Effect of dietary aromatic isothiocyanates fed subsequent to the administration of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone on lung tumorigenicity in mice

Naturally-occurring aromatic isothiocyanates, benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC), were tested for their post-treatment effects on lung tumorigenicity by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice. Mice at 7 weeks of age were administered a single i.p. dose of NNK (10 mumol/mouse). One week after NNK dosing, mice were placed on AIN-76A diet containing 1 or 3 mumol/g diet of BITC or PEITC. The control group was maintained on AIN-76A diet after NNK administration. Mice were killed 16 weeks after NNK treatment and lung adenomas were counted. The results showed mice fed control diet developed 7.8 tumors/mouse. Mice fed PEITC at concentrations of 1 or 3 mumol/g diet had 8.2 or 6.1 tumors/mouse, respectively. Feeding BITC at 1 mumol/g diet resulted in a tumor yield of 8.0 tumors/mouse, whereas BITC diet at 3 mumol/g diet gave 5.2 tumors/mouse, a small but significant inhibition. However, in the high BITC dose group, a loss in weight gain due to reduced food intake was noted. The results of this study showed that post-treatment of aromatic isothiocyanates had little, if any, effect on NNK lung tumorigenicity in A/J mice. This is in contrast to our previous findings in which pretreatment with PEITC greatly inhibited lung tumor induction by NNK in A/J mice and suggests that tumor inhibition by PEITC is due to inhibition of NNK metabolic activation.     

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.     

Pharmacokinetics of N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in laboratory animals

The pharmacokinetics of N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the Syrian golden hamster, the CD-1 mouse, and the baboon were compared to the pharmacokinetics in the Fischer rat. The formation and biological half-life of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), the major metabolite of NNK, was also studied in these animal species. The biological half-life of NNN in these 4 animal species ranged from 0.24 h to 3.06 h, that of NNK from 0.21 h to 0.43 h and NNAL from 0.48 h to 2.9 h. The pharmacokinetic data obtained in the baboon suggest that treatment with NNN and NNK causes an enzyme induction which accelerates the rate of elimination of these compounds.     

O6-methylguanine is a critical determinant of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone tumorigenesis in A/J mouse lung

The relative importance of the two alpha-hydroxylation pathways in the tumorigenicity of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), was examined in the A/J mouse lung. Methyl hydroxylation, which results in DNA pyridyloxobutylation, was investigated with 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc) and N'-nitrosonornicotine. Methylene hydroxylation, which leads to DNA methylation, was studied by using acetoxymethyl-methylnitrosamine (AMMN). The tumorigenic activities of these compounds were compared to that of 10 mumol NNK at doses that yielded similar or greater adduct levels 24 h after exposure. The methylating agent AMMN was more tumorigenic than the pyridyloxobutylating agents, NNKOAc and N'-nitrosonornicotine. NNKOAc enhanced the tumorigenic activity of AMMN when the two compounds were given in combination. These results suggested that DNA methylation was more important than DNA pyridyloxobutylation in A/J mouse lung tumor induction by NNK and that pyridyloxobutylation enhanced the activity of the methylation pathway. However, the tumorigenicity of 10 mumol NNK could not be reproduced by AMMN +/- NNKOAc at doses that yielded similar levels of DNA adducts 24 h after exposure. Therefore, a second study was conducted in which the persistence of O6-methylguanine in lung DNA following various doses of NNK or AMMN +/- NNKOAc was compared to the tumorigenicity of these treatments. A strong correlation was observed between lung tumor yield and levels of O6-methylguanine at 96 h for NNK and AMMN +/- NNKOAc (r = 0.98). The ability of NNKOAc to increase the tumorigenic activity of AMMN was attributed to its ability to enhance the persistence of O6-methylguanine in lung DNA. These results demonstrate that the formation and persistence of O6-methylguanine are critical events in the initiation of A/J mouse lung tumors by NNK. They also suggest that DNA pyridyloxobutylation by NNK can increase the persistence of this promutagenic base in lung DNA.     

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.     

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.     

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.     

