Tumorigenicity of the tobacco-specific carcinogen 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone in infant mice.

The tobacco-specific nitrosamine, 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a potent carcinogen in adult rodents and variably effective transplacentally, depending on species. In pursuit of the thesis that human infants may be especially vulnerable targets for tumor initiation by tobacco smoke constituents, we tested the efficacy of NNK as a tumor initiator in infant mice. Cr:NIH(S) (NIH Swiss outbred) mice were given 50 mg/kg NNK i.p. on postnatal days 1, 4, 7, 10 and 14, with saline to controls. At an average age of 13-15 months, 57% of the NNK-exposed male offspring had hepatocellular tumors, with a multiplicity of 1.15 +/- 1.4, including 4 with carcinoma. Liver tumors including 2 carcinomas were found in 8 (14%) of the NNK-exposed female offspring. There were no hepatocellular neoplasms in any control. A significant increase in primary lung tumors also occurred in the NNK-treated males, with an incidence of 30/55 (57%) and a multiplicity of 0.7 +/- 0.2, vs. 7/33 (21%), multiplicity 0.3 +/- 0.6, in controls (P less than 0.025). An apparent increase in the incidence of lung tumors in NNK-treated females, 21/57 (37%) vs. 7/32 (22%) in controls, approached significance (P less than 0.1). Thus NNK was a moderately potent neonatal carcinogen for liver and lung in infant Swiss mice and more efficacious in this regard than when received transplacentally by mice of the same strain.     

Chronic nicotine consumption does not influence 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis

Nicotine replacement therapy is often used to maintain smoking cessation. However, concerns exist about the safety of long-term nicotine replacement therapy use in ex-smokers and its concurrent use in smokers. In this study, we determined the effect of nicotine administration on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumors in A/J mice. Female mice were administered a single dose of NNK (10 μmol) and 0.44 μmol/mL nicotine in the drinking water. Nicotine was administered 2 weeks prior to NNK, 44 weeks after NNK, throughout the experiment, or without NNK treatment. The average weekly consumption of nicotine-containing water was 15 ± 3 mL per mouse, resulting in an estimated daily nicotine dose of 0.9 μmol (0.15 mg) per mouse. Nicotine administration alone for 46 weeks did not increase lung tumor multiplicity (0.32 ± 0.1 vs. 0.53 ± 0.1 tumors per mouse). Lung tumor multiplicity in NNK-treated mice was 18.4 ± 4.5 and was not different for mice consuming nicotine before or after NNK administration, 21.9 ± 5.3 and 20.0 ± 5.4 tumors per mouse, respectively. Lung tumor multiplicity in animals consuming nicotine both before and after NNK administration was 20.4 ± 5.4. Tumor size and progression of adenomas to carcinomas was also not affected by nicotine consumption. In addition, nicotine consumption had no effect on the level of O(6)-methylguanine in the lung of NNK-treated mice. These negative findings in a commonly used model of human lung carcinogenesis should lead us to question the interpretation of the many in vitro studies that find that nicotine stimulates cancer cell growth.     

Inhibition of tobacco-specific nitrosamine 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) tumorigenesis with aromatic isothiocyanates

4-(N-Nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific carcinogenic nitrosamine. At low doses, it induces primarily lung tumours in mice, hamsters and rats, regardless of the route of administration. Its unique organ specificity and potency suggest its possible role in the high incidence of lung cancer in smokers. The goal of this study was to find agents that would potentially prevent NNK tumorigenesis. Previous results led us to test phenethyl isothiocyanate (PEITC) on NNK tumorigenesis in a two-year bioassay in Fischer 344 rats. The NNK-treated group developed 80% lung tumour incidence, whereas NNK-treated rats fed PEITC diets had only 40% lung tumour incidence. Incidences in other organs were not affected by this treatment. We also tested PEITC in a 16-week, short-term bioassay against NNK-induced lung adenomas in A/J mice. Pretreatment of mice with PEITC by gavage at four daily doses of 5 mumol or 25 mumol reduced the formation of NNK-induced lung adenomas by 70% or 100%, respectively. Interestingly, benzyl isothiocyanate and phenyl isothiocyanate, the lower homologues of PEITC, were inactive in this bioassay. Using a protocol similar to that used in the bioassays, PEITC was shown to decrease DNA methylation by NNK in the lungs of rats and mice and suppress the metabolism of NNK by mouse lung microsomes. These results are consistent with the previous data, suggesting that the inhibition of NNK-induced lung tumour formation by PEITC is a consequence of reduced DNA methylation caused by inhibition of NNK metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lack of correlation between DNA methylation and hepatocarcinogenesis in rats and hamsters treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

