Acrolein initiates rat urinary bladder carcinogenesis.

Acrolein, a reactive, alpha,beta-unsaturated aldehyde which is ubiquitous in the environment, forms DNA adducts, is mutagenic, and is teratogenic. However, studies have not indicated a carcinogenic effect in rodent bioassays. Since it is present in cigarette smoke and is the toxic metabolite of cyclophosphamide with respect to the urinary tract, we investigated the possibility that acrolein might have carcinogenic activity toward the rat urinary bladder. We also evaluated whether it possessed initiating and/or promoting activity. To evaluate initiating activity, acrolein was administered at a dose of 2 mg/kg i.p. twice a week for 6 weeks followed by uracil as 3% of the diet for 20 weeks and then control diet for 6 weeks. N-[4-(5-Nitro-2-furyl)-2-thiazolyl]formamide (FANFT) as 0.2% of the diet followed by uracil was used as a positive control, and a negative control group was administered solvent control (water) i.p. during the 6-week initiation period followed by uracil. Acrolein followed by uracil produced an incidence of 18 of 30 rats (60%) with papilloma compared to 8 of 30 rats (27%) treated with solvent control followed by uracil. FANFT followed by uracil produced an incidence of 70% carcinomas and 30% papillomas, clearly indicating that it is a much more potent initiating agent than acrolein. Acrolein for 6 weeks followed by control diet produced no tumors. To evaluate promoting activity, groups of rats were fed FANFT for 6 weeks followed by acrolein. Acrolein administered during the initial 6 weeks and continued for the second phase of the experiment (to evaluate complete carcinogenic activity) resulted in severe toxicity. Administration of acrolein had to be terminated after 21 weeks of the experiment. The animals were maintained for 53 weeks of the experiment without further chemical treatment, and there was no evidence of papilloma or carcinoma development. This study clearly indicates that acrolein has initiating activity for the urinary bladder when administered by i.p. injection to the male F344 rat, but toxicity precluded evaluation of its promoting or complete carcinogenic activity.

Enhanced immunoreactivity of ras oncogene p21 protein in urinary bladder epithelium of rats treated with N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide.

Normal urothelium and various lesions of the rat urinary bladder induced by the dietary administration of 0.2% N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) (up to 77 weeks) or by the combination of 0.2% FANFT and the subsequent administration of 5% sodium saccharin or 2% DL-tryptophan (up to 104 weeks) were evaluated for immunoreactivity with monoclonal antibody to ras p21 by avidin-biotin immunohistochemistry. Seventy-one to 100% of transitional cell carcinomas showed strong reactivity to the antibody to ras p21 depending on treatment with long-term administration of FANFT or by 6 weeks administration of FANFT followed by sodium saccharin or DL-tryptophan. Focal reactivity to the ras p21 antibody was frequently observed in the hyperplastic (57-96%) or normal appearing urinary bladder epithelium (50-100%) in rats treated with FANFT (FANFT alone or in combination with sodium saccharin or tryptophan) but not in hyperplasia or normal epithelium in rats given sodium saccharin or tryptophan alone, without pretreatment with FANFT or in untreated controls. The present results show that there is a close association of enhanced immunoreactivity with ras p21 antibody in the urinary bladder epithelium to FANFT treatment, and that ras p21 is expressed in normal, hyperplastic and neoplastic lesions of the bladder of rats treated with FANFT. These results suggest that enhanced immunoreactivity with ras p21 is observed as a consequence of the treatment with FANFT but it alone does not reflect the progression from benign to malignant lesions.

Oncogene expression of FANFT- or BBN-induced rat urothelial cells.

Proto-oncogene expression by cultured urothelial cells prepared from the bladders of male F344 rats that had been treated with N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide (FANFT) or N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) were examined. Although all of the cultured cells showed varying degrees of anchorage-independent growth, only 9 of them were transplantable into nude mice. A Northern blot technique was employed for the detection of proto-oncogene transcripts. The c-Ha-ras transcripts were detected in all the cultured urothelial cells prepared from the carcinogen-treated rats and in normal urothelial cells. However, the transcript levels were several-fold higher in the former than in normal cells. Increased expression of p21, as determined by immunohistochemical techniques, was also observed in all the original bladder tissues from which the cultures were derived. c-myc transcripts were detected in the cells from carcinogen-treated rats but not in the normal cells. The presence of myc product in hyperplastic urothelial lesions and carcinomas of original bladder tissues was confirmed by immunohistochemical methods. Transcripts of mos, erb B, Ki-ras, abl and src were not detected. Since increased expression of c-myc and c-Ha-ras were present in both transplantable and non-transplantable cell lines, and the expression of p21 occurs in preneoplastic cells, this suggests that elevated expression of these 2 genes may be an early genetic event during bladder carcinogenesis in the rat and further alteration of these 2 genes or mutation of additional genes may be required for the completion of malignant transformation.