Structure-activity relationships for inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone lung tumorigenesis by arylalkyl isothiocyanates in A/J mice

Phenethyl isothiocyanate (PEITC), 3-phenylpropyl isothiocyanate (PPITC), 4-phenylbutyl isothiocyanate (PBITC), and the newly synthesized 5-phenylpentyl isothiocyanate (PPeITC), 6-phenylhexyl isothiocyanate (PHITC), and 4-(3-pyridyl)butyl isothiocyanate (PyBITC) were tested for their abilities to inhibit tumorigenicity and DNA methylation induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the lungs of A/J mice. Mice were administered isothiocyanates by gavage for 4 consecutive days at doses of 5, 1, or 0.2 mumol/day prior to administration of 10 mumol of NNK by i.p. injection. Mice were sacrificed 16 weeks after NNK administration and pulmonary adenomas were quantitated, PEITC effectively inhibited NNK-induced lung tumors at a dose of 5 mumol/day but was not inhibitory at doses of 1 or 0.2 mumol/day. PPITC, PBITC, PPeITC, and PHITC were all considerably more potent inhibitors of NNK lung tumorigenesis than PEITC. While virtually no differences in inhibitory activity could be ascertained for PPITC, PBITC, and PPeITC, PHITC appeared to be the most potent tumor inhibitor of all of the compounds. At a dose of 0.2 mumol/day, PHITC pretreatment reduced tumor multiplicity by 85%. PyBITC, an analogue of both NNK and PBITC, was ineffective as an inhibitor. Using the same protocol, the compounds were found to have qualitatively similar inhibitory effects on NNK-induced DNA methylation when administered at 1 mumol/day. These results extend our previous findings that increased alkyl chain length enhances the inhibitory activity of an arylalkyl isothiocyanate toward NNK lung tumorigenesis and demonstrate the exceptional chemopreventive potentials of two new isothiocyanates, PPeITC and PHITC.     

K-ras and p53 point mutations in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced hamster lung tumors

Lung tumors were induced in Syrian golden hamsters by s.c. injectionof 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). After40 weeks lung tumor tissue was isolated. Administration of theNNK and exposure of the animals to an atmosphere of 65% oxygenresulted in a statistically significant reduction in tumor sizebut did not alter the histological tumor type or tumor incidencewhen compared with carcinogen treated animals maintained underambient air. Histologically, lung tumors had the morphologicfeatures of adenomas and adenocarcinomas with     

Tumorigenicity and metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol enantiomers and metabolites in the A/J mouse.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a major metabolite of the tobacco-specific pulmonary carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), has a chiral center but the tumorigenicity of the NNAL enantiomers has not been previously examined. In this study, we assessed the relative tumorigenic activities in the A/J mouse of NNK, racemic NNAL, (R)-NNAL, (S)-NNAL and several NNAL metabolites, including [4-(methylnitrosamino)-1-(3-pyridyl)but-(S)-1-yl] beta-O-D-gluco-siduronic acid [(S)-NNAL-Gluc], 4-(methylnitrosamino)-1-(3-pyridyl N-oxide)-1-butanol, 5-(3-pyridyl)-2-hydroxytetrahydrofuran, 4-(3-pyridyl)butane-1,4-diol and 2-(3-pyridyl) tetrahydrofuran. We also quantified urinary metabolites of racemic NNAL and its enantiomers and investigated their metabolism with A/J mouse liver and lung microsomes. Groups of female A/J mice were given a single i.p. injection of 20 micromol of each compound and killed 16 weeks later. Based on lung tumor multiplicity, (R)-NNAL (25.6 +/- 7.5 lung tumors/mouse) was as tumorigenic as NNK (25.3 +/- 9.8) and significantly more tumorigenic than racemic NNAL (12.1 +/- 5.6) or (S)-NNAL (8.2 +/- 3.3) (P < 0. 0001). None of the NNAL metabolites was tumorigenic. The major urinary metabolites of racemic NNAL and the NNAL enantiomers were 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid), NNAL-N-oxide and NNAL-Gluc, in addition to unchanged NNAL. Treatment with (R)-NNAL or (S)-NNAL gave predominantly (R)-hydroxy acid or (S)-hydroxy acid, respectively, as urinary metabolites. While treatment of mice with racemic or (S)-NNAL resulted in urinary excretion of (S)-NNAL-Gluc, treatment with (R)-NNAL gave both (R)-NNAL-Gluc and (S)-NNAL-Gluc in urine, apparently through the metabolic intermediacy of NNK. (S)-NNAL appeared to be a better substrate for glucuronidation than (R)-NNAL in the A/J mouse. Mouse liver and lung microsomes converted NNAL to products of alpha-hydroxylation, to NNAL-N-oxide, to adenosine dinucleotide phosphate adducts and to NNK. In lung microsomes, metabolic activation by alpha-hydroxylation of (R)-NNAL was significantly greater than that of (S)-NNAL. The results of this study provide a metabolic basis for the higher tumorigenicity of (R)-NNAL than (S)-NNAL in A/J mouse lung, namely preferential metabolic activation of (R)-NNAL in lung and preferential glucuronidation of (S)-NNAL.     