Previous studies have demonstrated that the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induced liver tumors in F344 rats but not in Syrian golden hamsters. The aim of this study was to determine whether there was a correlation between the persistence of O6-methylguanine (O6-mGua) adducts and the rate of recovery of O6-methylguanine-DNA methyltransferase (O6-mGuaT) after depletion in the liver and susceptibility to NNK in F344 rat and Syrian golden hamster injected s.c. with NNK (80 mg/kg). The levels of both 7-methylguanine and O6-mGua reached a maximum 24 h after NNK treatment. O6-mGua in NNK-treated rat liver was undetectable after 48 h. In the rat, the depletion of O6-mGuaT activity occurred within 4 h following NNK treatment. A subsequent rapid recovery of enzyme activity was observed 36 h after NNK exposure. In contrast, high levels of O6-mGua persisted in hamster liver DNA and no O6-mGuaT activity was detected up to 336 h after NNK injection. Thus, the persistence of O6-mGua in hamster liver is most likely related to a lack of recovery of the O6-mGuaT. These results suggested that factors other than O6-mGua may be determining NNK-induced hepatocarcinogenesis in rats. An aldehyde generated by alpha-hydroxylation of NNK, 4-oxo-4-(3-pyridyl)butanal, inhibited O6-mGuaT activity in rat hepatocytes, suggesting that this aldehyde contributes to the carcinogenicity of NNK by inhibiting this repair enzyme.     

Effects of indole-3-carbinol on lung tumorigenesis and DNA methylation induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and on the metabolism and disposition of NNK in A/J mice

The effects of indole-3-carbinol (I3C) on lung neoplasia induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were assessed in an A/J mouse pulmonary adenoma bioassay. Mice were administered corn oil or I3C (25 or 125 mumol/mouse/day) by gavage for 4 consecutive days. Two h after the final pretreatment, mice were administered a single dose of NNK (10 mumol/mouse) i.p. Pulmonary adenomas were quantitated 16 wk after NNK dosing. Mice pretreated with corn oil developed 10.7 tumors/mouse; I3C pretreatment at either dose level inhibited tumor multiplicity by approximately 40%. The effects of I3C on NNK-induced DNA methylation in the lungs and livers of A/J mice were assessed using the same dosing regimen as in the bioassay. Both dose levels of I3C inhibited pulmonary O6-methylguanine formation by at least 50%, but enhanced hepatic DNA methylation at 2 or at 6 h after NNK administration. The effects of I3C pretreatment on NNK metabolism were also investigated. Hepatic microsomes of I3C-pretreated mice showed increased formation of alpha-hydroxylation products, while no significant effect of I3C pretreatment was observed in pulmonary microsomes. The effects of I3C on [5-3H]NNK disposition were also evaluated. I3C pretreatment produced lower levels of total radioactivity in the lung when compared with controls. Additionally, lower proportions of NNK and its carcinogenic metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol were found in the lungs of I3C-pretreated mice. These results demonstrate that I3C inhibits NNK-induced lung neoplasia in A/J mice and suggest that the basis of this inhibition is the decrease in O6-methylguanine formation in A/J lung caused by I3C pretreatment. This decrease in lung DNA methylation appears to be due to the decreased bioavailability of NNK and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in the lungs of I3C-treated mice which, in turn, may be a result of increased metabolic alpha-hydroxylation of NNK by the liver.     