Inhibition by aspirin of N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide-induced bladder carcinogenesis and enhancement of forestomach carcinogenesis.

N-[4-(5-Nitro-2-furyl)-2-thiazolyl]formamide (FANFT) is a potent urinary bladder carcinogen in the rat, and it can be metabolically activated in vitro by a variety of enzyme systems including aerobic cooxidation by prostaglandin H synthase. The latter enzyme is present in the rat bladder mucosa and can be inhibited by the oral administration of aspirin (ASA). To determine if ASA could inhibit the bladder carcinogenicity of FANFT, FANFT (0.2%) was co-administered in the diet with ASA (0.5%) for 12 weeks followed by 1 week of ASA only and then 56 weeks on control diet. 0.2% FANFT followed by control diet induced bladder carcinomas in 18 of 21 (87%) rats, but when ASA was co-administered, only 10 of 27 (37%) rats developed bladder carcinoma (p less than 0.001). However, forestomach tumors, not seen in rats fed only FANFT, developed in 7 rats fed FANFT plus ASA. No tumors occurred in control rats or those fed only ASA. Possible mechanisms, including the role of prostaglandin H synthase in FANFT metabolism, are discussed.

The relationship of histamine H2 receptor-bearing suppressor cells with the growth and metastasis of FANFT-induced bladder cancer.

A poorly differentiated transitional cell carcinoma in C3H/He mice results from the oral ingestion of the urinary tract carcinogen FANFT. This model, designated MBT2, is readily transplantable into syngeneic animals and has proven to be very useful in the development of chemotherapy. Prior to the use of this model for the testing of potential immunotherapeutic strategies, we have attempted to characterize the immunobiology of this tumor line. We report that the primary growth of this tumor in the footpad and its metastasis to lung are correlated with the development of increased numbers of suppressor cells, characterized by the expression of a surface histamine H2 receptor. These cells are originally evident in spleen and become maximal approximately 4 weeks after tumor implantation. This is followed by the migration of these cells from spleen to peripheral blood, an event that parallels the growth and eventual metastasis of this implanted transitional cell carcinoma. These events may have important significance for the development of immunomodulating therapy against bladder cancer.

Multistage carcinogenesis in the urinary bladder.

The induction of cancer of the urinary bladder is a multi-stage process involving multiple exogenous and endogenous factors. Based on the classical initiation-promotion model, we have used N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) as initiator and sodium saccharin (SAC) or tryptophan as promoters. These latter chemicals have the properties expected of promoters: induction of hyperplasia, reversibility and nonmutagenicity. Also, tumors were induced whether the promoter was administered immediately after FANFT or beginning 6 weeks after FANFT was discontinued, but no tumors resulted if either promoter was given without initiation with FANFT. Factor(s) present in normal urine also are involved in the promotion process, in addition to the role of urine as a carrier of carcinogens. However, administration of SAC to animals with a rapidly proliferating bladder mucosa, induced by ulceration, pellet insertion, or in utero, resulted in bladder tumor induction, even without prior initiation with FANFT. To better understand the complex interaction of the multiple variables in bladder carcinogenesis, a stochastic computer model has been formulated based on long-term carcinogenicity and tissue kinetic studies in vivo. This model indicates the importance of cell proliferation and the development of hyperplasia in carcinogenesis.

In vitro characterization of four N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide (FANFT) induced mouse bladder tumors.

Four longterm murine bladder tumor cell lines were established in vitro. The 4 lines were initiated from primary N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide (FANFT) induced murine bladder tumors arising in C3H/He mice. Each was maintained as a solid tumor in syngeneic mice for at least 30 generations before initiation in tissue culture. The cell lines MBT-2, MBT-8, MBT-409 and MBT-683, have been subcultured over 75 times in vitro for 18 months. They are all epithelial, grow in islands on plastic Petri dishes before confluent growth and form colonies in soft agar suspension culture. Morphologic studies indicate that all 4 lines have epithelial characteristics and karyotypic studies indicate that all lines have polyploidy and marker chromosomes. Population doubling times range from 10 to 26 hours and are consistent for each line.

