In vitro neoplastic transformation of epithelial cells of rat urinary bladder by nitrosamines

ORAL administration of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) selectively induces tumours in the urinary bladder of rats and mice1–3. This organ specificity can be explained to some extent by the metabolic fate of the compound and the carcinogenic effect of its major urinary metabolite on the bladder; Okada and Suzuki4 found that BBN given to a rat was soon excreted as its carboxy derivative, N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN). Subsequent tests for carcinogenicity of BCPN showed that it too caused bladder tumour in rats5,6. Furthermore, BBN can be converted to BCPN when incubated with bladder mucosa or liver tissue7. These findings suggest that BCPN is responsible for carcinogenesis of the bladder by BBN, or that both BCPN and BBN are directly carcinogenic to bladder epithelium. We have therefore studied the in vitro effect of both BCPN and BBN on epithelial cells of rat bladder. In preliminary experiments we had failed to induce growth of these cells, but we found that addition of urea to medium containing a nitrosamine induced growth. We have now established epithelial cell strains from normal cell by culturing them in the presence of BCPN plus urea or BBN plus urea and found that they grow as cancers in syngeneic animals.