Transformation of hamster pancreatic duct cells by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), in vitro

The tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK) is a potent carcinogen in laboratory animals. In the presentstudy, in vitro transformation of spontaneously immortal hamsterpancreatic duct cells following exposure to 20 mM NNK for 1,3,5and 7 days is described. NNK imparted a dose-dependent and time-dependenttoxicity to pancreatic duct cells in vitro. After NNK treatment,duct cells were grown either in complete duct medium (CDM) orin the absence of bovine pituitary extract, epidermal growthfactor and Nu-seruin (incomplete duct medium, 1DM). Additionof NNK to the culture for 1 and 3 days did not affect the growthof the cells, whereas exposure of the cells for 5 and 7 dayswas inhibitory. One and 3 day NNK-treated cells were able togrow in the absence of growth factors and serum immediatelyafter the treatment without any inhibition of growth. Untreatedcells grew as a monolayer consisting of tightly packed polygonalcells with single nuclei. NNK treated cells also grew as a monolayerwith numerous mitotic figures and multi-nucleated large cells.The doubling time between the untreated (16 h) and NNK-treatedcells (14 h) was not significantly different prior to injectioninto the nude mice. NNK treated cells grown in 1DM displayedanchorage independency in soft-agar. The tumorigenicity of theuntreated and NNK treated cells (5x10⁶) was determined in nudemice. One and 3 day NNK-treated cells grown in CDM producedwell-differentiated, mucinous tumors with a lower frequency(2/4 sites) and longer duration, but produced tumors at a higherfrequency (4/4 sites) and shorter duration when grown in IDM.Five and 7 day NNK-treated cells grown in CDM did not produceany tumors; however, they produced tumors when grown in CDMfollowed by IDM (5/8 and 6/8 sites) with a shorter durationin nude mice. Analysis of DNA for k-ras mutation at codons 12,13 and 61 showed G–A transition at codon 12 of the k-rasoncogene in tumor cells of 1 and 3 day NNK treatment. No mutationwas detected in tumor cells from 5 and 7 day treatment.     

Spontaneous and urethan-induced tumor incidence in B6C3F1 versus B6CF1 mice

The incidences of spontaneous tumors of the murine hybrids (C57BL/6J X C3Hf)F1 (B6C3F1) and (C57BL/6J X BALB/c)F1 (B6CF1) were compared in untreated mice kept until 110 weeks of age. Male B6C3F1 and B6CF1 mice had respectively 16% and 20% incidence of lymphomas, 26% and 4% of liver tumors and 12% and 22% of lung tumors. Among B6C3F1 and B6CF1 females, a 36% and 12% incidence of lymphomas, a 6% and zero incidence of liver tumors, and a 4% and 16% of lung tumors were observed. A few other tumors were seen in both hybrids. Groups of male and female mice of the 2 hybrids received 5 i.p. injections of 1000 mg/kg urethan once every other day starting at 10 days of age, and were kept under observation until 65-80 weeks of age. Treated B6C3F1 mice had an earlier mortality than B6CF1 mice due to tumor development. The statistical analysis, allowing for survival, showed a significantly higher lymphoma incidence in male and female B6C3F1 than B6CF1 mice, which had instead a higher incidence of lung tumors. Hepatocellular tumors were seen in both sexes of the 2 hybrids, with a higher frequency in B6C3F1 mice. Male mice of both hybrids had a higher incidence of liver tumors than females.     

Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced DNA adduct formation and tumorigenicity in the lung of F344 rats by dietary phenethyl isothiocyanate

F344 rats fed diets containing phenethyl isothiocyanate (PEITC, 3 mumol/g diet), a cruciferous vegetable component, before and during treatment with the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), developed about 50% fewer lung tumors than NNK-treated rats fed control diets. NNK-induced liver and nasal cavity tumors in rats were, however, not affected by this dietary treatment. The effects of PEITC diets on the formation of DNA adducts by NNK were also investigated in these target tissues. DNA methylation and pyridyloxobutylation by NNK were both decreased by 50% in lung of rats fed PEITC diets compared to that of rats fed control diets, but the levels of DNA methylation were not affected in liver and nasal mucosa. These results correlated with those from the carcinogenicity bioassay, suggesting that DNA alkylations could be used as indicators for screening inhibitors of NNK tumorigenesis. A slight increase in the number of tumors of the exocrine pancreas was observed in PEITC-fed rats with or without NNK treatments. However, these incidences were not statistically significant when compared to the control groups. The potential toxicity of PEITC at concentrations ranging from 0.75 mumol to 6 mumol/g diet was evaluated in a 13-week study. The only toxicity caused by this treatment was minimal fatty metamorphosis in the liver. Considering the widespread human exposure to NNK through tobacco use, it is of practical importance to demonstrate inhibition of lung tumors induced by this carcinogen. These results provide a basis for studies designed to discover agents of better efficacy for the prevention of NNK-induced tumorigenesis.     

Comparison of transplacental and neonatal initiation of mouse lung and liver tumors by N-nitrosodimethylamine (NDMA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and promotability by a polychlorinated biphenyls mixture (Aroclor 1254)

We have previously shown a positive tumor-promoting effect ofa single dose of Aroclor 1254 on lung and liver tumors initiatedneonatally in the mouse by N-nitrosodimethylamine (NDMA). Inthis study, we have confirmed and extended this observationwith NDMA and the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyI)-1-butanone(NNK) given either transplacentally or postnatally, followedby a single dose of Aroclor 1254 on day 56. This polychlorinatedbiphenyl (PCB) mixture was an effective promoter of both lungand liver tumors; however, there were specific initiator andsex-related differences in this response. Aroclor administrationsignificantly increased the incidence of lung tumors initiatedtransplacentally by NDMA or NNK in male mice. Neither nitrosamineinitiated tumors transplacentally in females, but lung tumorsinitiated with NNK and liver tumors caused by NDMA in neonatalfemales were promoted by PCBs. Both liver and lung tumors initiatedneonatally by NDMA in male animals, but not NNK-initiated tumors,were promoted by PCBs. These data confirm that PCBs are ableto promote both NDMA- and NNK-initiated tumors, but with chemical-,sex- and age-dependent difference; this suggests influencesof both quantitative and qualitative factors in susceptibilityto tumor promotion.     

Tumorigenic activity of the tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) and N'-nitrosonornicotine (NNN) on topical application to Sencar mice

The tumor-initiating activities of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-4-(3-pyridyl)-1-butanol (iso-NNAL) and N'-nitrosonornicotine (NNN) were evaluated on the skin of female SENCAR mice. A total initiator dose of 28 mumol/mouse of each nitrosamine was applied in 10 subdoses administered every second day. Promotion commenced 10 days after the last initiator dose and consisted of twice weekly application of 2.0 micrograms of tetradecanoylphorbol acetate for 20 weeks. NNK induced a 79% incidence of skin tumors with an average of 1.6 tumors/mouse and a 59% incidence of lung adenomas. In contrast, iso-NNAL and NNN were not active as tumor initiators in either the skin or lung of mice. The tumorigenic activity of NNK on SENCAR mouse skin was evaluated at several doses. At a total initiator dose of 28 and 5.6 mumol/mouse, NNK exhibited significant activity (P less than 0.005) inducing a 59% and 24% incidence of skin tumors, respectively. In this dose response bioassay, NNK at a total initiator dose of 28 mumol induced a 63% incidence (P less than 0.005) of lung adenomas. The numbers of lung adenomas induced at the lower doses employed were not significant. NNK, at a total initiation dose of 1.4 mumol, did not exhibit significant tumorigenic activity (P greater than 0.05). Analysis of DNA from the skin of mice treated with NNK using HPLC with fluorescence detection failed to detect O6- and N-methylguanine (O6-MG and N7-MG) adducts. These data indicate that NNK can exert a contact carcinogenic effect and suggest that mechanisms other than DNA methylation may be involved in its activation to a tumorigenic agent in mouse skin.     

