Metabolic activation and detoxification of tobacco-specific nitrosamines--a model for cancer prevention strategies.
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Animals↗
Biomedical subjects
S S Hecht
Publications and source records attributed to S S Hecht.
Tobacco-specific nitrosamines.
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Animals↗
A tobacco-specific lung carcinogen in the urine of men exposed to cigarette smoke.
BACKGROUND: Environmental tobacco smoke has been classified by the Environmental Protection Agency as a carcinogen causally associated with lung cancer in adults, but there have been no reports of lung carcinogens or their metabolites in the body fluids or tissues of nonsmokers exposed to environmental tobacco smoke. METHODS: Five male nonsmokers were exposed to sidestream cigarette smoke generated by machine smoking of reference cigarettes for 180 minutes on each of two days, six months apart. Sidestream smoke is the smoke that originates from the smoldering end of a cigarette between puffs. Twenty-four-hour urine samples were collected before and after exposure. The urine samples were analyzed for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronide, which are metabolites of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a powerful lung carcinogen in rodents. NNAL is also a lung carcinogen in rodents. RESULTS: The urinary excretion of the metabolites increased after exposure to sidestream smoke in all the men. The mean (+/- SD) amount of NNAL and NNAL glucuronide was significantly higher after exposure than at base line (33.9 +/- 20.0 vs. 8.4 +/- 11.2 ng per 24 hours [127 +/- 74 vs. 31 +/- 41 pmol per day], P < 0.001) and was correlated with urinary cotinine excretion (r = 0.89, P < 0.001). The nicotine concentrations in the air to which the men were exposed were comparable to those in a heavily smoke-polluted bar. CONCLUSIONS: Nonsmokers exposed to sidestream cigarette smoke take up and metabolize a lung carcinogen, which provides experimental support for the proposal that environmental tobacco smoke can cause lung cancer.
Adult↗
Effects of isothiocyanates on tumorigenesis by benzo[a]pyrene in murine tumor models.
Previous studies have shown that benzyl isothiocyanate (BITC) inhibited lung tumorigenesis induced in A/J mice by benzo[a]pyrene (BaP), but other experiments using a somewhat different protocol demonstrated that phenethyl isothiocyanate (PEITC) had no effect on lung tumorigenesis induced by BaP in this strain. In contrast, PEITC but not BITC had been shown to inhibit lung tumorigenesis induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice. Therefore, one goal of this study was to directly compare the chemopreventive activities of BITC and PEITC on BaP-induced lung tumorigenesis in A/J mice. In the same experiment we also compared the tumorigenic activities of BaP and NNK. Either BITC or PEITC was administered by gavage 15 min before gavage of BaP. This regimen was carried out three times at 2-week intervals, and the mice were sacrificed 26 weeks after the first treatment. As assessed by tumor multiplicity, BITC but not PEITC significantly inhibited lung tumorigenesis by BaP, whereas PEITC but not BITC significantly inhibited forestomach tumorigenesis. Comparison of the tumorigenic activities of NNK and BaP demonstrated that NNK was about ten times more potent than BaP as a lung tumorigen, while BaP but not NNK induced forestomach tumors. In a second set of experiments we evaluated the effects of isothiocyanates on the mouse skin tumor-initiating activity of BaP. The isothiocyanates tested were BITC, PEITC, 6-phenylhexyl isothiocyanate (PHITC) and a series of isothiocyanates structurally related to polynuclear aromatic hydrocarbons: 9-phenanthryl isothiocyanate (9-PhenITC), 9-phenanthrylmethyl isothiocyanate (9-PhenMeITC), 6-chrysenyl isothiocyanate (6-ChrysITC) and 6-benzo[a]pyrenyl isothiocyanate (6-BaPITC). None of the isothiocyanates inhibited tumor development by BaP, and three of them--PHITC, 9-PhenITC and 9- PhenMeITC--enhanced skin tumor multiplicity. Taken together with available literature data, the results of this study suggest that different isothiocyanates selectively inhibit cytochrome P450 enzymes involved in the metabolic activation or detoxification of BaP and therefore have differing effects on BaP tumorigenesis.