Comparative metabolism of N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by cultured F344 rat oral tissue and esophagus

The metabolism and DNA binding of N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by cultured F344 rat oral tissue and esophagus were investigated over a range of concentrations. The metabolites present in the culture media were separated by high performance liquid chromatography and were identified by comparison to standards. alpha-Hydroxylation of NNN, an esophageal carcinogen, was the major pathway for metabolism of this nitrosamine in both tissues. The metabolites formed from 2'-hydroxylation were between 3.0 and 3.9 times those formed from 5'-hydroxylation. 2'-Hydroxylation results in a pyridyloxobutylating species. DNA from esophagus cultured with [5-3H]NNN contained a pyridyloxobutylated adduct which upon acid hydrolysis released 3.8 pmol [5-3H]-4-hydroxy-1-(3-pyridyl)-1-butanone/mumol guanine. DNA from oral tissue cultured under the same conditions, where the extent of metabolism was the same, contained no measurable [5-3H]NNN DNA adduct. This suggests that factors, as yet unknown, cause the DNA of oral cavity tissue to be protected from pyridyloxobutylation by NNN. The metabolism of NNK by alpha-hydroxylation was as much as 10-fold less than the metabolism of NNN by this pathway in both tissues. alpha-Hydroxylation of NNK results in either a methylating species or a pyridyloxobutylating species. DNA from oral tissue cultured with [C3H3]NNK contained between 1.7 and 4.3 pmol 7-methylguanine/mumol guanine, respectively. No pyridyloxobutylated DNA (less than 0.2 pmol/mumol guanine) was detected in oral tissue incubated with [5-3H]NNK. The DNA from esophagi incubated with [C3H3]NNK contained no 7-methylguanine (less than 0.4 pmol/mumol guanine). The level of pyridyloxobutylation of DNA from esophagi incubated with [5-3H]NNK was 0.17 pmol/mumol guanine. The ability of the esophagus to metabolize NNN to a greater extent than NNK to a reactive species which pyridyloxobutylates DNA may be important in determining the carcinogenicity of NNN in the esophagus. In contrast, the metabolism of NNK to a methylating species by oral cavity tissue suggests that this tobacco-specific nitrosamine is important in tobacco-related oral cavity carcinogenesis.     

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.     

Mutagenesis induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N-nitrosonornicotine in lacZ upper aerodigestive tissue and liver and inhibition by green tea

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and nitrosonornicotine (NNN) were administered to lacZ mice (MutaMouse) at equal concentrations in drinking water (2 weeks at 0.1 followed by 2 weeks at 0.2 mg/ml) over a 4 week period, for a total estimated dose of 615 mg/kg) and mutagenesis in a number of organs was measured. For mutagenesis induced by NNK the potency order was: liver > lung> pooled oral tissues kidney > esophagus > tongue. The mutant fraction varied from approximately 6 to 40 mutants per 10(-5) plaque forming units This corresponds to approximately 2-13 times the background levels. A somewhat different pattern was observed with NNN, where the order was liver > esophagus oral tissue approximately tongue > lung > kidney. The potency of NNK was about twice that of NNN in liver and lung, but somewhat less in aerodigestive tract tissue. When compared with results previously obtained for a similar administered dose of benzo[a]pyrene, NNK was approximately 10-100% as mutagenic in the corresponding organs. Reported target organs for carcinogenesis by NNN and NNK in rodents were targets for mutagenesis, but mutagenesis was also observed at other sites, suggesting that these sites are initiated. The effect of green tea consumption on mutagenesis by NNK was also investigated. Green tea reduced mutagenesis by approximately 15-50% in liver, lung, pooled oral tissue and esophagus.     

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.     