Carcinogenicity of o-ethoxybenzamide in (C57BL/6N X C3H/HeN)F1 mice

The carcinogenicity of o-ethoxybenzamide (CAS: 938-73-8), which is also called ethenzamide and which is widely used as an antipyretic anodyne in Japan, was examined in 298 (C57BL/6N X C3H/HeN)F1 mice. Groups of males and females were fed a diet containing 0 (control), 0.4, or 1.2% o-ethoxybenzamide for 96 weeks and sacrificed at the 100th week. Among the male mice fed the higher dose of the drug, the total incidence of liver cell tumors was 68%, with 18% of the mice developing hepatocellular carcinomas; both yields were significantly higher than those in the controls. In o-ethoxybenzamide-treated male mice the multiplicities of the hepatic cell tumors were also significantly higher than the multiplicity of the hepatic tumor in male control mice. A dose-response relationship with regard to both incidence and multiplicities of hepatic cell tumors in male mice was observed. In female mice fed o-ethoxybenzamide the incidence and multiplicities of the liver cell tumors were increased compared to those of the controls, but statistical significance was observed only in the multiplicity of tumors in mice given the lower dose. In both sexes hepatic cell tumors developed earlier than in the controls. These results show that o-ethoxybenzamide enhances the development of hepatic cell tumors in male (C57BL/6N X C3H/HeN)F1 mice.     

Inhibitory effects of cinnamaldehyde on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung carcinogenesis in rasH2 mice.

Previously we reported a lack of modification by cinnamaldehyde (CNMA) of development of lung proliferative lesions induced by urethane in CB6F1-TgHras2 (rasH2) mice. In the present study, we re-evaluated CNMA effects using the same rasH2 strain and non-transgenic littermates initiated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Sixteen mice/strain/sex received intraperitoneal NNK injections at a dose of 3 mg/mouse once a week for 2 weeks followed by free feeding of commercial diet containing 5000 ppm CNMA for 26 weeks. Additional groups were maintained without NNK injection and/or CNMA feeding for 28 weeks. Lung tumors were induced by NNK in both rasH2 and non-Tg males and females at incidence ranging from 63 to 100%. CNMA treatment significantly reduced the combined incidence of adenomas and carcinomas from 86 to 31% in rasH2 males (P<0.05), but no significant influence was evident in females. The multiplicity of NNK-induced lung tumors was also significantly reduced in rasH2 males given CNMA (P<0.01). Similar effects were also observed in non-Tg females given CNMA after NNK initiation. The results of our study strongly indicate that CNMA is capable of inhibiting development of NNK-initiated pulmonary tumorigenesis in rasH2 and non-Tg mice.     

Comparative tumorigenicity and DNA methylation in F344 rats by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N-nitrosodimethylamine

The tumorigenic activities and DNA methylating abilities in F344 rats of the tobacco specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and the structurally related nitrosamine N-nitrosodimethylamine (NDMA) were compared. Groups of 30 male rats were given 60 s.c. injections of 0.0055 mmol/kg of either NNK or NDMA over a 20-week period (total dose, 0.33 mmol/kg). The experiment was terminated after 104 weeks. The numbers of rats with tumors were as follows for NNK and NDMA, respectively: liver, 10 and 6; lung 13 and 0; and nasal cavity, 6 and 1. NNK was significantly more tumorigenic than was NDMA toward the lung (P less than 0.01) and nasal cavity (P less than 0.05). Groups of rats were treated with a single s.c. injection of 0.39 mmol/kg or 0.055 mmol/kg of NNK or NDMA and the levels of 7-methylguanine and O6-methylguanine were measured in liver, lung, and nasal mucosa 1-48 h after treatment. In liver and lung, levels of 7-methylguanine and O6-methylguanine in DNA were 3-22 times (P less than 0.001) greater in NDMA treated rats than in NNK treated rats. Levels of methylation induced by NDMA and NNK in the nasal mucosa were similar. The results of this study demonstrate that NNK is a more potent tumorigen than NDMA in the F344 rat and suggest that DNA methylation alone does not account for its strong tumorigenicity in rat lung and nasal mucosa.     

