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.     

Initiation activity of endogenously synthesized N-nitrosobis(2-hydroxypropyl)amine in the rat liver

The initiation potential of N-nitrosobis(2-hydroxypropyl)amine (NDHPA) endogenously synthesized from bis(2-hydroxypropyl)amine (DHPA) or tris(2-hydroxypropyl)amine (THPA) in the presence of sodium nitrite (NaNO2) was investigated in the rat liver by quantitation of hepatocellular foci showing phenotypic expression of glutathione S-transferase placental form (GST-P). The investigation consisted of two experiments. In the first, male Wistar rats were divided into six groups as follows: group 1 was non-treated; groups 2 and 3 received 0.15% and 0.3% NaNO2, respectively; group 4 received 1% DHPA; groups 5 and 6 received 1% DHPA together with 0.15% and 0.3% NaNO2, respectively. In experiment 2, the same protocol was used except that 2% THPA was substituted for 1% DHPA. The treatments were continuous until sacrifice at week 94 in experiment 1 and week 104 in experiment 2. As a result putative preneoplastic GST-P-positive foci observed in the liver and increased dose-dependently in rats from groups 5 and 6 which had received DHPA and NaNO2, but not in rats administered THPA and NaNO2. The results indicate that endogenously synthesized NDHPA from DHPA and NaNO2 is capable of initiating neoplastic development in the rat liver.     

Carcinogenicity of N-nitrosobis(2-hydroxypropyl)amine and N-nitrosobis(2-oxopropyl)amine in MRC rats.

Weekly sc injections of equitoxic doses of N-nitrosobis(2-hydroxypropyl)amine (BHP) and N-nitrosobis(2-oxopropyl)amine (BOP) to Wister-derived MRC rats induced tumors. The incidence, latency, multiplicity, morphologic type, and distribution of these tumors varied according to the compound given. The esophagus was the main target organ for BHP (100%), followed by the respiratory tract (87%), pharynx (80%), colon and liver (each 73%), kidneys (20%), thyroid gland (20%), and urinary bladder and urethra (each 7%). BOP was ineffective in the esophagus and pharynx but induced a higher incidence of tumors in the kidneys (27%), thyroid gland (60%), urinary bladder (33%), and urethra (73%) and fewer neoplasms in the respiratory tract (20%), colon (67%), and liver (53%). In addition, BOP caused a few, apparently primary, prostate squamous cell carcinomas. The results are compared with results of BHP treatment in Sprague-Dawley rats and with results of BHP and BOP treatment in Syrian golden hamsters.     

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.     

Carcinogenic potency of N-nitrosomethyl(2-hydroxypropyl)amine and other metabolic relatives of N-nitrosobis(2-hydroxypropyl)amine by single intraperitoneal injection on the lung of rats

The carcinogenic effects of a single intraperitoneal injection of N-nitrosobis(2-hydroxypropyl)amine (BHP) or its metabolic relatives, N-nitrosomethyl(2-hydroxypropyl)amine (MHP), N-nitrosobis(2-oxopropyl)amine (BOP), N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) and N-nitroso-2,6-dimethylmorpholine (NDMM), were studied in male Wistar rats. The main target organ of these nitrosamines proved to be the lung, followed by the thyroid. Lung lesions were induced in a dose-dependent manner with total lung tumor incidences reaching 55% to 100%. BHP, MHP, HPOP and NDMM all caused lung carcinomas to develop (22% to 44% incidence), whereas BOP was only associated with adenomas. On the basis of dose administered and incidence of carcinomas, MHP appeared to be the most potent lung carcinogen of the five nitrosamines investigated. Smaller numbers of neoplasms were also induced in the kidney, urinary bladder, esophagus and intestine at differing rates by these nitrosamines.     

Initiation of hepatocarcinogenesis by endogenously formed N-nitrosobis(2-hydroxypropyl)amine, N-nitrosodiethanolamine and N-nitroso-2,6-dimethylmorpholine in rats

Initiation activities of endogenously formed N-nitrosobis(2-hydroxypropyl)amine(NBHPA), N-nitrosodiethanolamine (NDELA) and N-nitroso-2,6-dimethylmorpholine(NDMM) were investigated in a modified short-term assay forrat hepatocarcinogenesis. Male Wistar rats were fed 1 % bis(2-hydroxypropyl)amine,0.5% diethanolamine or 0.25% 2,6-dimethylmorpholine in the dietplus 0.3% sodium nitrite in the drinking water. Two weeks afterstarting the experimental regimen they underwent 2/3 partialhepatectomy and were then maintained on the respective dietsfor a further week. Following a 2 week recovery period on basaldiet the rats were subjected to a resistant hepatocyte regimenconsisting of 0.02% 2-acetylaminofluorene in the diet for 2weeks and 1 mg carbon tetrachloride/kg body wt by gavage atthe midpoint. Initiation activity was assayed by measuring hepaticfoci positive for     

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.     

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.     

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.     

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.     

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.     

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-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.     

Alkylation of rodent tissue DNA induced by N-nitrosobis(2-hydroxypropyl)amine.

Levels of methyl and hydroxypropyl adducts induced by single s.c. injections of various doses of tritium-labeled N-nitrosobis(2-hydroxypropyl)amine ([1-3H]BHP) were determined in the liver, pancreas, kidney and lung of hamsters and rats. At doses of BHP used in carcinogenesis studies (100-500 mg/kg), methylation of DNA was more extensive than its hydroxypropylation; however, it did not increase proportionally with the dose and gradually became secondary to hydroxypropylation at higher doses of the carcinogen. Ratios of hydroxypropyl versus methyl adducts also varied significantly depending on the tissue and species. In both species ratios of N7-hydroxypropylguanine (N7-HpG) versus N7-methylguanine (N7-MeG) were greater in kidney and pancreas than in liver or lung. Due to apparent differences in the repair of O6-methylguanine (O6-MeG) and O6-hydroxypropylguanine (O6-HpG), and the propensity of 2-hydroxypropylating as compared to methylating agents to yield a greater percentage of oxygen adducts, ratios of O6-HpG versus O6-MeG were markedly greater than those of N7-HpG versus N7-MeG. Levels of O6-HpG were greater than those of O6-MeG in rat liver, pancreas and kidney and also in hamster kidney, while such levels were similar in rat lung and also in hamster liver, pancreas and lung. Like N-nitrosobis(2-oxopropyl)amine (BOP) and N-nitroso(2-hydroxypropyl) (2-oxopropyl)amine (HPOP), BHP was activated primarily in the liver and induced substantially greater DNA damage in this than in any other tissue examined. However, unlike BOP and HPOP, which induced similar levels of hepatic DNA damage in the above two species, BHP methylated and hydroxypropylated hamster liver DNA more extensively than that of the rat. Differences between BOP and BHP were also observed regarding levels and distribution of DNA adducts in extrahepatic tissues. In rats, BHP induced greater levels of methylation and hydroxypropylation in lung than in kidney, while the reverse was observed with BOP. Apparently reduction of the beta-carbon of pancreas-specific nitrosamine carcinogens results in a shift of alkylation from kidney to the lung. Excretion of HPOP in the urine of BHP-treated animals and the observed saturation of DNA methylation at high doses of BHP, supported the hypothesis that the BHP-induced methylation of DNA proceeded via the intermediate formation of HPOP. This was further supported by the observation that both excretion of HPOP and levels of methyl adducts were greater in hamsters than in rats.(ABSTRACT TRUNCATED AT 400 WORDS)