Prostatic cancer induced in MRC rats by N-nitrosobis(2-oxopropyl)-amine and N-nitrosobis(2-hydroxypropyl)amine

Weekly intragastric treatment with N-nitrosobis(2-oxo-propyl)amineor N-nitrosobis(2-hydroxypropyl)amine induced hyperplastic,preneoplastic and neoplastic prostatic changes in >80% ofMRC rats. The lesions initially appeared as focal or multifocalproliferations of alveolar epithelium in a cribriform patternwhich, in all but one case, underwent progressive changes, oftentending toward squamous cell formation. Tumors, found primarilyin the ventral prostate, demonstrated various degrees of differentiationand invasive growth. A few neoplasms developed in the seminalvesicles; however all were of a glandular type. The sequentialalteration of induced lesions is described and the possiblereasons for the squamous cell character of most tumors discussed.Prostatic cancer induction by systemic application of specificnitrosamines could provide a unique tool for investigating importantaspects of the disease.     

Induction of pancreatic DNA damage and nodules in rats treated with N-nitrosobis(2-oxopropyl)amine and N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine

DNA damage was demonstrated by alkaline elution analysis inpancreas of rats treated with 100 mg/kg N-nitrosobis(2-oxopropyl)amine(BOP) and 20 mg/kg N-nitroso(2-hydroxy-propyl)2-oxopropyl)amine(HPOP). Young rats were treated with a single dose of BOP orHPOP and autopsied 4 months later. Histologically the pancreasescontained multiple foci and nodules of atypical acinar cellswhich were not seen in controls.     

Carcinogenicity of N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine, N-nitrosobis(2-hydroxypropyl)amine and cis-N-nitroso-2,6-dimethylmorpholine administered continuously in the Syrian hamster, and the effect of dietary protein on N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine carcinogenesis

The effect of continuous week-long administration of the threepancreatic carcinogens N-nitroso(2-hydroxypropyl)(2-oxo-propyl)amine(HPOP), N-mtrosobis(2-hydroxypropyl)amine (BHP), and cis-N-nitroso-2,6-dimethylmorpholine(cis-NNDM), by a s.c. implanted osmotic pump, was examined inSyrian hamsters. HPOP at total doses of 220–250 mg/kgbody weight induced ductal adenocarcinomas in the pancreas (41%),and cholangiomas (18%) and cholangiocarcinomas (18%) in theliver, 25 weeks following the initiation of treatment. Higherdoses of HPOP resulted in severe hepatic injury and increasedmortality (LD₅₀=280 mg/kg). Cis-NNDM and BHP were less toxicthan HPOP and induced pancreatic lesions at doses of 950 mg/kg.These data document that a week-long schedule of continuousadministration of HPOP for the induction of pancreatic cancercompares favorably with those involving weekly injections. Applicationof this model to study the effect of dietary protein in HPOP-inducedcarcinogenicity showed that the number of cystic, intermediateand tubular complexes in the pancreas was significantly higherin animals fed a 20% as compared to an 8% protein diet 2 weeksprior to HPOP administration. Furthermore, the incidence ofpancreatic adenocarcinomas and in situ carcinomas was only 13%in the hamsters fed the low-protein diet as compared to 46%in those fed the high-protein diet.     

Carcinogenicity of N-nitrosobis (2-hydroxypropyl) amine after administration to Syrian hamster skin

Weekly application of N-nitrosobis (2-hydroxypropyl) amine (BHP) at a dose of 50 mg/application to the skin of the neck and flank organ of male Syrian golden hamsters induced no local lesions. However, nearly all treated animals developed internal tumors, primarily of pancreatic, hepatic, respiratory and colorectal origins. Results from this and previous studies indicate that the local carcinogenic effect of nitrosamines depends on the molecular structure of the carcinogen, rather than on tissue specificity. Furthermore, some common types of human cancer could be induced by absorption of specific nitrosamines through skin.     

Carcinogenic effect of N-nitrosobis (2-oxopropyl)amine in newborn rats

N-nitrosobis(2-oxopropyl)amine (BOP) was administered twiceweekly for a total of 11 doses to 2-day-old Fischer F-344 ratsof both sexes to ascertain the spectrum of tissues sensitiveto its carcinogenic effects. At 26 weeks, the following incidenceof neoplasms were encountered in male and females, respectively;hepatocellular carcinoma (53 and 46%); nephroblastoma (21 and11%); and in males gonodal stromal tumors of testis (68%). Althoughacidophilic and basophilic acinar cell foci were encounteredin pancreas, these were few in number and microscopic. Thesefindings indicate that in newborn Fischer rats, hepatocytes,epithelial and mesenchymal cells of the kidney, and mesenchymalcells of testis are more sensitive to BOP than those of exocrinepancreas.     

