Chemopreventive Potential of Myrtenal against Nitrosamine-Initiated, Radiation-Promoted Rat Bladder Carcinogenesis.

The present study was undertaken to evaluate the chemopreventive activity of myrtenal, a natural monoterpene, against bladder carcinoma in rats induced with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) and promoted with γ-ionizing radiation (γ-IRR) as well as to assess the involvement of inflammation, apoptosis and oxidative damage in tumor development. Histopathological examination of rat bladder revealed the presence of noninvasive papillary transitional cell carcinoma (Grade 2) in sections from BBN group indicating the credibility of the applied carcinogenesis model. Myrtenal treatment caused improvement in urinary bladder mucosa with cells more likely in Grade 1. Administration of myrtenal to BBN-treated rats exhibited downregulation in the expressions of COX-2, NF-kB and STAT-3 associated with suppression of inflammatory cytokines levels of TNF-α and IL-6 as well as biomarkers of oxidative damage (MDA & NO). In addition, myrtenal treatment caused a significant increase in caspase-3 activity and Bax/Bcl-2 ratio. Data obtained suggested that the anti-inflammatory effect and the induction of apoptosis contributed largely to the beneficial antitumor effects of myrtenal in rats with BBN/γ-IRR-induced bladder carcinoma. Present findings, in addition to benefits described in other pathologies, indicated myrtenal as a potential adjuvant natural compound for the prevention of tumor progression of bladder cancer.

Gut microbiome and bladder cancer: A new link through nitrosamine metabolism.

A recent Nature paper1 reveals that gut microbes metabolize N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) into the bladder carcinogen N-n-butyl-N-(3-carboxypropyl)-nitrosamine (BCPN) in the intestines, establishing a direct link between gut microbial activity and the development of bladder cancer.

Mitochondrial and liver oxidative stress alterations induced by N-butyl-N-(4-hydroxybutyl)nitrosamine: relevance for hepatotoxicity.

The most significant toxicological effect of nitrosamines like N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) is their carcinogenic activity, which may result from exposure to a single large dose or from chronic exposure to relatively small doses. However, its effects on mitochondrial liver bioenergetics were never investigated. Liver is the principal organ responsible for BBN metabolic activation, and mitochondria have a central function in cellular energy production, participating in multiple metabolic pathways. Therefore any negative effect on mitochondrial function may affect cell viability. In the present work, ICR male mice were given 0.05% of BBN in drinking water for a period of 12 weeks and were sacrificed one week later. Mitochondrial physiology was characterized in BBN- and control-treated mice. Transmembrane electric potential developed by mitochondria was significantly affected when pyruvate-malate was used, with an increase in state 4 respiration observed for pyruvate-malate (46%) and succinate (38%). A decrease in the contents of one subunit of mitochondrial complex I and in one subunit of mitochondrial complex IV was also observed. In addition, the activity of both complexes I and II was also decreased by BBN treatment. The treatment with BBN increases the susceptibility of liver mitochondria to the opening of the mitochondrial permeability transition pore. This susceptibility could be related with the increase in the production of H2 O2 by mitochondria and increased oxidative stress confirmed by augmented susceptibility to lipid peroxidation. These results lead to the conclusion that hepatic mitochondria are one primary target for BBN toxic action during liver metabolism.

Persistent γ-H2AX Formation and Expression of Stem Cell Markers in N-Butyl-N-(4-Hydroxybutyl)Nitrosamine-Induced Bladder Carcinogenesis in Rats.

