2, 0.1, 0.05, 0.01, 0.005 and 0.001%, respectively. There was an inverse relationship between dose and latency period. There was no increased incidence of tumors of other tissues.     

The influence of antipyrene on N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide-induced urinary tract carcinogenesis

Human over-use of analgesics containing phenacetin, antipyrene(phenazone) and caffeine has been associated with the developmentof both renal pelvic and bladder tumors. In Sprague-Dawley ratsantipyrene has been shown to be a weak complete urinary tractcarcinogen. The present study was designed to evaluate the promotingcapacity of antipyrene in N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide(FANFT)-induced urinary tract carcinogenesis. One hundred andeighty male Sprague-Dawley rats were divided into groups of30 and were treated with the following chemicals in the diet:group 1 received a control diet without chemicals; group 2 wastreated with 0.2% FANFT in the diet for five weeks followedby control diet; group 3 received 0.2% FANFT for five weeksfollowed by 0.535% antipyrene in the diet; group 4 was treatedwith 0.535% antipyrene; group 5 was treated with 0.102% caffeine;and group 6 was treated with 0.535% antipyrene and 0.102% caffeinein the diet. Ten of 27 rats in group 3(37%) developed urinarytract tumors (P> 0.001, five of which were renal pelvic tumorsand five were bladder tumors. The majority of the tumors werewell differentiated non-invasive urothelial carcinomas. Noneof the rats in other groups developed urinary tract tumors.In addition, renal papillary necrosis (RPN) was found in 33%of the rats in group 3, 50% in group 4, and 10% in group 6.The present study clearly shows that antipyrene acts as a promoterof FANFT-induced urinary tract carcinogenesis and that it isnephrotoxic to the renal papilla resulting in renal papillarynecrosis.     

Effect of aspirin on urinary bladder carcinogenesis initiated with N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide in rats

N-[4-(5-Nitro-2-furyl)-2-thiazolyl]formamide (FANFT), a potent urinary bladder carcinogen, is metabolically activated in vitro by a variety of enzyme systems including aerobic cooxidation by prostaglandin H synthase which is present in the rat bladder mucosa. In a previous experiment, aspirin coadministered with FANFT for 12 weeks inhibited FANFT-induced bladder carcinogenesis and enhanced forestomach carcinogenesis. To further evaluate the effects of aspirin on FANFT carcinogenesis, male F344 rats were fed either FANFT (0.2% of the diet) for 12 weeks (Group 4), aspirin (0.5% of the diet) simultaneously with FANFT for 12 weeks (Group 2), aspirin simultaneously with FANFT for 12 weeks and then subsequently to the end of the experiment (Group 1), or FANFT only followed by aspirin (Group 3). The incidence of bladder carcinoma was significantly higher when aspirin was fed after FANFT treatment (87%) compared to FANFT followed by control diet (48%) and was higher in rats given aspirin plus FANFT followed by aspirin (73%) compared to aspirin plus FANFT followed by control diet (47%). Aspirin alone given for 13 weeks (Group 6) or throughout the experiment (68 weeks) (Group 5) did not induce bladder cancer. However, in all groups administered aspirin long-term, renal papillary necrosis and renal pelvic hyperplasia and atypia were frequently observed. Only a single forestomach tumor was observed. In the present experiment, aspirin appeared to exhibit promoting activity for bladder carcinogenesis in the rat.     

The influence of urinary tract infection on the incidence of urinary tract tumors in N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide induced carcinogenesis in male Sprague-Dawley rats

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.     

Effect of dose on urinary bladder carcinogenesis induced in F344 rats by N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide.