Distribution and metabolism of N-nitrosobis(2-hydroxypropyl)amine in rats

The metabolic fate of the lung carcinogen N-nitrosobis(2-hydroxypropyl)amine (ND2HPA) in male Wistar rats was studied. The blood level after a single intraperitoneal (i.p.) injection of [1-14C]-ND2HPA at a dose of 3 g/kg body weight reached a maximum within 1 h. Most of the administered 14C was eliminated via the urine; 90.8% of the 14C was excreted in urine within 24 h, 5.5% in faeces, and 3.2% in expired air. About 11% of the 14C was detected in bile collected over 24 h. A relatively high concentration of 14C was found in the blood and target organs, such as the lung, liver, thyroid gland and kidney 1 h after treatment. Analysis by high-pressure liquid chromatography showed that the 14C in the blood and urine was mostly accounted for by unchanged ND2HPA, together with smaller amounts of N-nitroso-(2-hydroxypropyl)(2-oxopropyl)amine (N2HP2OPA). ND2HPA and N2HP2OPA were also detected in the lung and liver of rats 30 min to 12 h after the administration and were present in higher concentrations in the blood and lung than in the liver and pancreas. Besides ND2HPA and N2HP2OPA. N-nitrosomethyl(2-hydroxypropyl)amine (NM2HPA) was also found in urine collected over 6 h. ND2HPA, N2HP2OPA and NM2HPA showed mutagenicity in the Salmonella assay system with metabolic activation by a 9000 X g supernatant of rat liver, and N2HP2OPA was also mutagenic in the presence of a rat lung preparation. These data suggest that N2HP2OPA and NM2HPA might be important intermediates in the metabolic activation of ND2HPA to its ultimate carcinogenic form in rats.     

The formation of N-nitrosomethyl(2-oxopropyl)amine from N-nitrosobis(2-oxopropyl)amine in vivo

After injection of N-nitrosobis(2-oxopropyl)amine (BOP) (10mg/kg) to male Syrian golden hamsters there were higher concentrationsof BOP and many of its metabolites in the hamster pancreas comparedwith the liver and salivary gland. Also, a potential methylatingmetabolite of BOP, N-nitrosomethyl(2-oxopropyl)amine, was foundin both tissues.     

Carcinogenic activity of endogenously synthesized N-nitrosobis(2-hydroxypropyl)amine in rats

The carcinogenic activity of endogenously synthesized N-nitroso-bis(2-hydroxy-propyl)amine (NDHPA) was investigated in male Wistar rats administered bis(2-hydroxypropyl)amine (DHPA), mixed into a powdered diet at a concentration of 1%, and NaNO2 dissolved in distilled water at concentrations of 0.15% and 0.3%, for 94 weeks. Urinary excretion of NDHPA clearly demonstrated its endogenous synthesis in rats given 1% DHPA and 0.3% NaNO2, but not in the groups receiving either of these precursors alone. Tumours of the nasal cavity, lung, oesophagus, liver and urinary bladder were found in rats treated with 1% DHPA and 0.15% or 0.3% NaNO2. The incidences of nasal cavity and lung tumours reached 74% and 58% respectively, in rats given 1% DHPA and 0.3% NaNO2. The tumour distribution was almost the same as that seen in rats given NDHPA. These results indicate that endogenously synthesized NDHPA has similar carcinogenic activity to exogenously administered NDHPA in rats.     

Species specificity in the metabolism of N-nitrosobis(2-oxopropyl)amine and N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine to mutagens by isolated rat and hamster hepatocytes

