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PubMed1990

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.

Animals↗
PubMed1990

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.

Adenoma↗
PubMed1990

Effect of nicotine and tobacco-specific nitrosamines on the metabolism of N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by rat oral tissue.

The effect of N'-nitrosoanatabine (NAT) and nicotine on the metabolism of N'-nitrosonornicotine (NNN) and 4-(methyl-nitrosamino)-1-(3- pyridyl)-1-butanone (NNK) by cultured rat oral tissue was investigated. The effect of NNN on NNK metabolism and the effect of NNK on NNN metabolism was also determined. NNK inhibited NNN metabolism more than NNN inhibited NNK metabolism. NAT inhibited the metabolism of NNK but not of NNN. Nicotine, which is present at greater than 500 times the levels of NNN and NNK in smokeless tobacco, inhibited the metabolism of both nitrosamines. Inhibition of 1 microM NNN metabolism was greater than that of 1 microM NNK when the concentration of nicotine was 1, 10 or 100 microM. Nicotine at 100 microM inhibited the formation of all metabolites of NNN by 85-92%. These results suggest that NNN and nicotine may be metabolized by a common enzyme.

Animals↗
PubMed1991

Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone carcinogenesis in mice by D-limonene and citrus fruit oils.

The effects of D-limonene and citrus fruit oils, i.e. orange oil and lemon oil, on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced neoplasia of the lungs and forestomach of female A/J mice were investigated. D-Limonene and the citrus fruit oils given p.o. 1 h prior to NNK, also administered p.o., inhibited pulmonary adenoma formation and the occurrence of forestomach tumors. In an additional experiment, D-limonene given p.o. 1 h prior to NNK administered i.p. again showed pronounced inhibition of pulmonary adenoma formation. This study provides additional data demonstrating that non-nutrient constituents of the diet can inhibit carcinogen-induced neoplasia when administered at a short time interval prior to carcinogen challenge.

Adenoma↗
PubMed1991

Comparison of DNA alkali-labile sites induced by 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone and 4-oxo-4-(3-pyridyl)butanal in rat hepatocytes.

4-Oxo-4-(3-pyridyl)butanal (OPB) is an aldehyde formed during the activation of the tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Using the DNA alkaline elution technique, the properties of DNA alkali-labile sites induced in the isolated rat hepatocytes by NNK and OPB were compared. The DNA single-strand break (SSB) frequencies in vitro, as measured by the elution rate (ER), ranged from 0.015 to 0.479 and were proportional (r2 = 0.991) to the dose (0-2 mM) of OPB. These concentrations, however, were slightly cytotoxic. For example, the LC50 after 4 h of exposure was 2.8 mM. This suggests that OPB-induced DNA SSB result from additive effects of OPB-DNA interaction and the indirect DNA damage associated with OPB cytotoxicity. NNK induced a significant and dose-dependent increase of DNA fragmentation at concentrations ranging from 0.5 to 5.0 mM with ER values ranging from 0.012 to 0.274 (r2 = 0.951). Genotoxicity as measured by the DNA-damaging potency coefficient (DDP) was 810, 345, 131 and 75 for N-methyl-N-nitrosourea (MNU), N-nitrosodimethylamine (NDMA), OPB and NNK respectively. Both MNU- and NNK-induced DNA lesions showed increased lability with increased pH (from 12.1 to 12.5) of the eluting buffer (r2 = 0.979 and 0.967 respectively). In contrast, the number of OPB-induced labile sites were not affected by increases in the pH. These results indicate that OPB is not the metabolite contributing the majority of alkali-labile sites generated by NNK. The filter elution procedure was used to study the in vitro rejoining of SSB in DNA induced by NNK. The extent of DNA SSB rejoining after 18 h of culture of hepatocytes in NNK-free medium were dependent on the concentration of NNK (0.5, 2.0 and 5.0 mM) and ranged from 50 to 90%. Rats were injected s.c. with NNK (0.39 mmol/kg). SSB frequency in liver DNA increased rapidly and reached a maximum 12 h after injection. DNA SSB frequency declined during the next 2 weeks with biphasic kinetics. The fast phase (75% rejoining of DNA SSB between 12 h and 2 days) was followed by a slow one (25% of DNA SSB maintained during the next 5 days but not present after 2 weeks). The results of this study better define the role of OPB-induced DNA damage. The persistence of DNA SSB in the liver of NNK-treated rats reflects the inability of this tissue to repair all DNA lesions.

