Comparative carcinogenicity of N-butyl-N-(3-carboxypropyl)-nitrosamine and N-butyl-N-(4-hydroxybutyl)nitrosamine for the urinary bladder of (C57BL/6 X DBA/2)F1 mice.

The carcinogenicity of N-butyl-N-(3-carboxypropyl)-nitrosamine [CAS: 38252-74-3; 4-(N-butyl-N-nitrosamino)butyric acid] in male and female (C57BL/6 X DBA/2)F1 mice was determined. N-Butyl-N-(3-carboxypropyl)nitrosamine given in the drinking water at a concentration of 3 mM (0.056%) for 13 weeks induced only carcinoma of the urinary bladder in both sexes. At 22-28 weeks, the incidences of bladder cancer in the male and female mice were 100 and 88%, respectively. These bladder tumors were classified histologically according to the frequency (%) of tumor type: pure transitional cell carcinoma, 42%; mixed (transitional cell carcinoma with squamous or glandular differentiation, or both), 28%; squamous cell carcinoma, 27%; and carcinoma in situ, 3%. No significant sex differences were observed. In comparative studies, the incidence of bladder cancer was 100% for both sexes after administration of 3 mM (0.052%) N-butyl-N-(4-hydroxybutyl)nitrosamine [CAS: 3817-11-6; 4-(butylnitrosoamino)-1-butanol] in the drinking water. The frequency of pure transitional cell carcinoma was 47%, which was not significantly different from that observed for the carboxypropyl compound. The frequencies of other types of bladder carcinoma induced by N-butyl-N-(4-hydroxybutyl)nitrosamine were the following: mixed, 8%; squamous cell carcinoma, 42%; and carcinoma in situ, 3%.

Biochemically detectable tumor markers in urine of bladder cancer patients.

Potential biochemical markers excreted in the urine of bladder cancer patients have been considered, with the conclusion that none alone has yet proven to be useful as a screening procedure for the detection of urothelial cancer. Quantitative fluctuations in urinary levels of several of these markers in combination, such as pseudouridine, beta-amino-isobutyric acid, and fibrinogen degradation products, appear to be valuable in the assessment of the treatment of bladder cancer patients and in helping to predict recurrences in these patients.

Comparative toxicity of N-hydroxy-2-acetylaminofluorene in several strains of rats.

Male Spraque-Dawley rats were 6 or 7 times more susceptible than females to the acute toxic effects of a single i.p. injection of N-hydroxy-2-acetylaminofluorene. The N-hydroxy compound were equally toxic in male and female Fischer rats and about twice as toxic to male as to female Wistar rats. A negative correlation between the 50% lethal dose of N-hydroxy-2-acetylaminofluorene and hepatic N-hydroxy-2-acetylaminofluorene sulfotransferase activity was found. These data substantiate earlier indications that the level of the liver sulfotransferase is an important factor in determining the degree of toxicity of N-hydroxy-2-acetylaminofluorene. It is suggested that the reported sex difference in the hepatocarcinogenicity of N-hydroxy-2-acetylaminofluorene might be peculiar to the Sprague-Dawley rat.

Influence of the aryl group on the reaction of glucuronides of N-arylacethydroxamic acids with polynucleotides.

The reactions of the glucuronide conjugates of the carcinogens N-hydroxy-2-acetylaminofluorene (N-hydroxy-AAF), N-hydroxy-4-acetylaminostilbene (N-hydroxy-AAS), N-hydroxy-4-acetylaminobiphenyl (N-hydroxy-AABP), and N-hydroxy-2-acetylaminophenanthrene (N-hydroxy-AAP) with transfer RNA, ribosomal RNA, DNA, polyadenylate, polyguanylate, polyuridylate, polycytidylate, poly(adenylate, guanylate), and poly(guanylate, uridylate) were studied. The relative order of reactivity of these glucuronides with nucleic acids, measured by the covalent binding of the aryl group labeled with 3H or 14C, was glucuronide of N-hydroxy-AAF greater than glucuronide of N-hydroxy-AAS greater than glucuronide of N-hydroxy-AABP greater than glucuronide of N-hydroxy-AAP. The glucuronide of N-hydroxy-AAP showed only marginal or negligible reactivity. The glucuronide of N-hydroxy-AAF showed greater reactivity with polyguanylate than with polyadenylate, but the reverse was true for the glucuronide of N-hydroxy-AAS. Both of these glucuronides had much lower extents of reaction with polyuridylate and polycytidylate. Except for the reaction of the glucuronide of N-hydroxy-AABP with polyadenylate, there was no detectable reaction of this glucuronide or the glucuronide of N-hydroxy-AAP with the homopolynucleotides. Under comparable conditions the glucuronide conjugates of N-hydroxy-AAF, N-hydroxy-AAS, and N-hydroxy-AABP demonstrated greater reactivity with poly(adenylate, guanylate) and poly(guanylate, uridylate) than with the homopolynucleotides. Furthermore, the synthesis of two new glucuronide conjugates, those of N-hydroxy-AAS and N-hydroxy-AAP, is described.