Inhibitory effects of propolis granular A P C on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis in A/J mice

We examined the effect of propolis granular A. P. C on lung tumorigenesis in female A/J mice. Lung tumors were induced by the tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) administered in drinking water for 7 weeks in mice maintained on an AIN-76A semi-synthetic diet. Propolis granular A. P. C (100 mg/kg body wt.) was administered orally daily for 6 days/week from 1 week before NNK administration and throughout the experiment. Sixteen weeks after the NNK treatment, the mice were killed and the number of surface lung tumors was measured. The number of lung tumors in mice treated with NNK alone for 7 weeks (9.4 mg/mouse) was significantly more than in that observed in control mice. Propolis granular A. P. C significantly decreased the number of lung tumors induced by NNK. These results indicate that propolis granular A. P. C is effective in suppressing NNK-induced lung tumorigenesis in mice.     

Inhibitory effects of diallyl sulfide on the metabolism and tumorigenicity of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mouse lung.

Diallyl sulfide (DAS), a component of garlic oil, has been shown to inhibit tumorigenesis by several chemical carcinogens. Our previous work demonstrated that DAS inhibited the metabolic activation of carcinogenic nitrosamines, including the tobacco-specific 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), in rat lung and nasal mucosa microsomes. In the present study, the effects of DAS on the tumorigenicity and the metabolism of NNK in A/J mouse lung were examined. Female A/J mice at 7 weeks of age were pretreated with DAS (200 mg/kg body wt in corn oil, p.o) daily for 3 days. Two hours after the final DAS treatment, the mice were either given a single dose of NNK (2 mg/mouse, i.p.) and kept for an additional 16 weeks for determining the production of pulmonary tumors, or were killed immediately so as to measure the microsomal activity in metabolizing NNK. In comparison to the vehicle control group, DAS pretreatment significantly decreased the incidence of NNK-induced lung tumors (37.9 versus 100%) and the tumor multiplicity (0.6 versus 7.2 tumors/mouse). In pulmonary metabolism of NNK, DAS pretreatment reduced the rates of formation of keto aldehyde, keto alcohol, NNAL-N-oxide, and NNK-N-oxide by 70-90%. In addition, the formation of NNK oxidative metabolites from NNK in the liver microsomes from DAS-pretreated mice was remarkably reduced. DAS also inhibited the metabolism of NNK in mouse lung microsomes in vitro. These results demonstrate that DAS is an effective chemopreventive agent against NNK-induced lung tumorigenesis, probably by inhibiting the metabolic activation of NNK.     

Characterization of a glucuronide metabolite of 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its dose-dependent excretion in the urine of mice and rats