Correlation between the half-wave reduction potentials of nitroheterocycles and their mutagenicity in Chinese hamster V79 spheroids.

Toxicity and DNA damage by nitroheterocycles has previously been correlated with their redox potentials. Resistance to 6-thioguanine was measured using Chinese hamster V79 cells grown in suspension culture as three-dimensional cell clusters of "spheroids". Since diffusion gradients of oxygen and other nutrients are largely responsible for the growth properties of spheroids, cells grown as spheroids might better simulate cells exposed to mutagens in vivo. The log of the concentration inducing 10 mutants/plate or 1.6 X 10(5) clonogenic cells from spheroids (equivalent to about 300 rad), was correlated with the half-wave reduction potential of a series of nitroheterocycles. FANFT and 4NQO were more mutagenic than predicted on the basis of redox potential.

Genetic analysis of ad-3 mutants induced by AF-2 and two other nitrofurans in Neurospora crassa.

Our previous studies have shown that the nitrofurans AF-2, SQ18506, and FANFT are potent mutagens in Neurospora crassa. The genetic damage produced by these chemicals at the ad-3 region in N crassa has been characterized by a series of genetic tests. The results of these tests indicate that all three agents induce a high frequency of point mutations and probably a low frequency of multilocus deletions. A comparison of the complementation patterns among the AF-2--induced ad-3B mutants and those induced by other chemical agents indicates that the spectra of intragenic alterations induced by AF-2 in N crassa are similar to those induced by monofunctional alkylating agents.

Early induction of mouse urinary bladder ornithine decarboxylase activity by rodent vesical carcinogens.

The responses of mouse urinary bladder ornithine decarboxylase (EC 4.1.1.17) and S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50) activities were studied following topical intravesical administration of N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide (FANFT) or 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT), potent rodent bladder carcinogens. A single bladder topical application of ANFT or FANFT resulted in a significant increase over controls of ornithine decarboxylase activity within 5 hr, with a return to control levels by 10 hr. S-Adenosyl-L-methionine decarboxylase activity demonstrated a lesser response to topical ANFT or FANFT, achieving a level 2 or 3 times that of controls at 5 to 8 hr, followed by a gradual decline to control levels. Stimulation of activities of both enzymes was dose dependent over a range of 4.6 to 460 nmol of ANFT. ANFT-induced ornithine decarboxylase activity was principally localized in the bladder epithelium and was inhibited in a linear dose-response relationship by the synthetic retinoid, 13-cis-retinoic acid. Mice given FANFT p.o. demonstrated a significant increase over controls in ornithine decarboxylase activity within 12 hr, followed by a gradual decline to control levels by 72 hr.

Galactosyl transferase activity in rat bladder transitional cell carcinoma lines and in exfoliated cells in urine during carcinogenesis and reversible hyperplasia.

Cultured cells of rat bladder transitional cell carcinoma line AY-27, in suspension, were assayed for galactosyl transferase (GT) by measurement of the transfer of [3H]galactose from uridine diphosphate-[3H]galactose to desialylated ovine submaxillary mucin (OSM-NANA). The assay was optimized with respect to time and to protein, uridine diphosphate galactose, OSM-NANA and Triton X-100 concentrations. This assay was then applied weekly to suspensions of exfoliated bladder cells collected from urines of rats fed the bladder carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide, and of control rats. Increases in activity over controls appeared 42 weeks after feeding the carcinogen, at a stage when bladder tumors were already microinvasive or deeply invasive, and activities at 52 weeks were about 10-fold greater than normal values. In contrast, a bladder cytotoxic agent inducing reversible hyperplasia was injected into rats, and exfoliated cells were collected from urines: these cells showed no greater GT activity than normal. Bladder tumor tissue from a transplanted tumor had the same high specific enzymatic GT activity as exfoliated cells from tumor-bearing rats.

Topography and numerical densities of intramembrane particles in chemical carcinogen-induced urinary bladder carcinomas in Fischer rats.