We investigated γ-H2AX formation, a biomarker of DNA damage, and expression of stem cell markers (SCMs), including cytokeratin 14, aldehyde dehydrogenase 1A1 (ALDH1A1), and CD44, in the development of rat bladder tumors induced by short-term administration of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Histopathological examination showed that diffuse simple hyperplasia of the bladder urothelium induced by BBN recovered to the normal-appearing urothelium after withdrawal, whereas focal proliferative lesions were newly developed and subsequently progressed to benign papilloma and carcinoma. Immunohistochemical analysis revealed that BBN-induced γ-H2AX formation and ALDH1A1 and CD44 expression persisted at higher levels in the normal-appearing urothelium than those in the control group for long periods after withdrawal. Since persistent chronic inflammation was observed even after withdrawal, targeted gene expression analysis of inflammation-related factors revealed 101 genes, including Stat3 and Myc, that showed persistent high expression. Pathway analysis suggested that Stat3 and/or Myc activation may be associated with SCM expression. We focused on hepatocyte growth factor (Hgf), one of the genes predicted in relation to Stat3/Myc, and confirmed that HGF-positive cells increased by BBN persisted in the normal-appearing urothelium after withdrawal and colocalized with γ-H2AX and SCMs. These results suggested that the long-term persistence of γ-H2AX formation and SCM expression, which occurred during the early stages of bladder tumorigenesis, is not a transient response to exposure and might contribute to bladder tumorigenesis. Although further studies are needed, BBN-induced rat bladder tumors may originate from focal hyperplasia arising from SCM-positive cells via activation of the STAT3/MYC pathway after DNA damage involving γ-H2AX formation.

Chemopreventive Efficacy of Thymoquinone in Chemically Induced Urinary Bladder Carcinogenesis in Rat.

The effects of thymoquinone (TQ) in a carcinogen-based models of urinary bladder cancer were evaluated, using 45 male rats in five groups. In negative control (n = 10), only tap water was given. In positive control (n = 10), the rats received 0.05% N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) in drinking water for 9 weeks. In preventive groups with 25 mg/kg (n = 10) and 50 mg/kg (n = 10), oral TQ was concurrently given with 0.05% BBN for 9 weeks and continued for one more week after cessation of BBN. Preventive-treatment group (n = 5) received 50 mg/kg TQ orally for 20 weeks. Five rats from each group were sequentially sacrificed in two phases: the induction phase at 12th week (except the last group) and the rest in postinduction phase at 20th week. The bladders were examined macroscopically for lesion formation, and the masses were submitted for histopathological evaluation. Markers for total oxidant status (TOS), inflammation (nuclear factor kappa B (NF-κB)), and angiogenesis (vascular endothelial growth factor (VEGF)) were also assessed. There was a reduced number of bladder lesions in the TQ groups versus the carcinogen group at both phases. Histopathological findings demonstrated a significant improvement in the abnormal morphological changes in the urothelium of the TQ-treated groups. Thymoquinone exerted a significant antioxidant and anti-inflammatory effect by a decrease in serum level of TOS and NF-κB at week 12 which was maintained low in phase two at week 20. The serum level of VEGF was also alleviated in the induction phase at week 12 and maintained low in postinduction period. In TQ preventive-treatment approach, a nonsignificant elevation of serum level of TOS and NF-κB and slight reduction in VEGF were observed at the end of the experiment. These data suggest that TQ may be effective in preventing bladder carcinogenesis, and the suggested mechanisms might be related to antioxidant, prooxidant, and anti-inflammatory properties of TQ.

Pharmacokinetic profile and metabolism of N-nitrosobutyl-(4-hydroxybutyl)amine in rats.