Analysis of Variance↗
PubMed1991

Mutational specificities of environmental carcinogens in the lacI gene of Escherichia coli: IV. The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

We have determined the mutational specificity of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by the characterization of 58 induced Escherichia coli lacI-d mutants at the DNA sequence level. Metabolic activation of NNK was carried out using the S9 fraction from Aroclor 1254-treated rats. G:C----A:T transitions dominated the spectrum, accounting for 55% of the mutations recovered. The other base substitutions recovered include three A:T----G:C transitions as well as two A:T----T:A, three A:T----C:G, five G:C----C:G and five G:C----T:A transversions. Other classes of mutational events included two deletions, three duplications and three frame-shifts. The complexity of the NNK mutational spectrum appears consistent with a model that this compound induces mutations by both the methylation and the pyridoloxobutylation of DNA.

Bacterial Proteins↗
PubMed1991

A tobacco smoke-derived nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, is activated by multiple human cytochrome P450s including the polymorphic human cytochrome P4502D6.

We have developed a human B-lymphoblastoid cell line, designated 2D6/Hol, which stably expresses human cytochrome P450 CYP2D6 cDNA. This cell line exhibits bufuralol 1'-hydroxylase activity and immunologically detectable CYP2D6 protein. The specific activity of (+)-bufuralol 1'-hydroxylase in microsomes from 2D6/Hol cells was comparable to that observed in human liver microsomes. This cell line was used to examine the mutagenicity activation of three tobacco smoke-derived nitrosamines, N-nitrosonornicotine (NNN), 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal) (NNA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), by CYP2D6. Exposure of 2D6/Hol cells to NNK concentrations of 30-90 micrograms/ml induced a concentration-dependent decrease in relative survival and increase in mutant fraction at the hypoxanthine guanine phosphoribosyl transferase (hprt) locus. In contrast, NNK was non-mutagenic and non-cytotoxic to control cells at exposure concentrations up to 150 micrograms/ml. NNK mutagenicity in 2D6/Hol cells was compared to the responses observed in isogenic cell lines expressing human CYP1A2 (1A2/Hol), human CYP2A3 (2A3/Hol) and human CYP2E1 (2E1/Hol). These three additional human cytochrome P450-expressing cell lines were also found to be sensitive to NNK-induced mutagenicity and cytotoxicity. We found no evidence for CYP2D6-mediated activation of NNN or NNA. NNN was non-cytotoxic and non-mutagenic to both control and 2D6/Hol cells. NNA was equally cytotoxic and mutagenic to control cells and 2D6/Hol cells. The activation of NNA to a mutagen may have been carried out by P450 native to the AHH-1 TK +/- cell line. The 2D6/Hol cell line, in conjunction with the control cell line and other isogenic cell lines expressing other human cytochrome P450 cDNAs provides a useful system for the examination of the role of the polymorphic CYP2D6 in human procarcinogen activation and drug metabolism.

Biotransformation↗
PubMed1991

Inhibition of the metabolism and genotoxicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in rat hepatocytes by (+)-catechin.

(+)-Catechin is a plant flavonoid which decreases the mutagenicity of several mutagens and carcinogens. In this study, we have investigated how (+)-catechin could inhibit the metabolism and DNA damage induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific carcinogen. Addition of 5 to 1000 microM (+)-catechin to rat hepatocytes cultured with 4.5 mM NNK caused a concentration-dependent reduction of alpha-carbon hydroxylation which is the activation pathway of NNK. Under the same conditions, (+)-catechin had a less significant effect on pyridine N-oxidation, which is a deactivation pathway. Reduction of the carbonyl group of NNK was not inhibited by (+)-catechin. We had previously shown that NNK induced single-strand breaks (SSBs) in primary culture of hepatocytes. In this study, we observed that 1.0 mM (+)-catechin inhibited the DNA SSBs induced by 1 mM NNK by 31%. With 1 mM N-nitrosodimethylamine, the inhibition of DNA SSBs was 30%. We concluded that (+)-catechin selectively inhibits the enzymes involved in the activation of NNK. Rats were gavaged with (+)-catechin (1.5 mmol/kg), injected s.c. 1 h later with NNK (0.39 mmol/kg) and killed 4 h after NNK treatment. (+)-Catechin significantly reduced DNA SSBs induced by NNK. Rats were injected s.c. with 0.39 mmol/kg NNK. (+)-Catechin reduced the methylation of liver DNA at the O6-guanine and N-7 guanine sites by 28 and 34% respectively. These results demonstrate that (+)-catechin inhibits the formation of DNA-damaging intermediates by selectively impairing the enzymatic activation of NNK. They suggest that (+)-catechin could be an effective preventive agent against NNK hepatocarcinogenicity.