Integral membrane proteins are visualized as intramembrane particles (IMP) at the cleaved surfaces of freeze-fractured plasma membranes. Topographical distributions of the IMP of the urinary bladder epithelial cells membranes in normal Fischer rat bladder and noninvasive and invasive N-(4-(5-nitro-2-furyl)-2-thiazolyl)formamide (FANFT)-induced bladder tumors are shown to be significantly different. Using several statistical methods that test IMP topography vis-a-vis the Poisson (random) hypothesis, it is demonstrated that IMP are mathematically randomly distributed in the large majority of plasma membranes of cells in normal rat bladder epithelium and in invasive N-(4-(5-nitro-2-furyl)-2-thiazolyl)formamide tumors. In noninvasive rat bladder carcinomas, IMP are in a lattice-like arrangement in half of the tumor cells and randomly distributed in the remainder. IMP numerical densities are also altered in the course of neoplastic transformation. IMP are equally increased above control values in both noninvasive and invasive N-(4-(5-nitro-2-furyl)-2-thiazolyl)formamide tumors. Although transformation into noninvasive tumors is associated with increased numbers of IMP, there is no evidence that this parameter is specifically related to tumor biologic behavior in this model system.

The influence of N-[4-(5-nitro-2-Furyl)-2-thiazolyl]formamide and phenacetin on the immune status in male Fischer rats.

The effect of N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANTF) and phenacetin on the immune status of Fischer rats was determined utilizing the spleen plaque and phytohemagglutinin blastogenesis assays 6, 10, and 15 weeks after administration at doses of 0.2 per cent and 0.535 per cent, respectively. Azathioprine (0.02 per cent of the diet) was tested as a known immunosuppressive chemical for comparison, and a negative control group, fed a control diet without added chemicals, was also tested. Immunosuppression was shown in the rats receiving azathioprine at all times by both assays, whereas in FANFT-fed rats immunosuppression was greater than in the control group (not statistically significant). Results from rats fed phenacetin were not significantly different than those of the control group. These results show that immunosuppression does not occur in rats administered phenacetin for up to 15 weeks, nor in FANFT-induced bladder carcinogenesis at the time of reversible bladder mucosal hyperplasia (6 weeks), or when the mucosal lesions have become irreversible, non-invasive carcinomas (10 and 15 weeks).

Uridine 5'-diphosphate galactose: glycoprotein galactosyl transferase activity in exfoliated bladder epithelial cells in rats fed N-(4-(5-nitro-2-furyl)-2-thiazolyl) formamide.

Urine samples of normal male Fischer rats or rats fed 0.2% N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide for 6,8 or 30 weeks were collected and centrifuged 50 weeks after beginning treatment. After being sonicated and assayed (with purified desialylated ovine submaxillary mucin as acceptor glycoprotein), the exfoliated bladder cells obtained from the urines of treated rats showed uridine 5'-diphosphate galactose:glycoprotein transferase activity. The specific enzymatic activity of the enzyme from cells of 30-week-treated rats was about 10 times higher than from normal rats. The enzyme from cells of hyperplastic rats (treated 6 or 8 weeks) was only slightly higher in specific activity than that of normal rats. A similar was obtained at a later stage of bladder tumor induction, when the urines from 30-week-treated rats contained blood. A correction was made for protein contributed by the blood clot. The possibility that the blood clot contributed galactosyl transferase activity was excluded. Activity of the enzyme was detected in normal rat bladder tissue and in normal human urine.

Experimental bladder tumor induction, propagation, and therapy.

The development of animal bladder tumor models as a research tool for different modes of therapy has been widely evaluated. Recently these tumors have either spontaneously grown or have been propagated in inbred strains. Bladder tumors have also been chemically produced by N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide (FANFT) when orally administered over a long period of time. It has been further reported that these tumors have been inhibited by various chemotherapy regimens. The availability of an experimental bladder tumor model offers an opportunity to evaluate the effectiveness of a prescribed treatment. In our studies FANFT was noted to produce only from 33 to 40% bladder tumors in several experiments in an inbred strain of rats conducted over several years. Reproducible transplantability of these tumors was not demonstrable in the same inbred strain. In addition, treatment with mitomycin C an an effective chemotherapeutic agent was not detectable in part, since comparably the percentage of control bladder tumor growth was low. These findings of a three-year study should be carefully considered when evaluating recommendations for clinical adjuvant chemotherapy based on results obtained with FANFT.