Samuel M. Cohen - US grants

A reliable, highly reproducible model of bladder cancer in Fischer rats using the carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide (FANFT) was developed. The natural history and the reversibility or irreversibility of early lesions were determined. It was also demonstrated that the carcinogenic process could be divided into 2-stages with properties similar to the classical murine skin carcinogenesis model of initiation and promotion. FANFT fed for 6 weeks was used as initiator and sodium saccharin and DL-tryptophan demonstrated promoting properties in this model. We propose to evaluate this model with regard to other criteria for initiation and promotion in urinary bladder carcinogenesis including irreversibility of initiation, reversal of order of administration of initiator and promoter, and length of time that promotion administration is required. The possible co-carcinogenicity of sodium saccharin and L-tryptophan will be evaluated. Mechanisms involved in initiation and promotion will be evaluated. Induction of bladder epithelial proliferation appears to be a prerequisite for initiation. Moreover, increasing the level of cell proliferation enhances the effectiveness of a given dose of initiator. Attempts will be made using increased proliferation to develop a model requiring a single dose of orally administered initiator. The anti-promoting activity of 13-cis-retinoic acid will be evaluated. A marker of abnormal cell proliferation in this model is the appearance of pleomorphic microvilli on cell surfaces as detected by scanning electron microscopy. Development of a short term assay for evaluating chemicals for activity toward the bladder will be attempted using pleomorphic microvilli as a marker. Identification of other markers, particularly histochemical assays for use in evaluating the carcinogenic and differentiation process in the bladder, will be attempted.

Cigarette smoking has been identified as a major etiologic factor for urinary bladder cancer in the United States and is estimated to account for approximately 50% of all cases. Although several carcinogens, co-carcinogens, and promoters have been identified in cigarette smoke, which of these are relevant to the development of human bladder cancer is unknown. Acrolein is present in relatively large quantities in cigarette smoke (10-140 Mug/cigarette) and is also an economically important intermediate in the chemical industry, where process workers often have an increased incidence of bladder cancer. Cyclophosphamide, a chemotherapeutic alkylating agent used in the treatment of neoplastic and autoimmune diseases, has as a side-effect the induction of hemorrhagic cystitis and eventually carcinoma of the bladder. Data from human and animal studies have identified acrolein (a metabolite of cyclophosphamide) as the agent responsible for the hemorrhagic and the carcinogenic effects of cyclophosphamide on the bladder. We propose to investigate the interactions of acrolein on the bladder epithelium in vitro and in vivo. Short-term assays will be performed utilizing scanning electron microscopy and autoradiography to determine a dose response for the effects on acrolein on urothelial cytotoxicity and consequent regenerative proliferation. Based on the short-term studies, long-term bioassays will be performed evaluating the possible carcinogenic, initiating, and promoting activity of acrolein for the bladder. To develop an understanding of the molecular events which underlie such changes in morphology, extensive biochemical investigations are also proposed. Acrolein will be reacted with deoxynucleotides in vitro and the products identified. These will be used for the qualitative and quantitative determination of adducts after reaction of acrolein with DNA in vitro and after exposure of cultured cells to acrolein. Ultimately, these studies will be extended to the detection and quantitation of adducts formed in the bladder epithelium of the rat following acrolein administration. The miscoding potential of acrolein adducts in DNA will also be evaluated. In addition, the amounts of acrolein in biological fluids, particularly urine, will be determined by methods that will be developed. Ultimately, these analytical procedures should be useful for examination on humans exposed to acrolein.

A reliable, highly reproducible model of bladder cancer in Fischer rats using the carcinogen N-[4-(5-nitro-2-furyl)-2-thiazolyl] formamide (FANFT) was developed. The natural history and the reversibility or irreversibility of early lesions were determined. It was also demonstrated that the carcinogenic process could be divided into 2-stages with properties similar to the classical murine skin carcinogenesis model of initiation and promotion. FANFT fed for 6 weeks was used as initiator and sodium saccharin and DL-tryptophan demonstrated promoting properties in this model. We propose to evaluate this model with regard to other criteria for initiation and promotion in urinary bladder carcinogenesis including irreversibility of initiation, reversal of order of administration of initiator and promoter, and length of time that promotion administration is required. The possible co-carcinogenicity of sodium saccharin and L-tryptophan will be evaluated. Mechanisms involved in initiation and promotion will be evaluated. Induction of bladder epithelial proliferation appears to be a prerequisite for initiation. Moreover, increasing the level of cell proliferation enhances the effectiveness of a given dose of initiator. Attempts will be made using increased proliferation to develop a model requiring a single dose of orally administered initiator. The anti-promoting activity of 13-cis-retinoic acid will be evaluated. A marker of abnormal cell proliferation in this model is the appearance of pleomorphic microvilli on cell surfaces as detected by scanning electron microscopy. Development of a short term assay for evaluating chemicals for activity toward the bladder will be attempted using pleomorphic microvilli as a marker. Identification of other markers, particularly histochemical assays for use in evaluating the carcinogenic and differentiation process in the bladder, will be attempted.

Human urinary bladder cancer is associated with exposure to chemical carcinogens, and several experimental rodent models are available which closely mimic the disease. These models involve administration of genotoxic chemicals, such as N-[4-5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) or N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), or non-genotoxic chemicals, such as uracil or sodium saccharin (NaSac). Increased cell proliferation of the urothelium is a major factor which affects the tumor dose-response of genotoxic chemicals and has a primary role in nongenotoxic chemical carcinogenesis. The ultimate consequence of either group of chemicals is generation of errors in specific genes (oncogenes and suppressor genes) which lead to the development of cancer. Mutations in the ras family of genes have been demonstrated following FANFT treatment, and more recently we have demonstrated that mutations occur in the gene for p53. We plan to further evaluate mutations in p53 during different stages of bladder carcinogenesis, comparing the changes during administration of genotoxic (FANFT and BBN) or nongenotoxic (uracil and NaSac) carcinogens. It has been hypothesized that tumors arising secondary to treatment with nongenotoxic agents more frequently involve mutations in CpG islands than is the case for tumors arising from treatment with genotoxic agents, even though ultimately, the same genes are affected. The spectrum of mutations will be compared in the above models. NaSac is a nongenotoxic carcinogen in rats, with males more susceptible than females, but bladder cancer occurs only if NaSac is administered beginning at birth and continuing for the life of the animal. Increased cell proliferation is a key factor by which NaSac acts, and this appears to occur secondary to toxicity caused by the formation of silicate- containing precipitate and/or crystals following treatment with high doses of NaSac. The role of these silicates in saccharin carcinogenesis will be investigated including the role of dose, comparison with other sodium salts and their related acids, other classes of bladder tumor promoters, and comparison of rat strains and other species. The mechanism by which these precipitates and/or crystals form in the urine and the multiple factors by which influence their formation will be studied. By testing this hypothesis further, a more rational basis for extrapolation of the findings in rats to a possible risk for humans can be made.