Effect of N-methyl-N-nitrosourea on the DNA of rat bladder epithelium.

Bladder cancer can be induced in the rat by the intravesicular administration of N-methyl-N-nitrosourea. DNA damage in rat bladder epithelial cells after administration of methylnitrosourea has been examined by measuring the change in sedimentation of the DNA in alkaline sucrose gradients. A dose response of DNA damage in the urothelium was observed with single intravesicular doses of 0.1, 0.3, and 0.5 mg of methylnitrosourea. Larger doses of methylnitrosourea damaged the epithelium so extensively, that biochemical studies were not feasible. DNA repair, measured by the return to a normal sedimentation pattern of DNA on alkaline sucrose gradients, was followed over a period of 9 days with the use of 0.5 mg of methylnitrosourea to initiate the damage. Bladder epithelial cells were able to repair the DNA damage induced by methylnitrosourea. However, the possibility of persistent damage not detectable by sedimentation of DNA on alkaline sucrose gradients cannot be ruled out.

Damage and repair of DNA in various tissues of the rat induced 4-nitroquinoline 1-oxide.

4-Nitroquinoline 1-oxide induces pulmonary tumors when given by a s.c. route or skin cancer by repeated local applications. This carcinogen is absorbed by the lung more readily than other tissues. Therefore, we have compared the ability of 4-nitroquinoline 1-oxide to damage DNA of the liver, lung, and kidney in the intact animal. A differential effect of DNA damage was detected in all three organs, with the lung showing the greatest amount of damage. All three tissues were able to repair the damaged DNA. The preferential damage of rat lung DNA by 4-nitroquinoline 1-oxide correlates with the specificity of this carcinogen to induce pulmonary tumors.

Inhibition of DNA methylation by S-adenosylethionine with the production of methyl-deficient DNA in regenerating rat liver.

Ethionine, a liver carcinogen, was administered p.o. (300 mg/kg) to rats 17 hr after partial hepatectomy. At 6 hr after administration of the ethionine, hepatic S-adenosylethionine levels were 30- to 40-fold greater than the hepatic level of S-adenosylmethionine. A 10-fold ratio of S-adenosylethionine to S-adenosylmethionine still persited at 24 hr after ethionine administration. When given at 17 hr after partial hepatectomy, ethionine produced a 30% inhibition of DNA synthesis, measured by the incorporation of [methyl-3H]thymidine at 23 to 24 hr after partial hepatectomy (6 to 7 hr after ethionine administration). DNA synthesized during this interval was methyl deficient as judged by the reduced incorporation of radioactivity from L-[methyl-3H]methionine into 5-methylcytosine residues of DNA. In an assay for DNA methylation in vitro using whole nuclei, the methyl-deficient DNA was methylated by S-adenosylmethionine 8 times more than was control DNA; the DNA methylation was competitively inhibited by S-adenosylethionine. These data suggest that S-adenosylethionine, formed in vivo from ethionine, competitively inhibits the methylation of DNA in vivo by S-adenosylmethionine, resulting in the production of methyl-deficient DNA.

Inhibition of N-n-butyl-N-(4-hydroxybutyl)nitrosamine-induced urinary bladder cancer in rats by administration of disulfiram in the diet.