Animals↗
PubMed1992

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 excretion of NNK was therefore studied in anesthetized female Sprague-Dawley rats following i.p. administration of 0.7 mumol/kg [carbonyl-14C]NNK. The concentration of radioactivity peaked within 30 min and decreased thereafter exponentially. Cumulative excretion of radioactivity reached a plateau at 6-9% of the total dose. HPLC analysis revealed the presence of 4-hydroxy-4-(3-pyridyl)butyric acid (hydroxy acid), 4-oxo-4-(3-pyridyl)-butyric acid (keto acid), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butyl beta-D-glucopyranosiduronic acid (NNAL Glu), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and NNK. NNAL Glu was the major metabolite contributing 34 +/- 4% of total radioactivity in bile at 30 min and 58 +/- 4% at 5 h. The percentage of acidic metabolites remained constant at approximately 20%. In contrast, the percentage of NNK and NNAL decreased within the first 2 h to < 5% and < 10% respectively. The elimination kinetics of NNK and its metabolites fitted into a one-compartment model with a half-life of 37 min for NNK, 52 min for NNAL and 110 min for NNAL Glu and acidic metabolites. In three rats dosed with 240 mumol/kg NNK i.p., the concentration of radioactivity peaked after 1-2 h and decreased very slowly thereafter. After 5-8 h a total of 12-17% of the dose has been excreted in the bile with no indication of a plateau. At all time points NNAL Glu was the major metabolite contributing up to 95% of total radioactivity in bile. The percentage of acidic metabolites was < 5% throughout the experiment. Whereas NNK contributed one-third of the radioactivity at 30 min and decreased rapidly, the percentage of NNAL in bile remained rather constant at approximately 5-10%. In conclusion, the detection of NNK, NNAL and NNAL Glu gives support to the hypothesis that tobacco-specific carcinogens could reach the pancreas retrograde from the bile, especially at high NNK concentrations.

Animals↗
PubMed1992

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.

Animals↗
PubMed1992

Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a tobacco-specific carcinogen, by rabbit nasal microsomes and cytochrome P450s NMa and NMb.

Rabbit nasal olfactory and respiratory microsomes were found to catalyze the alpha-hydroxylation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) with specific activities of 262 and 136 pmol/min/mg protein in the formation of keto aldehyde, and of 318 and 190 pmol/min/mg protein in the formation of keto alcohol respectively. The formation of NNK-N-oxide was observed in experiments with rabbit olfactory and respiratory microsomes, but not with rat nasal microsomes. However, the rat nasal microsomes had higher activity in catalyzing the alpha-hydroxylation of NNK. In a reconstituted system, rabbit P450NMa, a major constitutive P450 isozyme in nasal microsomes, displayed high activities in the formation of the keto aldehyde and the keto alcohol with apparent Km values of 15 and 9 microM respectively. In comparison, rabbit olfactory specific P450NMb had a low activity in catalyzing the formation of keto aldehyde (Km = 186 microM) and no activity in the formation of keto alcohol. The P450NMa-catalyzed oxidation of NNK was inhibited by nicotine and diallyl sulfide. Kinetic studies indicated that nicotine is a competitive inhibitor. These results demonstrate that enzymes in rabbit nasal microsomes, especially P450NMa, efficiently catalyze the bioactivation of NNK.

Animals↗
PubMed1992

Effects of sulindac and oltipraz on the tumorigenicity of 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone in A/J mouse lung.

The efficacies of the non-steroidal, anti-inflammatory drug sulindac and the schistosomicidal agent oltipraz in inhibiting lung tumorigenesis was measured in A/J mice. Lung tumors (15.7 tumors/mouse) were induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK; 9.1 mg/mouse) administered in drinking water for 7 weeks. Feeding mice with sulindac (123 mg/kg diet), 2 weeks before carcinogen treatment until they were killed reduced tumor multiplicity by 53%. Oltipraz (250 mg/kg diet), however, has no effect on tumorigenesis. The absorption and metabolism of NNK were compared in the stomachs and intestines isolated from mice fed AIN-76A diet or sulindac + diet. Sulindac had no effect on alpha-carbon hydroxylation, pyridine N-oxidation or carbonyl reduction of NNK. Mouse lung explants were cultured with 4.7 microM [5-3H]NNK for 4 or 8 h. The addition of 1 mM sulindac to the culture medium reduces the alpha-carbon hydroxylation and pyridine N-oxidation of NNK. However, the administration of sulindac in the diet prior to the excision of the lung explants had no effect on these two metabolic pathways. We compared the levels of sulindac and its sulfide and sulfone metabolites in the lungs, livers and plasma of mice fed an AIN-76A diet containing 130 mg sulindac/kg for 2 weeks. The sulfide metabolite was the most abundant of the three compounds in plasma (17.6 pmol/microliters) and liver tissues (17.7 pmol/mg) but it could not be detected in lung tissues. These results show that non-steroidal anti-inflammatory drugs constitute a new class of chemopreventive agents in lung tumorigenesis. The tumor chemopreventive activity of sulindac is not mediated by the sulfide metabolite responsible for its anti-inflammatory activity.