The metabolic activation of the carcinogens N-nitrosobis(2-oxopropyl)amine (BOP) and N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) by Fischer rat and Syrian hamster hepatocytes was investigated in order to determine the existence of species differences in the induction of cell mutation. The conversion of BOP and HPOP into forms mutagenic to V79 cells was studied by using the hepatocyte-mediated mutagenicity assay. Mutations at the hypoxanthine:guanine phosphoribosyltransferase locus and the Na-K-ATPase locus were scored by the induction of 6-thioguanine resistance (TGr) or ouabain resistance (Ouar), respectively. Hepatocytes of both species were capable of converting BOP and HPOP to mutagens for V79 cells in a dose-dependent manner. Metabolism of BOP by rat hepatocytes resulted in higher mutation frequencies than that by hamster hepatocytes. At a BOP concentration of 240 microM, rat hepatocyte metabolism yielded 90.7 TGr mutants and 19.5 Ouar mutants per 10(5) V79 cells. At the same concentration, hamster hepatocyte metabolism of BOP yielded 54.1 TGr mutants and 13.0 Ouar mutants per 10(5) V79 cells. These results did not correlate with the known carcinogenic potency of BOP in the hamster as compared to the rat. Hamster hepatocytes carried out the catabolism of BOP to CO2 at faster rates than rat hepatocytes; therefore, the species difference in mutagenic activation was not due to a defect in BOP uptake or metabolism by hamster hepatocytes. In contrast, metabolism of HPOP by hamster hepatocytes resulted in significantly higher mutation frequencies than that by rat hepatocytes. At an HPOP concentration of 240 microM, hamster hepatocyte metabolism yielded 83.5 TGr mutants per 10(5) V79 cells; rat hepatocyte metabolism yielded only 19.8 TGr mutants per 10(5) V79 cells. This species difference in mutagenic activation correlated well with the known potency of HPOP as a carcinogen for the hamster as compared to the rat. Since hamster pancreatic cells and subcellular fractions are known to have very limited capacity to perform the metabolic activation of HPOP, the results of this study imply that liver metabolism plays an important role in the conversion of HPOP to an agent(s) which subsequently affects the hamster pancreas. The mutagenic potency of BOP versus HPOP was compared after metabolism by hepatocytes from both species. Following their metabolism by hamster hepatocytes, the two compounds were nearly equivalent in mutagenic potency. After metabolism by rat hepatocytes, BOP was significantly more potent mutagen than HPOP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Induction of benign and malignant lip tumors in Syrian hamsters by topical application of N-nitrosobis(2-oxopropyl)amine and N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine

Weekly topical application of equitoxic doses of N-nitrosobis(2-oxopropyl)amine (BOP) or N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) to lip and/or vagina of female Syrian hamsters led to the development of papillomas and carcinomas of the lip, papillomas of the vagina, and tumors of internal organs. The relative incidence of the tumor types is affected by the dose of BOP or HPOP administered. BOP is excreted unchanged and as HPOP in the saliva of Syrian hamsters injected subcutaneously with BOP and pilocarpin. This result may help to explain preliminary observations that subcutaneously injected BOP and pilocarpin also lead to lip tumors.     

Mutagenicity in V79 cells of N-nitrosobis(2-hydroxypropyl)amine and N-nitrosobis(2-oxopropyl)amine activated by tissues from hamsters fed low and high fat diets.

Hepatocytes and pancreas duct tissues from male Syrian hamsters fed high-fat (HFD) and low-fat (LFD) diets were used to activate N-nitrosobis(2-hydroxypropyl)amine (BHP) and N-nitrosobis(2-oxopropyl)amine (BOP) in the V79 cell mutagenicity assay. V79 DNA alkylation by BHP and BOP was also measured. There was a 3.5-fold increase in BHP mutagenicity but only a 1.4-fold increase in BOP mutagenicity when hepatocytes from HFD-fed hamsters were used over the mutagenicity when hepatocytes from LFD-fed hamsters were used. When pancreas duct tissue was the activating system there was a 2-fold increase in BHP and BOP mutagenicity. O6-Methylguanine levels in V79 DNA rose 4-fold when hepatocytes from HFD-fed hamsters were used to activate BOP but they declined when BHP was the alkylating agent.     

Carcinogenic activity of endogenously synthesized N-nitrosobis(2-hydroxypropyl)amine in rats administered bis(2-hydroxypropyl)amine and sodium nitrite

The carcinogenic activity of endogenously synthesized N-nitrosobis(2-hydroxypropyl)amine(BHP) was investigated in male Wistar rats administered bis(2-hydroxypropyl)amine(BHPA) mixed in powder diet at a concentration of 1%, and sodiumnitrite (SN) dissolved in distilled water at concentrationsof 0.15 and 0.3%, for 94 weeks. Urinary excretion of BHP wasdetected in rats given 1% BHPA and 0.3% SN but not in the groupsreceiving either of these precursors alone. Nasal cavity, lung,esophagus, liver and urinary bladder tumors were found in animalstreated with combinations of 1% BHPA and 0.15 or 0.3% SN, suggestingthat the target organs of the endogenously synthesized BHP aresimilar to those affected when the carcinogen is administeredexogenously. The incidences of nasal cavity and lung tumorsreached 74 and 58% in rats given 1% BHPA and 0.3% SN, respectively.Tumors at sites other than target organs were only found atlevels similar to those previously reported for spontaneoustumors in male Wistars. The present results clearly indicatedthe tumor inducibility of a nhrosatable amine, BHA, throughan endogenous nitrosation by feeding to rats in conjunctionwith nitrite, and provide further suggestive evidence that endogenousnitrosations of environmental nitrosatable amines can be a potentialrisk factor in human cancer development.     