Effects of long term dietary phenethyl isothiocyanate on the microsomal metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanol in F344 rats

Phenethyl isothiocyanate (PEITC), a cruciferous vegetable component, inhibits lung tumor induction by the tobacco specific nitrosamine, 4- (methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). To gain insight into the mechanism of PEITC lung tumor inhibition, we examined, in male F344 rats, the effects of dietary PEITC (3 micromol/g NIH-07 diet) in combination with NNK treatment (1.76 mg/kg, s.c., three times a week) for 4, 12 and 20 weeks on liver and lung microsomal metabolism of NNK and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a major metabolite of NNK and also a lung carcinogen. This was compared with rats fed NIH-07 diet, without PEITC, and treated with NNK alone or saline. The protocol was identical to that employed for inhibition of lung tumorigenesis by PEITC. We observed decreased rates of alpha- hydroxylation of NNK and NNAL in lung microsomes of 4-, 12- and 20-week PEITC + NNK treated rats compared with those treated with NNK or saline. NNK treatment alone also decreased lung alpha-methylene hydroxylation of NNK. Long-term NNK + PEITC administration did not significantly affect liver oxidative metabolism of NNK or NNAL, and did not affect the rate of glucuronidation of NNAL in liver microsomes when compared with rats treated with NNK or saline. Thus, PEITC selectively inhibited lung metabolic activation of NNK and NNAL. These results support the hypothesis that PEITC inhibits NNK-induced lung tumors by inhibiting metabolic activation of NNK in the lung. This study also demonstrated that PEITC inhibits lung alpha-hydroxylation of NNAL; this may play a role in PEITC inhibition of lung tumorigenesis by NNK.      

Effects of {alpha}-deuterium substitution on the tumorigenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in F344 rats

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and itsanalogues substituted with deuterium at the methylene carbon,4,4-dideutero-4-(methylnitrosamino)-1-(3-pyndyl)-1-butanone[4,4-D₂)NNK], and the methyl carbon, 4-(trideuteromethylnitrosamino)-1-(3-pyridyl)-1-butanone[(CD₃NNK) adjacent to the N-nitroso group were tested for tumorigenlcityin F344 rats. Each compound was administered by 60 s.c. injectioiisover a 20-week period such that the total doses were either1.0 or 0.33 mmol/kg. The experiment was terminated after 104weeks. Survival of the rats treated with the higher dose of(4,4-D₂)NNK was significantly less than survival the groupstreated with the same doses of NNK or (CD₃)NNK Target tissueswere liver, lung and nasal cavity for all three compounds. Thehigher dose of (4,4-D₂)NNK induced higher numbers of nasal tumorsand malignant nasal tumors than did NNK. The lower dose of (4,4-D₂)NNKinduced a higher number of nasal tumors than did NNK. No othersignificant differences in tumor incidence were observed. Theresults suggest that 4-(3-pyridyl)-4-oxobutylation DNA mightbe important in induction of nasal cavity tumors by NNK.     

Biliary excretion of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in the rat

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK) is a potent pancreas carcinogen in rats. The biliary excretionof NNK was therefore studied in anesthetized female Sprague— Dawley rats following i.p. administration of 0.7 µmol/kg[carbonyl-¹⁴C]NNK. The concentration of radioactivity peakedwithin 30 min and decreased thereafter exponentially. Cumulativeexcretion of radioactivity reached a plateau at 6–9% ofthe total dose. HPLC analysis revealed the presence of 4-hydroxy-4-(3-pyridyl)butyricacid (hydroxy acid), 4-oxo-4-(3-pyridyl)-butyric acid (ketoacid), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butyl ß-D-glucopyranosiduronicacid (NNAL Glu), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol(NNAL) and NNK. NNAL Glu was the major metabolite contributing34 ± 4% of total radioactivity in bile at 30 min and58 ± 4% at 5 h. The percentage of acidic metabolitesremained constant at     

Effects of aromatic isothiocyanates on tumorigenicity, O6-methylguanine formation, and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in A/J mouse lung

Phenethyl isothiocyanate (PEITC), benzyl isothiocyanate (BITC), and phenyl isothiocyanate (PITC) were tested for their abilities to inhibit lung tumorigenesis and O6-methylguanine formation in lung DNA induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice. Pretreatment with PEITC for 4 consecutive days at daily doses of 5 or 25 mumol inhibited tumor multiplicity induced by a single 10-mumol dose of NNK by approximately 70% or 97%, respectively. The 25-mumol daily dose of PEITC also reduced the percentage of animals that developed tumors by 70%. In contrast, both BITC and PITC failed to significantly reduce tumor multiplicity or the percentages of mice that developed tumors. Using an identical dosing regimen, parallel results were observed in the effects of these isothiocyanates on O6-methylguanine formation in the lung, in which PEITC at either dose resulted in considerable inhibition at 2 or 6 h after NNK administration, while BITC or PITC had little effect. PEITC was further tested for its ability to inhibit lung microsomal metabolism of NNK. A single administration of PEITC (5 or 25 mumol) resulted in 90% inhibition of NNK metabolism. These results in conjunction with recent results obtained using F344 rats firmly establish PEITC as an effective inhibitor of NNK lung tumorigenesis and suggest that the basis of this inhibition is the reduction of DNA adduct formation caused by the inhibition of enzymes responsible for NNK activation.     