N-Nitrosodibutylamine and its omega-hydroxylated metabolite N-nitrosobutyl(4-hydroxybutyl)amine (NB4HBA) induce tumors in the urine bladder of different animal species through their common urinary metabolite N-nitrosobutyl(3-carboxypropyl)amine (NB3CPA), resulting from the oxidation of the alcoholic group of NB4HBA to a carboxylic group. NB4HBA disappearance from blood, the formation of its main metabolites, NB3CPA and NB4HBA-glucuronide (NB4HBA-G), and their urinary excretion, were investigated in rats after an i.v. dose of 1 mg/kg (5.7 mumol/kg). NB3CPA and NB4HBA-G formation was readily detectable 2 min after treatment and levels were still measurable at 120 and 30 min, respectively. The parent compound disappeared from blood 90 min after injection. The NB4HBA blood concentration-time profile was adequately described by a one-compartmental linear model. NB4HBA half-life was 8 min, total body clearance and renal clearance were 86.1 and 0.22 ml/min/kg, respectively. The 0-96-h urinary excretion of NB4HBA was 0.3% of the administered dose. NB3CPA half-life was 15 min; NB3CPA and NB4HBA-G urinary excretion were 36 and 11.7%, respectively, urinary excretion of known compounds accounting for less than 50%. After i.v. injection of NB3CPA equimolar to the NB4HBA dose, only 50% of unchanged compound was recovered in the urine and after NB4HBA-G, 41% of the administered dose was excreted unchanged, NB3CPA accounting for 10%. Thus NB3CPA and NB4HBA-G might undergo further biotransformation, suggesting that NB3CPA may not be the ultimate carcinogen responsible for urinary bladder tumor induction.

Development of an experimental model for studying bladder carcinogen metabolism using the isolated rat urinary bladder.

The isolated rat urinary bladder was used to study this organ's capacity to metabolize chemical carcinogens. In our experimental conditions, the urinary bladder carcinogen N-nitrosobutyl(4-hydroxybutyl)amine was oxidized to N-nitrosobutyl(3-carboxypropyl)amine. A time-dependent increase was observed in the amount of N-nitrosobutyl(3-carboxypropyl)amine formed and simultaneous disappearance of N-nitrosobutyl(4-hydroxybutyl)amine added, indicating that the bladder can metabolize N-nitrosobutyl(4-hydroxybutyl)amine to the metabolite considered responsible for tumor induction in the urinary bladder of laboratory animals. At 15, 30, 60, and 120 min the percentages of N-nitrosobutyl(3-carboxypropyl)amine formed were 11, 22, 36, and 64%, respectively, and 62, 48, 37, and 26% of N-nitrosobutyl(4-hydroxybutyl)amine remained unchanged. When N-nitrosodibutylamine was introduced into the isolated urinary bladder and incubated for 120 min, its oxidized metabolites N-nitrosobutyl(4-hydroxybutyl)amine and N-nitrosobutyl(3-carboxypropyl)amine were formed, amounting to, respectively, 0.13 and 0.06% of the substrate added. The glucuronide of N-nitrosobutyl(4-hydroxybutyl)amine was incubated in the isolated rat urinary bladder both as a buffer and as a urine solution in order to detect cellular and urinary beta-glucuronidase activity. In both systems N-nitrosobutyl(4-hydroxybutyl)amine released was about 1% at 4 h and this percentage did not increase at 6 h. N-Nitrosobutyl(3-carboxypropyl)amine was detectable at 2 h and reached 0.2% of the substrate incubated at 6 h. The results indicate that the urinary bladder may play a role in activating bladder carcinogens.

Mutagenicity of N-butyl-N-(4-hydroxybutyl)nitrosamine, a bladder carcinogen, and related compounds.

N-Butyl-N-(4-hydroxybutyl)nitrosamine (BBN), which specifically induces bladder tumors, was shown to be mutagenic to Salmonella typhimurium strains TA 1535 and TA100 in the presence of an S-9 mix prepared from the liver of rats treated with polychlorinated biphenyl. Reduced nicotinamide adenine dinucleotide was a more effective cofactor than reduced nicotinamide adenine dinucleotide phosphate in the activation of BBN by the rat liver S-9 fraction, N-Butyl-N-(3-carboxypropyl)nitrosamine, reported to be the main urinary metabolite of BBN as well as of N,N-dibutylnitrosamine and to induce urinary bladder tumors specifically, was found to be mutagenic without metabolic activation by the S-9 mix. The mutagenicities of 31 compounds related structurally or metabolically to BBN and N,N-dibutylnitrosamine were tested. Of these compounds, 13 have previously been demonstrated to be carcinogenic, and nine have been shown to be noncarcinogenic. All the carcinogenic compounds were found to be mutagenic to strain TA1535 with or without the S-9 mix. Four of the nine noncarcinogenic compounds were also mutagenic. These "false-positive" compounds were predicted, in fact, to be carcinogenic.