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Induction of mammary cancer by 6-nitrochrysene in female CD rats.
6-Nitrochrysene (6-NC) is a pollutant generated in diesel exhaust. In order to study its propensity to induce mammary cancer, we injected 6-NC into the mammary glands of female CD rats. 4-Nitropyrene (4-NP), the most active mammary carcinogen among the mononitropyrene isomers, was used as a positive control (K. Imaida et al., Cancer Res., 51: 2902-2907, 1991). A total of 2.04 mumol of each compound in dimethyl sulfoxide was injected into each of the six mammary glands on the left side of weanling rats. The corresponding glands on the right side received injections of dimethyl sulfoxide. The thoracic glands were treated on Day 1 and those located in the inguinal area were treated on Day 2. Rats were sacrificed after 43 wk. 6-NC induced fibroadenomas, adenocarcinomas, and spindle cell sarcomas of the mammary glands in a high percentage of the rats. The numbers of animals with mammary tumors and the numbers of malignant tumors were significantly higher in the group treated with 6-NC than in those receiving 4-NP or dimethyl sulfoxide alone. The results of this study, taken together with those of previous bioassays, demonstrate the remarkable activity of 6-NC as a mammary, colon, and lung carcinogen in rodents.
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Pyridyloxobutyl DNA adducts inhibit the repair of O6-methylguanine.
The carcinogenic tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), both methylates and pyridyloxobutylates DNA. O6-Methylguanine (O6-mG) persistence has been correlated to NNK-induced lung tumor formation in A/J mice. The pyridyloxobutylation pathway enhances the tumorigenicity of the methylation pathway. In this paper we test the hypothesis that DNA pyridyloxobutylation increases O6-mG persistence by inhibiting the repair protein O6-alkylguanine-DNA alkyltransferase (AGT). Pyridyloxobutylated DNA was generated by reacting calf thymus DNA with the model pyridyloxobutylating agent 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc) in the presence of esterase. The alkylated DNA inhibited the ability of partially purified rat liver AGT to repair O6-mG when it was incubated with AGT prior to the addition of 3H-methylated DNA. The extent of inhibition was dependent on the amount of NNKOAc reacted with DNA. The ability of NNKOAc-treated DNA to inhibit AGT was destroyed when the DNA was subjected to neutral thermal hydrolysis. Approximately 1 pmol of AGT was inhibited for every 25 to 50 pmol of 4-hydroxy-1-(3-pyridyl)-1-butanone- releasing adducts present in NNKOAc-treated DNA. The inhibitory activity of this alkylated DNA was relatively stable under physiological conditions (pH 7.4, 37 degrees C). Only 13% of the AGT reactive activity was lost after 7 days. When pyridyloxobutylated DNA was incubated simultaneously with 3H-methylated DNA and AGT, a significant reduction in [3H]methyl transfer to AGT was observed. The levels of reduction were similar to those observed when unlabeled methylated DNA containing comparable levels of O6-mG was substituted for NNKOAc-treated DNA. Based on these results, a cocarcinogenic role for pyridyloxobutylation in NNK-induced lung tumorigenesis is proposed in which pyridyloxobutyl DNA adduct(s) compete with O6-mG for reaction with AGT resulting in sustained levels of O6-mG. These enhanced levels then increase the probability of tumor induction by NNK.
Alkylation↗
Environmental carcinogenesis and its prevention: the head and neck cancer model.
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DNA Repair↗
Metabolites of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in smokers' urine.
Metabolites of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a potent pulmonary carcinogen, have been quantified in the urine of 11 smokers. They were not detected in nonsmokers' urine. The metabolites, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and its glucuronide, were detected in quantities of 0.23-1.0 and 0.57-6.5 micrograms/24 h, respectively. The results of this study provide the first evidence for metabolites of tobacco-specific nitrosamines in human urine.
Adult↗
Carcinogen biomarkers related to smoking and upper aerodigestive tract cancer.