Because of the utility of the N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) rat model in the study of bladder cancer, the effect of dose on FANFT-induced bladder carcinogenesis was evaluated. Weaning male F344 rats were given FANFT in the diet at doses of 0.1, 0.05, 0.01, 0.005, 0.001, and 0.0005% for 30 weeks and then a control diet for 22 weeks. A control group received only the control diet throughout the experiment. Papillary tumors were present at the higher doses, hyperplasia of various degrees of severly was present at the intermediate doses, and minimal hyperplasia was observed in 4 of 16 rats at the 0.005% dose; no mucosal abnormalities were observed at the two lower doses or in the control group. Bladder epithelium from selected animals was also examined by scanning electron microscopy (SEM) after 10 weeks and again at the end of the experiment. Hyperplastic mucosa with pleomorphic microvilli similar to that previously demonstrated for 0.2% FANFT was observed at 10 weeks in rats fed 0.1% FANFT. Hyperplastic mucosa with pleomorphic microvilli was also observed at 52 weeks in rats fed 0.1% and 0.05% FANFT. Hyperplastic mucosa without pleomorphic microvilli was observed in rats fed 0.01 and 0.005% FANFT. The bladder appeared normal by light microscopy and SEM at the two lower doses and in the control group at both the 10- and 52-week intervals. A dose relationship was thus demonstrated for FANFT-induced bladder carcinogenesis in male F344 rats, and more severe surface changes were observed by SEM as the dose increased.     

Potentiation of 1-2-dimethylhydrazine-induced bowel carcinogenesis by the urinary bladder carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide

Male inbred 10–12 week old Wistar/Furth rats receivedeither no carcinogen, or 1-2-dimethylhydrazine (DMH) 20 mg/kgbody weight s.c. once weekly for 16 weeks, or N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide(FANFT) 0.2% of the feed for 16 weeks, or DMH and FANFT concurrently.Thirty-three weeks after carcinogen exposure, all survivingtreated and control animals were killed and examined for boweland urinary bladder tumors. Adenocarcinomas of the large andsmall bowel occurred in approximately 33% of DMH-treated animals,and transitional cell carcinomas of the urinary bladder in approximately33% of the FANFT-treated animals. After concurrent exposureto both carcinogens, no increased incidence of bladder tumorswas noted compared to FANFT treatment alone. However, the numberof animals with one or more adenocarcinomas of the bowel (22/30versus 17/50, p<0.001), the mean number of tumors per animal(2.1 ± 0.2 versus 1.1 ± 0.1, p <0.01), andthe invasiveness of the tumors through the bowel wall were allsignificantly increased after DMH + FANFT compared to DMH exposurealone.     

Co-carcinogenicity of sodium saccharin and N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide for the urinary bladder

The co-carcinogenic activity of sodium saccharin and N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide(FANFT) was evaluated in male Fischer rats by co-administeringthem as 5% and 0.005% of the diet, respectively, for 2 years.The effect of simultaneous administration of two urinary bladderpromoting substances, sodium saccharin and L-tryptophan as 5%and 2% of the diet, respectively, was also evaluated. Five of16 rats administered sodium saccharin plus FANFT developed bladdertumors whereas none of the rats administered FANFT, sodium saccharin,or L-tryptophan alone, sodium saccharin plus L-tryptophan, orthe control diet developed bladder tumors. Possible mechanismsfor the co-carcinogenic activity of sodium saccharin and FANFTare discussed.     

Production of urinary tract tumors by co-administration of uracil and N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide in F344 rats

Co-administration of uracil and N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) to weanling female Fischer rats produced uracil stones in the bladder and significantly reduced the incidence of bladder tumors. Contrary to bladder tumors, the incidence of renal pelvic and ureteric tumors was increased by this regimen. Feeding of uracil alone produced bladder tumors, in addition to the hyperplasia of renal pelvis, ureter and bladder. The mechanism of uracil's effect on FANFT carcinogenesis is not known.     

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.     