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.

Assessment of L-ascorbic acid requirement for prolonged survival in ODS rats and their susceptibility to urinary bladder carcinogenesis by N-butyl-N-(4-hydroxybutyl)nitrosamine.

Assessment of L-ascorbic acid requirement for prolonged survival in ODS (genotype: od/od) rats and their susceptibility to urinary bladder carcinogenesis by N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) were examined. In ODS rats without L-ascorbic acid synthesizing ability, the 50 ppm dietary total ascorbic acid (TAA) was insufficient to survive for 4 weeks, the 250 ppm dietary TAA was sufficient to survive for 36 weeks. In examination of BBN treatment, ODS rats--although showing a lower availability of TAA than the heterozygotes (+/od) and normal (+/+) rats with L-ascorbic acid synthesizing ability--were equally susceptible to bladder carcinogenesis.

Inhibitory effect of partial cystectomy on experimental carcinogenesis in the urinary bladder.

It was our aim in the present animal experiments to study the influence of stimulation of proliferative activity on carcinogenesis in the urinary bladder. Stimulation of urothelial proliferation was achieved by a one-third resection of the bladder. N-butyl-N-(4-hydroxybutyl)- nitrosamine (BBN), which was used as a carcinogen, was administered by gavage in three fractionated doses when proliferative activity was highest at 30, 45, and 70 h postoperatively. Contrary to our working hypothesis, the incidence of urinary bladder tumors proved to be significantly reduced by partial cystectomy. After administration of a low total dose of BBN (300 mg/kg bodyweight) and an experimental period of 6, 12, and 18 months, only 2.6% of the rats with a partial cystectomy, but 12.6% of the control animals with an intact bladder had developed papillomas and noninvasive papillary transitional cell carcinomas. Following administration of BBN at a higher total dose (1,300 mg/kg bodyweight), bladder tumors occurred after an induction period of 4, 6, and 12 months in 27.4% of the partially cystectomized and 48.1% of the nonoperated rats. Multiple tumors were found more frequently in the controls than in the operated animals. The reduction in the tumor incidence following one-third resection of the bladder evidently does not depend on a prolongation of the latency period or induction time. From findings in analogous experimental models it is conceivable that the observed inhibition of experimental bladder carcinogenesis is brought about by an increased capacity of the proliferating urothelial cells to repair carcinogen-induced DNA damage. Further studies are required to elucidate the significance of a stimulated proliferation for the repair system and neoplastic transformation of the urothelium.

Effect of butylated hydroxyanisole added in vitro or administered to rats on N,N-dibutylnitrosamine and N-butyl-N-(4-hydroxybutyl)nitrosamine metabolism by post-mitochondrial supernatant of liver homogenates.

The effect of butylated hydroxyanisole (BHA) on P-450-dependent omega-hydroxylation of N,N-dibutylnitrosamine (NDBA) to N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), and the further oxidation of BBN to N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN) by the alcohol/aldehyde dehydrogenase system was investigated using the post-mitochondrial supernatant of liver homogenates (S9) from acutely and chronically BHA pretreated animals or S9 fractions from untreated rats with BHA added. Acute oral BHA (50 and 250 mg.kg-1) did not change NDBA omega-oxidation, which was reduced by 35% only when the compound was administered 0.5% in the diet for 3 weeks. BCPN formation from BBN was unaffected by acute and chronic BHA pretreatment. In order to verify whether BHA or its metabolite(s) had a direct effect on NDBA and BBN oxidation, the compound was added to S9 fractions from untreated rats at various concentrations. Only when BHA concentrations were equimolar or in a 10-fold molar excess to the substrate concentration, we observed 30-50% inhibition of BBN formation and a reduced BCPN formation (60-80% of control values), from BBN. Thus, only at very high BHA concentrations could we confirm the inhibition of P-450-dependent mixed function oxidase and alcohol dehydrogenase activities involved in the metabolism of NDBA and BBN.