Smoking is the major cause of upper aerodigestive tract cancers. Among the many constituents of tobacco smoke, polynuclear aromatic hydrocarbons and tobacco-specific nitrosamines are strongly implicated as causative factors for these cancers. The probability that these compounds will induce cancer in a given individual will depend on that person's ability to metabolically activate or detoxify them. Chronic production of DNA damage by these metabolically activated carcinogens is consistent with current concepts of carcinogenesis in which multiple genetic changes, such as activation of oncogenes or inactivation of tumor suppressor genes, appear to be critical. Chemopreventive agents which decrease the level of DNA damage should therefore decrease the risk for cancer. Biomarkers such as carcinogen-DNA adducts, carcinogen-hemoglobin adducts, and urinary metabolites of carcinogens will indicate the amount of metabolically activated carcinogen which may damage DNA in an individual and can therefore be used as an index of risk. Selected biomarkers are discussed in this paper. These biomarkers of internal dose have great potential for application in chemoprevention trials.
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Tumor-initiating activity on mouse skin of bay region diol-epoxides of 5,6-dimethylchrysene and benzo[c]phenanthrene.
Previous metabolism and DNA-binding studies indicated that 5,6-dimethylchrysene (5,6-diMeC) is metabolically activated in mouse skin through formation of its 1,2-dihydrodiol (5,6-diMeC-1,2-diol) and bay region diol-epoxide (anti-5,6-diMeC-1,2-diol-3,4-epoxide). These metabolites were tested as tumor initiators on mouse skin. Included for comparison were syn-5,6-diMeC-1,2-diol-3,4-epoxide and anti-4,3-dihydroxy-2,1-epoxy- 4,3,2,1-tetrahydrobenzo[c]phenanthrene (anti-B[c]Ph-4,3-diol-2,1-epoxide). At an initiating dose of 100 nmol/mouse, 5,6-diMeC-1,2-diol and anti-5,6-diMeC-1,2-diol-3,4-epoxide were significantly more tumorigenic than 5,6-diMeC, inducing 7.1 and 3.9 skin tumors per mouse respectively compared to 1.1 induced by 5,6-diMeC. Similar results were obtained at an initiating dose of 33 nmol/mouse. This is the first example of a methylated polynuclear aromatic hydrocarbon bay region diol-epoxide which is more tumorigenic than its parent hydrocarbon on mouse skin. syn-5,6-diMeC-1,2-diol-3,4-epoxide was only weakly tumorigenic. Comparisons of anti-5,6-diMeC-1,2-diol-3,4-epoxide and anti-B[c]Ph-4,3-diol-2,1-epoxide demonstrated that the latter was a stronger tumor initiator. The results of this study confirm the bay region diol-epoxide metabolic activation pathway of 5,6-diMeC but do not provide an explanation for the relatively weak tumorigenicity of this hydrocarbon on mouse skin.
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G to A transitions and G to T transversions in codon 12 of the Ki-ras oncogene isolated from mouse lung tumors induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and related DNA methylating and pyridyloxobutylating agents.
Lung tumors were induced in A/J mice by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and the related compounds acetoxymethylmethylnitrosamine (AMMN) and 4-acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc). NNK both methylates and pyridyloxobutylates DNA while AMMN and NNKOAc only methylate or pyridyloxobutylate DNA, respectively. The lung tumors were analyzed for mutations in the Ki-ras oncogene by PCR amplification followed by either restriction fragment length polymorphism, hybridization, or sequencing procedures. NNK induced GGT to GAT mutations in codon 12 (26 of 28 samples analyzed). AMMN induced GGT to GAT mutations in 18 of 18 samples. In contrast, NNKOAc induced a variety of changes including GGT to GAT (8/21), GGT to TGT (5/21) and GGT to GTT (4/21) mutations. These results demonstrate that DNA methylation causes mainly G to A transitions in the Ki-ras gene of A/J mouse lung tumors, consistent with previous results and a role for O6-methyl-guanine, while DNA pyridyloxobutylation induces G to A transitions as well as G to T transversions, perhaps due to the steric bulk of the adducts which are formed. The results are discussed with respect to mutations observed in rodent and human lung tumors.