Following analysis by reversed-phase HPLC, a previously uncharacterized metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was found in the urine of A/J mice treated with NNK. Treatment with beta-glucuronidase converted the metabolite to a peak that co-eluted with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). Treatment with sulfatase or beta-glucuronidase plus saccharic acid 1,4-lactone did not change the retention time of the metabolite. These data suggested that the unknown metabolite was a glucuronic acid conjugate of NNAL. Upon isolation and purification of larger quantities of the metabolite from the urine of A/J mice, CD-1 mice and F344 rats, 1H and 13C NMR and MS confirmed that the unknown metabolite was 4-(methylnitrosamino)-1-(3-pyridyl)-1-butyl beta-D-glucopyranosiduronic acid (NNAL Glu). To determine the quantitative relationship between NNK dose and NNAL Glu production and to compare the importance of glucuronidation relative to other metabolic pathways, [5-3H]NNK was administered to F344 rats and A/J mice at doses of 500-0.005 mumol/kg. At 500 mumol/kg, NNAL Glu accounted for 22% of the total urinary excretion of NNK in A/J mice, and for 8% in F344 rats 48 h after dosing. The proportions of excreted glucuronide and NNAL decreased with diminishing doses of NNK, yielding undetectable levels of each metabolite in both mice and rats at a dose of 0.005 mumol/kg NNK. Since substantial amounts of metabolites formed via alpha-hydroxylation and N-oxidation pathways were observed at the lower doses of NNK, these data demonstrate that NNAL glucuronidation is a quantitatively unimportant metabolic pathway at low doses of NNK.     

Investigations of metabolic precursors to hemoglobin and DNA adducts of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

Levels of DNA and/or hemoglobin pyridyloxobutylation were compared in A/J mice or F344 rats treated with a single dose of [5-3H]4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone ([5-3H]NNK), [5-3H]4-hydroxy-1-(3-pyridyl)-1-butanone ([5-3H]4-HPB) or [5-3H]4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone ([5-3H]NNKOAc), a compound that generates the proposed pyridyloxobutylating agent in situ upon esterase hydrolysis. The lung and liver DNA samples isolated from A/J mice treated with the various compounds were subjected to acid hydrolysis and the hydrolysates were analyzed for the presence of [5-3H]4-HPB. No detectable levels were found in the lung DNA isolated from [5-3H]4-HPB-treated animals, whereas significant amounts of [5-3H]4-HPB were released from lung and liver DNA isolated from [5-3H]NNK- and [5-3H]NNKOAc-treated mice. The levels of total binding and [5-3H]4-HPB released from the globin isolated from these animals showed a similar trend. That is, low binding levels were detected in the globin isolated from [5-3H]4-HPB-treated animals and significantly higher levels of binding were detected in the globin from the [5-3H]NNKOAc- and [5-3H]NNK-treated animals. Comparable findings were obtained in the rat experiments. These studies clearly demonstrate that methyl hydroxylation of NNK leads to a species that is capable of reacting covalently with nucleophiles in DNA and protein. Thus, the levels of 4-HPB released from DNA and globin can be attributed to the activation of NNK and not to the direct binding of 4-HPB.     

K-ras mutations in lung tumors from A/J and A/JxTSG-p53 F1 mice treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and phenethyl isothiocyanate

The purpose of this study was to evaluate the effects of theloss of a p53 allele and phenethyl isothiocyanate (PEITC) pre-treatmenton the tumorigenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK) and K-ras mutation frequency in a hybrid mouse model.Male TSG-p53 ‘knock-out’ mice were bred with A/Jfemale mice to produce (A/JxTSG-p53) F₁ mice either homozygous(p53+/+) or heterozygous (p53+/–) for p53 alleles. Thesemice, together with female A/J mice, were treated at 6–8weeks of age with NNK or dosed with PEITC prior to administrationof NNK. The A/J mice treated with NNK had a 100% incidence oflung tumors, with 9.7 ± 3.4 tumors/mouse. A/J mice pre-treatedwith PEITC prior to NNK administration had 3.5 ± 2.1lung tumors/animal, although the incidence remained at 100%.In (A/JxTSG-p53 F₁ mice with either the p53(+/–) or p53(+/+)genotype PEITC pre-treatment significantly decreased tumor incidence(100 to 40 and 36%, respectively) and multiplicity (2.0 ±0.5 to 0.5 ± 0.4 and 2.1 ± 0.5 to 0.5 ±0.4, respectively), indicating that PEITC is an effective chemopreventiveagent in both A/J mice and (A/JxTSG-p53) F₁ mice. Analysis oflung tumor DNA from A/J mice treated with NNK or NNK/PEITC indicatedthat 15 of 17 (88%) and 20 of 23 (87%) of the tumors, respectively,contained G     