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.

In vitro metabolism of bladder carcinogenic nitrosamines by rat liver and urothelial cells.

In order to establish the importance of the target organ in the activation of bladder carcinogens, we compared rat liver and urothelial cell alpha-hydroxylation activities using as substrates N-nitrosobutyl(4-hydroxybutyl)amine and its metabolite N-nitrosobutyl(3-carboxypropyl)amine, two potent urinary bladder carcinogens in animals. Previous studies have shown that the production of molecular nitrogen can serve as an indicator of nitrosamine alpha-hydroxylation. The use of doubly 15N-labelled nitrosamines and the gas chromatography-mass spectrometric detection of 15N2 formed gives a measurement of the extent of this metabolic step. Various amounts of 15N-labelled substrates were incubated for 60 min at 37 degrees C with rat liver S9 preparations or urothelial cell homogenates in the presence of a NADPH generating system. Both enzyme sources metabolized 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine and N-nitrosobutyl(3-carboxypropyl)amine through the alpha-hydroxylation pathway. Using hepatic S9 fractions, 15N2 production from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine increased from 1.69 +/- 0.02 nmol/h per mg protein (mean +/- S.E.) to 5.78 +/- 0.5 with substrate concentrations ranging between 0.55 and 5.55 mM. 15N2 produced by urothelial cell homogenates was about 40-50% that of the liver S9. 15N-labelled N-nitrosobutyl(3-carboxypropyl)amine was also metabolized through the alpha-hydroxylation pathway both by hepatic S9 and urothelial cell homogenates, though to a lesser extent. 15N2 production was about 10-times less than from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine, but again urothelial cell 15N2 production was about 40-50% that of the liver. Treatment with phenobarbital resulted in a 2.7-fold increase in the 15N2 produced from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine by hepatic S9. No effect was observed with urothelial cell homogenates. Acetone treatment had no effect on 15N2 production from 15N-labelled N-nitrosobutyl(4-hydroxybutyl)amine by hepatic S9, but raised 15N2 production by urothelial cell homogenates 1.8 times. Although the liver has a greater capacity than the bladder for activating the 15N-labelled nitrosamines studied, the target organ can metabolize bladder carcinogens, thus increasing the possibility of a local toxic effect. Moreover, the distribution of P-450 isozymes might be different in the bladder and this could affect the metabolism of nitrosamines reportedly formed in the human bladder in some pathological conditions.

[Structure and metabolic fate of N-nitrosodialkylamines in relation to their organotropic carcinogenicity with special reference to induction of urinary bladder tumors].

The metabolic fates of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) and N,N-dibutylnitrosamine (DBN) were investigated in the rat and other animal species, to elucidate a possible relationship between metabolism and organotropic carcinogenicity to the urinary bladder of these N-nitrosamines. The principal urinary metabolite of BBN as well as of DBN in the rat was N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN), which was demonstrated to be the active form of these compounds as bladder carcinogen. The species difference in response to BBN or DBN is discussed on the basis of the urinary excretion rate of BCPN. Metabolism in vivo and carcinogenicity of a number of BBN analogues were investigated in the rat and a general scheme for biotransformation of N-alkyl-N-(omega-hydroxyalkyl)nitrosamines is given. A possible correlation of structure and metabolism with organotropic carcinogenicity of BBN analogues is discussed, with special reference to selective induction of urinary bladder tumors.

[An experimental study on bladder carcinogenesis in dogs by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) and its urinary metabolites].