Adenine↗
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.
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Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone tumorigenicity in mouse lung by the synthetic organoselenium compound, 1,4-phenylenebis(methylene)selenocyanate.
The chemopreventive effect of 5, 10 and 15 p.p.m. (as selenium) of 1,4-phenylenebis(methylene)selenocyanate (p-XSC) on lung tumor induction by the tobacco-specific 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was examined in female A/J mice by administering p-XSC in the diet. Sodium selenite (5 p.p.m. selenium) was given in the same manner for comparison with p-XSC. Mice were fed experimental diets containing the selenium compounds 1 week before i.p. injection of 10 mumol NNK in 0.1 ml saline and throughout the experiment until termination, 16 weeks after carcinogen administration. Body weights of the mice in the different dietary groups did not differ significantly. p-XSC significantly inhibited lung tumor multiplicity from 7.6 tumors per mouse in the control group to 4.1, 3.3 and 1.8 tumors per mouse in animals given 5, 10 and 15 p.p.m. of selenium respectively. In contrast, 5 p.p.m. sodium selenite had no protective effect against lung tumor induction. The results of this study clearly indicate that the structure of selenium-containing compounds is important in determining their efficacy as chemopreventive agents.
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Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone mouse lung tumorigenesis by arylalkynes, mechanism-based inactivators of cytochrome P450.
Arylalkynes such as 4-phenyl-1-butyne (PBY), 5-phenyl-1-pentyne (PPY) and 2-ethynylnaphthalene (2-EN) are suicide inhibitors of cytochrome P450 enzymes. Arylalkyl isothiocyanates such as 6-phenylhexyl isothiocyanate (PHITC) are structurally related to arylalkynes and are known to inhibit the cytochrome P450 mediated metabolic activation and tumorigenicity of a tobacco-specific lung carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In this study, we compared the ability of PBY, PPY, 2-EN and PHITC to inhibit A/J mouse lung tumorigenesis by NNK. Groups of 20 female mice were gavaged with 5 mumol of arylalkyne or PHITC in corn oil. Two hours later they were given a single i.p. injection of 10 mumol NNK. The mice were killed 16 weeks later. PPY and PHITC were both potent inhibitors of tumorigenesis by NNK, reducing lung tumor multiplicity from 8.35 tumors per mouse to 0.40 and 0.35 respectively. PBY and 2-EN also significantly inhibited tumor multiplicity. The results of this study demonstrate that arylalkynes and PHITC are potent inhibitors of NNK induced lung tumorigenesis in A/J mice, consistent with the hypothesis that inhibition of specific cytochrome P450 enzymes is involved in inhibition of tumorigenesis.
Alkynes↗
Evidence that a hemoglobin adduct used for dosimetry of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone is a carboxylic ester.
Hemoglobin adducts of the carcinogenic tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) upon mild base or acid hydrolysis. HPB has been detected in hydrolysates of hemoglobin from smokers and snuff dippers and has been proposed as a dosimeter of exposure to and metabolic activation of NNK in people exposed to tobacco products. In this study, labeling experiments were carried out with [18O]NaOH that provide strong evidence that the globin adduct that releases HPB upon hydrolysis is a carboxylic ester. Globin was isolated from rats treated with [5-3H]NNK. This globin was reacted with NaCNBH3, followed by hydrolysis at room temperature with 0.2 N NaOH. Analysis of the products demonstrated the presence of 4-hydroxy-1-(3-pyridyl)-1-butanol, but not HPB. These results demonstrate that the adduct in globin has a free carbonyl group and is not a Schiff base. This sequence of reactions was then carried out with [18O]NaOH under conditions that were shown to result in incorporation of 18O if nucleophilic displacement at C-4 had occurred. Analysis by GC-MS of the 4-hydroxy-1-(3-pyridyl)-1-butanol formed in this experiment demonstrated that there was no incorporation of 18O. These results are consistent only with the hydrolysis of an ester by a BAC2 mechanism. Therefore, the adduct releasing HPB upon mild base hydrolysis must be a 4-(3-pyridyl)-4-oxobutyl ester of aspartate, glutamate, or a terminal carboxylate.(ABSTRACT TRUNCATED AT 250 WORDS)
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Tobacco-specific nitrosamine adducts: studies in laboratory animals and humans.