High mutagenic activity of N-nitrosobis(2-oxopropyl)amine and N-nitrosobis(2-hydroxypropyl)amine in the host-mediated assay in hamsters: evidence for premutagenic methyl and hydroxylpropyl adducts

The carcinogenic nitrosamines N-nitrosobis(2-oxopropyI)-amine(BOP) and N-nitrosobis(2-hydroxypropyl)amine (BHP) were testedin excision-repair-deficient strains of his G46 Salmonella mutantsin the intrasanguinous host-mediated mutagenesis assay (HMA)in male Syrian hamsters. The major adducts produced by BOP inthe hamster are methylguanines, while BHP leads to hydroxypropylguaninesas well as methylguanines. Both nitrosamines were potent mutagensin bacteria recovered from the liver. On a comparison of administereddose, BOP was more potent, but when compared at doses producingsimilar levels of O⁶-methylguanine (O⁶MeG) in host liver DNA,or at equitoxic doses in the hamster, BHP was more potent. BHPwas 10 times less mutagenic in an excision-repair-proficientstrain of Salmonella, but the mutagenicity of BOP was not reduced.The effects of excision repair on in vitro mutagenesis inducedby the direct-acting analogs N-(2-oxopropyl)-N-nitrosourea (OPNU),a methylating agent, and N(2-hydroxypropyl)-N-nitrosourea (HPNU),a hydroxypropylating agent, were also examined. Mutagenesisby HPNU, but not OPNU was very sensitive to excision repair.Thus BOP appears to lead to mutagenesis via methylation, whilemutagenesis by BHP apparently proceeds via hydroxypropylation.BOP, BHP, OPNU and HPNU were several times less mutagenic inhisG428 than hisG46 strains. In contrast to hisG46 strains,which are reverted mainly by base-pair substitutions at G: Cbase pairs, hisG428 strains are generally more sensitive tomutagenesis at A: T base pairs. Taken together the above resultsand observations that >90% of the adducts from BOP and BHPwere alkylguanines, suggest that the major premutagenic adductsproduced from BOP and BHP are alkylguanines as opposed to otheralkylated bases. BOP and BHP were weak mutagens in the Salmonella/S-9mutagenesis assay using hamster liver S-9 fraction. When comparedwith results in the HMA, BOP and BHP were orders of magnitudeless mutagenic in vitro. This observation suggests: (i) thepathways or enzymes involved in the activation of these carcinogens(although uncertain) may be different in vivo and in vitro;or (ii) the pathways for the in vitro and in vivo metabolismmay be similar, but the conditions used for the in vitro activationof these nitrosamines are inadequate to generate significantlevels of nitrosamine metabolites.     

Distribution, metabolism and excretion of N-nitrosobis(2-hydroxypropyl)amine in Wistar rats

The metabolic fate of the carcinogen N-nitrosobis(2-hydroxypropyl)amine(BHP) in male Wistar rats was studied. The blood level of [1-¹⁴C]BHPafter a single intraperitoneal injection, administered at acarcinogenic dose of 3 g/kg body weight, reached a maximum within1 h. Whereas a relatively high concentration of ¹⁴C was foundin the blood and target organs, such as the lung, liver, thyroidgland and kidney 1 h after the treatment, most of the radioactivelabelling had disappeared from the tissues by 24 h after injection.Most of the administered ¹⁴C was eliminated via the urine; 90.8%was excreted in the urine within the 24 h period, 5.5% in thefeces and 3.2% by way of expired air. Studies in rats with exteriorizedbile flow demonstrated that about 11% of the intraperitoneallyadministered ¹⁴C was excreted via the bile in 24 h. Analysisby h.p.l.c. detected BHP (78.1% of the dose), HPOP (1.5%), glucuronidesof BHP (4.3%) and HPOP (0.16%), MHP (0.03%) and unknown metabolites(6.0%) in the urine 24 h after the treatment. Besides thesemetabolites, BOP and two unidentified metabolites were alsodetected in the blood, lung, liver or kidney of rats 3 h afterthe treatment. These results suggest the involvement of BHPmetabolites, HPOP, MHP and BOP, in carcinogenesis and in particularlung carcinogenesis induced by BHP in rats.     