Effect of aspirin on N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide-induced epithelial proliferation in the urinary bladder and forestomach of the rat

The co-administration of aspirin with N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide (FANFT) to rats resulted in a reduced incidence of FANFT-induced bladder carcinomas but a concomitant induction of forestomach tumors. An autoradiographic study was performed on male F-344 rats fed diet containing FANFT at a level of 0.2% and/or aspirin at a level of 0.5% to evaluate the effect of aspirin on the increased cell proliferation induced by FANFT in the forestomach and bladder. FANFT-induced cell proliferation in the bladder was significantly suppressed by aspirin co-administration after 4 weeks but not after 12 weeks. In the forestomach, and also in the liver, aspirin did not affect the FANFT-induced increase in labeling index. The present results are consistent with the carcinogenicity experiment suggesting that different mechanisms are involved in FANFT carcinogenesis in the bladder and forestomach, and that aspirin's effect on FANFT in the forestomach is not due to an irritant effect associated with increased cell proliferation. Also, there appears to be an adaptation by the rats to the chronic ingestion of aspirin.     

Effect of dose on the induction of urothelial proliferation by N-(4-(5-nitro-2-furyl)-2-thiazolyl)formamide and its relationship to bladder carcinogenesis in the rat

The effect on urothelial proliferation of a urinary bladdercarcinogen, N-(4-(5-nitro-2-furyl)-2-thiazolyl)formamide (FANFT),fed to male F344 rats at doses of 0.2, 0.1, 0.05, 0.01, 0.005and 0.001% of diet for 4 or 10 weeks was evaluated by autoradiography,using (³H-methyl)thymidine, and by histopathology. At week 4,hyperplasia was induced in 10/11 and 6/11 rats given 0.2% and0.1% FANFT, respectively. The dose-related increase of labelingindex in the bladder epithelium was significant for the groupsgiven 0.01% or higher doses of FANFT. At week 10, histopathologiclesions were observed in groups given 0.05% or higher dosesof FANFT. This was accompanied by a significant increase inlabeling index for these groups. The results are consistentwith the long-term carcinogenicity studies conducted with thesame dose levels of FANFT. The interrelationships between numbersof cells (hyperplasia), cell proliferation (labeling index)and cancer induction are discussed utilizing a computerizedmodel of bladder carcinogenesis.     

Biotransformation of the bladder carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide in mice

The biotransformation of N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT), a potent urinary bladder carcinogen, was studied in mice. About 82% of radioactivity was excreted as 14CO2 within 36 hr after intragastric administration of N-[4-(5-nitro-2-furyl)-2-thiazolyl]-[14C]formamide, suggesting its deformylation to 2-amino-4-(5-nitro-2-furyl)thiazole ( ANFT ). The latter was formed in vitro as a product following incubation of FANFT with mouse liver homogenates. Chromatographic analysis of mouse urine obtained 24 hr after the i.p. administration of N-[4-(5-nitro-2-furyl)-[2-14C]thiazolyl]formamide revealed excretion of ANFT and unmetabolized FANFT, suggesting the prevalence of the deformylation reaction in vivo. In addition, at least two more metabolites were present in urine. One of these metabolites exhibited chromatographic properties similar to those exhibited by a compound derived from the in vitro nitroreduction of ANFT . This metabolite was isolated from urine of FANFT-fed animals and from in vitro enzymatic reduction of ANFT with mouse liver homogenates. The isolated products had chromatographic and spectral properties and a mass spectral fragmentation pattern similar to that of a compound obtained by catalytic reduction of ANFT with palladium and activated carbon. Spectroscopic analyses established the structural identity of the chemical reduction product as 1-[4-(2-aminothiazolyl)]-3-cyano-1-propanone ( ATCP ). Since the chromatographic properties of the enzymatically derived product and the urinary metabolite were identical to those of a compound obtained by chemical reduction, they must be structurally the same and thus correspond to ATCP . About 5% of the urinary metabolites of FANFT is ATCP , and thus ATCP is quantitatively a minor excretory product. ATCP was far less active than was ANFT of FANFT in the Ames mutagenicity assay with Salmonella typhimurium TA.     

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.