This paper describes quantitation of human hemoglobin and DNA adducts of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN). NNK and NNN are believed to be involved in cancers of the lung, esophagus, oral cavity, and pancreas in people who use tobacco products. The adduct dosimetry method employs GC-MS for quantitation of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) released by mild base hydrolysis of hemoglobin or acid hydrolysis of DNA as a biochemical marker of the pyridyloxobutylation metabolic activation pathway. Approximately 22% of smokers (n = 101) had elevated levels of HPB released from hemoglobin (range, 200-1600 fmole/g Hb). Adduct levels in snuff dippers ranged from 200-1800 fmole/g Hb. HPB levels in nonsmokers were generally below the detection limit. Acid hydrolysis of lung and tracheal DNA obtained at autopsy and analysis for released HPB revealed levels ranging up to 50 fmole/mg DNA in smokers; the adduct was not detected in nonsmokers. These findings are consistent with data generated in studies of adduct formation by NNK in rats. The biological significance of the HPB-releasing DNA pyridyloxobutylation pathway was compared to that of the DNA methylation pathway in the A/J mouse. These studies demonstrated that the persistence of O6-methylguanine in lung DNA is critical for tumorigenesis by NNK and that pyridyloxobutylation enhances both persistence of O6-methylguanine and tumorigenesis by acetoxymethylmethylnitrosamine. In the rat, the relative roles of methylation and pyridyloxobutylation in lung tumorigenesis by NNK are not as clearly defined.(ABSTRACT TRUNCATED AT 250 WORDS)
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N-ethyl-N-nitrosourea induced brain tumors in rats monitored by nuclear magnetic resonance imaging, plasma proton nuclear magnetic resonance spectroscopy and microscopy.
Characteristic slow growing brain gliomas were induced in rats by a single subcutaneous injection of N-ethyl-N-nitrosourea (ENU) within 24 h of birth. A parallel control group of rats was injected with saline. Seven treated rats developed gliomas within 2 years. Periodic nuclear magnetic resonance imaging (MRI) of the brain in 3-mm slices at 1.5 Tesla and monthly plasma sampling for proton magnetic resonance spectroscopy (MRS) at 360 MHz were started 6 months after the injection of ENU. In the MRS experiments, the Fossel index, average of the line widths of the methylene and methyl peaks at 360 MHz, was determined from half-line widths of methyl and methylene peaks at 0.8 ppm and 1.3 ppm. In five of the ENU injected animals that developed histologically verified brain tumors, these were also observed by MRI without contrast agents. There was no consistent correlation between the imaged tumors and the Fossel index obtained through MRS during the course of the study where repeated observations were performed on individual animals, nor was there any consistent statistical difference in the Fossel index between ENU-treated and control animals. The results of this study demonstrate that slowly developing carcinogen-induced brain tumors in rats can be successfully and reliably monitored noninvasively by MRI but not by MRS of plasma.
Animals↗
DNA and hemoglobin adducts as markers of metabolic activation of tobacco-specific carcinogens.
Lung cancer is now the leading cause of excess mortality among smokers in the United States. The ability to identify smokers with the greatest risk of developing lung cancer would be an important step in reducing lung cancer mortality. Tobacco-specific nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine are important carcinogens in tobacco smoke. These carcinogens require metabolic activation to exert their carcinogenic effects. Methods are described for the measurement of DNA and hemoglobin adducts formed by the metabolites of these nitrosamines. Preliminary evidence is presented that shows that a subpopulation of smokers have elevated levels of DNA and hemoglobin adducts of tobacco-specific nitrosamines. Further work is in progress to test the hypothesis that smokers with elevated levels of tobacco-specific nitrosamine adducts are at increased risk of developing lung cancer.
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