The objective of this study was to determine if disulfiram would influence the induction of urinary bladder cancer in rats given N-n-butyl-N(4-hydroxybuty)nitrosamine (BHBN). Adult male Wistar rats were divided into: Group 1, control diet, 30 rats; Group 2, control diet plus 0.025% BHBN in the drinking water, 60 rats; Group 3, control diet containing 0.5% disulfiram, 30 rats; and Group 4, control diet containing 0.5% disulfiram plus 0.025% BHBN in the drinking water, 60 rats. The animals were kept on these regimens for 15 weeks and then were transferred to and maintained on control diet. The average total intake of BHBN was 1.21 g/rat for Group 2 and 1.23 g/rat for Group 4. The cumulative incidences of bladder cancer at 25 weeks after initial exposure to BHBN were: Group 1, 0 of 9; Group 2, 27 of 27; Group 3, 0 of 9; and Group 4, 0 of 27. At termination of the experiment (32 to 42 weeks), the final bladder cancer incidences were: Group 1, 0 of 30 (0%); Group 2, 57 of 57 ()00%); Group 3, 0 of 24 (0%); and Group 4, 7 of 55 (13%). Except for a carcinoma of the renal pelvis in one rat in Group 2 and the bladder tumors in Groups 2 and 4, tumors were not detected in other organs of any of these rats. It was concluded that disulfiram significantly inhibited the induction of bladder cancer in rats exposed to BHBN. The mechanism of action of disulfiram in this process is under investigation.

Polyamine-stimulated growth of cultured rat urinary bladder epithelial cells.

A technique for isolating and establishing long-term cultures of rat urinary bladder epithelium has been devised. Cells isolated and cultured by this method have been grown for 12 weeks without subculturing. Rat bladder epithelial cells require the addition of putrescine, spermine, and spermidine to attain maximum growth and long-term survival. Monolayer cultures have been subcultured and carried through five passages.

Synthesis and mutagenicity of 4-(N-butylnitrosamino)-4-hydroxybutyric acid lactone, a possible activated metabolite of the proximate bladder carcinogen N-butyl-N-(3-carboxypropyl)nitrosamine.

4-(N-Butylnitrosamino)-4-hydroxybutyric acid lactone (BBAL) was synthesized as a possible intermediate produced by metabolic activation of a selective bladder carcinogen, N-butyl-N-(3-carboxypropyl)nitrosamine. BBAL was stable in neutral sodium phosphate buffer (ionic strength, 0.2), having a half-life of more than 30 hr at 25 degrees. The mutagenic effects of BBAL were tested with the use of Salmonella typhimurium TA1535 and Escherichia coli B/rWP2-try-, WP2-try-hcr-, and Sd4. The gene-damaging effects were assayed by repair tests with Bacillus subtilis H17 (rec+) and M45 (rec-). BBAL showed potent effects in the mutation and repair tests on all the strains tested without activation. A possibility is suggested for the metabolic activation of N-butyl-N-(3-carboxypropyl)nitrosamine to BBAL by alpha-hydroxylation at the site of the 3-carboxypropyl chain followed by lactonization in target tissues prior to interaction with macromolecules to lead to carcinogenesis.

The effect of S-adenosylhomocysteine on DNA methylation in isolated rat liver nuclei.

DNA methylation was studied in vitro using whole nuclei from regenerating rat liver. Methyl incorporation from S-adenosyl-[Me-3H]methionine in nuclei from regenerating liver was four times higher than that of normal liver. The effect of S-adenosylhomocysteine on DNA methylation was examined, and it was found at equal molar concentrations of S-adenosylhomocysteine to to S-adenosylmethionine that DNA methylation was competitively inhibited 50%.

The development of polyploidy in two classes of rat liver nuclei.