Adenoma↗
PubMed1992

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.

Allyl Compounds↗
PubMed1992

Increased 8-oxodeoxyguanosine levels in lung DNA of A/J mice and F344 rats treated with the tobacco-specific nitrosamine 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone.

Evidence for the involvement of free radicals in nitrosamine carcinogenesis comes mainly from increased lipid peroxidation as a result of nitrosamine treatment. More direct evidence for nitrosamine-induced oxidative DNA damage has been lacking. In this study we examined the levels of 8-oxodeoxyguanosine or 8-hydroxydeoxyguanosine (8-OH-dG) in tissue DNA of mice and rats treated with the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Multiple doses of NNK (0.25 or 0.50 mg/mouse, 3 times weekly for 3 weeks) administered by gavage resulted in a significant elevation of 8-OH-dG in lung DNA, from 2.1 to 3.8 adducts/10(5) dG for the lower dose or to 6.6 adducts/10(5) dG for the higher dose, 2 h after the last NNK administration. A single dose treatment of NNK by gavage (4 mg/mouse) also resulted in an increase of this lesion in the lung DNA, however, the increase was not statistically significant. In liver, however, the increase was only significant by multiple doses at the higher dose, from 2.3 to 3.4 adducts/10(5) dG. This lesion appeared to be repaired efficiently. At 4 and 24 h after NNK treatment, the 8-OH-dG levels declined to the basal levels in both liver and lung. A single dose of NNK (20 mg/rat) also caused a significant increase of 8-OH-dG from 3.0 to 5.1 adducts/10(5) dG in rat lung DNA. An increase of 8-OH-dG in liver DNA was also seen, however, it was not statistically significant. Unlike the liver and the lung, the 8-OH-dG levels in rat kidney, a non-target tissue, were inert to NNK treatment. These results provide for the first time direct evidence supporting the role of oxidative DNA damage in NNK lung tumorigenesis.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1992

Kinetics and enzyme involvement in the metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in microsomes of rat lung and nasal mucosa.

The rat lung and nasal cavity are two target organs for carcinogenesis by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In order to characterize further the enzymes involved in the bioactivation of NNK, detailed kinetic and inhibitory studies were conducted with rat lung and nasal mucosa microsomes, and the results were compared with previous studies. The enzymes in rat lung microsomes catalyzed the alpha-hydroxylation, pyridine N-oxidation and carbonyl reduction of NNK. The apparent Km for the formation of the NNK-derived keto aldehyde, NNK-N-oxide, the NNK-derived keto alcohol and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol were 28.8, 10.4, 7.0 and 178.1 microM respectively. In rat nasal microsomes, alpha-hydroxylation was the predominant pathway and the rate was approximately 200 times higher than that in lung microsomes. The apparent Kms for keto aldehyde and keto alcohol formation in rat nasal microsomes were 9.6 and 10.1 microM respectively. The cytochrome P450 inhibitors metyrapone and carbon monoxide markedly inhibited the metabolism of NNK in both rat lung and nasal microsomes. In rat lung microsomes, alpha-naphthoflavone and monospecific antibodies against P450s 1A2, 2A1 and 2B1 inhibited the formation of keto aldehyde by 39, 46, 64 and 23% respectively. In rat nasal microsomes, alpha-naphthoflavone and antibodies against P450s 1A2, 2A1 and 3A inhibited the metabolism of NNK by 80, 35, 20 and 14% respectively. The results indicate that cytochromes P450 play a major role in the metabolic activation of NNK in rat lung and nasal microsomes, and that there are tissue-related differences in NNK metabolism.

Animals↗
PubMed1992

Modulation by glutathione of DNA strand breaks induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and its aldehyde metabolites in rat hepatocytes.