Placental transfer of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone instilled intratracheally in Syrian golden hamsters.

The tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK) is a potent tumorigen in adult Syrian golden hamsters and an active transplacental carcinogen in this species. In this study, we have investigated the biodistribution and metabolism of NNK in maternal and fetal hamster tissues as a function of the dose and the time after NNK treatment. Hamsters on day 15 of gestation were instilled intratracheally with single doses (0.05-100 mg/kg) of [5-3H]NNK and sacrificed 30 min later or treated with a single dose (25 mg/kg) of [5-3H]NNK and sacrificed at various times (5-360 min) after treatment. Total radioactivity was quantified in maternal tissues (liver, lung, kidney, placenta, and stomach), in whole fetus and in fetal tissues (liver and lung). NNK and its metabolites were extracted from selected tissues (maternal plasma, amniotic fluid, fetal liver, and lung) and assayed by high-performance liquid chromatography-scintigraphy. Thirty min after treatment, radioactivity associated with NNK and its metabolites showed similar widespread tissue distribution patterns at all doses, with a linear dose relationship observed in whole fetus and fetal tissues. NNK levels detected in maternal plasma, amniotic fluid, fetal liver, and lung were also related linearly to dose. At high doses (25 mg/kg or more) of NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol was the major metabolite detected in maternal plasma. Pyridine N-oxidation of NNK predominated at the lowest doses (0.05 and 0.5 mg NNK/kg). The toxicokinetics of NNK demonstrated that this carcinogen is rapidly absorbed from the maternal lung (less than 5 min), metabolized mainly to 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanol, and quickly distributed into the maternal-fetal compartment. Both NNK and its main metabolite 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanol were eliminated slowly from the amniotic fluid, with levels still detectable up to 6 h after NNK treatment. These results demonstrated that NNK instilled intratracheally in pregnant hamsters crossed the placental barrier even at low doses. Moreover, NNK quickly reached fetal tissues and amniotic fluid and was eliminated slowly from these tissues, resulting in an extended exposure of the fetus to this tobacco-specific carcinogen.     

Dose-response study of DNA and hemoglobin adduct formation by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in F344 rats

Levels of hemoglobin adducts and DNA adducts were measured in F344 rats after 4 consecutive daily i.p. injections of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The dose range was from 3 to 10,000 micrograms/kg/day. [5(-3)H]NNK and [C3H3]NNK were used to measure pyridyloxobutylation and methylation, in both globin and DNA, respectively. In globin, the level of binding increased linearly with dose. Total binding of [5(-3)H] NNK to globin was 3.2 to 8900 fmol/mg and total binding of [C3H3]NNK was 3.5 to 20,000 fmol/mg. The extents of pyridyloxobutylation of both DNA and globin were determined by measuring the amounts of 4-hydroxy-1-(3-pyridyl)-1-butanone released from each, over the dose range 15-5000 micrograms/kg/day. The levels of 4-hydroxy-1-(3-pyridyl)-1-butanone released were 3.2-650 fmol/mg globin, 18-3400 fmol/mg liver DNA, and 58-2180 fmol/mg lung DNA. The extents of DNA methylation in both lung and liver were greater than pyridyloxobutylation. When the dose range was 3-5000 micrograms/kg/day, the levels of 7-methylguanine were 0.22-246 pmol/mumol guanine (149-167,000 fmol/mg) in liver DNA and 0.23-78 pmol/mumol guanine (160-53,000 fmol/mg) in lung DNA. In the lung, the ratio of methylation to pyridyloxobutylation decreased as the dose decreased. In contrast to globin adduct formation, DNA adduct formation did not increase linearly with dose; adduct formation was greater at lower doses than would have been predicted by extrapolation from higher doses. Thus the results of this study demonstrate that there was not a linear relationship between globin adduct formation, neither pyridyloxobutylation nor methylation, and DNA adduct formation in the liver or the lung of rats treated with NNK.