Effects of deuterium substitution on the tumorigenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in A/J mice

Bioassays and DNA-binding studies of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its analogs with deuterium substitution at the positions alpha to the nitrosamino group ([4,4-D2]NNK and [CD3]NNK) were carried out in A/J mice in order to assess the potential importance of DNA methylation or pyridyloxobutylation in lung tumor induction. The tumorigenic activities of the major NNK metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its analog with deuterium at the carbinol carbon ([1-D]NNAL) were also determined. Groups of A/J mice were given single i.p. injections of either 10 or 5 mumol of NNK, [4,4-D2]NNK, [CD3]NNK, NNAL and [1-D]NNAL, and were killed 16 weeks later. Lung tumor multiplicities were as follows in mice treated with 10 mumol: NNK, 7.3 +/- 3.5; [4,4-D2]NNK, 1.4 +/- 1.6; [CD3]NNK, 11.7 +/- 5.4; NNAL, 3.2 +/- 2.0; [1-D]NNAL, 3.2 +/- 2.0. Similar relative tumorigenic activities were observed in mice treated with 5 mumol of these compounds. These results demonstrated that [4,4-D2]NNK was less tumorigenic than NNK and [CD3]NNK was more tumorigenic than NNK. NNAL was less tumorigenic than NNK; substitution of deuterium at the carbinol carbon did not affect its activity. Levels of O6-methylguanine (O6-mG) were measured in pulmonary DNA of A/J mice treated with 10 mumol of NNK, [4,4-D2]NNK or [CD3]NNK, and killed 2 or 24 h later. O6-mG levels were lower in mice treated with [4,4-D2]NNK than in those treated with NNK; no difference in O6-mG levels was observed between those treated with NNK and [CD3]NNK. The results of this study support the hypothesis that O6-mG formation in pulmonary DNA is the key step in lung tumor induction by NNK in A/J mice.     

Carcinogenicity studies on the two tobacco-specific N-nitrosamines, N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

The tobacc-specific N-nitrosamines (TSNA) have been implicatedin oral cancer. However, except for one study using rats, nostudy has shown the ability of TSNA in inducing oral tumoursin experimental animals. We have studied the carcinogenic potentialsof N'-nitrcssonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK) in mice and hamsters, wherein the nitrosamines were administeredon the tongues of the mice and the cheek pouches of the hamstersto simulate the exposure conditions of humans. It was observedthat in Swiss and BALB/c male mice, both NNN and NNK inducedtumours of lung, forestomach and liver. However, no oral tumourswere induced in mice. The effect of vitamin A depletion wastested in Swiss male mice. It was found that a low vitamin Astatus did not alter the percentage incidence of tumours inducedby both nitrosamines to a significant extent. In the studiesusing Syrian golden hamsters, long-term treatment of NNK tohamster cheek pouch induced tumours in the lung, liver, stomachand cheek pouch. Subsequently, the effed of hydrogen peroxide(H₂O₂) on NNK-induced carcinogenicity in hamsters was studied.It was observed that simultaneous administration of NNK andH₂O₂ to the animals increased the incidence of cheek pouch tumours.Another pertinent observation was that even whena small initiatordase of NNK was given followed by the application of H₂O₂, avery significant increase in the tumour incidence was observed.This observation suggeststhat H₂O₂ could act as a promoter toNNK-induced carcinogenesis. In conclusion it may be stated thatboth NNN and NNK do not show any strain or species specificity.They failed to produce tumours at the site of application inmice but in hamsters few cheek pouch turnours were seen or wereinduced when NNK was applied alone. The cheek pouch tumour incidenceincreased when H₂O₂ was given concurrently or when applied fora long period after a low initiator dose of NNK was administeredin the cheek pouch.