Activation of the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) produced methylating species and two aldehydes: formaldehyde and 4-oxo-4-(3-pyridyl)-butanal (OPB). We investigated the modulation by glutathione of single-strand breaks (SSB) generated by N-methyl-N-nitrosourea (MNU) and the two aldehydes. Hepatocytes were simultaneously exposed to 0.2 mM MNU and to 0-2.00 mM formaldehyde or OPB for 4 h. Both aldehydes induced SSB in a dose-dependent manner. Formaldehyde and OPB exerted a synergistic effect on the formation of DNA SSB by MNU. It is postulated that both aldehydes interfere with DNA repair processes and thus increase the genotoxic effect of DNA methylating species. We investigated whether glutathione (GSH) could protect DNA from NNK-derived intermediates. Formaldehyde (2 mM) and OPB (2 mM) decreased intracellular GSH contents to 60 and 86% of control respectively. DL-Buthionine-[S,R]-sulfoximine (BSO) treatment reduced the GSH contents of hepatocytes to 19% of control but did not reduce the content of cytochrome P450 nor the metabolism of NNK. The frequency of DNA SSB induced by NNK, formaldehyde or OPB was significantly higher in GSH-depleted hepatocytes. GSH repletion with GSH monoethyl ester returned NNK-induced SSB to its initial frequency. OPB but not NNK nor formaldehyde induced double-strand breaks. We conclude that OPB and formaldehyde inhibit the repair of DNA damage induced by methylating species and that GSH reduces the level of DNA damage induced by NNK-derived reactive metabolites.

Analysis of Variance↗
PubMed1993

Metabolism and macromolecular interaction of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in cultured explants and epithelial cells of human buccal mucosa.

Metabolism and macromolecular interaction of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were studied in human buccal mucosa in vitro. Microautoradiographic analysis of [5-3H]NNK-exposed explant cultures demonstrated a uniform distribution of bound radioactivity in the mucosal epithelium, without significant binding in the underlying connective tissue. The metabolism of [5-3H]NNK at concentrations of both 6 and 100 microM resulted in seven identified metabolites in both explant and epithelial cell cultures. Formation of 4-(methylnitrosamino)-1-(3-pyridyl)butan-1-ol by carbonyl reduction of NNK accounted for almost 95% of the total metabolism, whereas the proportions of other metabolites obtained by alpha-carbon hydroxylation and pyridine N-oxidation reactions varied from undetectable levels to approximately 2% in both experimental systems. A positive correlation between concentration and the metabolic route associated with the formation of DNA methylating intermediates (alpha-hydroxylation at the N-methylene carbon) was found, i.e. when the concentration of NNK was raised from 6 to 100 microM, keto-acid formation which in part reflects DNA methylation was increased preferentially over ketoalcohol production, an index of DNA pyridyloxobutylation. Both the total rate of NNK metabolism and the amount of protein adducts were higher in cells from primary cultures up to the third passage than in explants cultured for 1 day. Between 10(-9) to 10(-4) M, neither NNK nor its precursor alkaloid nicotine affected the colony forming efficiency of normal and tumorous buccal epithelial cells, although at 10(-3) M each agent inhibited this function. Taken together, the results demonstrate the capability of human buccal mucosal epithelium to metabolize NNK by three major pathways, including those involved in the formation of adducts with cellular macromolecules.

Autoradiography↗
PubMed1993

Metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in the patas monkey: pharmacokinetics and characterization of glucuronide metabolites.

The metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was examined in the patas monkey, in order to provide further information about NNK metabolic pathways in primates. Female patas monkeys were given i.v. injections of [5-3H]NNK, and metabolites in serum and urine were analyzed by HPLC. Metabolism by alpha-hydroxylation of NNK was rapid and extensive, and the products of this pathway, 4-hydroxy-4-(3-pyridyl)butyric acid and 4-oxo-4-(3-pyridyl) butyric acid, accounted for a relatively large proportion of serum and urinary metabolites at all time points. This is significant because the formation of these products is associated with modification of DNA by NNK. The other major metabolic pathway was carbonyl reduction to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which detected both unconjugated and diastereomeric O-glucuronides. One of these glucuronides had been previously identified in rat urine, but the other diastereomer, which was the more prevalent of the two in serum and urine, had not been observed in studies of NNK metabolism in rodents. It was characterized by its spectral properties, by enzymatic hydrolysis to NNAL, and by derivatization of the released NNAL enantiomer with (R)-(+)-alpha-methylbenzylisocyanate. The two NNAL glucuronides accounted for 15-20% of the urinary metabolites in monkeys given 0.1 micrograms/kg NNK, which is similar to a smoker's dose, suggesting their use as dosimeters of NNK exposure in humans. Pharmacokinetic parameters were consistent with those observed in previous studies of nitrosamines, and varied predictably with body weight of five species. The results of this study have provided new insights relevant to assessing human metabolism of NNK.

Animals↗