Two classes of nuclei from livers of Sprague-Dawley rats were isolated, one pelleting in 2.3 M sucrose (H nuclei) and the second class sedimenting through 1.6 and 1.8 M sucrose and banding at the 1.8/2.3 M sucrose interface (L nuclei) of a three-step discontinuous gradient. In younger animals, the L nuclear fraction was the major fraction, but the percentage of nuclei found in the L fraction decreased as the animals grew. Nuclear ploidy was determined by flow microfluorometry using propidium iodide as a DNA stain. Both the H and L nuclear fractions contained diploid, tetraploid and octaploid nuclei; but the degree of polyploidy was greater in the H fraction. Concomitant with the change in distribution of nuclei between the H and L fractions with increasing age was a progressive increase in the degree of polyploidy in the H fraction. Polyploidy did not increase linearly with age in the H nuclear fraction but increased in cycles marked by large changes in the numbers of nuclei found in H and L nuclear fractions. By 12 weeks of age, 4n-H nuclei were the largest single population of nuclei in rat liver. These observations suggested that the shift of liver nuclei from the L fraction to the H fraction was associated with the development of polyploidy and with the differentiation of hepatocytes.

O6-methylguanine accumulates in DNA of mammary glands after administration of N-methyl-N-nitrosourea to rats.

N-Methyl-N-nitrosourea (MNU) induces mammary carcinoma in female rats when given intravenously. After a single intravenous dose of N-methyl-N-nitrosourea (5 mg/100 g body wt.), we were unable to detect a shift of rat mammary gland DNA on an alkaline sucrose gradient. However, the alkylated products in DNA, 7-methylguanine and O6-methylguanine, were determined at various times following treatment with N-methyl-N-nitrosourea. O6-Methylguanine was removed from the DNA at a slower rate than 7-methylguanine and increased in the DNA with a second injection of N-methyl-N-nitrosourea. 3-Methyladenine was not detected in DNA from the mammary gland of the rat. These data support previous work with brain and bladder that suggest the persistence of O6-methylguanine in DNA might be involved in the induction of cancer by N-methyl-N-nitrosourea.

Influence of dose of N-methyl-N-nitrosourea on the induction of urinary bladder cancer in rats.

N-methyl-N-nitrosourea (MNU) was instilled by a urethral catheter into the urinary bladders of female Wistar rats in weekly doses of 0.5 mg for 1, 2, 3 and 4 weeks. At 75 weeks after the initial dose of MNU, the incidences of bladder cancer were 0, 7, 50 and 64% for the total doses of MNU of 0.5, 1.0, 1.5 and 2.0 mg, respectively. Control rats instilled with 0.9% NaCl only for 1--4 weeks did not develop bladder cancer by 75 weeks. Higher doses of MNU of 4.0 and 6.0 mg, given weekly in 0.5 mg amounts for 8 and 12 weeks, respectively, induced a higher incidence (nearly 90%) of urinary bladder cancer in rats at 22--28 weeks. However, it was shown that control rats given 12 weekly installations of solvent only developed a significant number (33%) of bladder cancers by 22--28 weeks.

Methylation of DNA in rat liver and intestine by dimethylnitrosamine and N-methylnitrosourea.

A chromatographic procedure for improved separation of deoxyribonucleosides and methylated deoxyribonucleosides is described. DNA was isolated from liver and small intestine of rats treated with [14C]dimethylnitrosamine ([14C]DMN) or N-[3H]methyl-N-nitrosourea ([3H]MNU), and the purified DNA was hydrolyzed enzymatically. The deoxyribonucleosides were chromatographed on an Aminex A-6 cation exchange column at 37 degrees C with 0.4 M ammonium formate, pH 4.5, as eluant. In addition to showing the presence of the expected alkylated products, N7-methyldeoxyguanosine (determined as N7-methylguanine) and O6-methyldeoxyguanosine, several other minor methylated products were found in liver and intestinal DNA of rats treated with DMN or MNU. Two of these products are believed to be N3-methylthymidine and O4-methylthymidine.

Induction of repair synthesis of DNA in primary cultures of rat urothelial cells by derivatives of the bladder carcinogen, N-n-butyl-N-(4-hydroxybutyl)nitrosamine.

Urinary metabolites of N-n-butyl-N-(4-hydroxybutyl)nitrosamine (BHBN) which have been identified thus far did not induce repair synthesis of DNA in cultured rat urothelial cells. Urine of rats which had been administered with BHBN or BCPN did not induce repair synthesis of DNA either. However, all the synthetic nitrosamines that can produce 1-hydroxyalkylnitrosamine intermediates induced repair synthesis of DNA. The results suggest that rat urothelial cells, at best, may only have very limited capability of activating nitrosamines.