Determination of N-nitrosobis(2-hydroxypropyl)amine in environmental samples

N-Nitrosobis(2-hydroxypropyl)amine (ND2HPA) is a potent pancreatic carcinogen in hamsters and induces gastrointestinal and respiratory tract cancer in rats. The precursor amines, diisopropanolamine (Di-PA) and triisopropanolamine (Ti-PA), are used in some manufacturing processes and in cosmetic preparations. We have found low levels of ND2HPA in commercial Ti-PA (21-270 ng/g) and in Di-PA (20-1 300 ng/g) and have demonstrated that ND2HPA is formed from Ti-PA and nitrite in a yield comparable to that observed for formation of N-nitrosodiethanolamine (NDELA) from triethanolamine under relatively mild conditions. After reaction for 4 h at 37 degrees C (10 mmol/L amine, 40 mmol/L nitrite, pH 3.0), the ND2HPA yield was 0.51%. The NDELA yield under the same conditions was 0.96%. ND2HPA was determined by gas chromatography-thermal energy analysis (GC-TEA) and GC-high-resolution mass spectrometry (GC-MS) selected ion monitoring of the tert-butyldimethylsilyl (t-BDMS) ether after extraction on a Celite 560 column. The t-BDMS ethers of ND2HPA and NDELA yielded intense, structurally significant peaks at m/z 333.2030 and 305.1716, respectively. The GC-MS procedure provides sensitivity and selectivity comparable to that of GC-TEA.     

Comparative carcinogenicity of N-nitrosobis(2-oxopropyl)-amine and N-nitrosomethyl(2-oxopropyl)amine following subcutaneous or oral administration to rats

The carcinogenicity of N-nitrosomethyl(2-oxopropyl)amine (MOP), a postulated proximate carcinogen of N-nitrosobis(2-oxopropyl)-amine (BOP), was tested after either a single subcutaneous (s.c.) injection or weekly intragastric (i.g.) administration in Wistar-derived MRC rats and was compared with the effect of BOP, given similarly and at equitoxic doses. Following i.g. administration, MOP induced a high incidence of neoplasms in the pharynx and esophagus which, however, were not affected by BOP; on the other hand, tumors of the thyroid, lungs, colon and urethra occurred in a greater incidence following BOP than after MOP, and renal neoplasms were found only following MOP, given s.c. Moreover, there were remarkable sex differences in the responses of the rats' respiratory and urothelial tissues to these two carcinogens: nasal cavity carcinomas, pulmonary adenomas, urinary and urethra papillomas were induced primarily or exclusively in male rats treated with BOP, either s.c. or i.g., whereas such sex differences were not found following either route of MOP administration. There were also differences in the spectrum of the neoplasms induced by BOP or MOP depending upon the route of their administration. For example, MOP was more effective in inducing nasal, esophageal and hepatic tumors when given orally, compared to its effect following the s.c. route, and thyroid and renal tumors were induced only after its s.c. injection. The results point to a complexity of nitrosamine carcinogenesis and also indicate that in some tissues activation of BOP, but not of MOP, depends on sex hormones.     

Prevention of rectal cancer induced by N-nitrosobis(2-oxopropyl)-amine by exogenous testosterone in MRC rats

Rectal carcinogenicity of N-nitrosobis(2-oxopropyl)amine (BOP)in male MRC Wistar rats was shown to be inhibited by exogenoustestosterone (T) when the hormone was given during, but notafter, administration of the carcinogen. This effect was independentof the dose and frequency of BOP, which was given either weeklyfor 20 weeks orally or daily for 3 days subcutaneously. Since,except for prostatic cancer, the incidence and the patternsof other BOP-induced tumors were not altered by T, this hormoneseems to play a specific role in the rectal carcinogenesis ofBOP.     

The effect of N-nitrosobis(2-oxopropyl)amine after oral administration to hamsters.

Oral administration of N-nitrosobis(2-oxopropyl)amine (BOP) in drinking water to Syrian golden hamsters for 90 days resulted in a high incidence of intra- and extrahepatic bile duct neoplasms. Only a few pancreatic neoplasms and no lung and kidney tumors developed, in contrast to results obtained after subcutaneous BOP administration.