Investigation of the reaction of myosmine with sodium nitrite in vitro and in rats.

Previous studies have shown that the minor tobacco alkaloid myosmine (5) reacts with NaNO2 in the presence of acid to yield 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB, 8) via 4-(3-pyridyl)-4-oxobutanediazohydroxide (7). Intermediate 7 is also formed in the metabolism of the tobacco-specific nitrosamines N'-nitrosonornicotine (NNN, 1) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 2), resulting in pyridyloxobutylation of DNA and Hb. These pyridyloxobutyl adducts can be quantified by analyzing HPB released upon acid treatment of DNA or base treatment of Hb. Quantitation of HPB-releasing DNA and Hb adducts has been used to assess the metabolic activation of NNN and NNK in smokers and smokeless tobacco users. Because myosmine is found in the diet as well as in tobacco products, it has been suggested that nitrosation of myosmine could lead to the formation of HPB-releasing adducts in people not exposed to tobacco products. We investigated the nitrosation of myosmine in vitro and in vivo in rats. The reaction of myosmine with NaNO2 under acidic conditions produced HPB, as previously reported. A new product was identified as 3'-oximinomyosmine (11) based on its spectral properties. NNN was not detected. Groups of rats were treated with NNN, NNK, myosmine, NaNO2, or combinations of myosmine and NaNO2. HPB-releasing Hb and DNA adducts were clearly detected in the rats treated with NNN or NNK, but we found no evidence for production of these adducts from the combination of myosmine plus NaNO2. The results of this study do not support the hypothesis that exposure to dietary myosmine could lead to HPB-releasing DNA or Hb adducts in humans.

Identification of cyanidin glycosides as constituents of freeze-dried black raspberries which inhibit anti-benzo[a]pyrene-7,8-diol-9,10-epoxide induced NFkappaB and AP-1 activity.

Dietary freeze-dried black raspberries inhibit tumor induction by N-nitrosomethylbenzylamine in the rat esophagus, but the constituents responsible for this chemopreventive activity have not been identified. We fractionated freeze-dried black raspberries and used mouse epidermal JB6 Cl 41 cells stably transfected with either a nuclear factor kappa B (NFkappaB)- or an activator protein 1 (AP-1)-luciferase reporter, and treated with racemic anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), to assess the inhibitory effects of the fractions. The ethanol and water extracts of the freeze-dried black raspberries had inhibitory activity and these extracts were fractionated by HPLC to give several bioactive fractions. Further HPLC analysis yielded multiple subfractions, some of which inhibited BPDE-induced NFkappaB activity. Major constituents of the most active subfractions were identified by their spectral properties and in comparison with standards as cyanidin-3-O-glucoside, cyanidin 3-O-(2(G)-xylosylrutinoside) and cyanidin 3-O-rutinoside. Analysis of freeze-dried black raspberries indicated that these three components comprised approximately 3.4% of the material by dry weight. Consistent with these results, standard cyanidin-3-O-glucoside and cyanidin chloride were also good inhibitors of BPDE-induced NFkappaB activity. The results of this study demonstrate that cyanidin glycosides of freeze-dried black raspberries are bioactive compounds which could account for at least some of the chemopreventive activity observed in animal models.

Effects of cruciferous vegetable consumption on urinary metabolites of the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in singapore chinese.