N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) was administered to beagle dogs to examine the urinary metabolites. After extraction and purification of the urine sample, the urinary metabolites were identified by gas-chromatography mass-spectrometry and measured using a gas chromatograph equipped with a thermal energy analyzer (GC-TEA). Five N-nitroso-compounds (N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN), N-butyl-N-(2-hydroxy-3-carboxypropyl)-nitrosamine (BHCPN), N-butyl-N-(carboxymethyl)-nitrosamine (BCMN), N-butyl-N-(2-oxopropyl) nitrosamine (BOPN) and BBN-glucuronide) were detected and identified by comparison with mass spectra of synthetic specimens. In the quantitative analysis by GC-TEA, BCMN greater than BHCPN greater than BCPN were larger part than BOPN and BBN-glucuronide. In early experimental period (0-25 wks) administered BBN (600 mg/day) to two dogs, small tumor was recognized in the urinary bladder of one dog earlier than the other. The former dog excreted larger amounts of BCPN in the urine than the latter. In late experimental period (100 wks approximately) administered BBN (400 mg/day) to five dogs, the dog that was found initial tumor earliest (103 wk) excreted the largest amounts of BCPN in the urine. Therefore, the excretion of BCPN had correlated with the carcinogenesis.

Direct alkyl chain cleavage after C-hydroxylation of dialkylnitrosamines in rats. A new pathway of the oxidative biotransformation.

Comparative in vivo investigations on the biotransformation of di-n-butylnitrosamine (DBNA) and (2-hydroxybutyl)-n-butylnitrosamine (HBBNA) showed the known omega-oxidation. However, a new metabolic mechanism of direct oxidative alkyl chain cleavage in the vicinity of the hydroxy group of HBBNA could be demonstrated.

Metabolic fate of N-butyl-N-(4-hydroxybutyl)nitrosamine homologs in the rat, in relation to their organotropic carcinogenicity to the urinary bladder.

The metabolic fate of alkyl homologs (alkyl=methyl, ethyl, propyl, pentyl, and tert-butyl) of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), a potent bladder carcinogen, was investigated in the rat, in order to elucidate any possible correlation of structure and metabolism with organospecific carcinogenicity to the urinary bladder of these N-nitrosamines. They were extensively metabolized in the rat, no unchanged compounds being found in the urine. The metabolic pattern of these alkyl homologs of BBN was essentially similar to that of BBN. Their principal urinary metabolite was the corresponding N-alkyl-N-(3-carboxypropyl)nitrosamine except in the case of the pentyl homolog. Minor metabolites characterized were subsequent transformation products of the principal metabolite by beta-oxidation according to the Knoop mechanism (i.e., N-alkyl-N-(2-hydroxy-3-carboxypropyl)nitrosamine, N-alkyl-N-(carboxymethyl)nitrosamine and N-alkyl-N-(2-oxopropyl)nitrosamine) and the glucuronic acid conjugate excretion of the N-alkyl-N-(c-carboxypropyl)nitrosamine with selective induction of bladder cancer by the N-alkyl-N-(4-hydroxybutyl)nitrosamine in rats is discussed.

Rapid assay of N-butyl-N-(3-carboxypropyl)nitrosamine in rat organs and urine by high-performance liquid chromatography after derivatization.

The concentration of N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN), which is the major metabolite of the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), was measured in the urine, thymus, liver, kidney, and bladder of rats orally administered with BBN. Since BCPN is a carboxylic acid, it forms an ester with 9-anthryldiazomethane (ADAM), which is a fluorescent labeling agent highly sensitive to carboxylic acids. Thus, BCPN and ADAM were reacted at 40 degrees for 1 hr, and the resulting ester was separated and measured by high-performance liquid chromatography (HPLC) with a reverse-phase type column. The range of measurement was 0 to 40 micrograms/ml, and the coefficient of variation (CV) was 3.8%. When 0.025% BBN was given orally to rats in tap water, the BCPN concentration in the urine was very high at 220 micrograms/ml, while it was 0.15 microgram/100 mg in the wet tissues of the thymus, 0.35 microgram/100 mg in the liver, 0.40 microgram/100 mg in the kidney, and 1.2 microgram/100 mg in the bladder. The BCPN concentration in the bladder, in which tumors are induced by the administration of BBN, was thus higher than those in the other organs.