Vegetable consumption, including cruciferous vegetables, is protective against lung cancer, but the mechanisms are poorly understood. The purpose of this study was to investigate the effects of cruciferous vegetable consumption on the metabolism of the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in smokers. The study was carried out in Singapore Chinese, whose mean daily intake of cruciferous vegetables is three times greater than that of people in the United States. Eighty-four smokers provided urine samples and were interviewed about dietary habits using a structured questionnaire, which included questions on consumption of nine commonly consumed cruciferous vegetables. Samples of these vegetables obtained in Singapore markets at three different times of year were analyzed for glucosinolates. Urine was analyzed for metabolites of NNK: 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronides (NNAL-Glucs). Glucobrassicins, which release indole-3-carbinols on chewing, were the major glucosinolates in seven of the nine cruciferous vegetables, accounting for 70.0% to 93.2% of all glucosinolates in these vegetables. There was a significant correlation (P = 0.01) between increased consumption of glucobrassicins and decreased levels of NNAL in urine after adjustment for number of cigarettes smoked per day; similar trends were observed for NNAL-Glucs (P = 0.08) and NNAL plus NNAL-Glucs (P = 0.03). These results are consistent with those of previous studies, which demonstrate that indole-3-carbinol decreases levels of urinary NNAL probably by inducing hepatic metabolism of NNK. The results are discussed with respect to the known chemopreventive activity of indole-3-carbinol against lung tumorigenesis by NNK in mice and the effects of isothiocyanates, which are also formed on consumption of cruciferous vegetables, on NNK metabolism. The results of this study demonstrate the complexities in assessing effects of cruciferous vegetables on carcinogen metabolism.

Stereospecific deuterium substitution attenuates the tumorigenicity and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK).

Stereochemical determinants of the tumorigenicity and metabolism of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were investigated using the stereospecifically deuterated isotopomers (4R)-[4-(2)H(1)]NNK and (4S)-[4-(2)H(1)]NNK. Upon ip administration to groups of 20 female A/J mice, NNK and (4S)-[4-(2)H(1)]NNK exhibited similar lung tumorigenicity at three different doses, whereas (4R)-[4-(2)H(1)]NNK was 2-fold less tumorigenic at all three doses. In a parallel experiment, levels of O(6)-methylguanine and 7-methylguanine were 2-fold lower in lung DNA of mice treated with (4R)-[4-(2)H(1)]NNK than in mice treated with NNK or (4S)-[4-(2)H(1)]NNK. To corroborate these in vivo data, the in vitro metabolism of these compounds was investigated using A/J mouse lung microsomes and Spodoptera frugiperda (Sf9)-expressed mouse cytochrome p450s 2A4 and 2A5. Kinetic isotope effects on the apparent V(max) ((D)V) for the product of NNK 4-hydroxylation, OPB, were 2.7 +/- 0.2 and 2.8 +/- 0.4 when (4R)- and (4S)-[4-(2)H(1)]NNK were incubated with mouse lung microsomes, respectively. The (D)V values for OPB formation were 3.2 +/- 0.2 and 2.2 +/- 0.2 when (4R)-[4-(2)H(1)]NNK was the substrate for p2A4 and 2A5, respectively, whereas they were 1.3 +/- 0.1 and 1.1 +/- 0.1 when (4S)-[4-(2)H(1)]NNK was the substrate for these respective enzymes. Analysis of an OPB derivative (10) for deuterium content by LC/MS confirmed the results from the kinetic assays and indicated that p450s 2A4 and 2A5 preferentially abstract the pro-R 4-hydrogen of NNK. The results obtained using Sf9-expressed p450s provide a rationale for the differences observed in the lung tumor and DNA adduct experiments, namely, that the attenuated tumorigenicity of (4R)-[4-(2)H(1)]NNK relative to (4S)-[4-(2)H(1)]NNK is due to prochiral selectivity during p450-catalyzed metabolic activation.

Effects of benzyl isothiocyanate and 2-phenethyl isothiocyanate on benzo[a]pyrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone metabolism in F-344 rats.

A mixture of dietary benzyl isothiocyanate (BITC) and 2-phenethyl isothiocyanate (PEITC) inhibits lung tumorigenesis by a mixture of benzo[a]pyrene (B[a]P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice. Previous studies indicated that inhibition of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) releasing DNA adducts of NNK by PEITC in the lung was responsible for inhibition of tumorigenicity. We have now extended these investigations to F-344 rats treated with 2 p.p.m. B[a]P in the diet and 2 p.p.m. NNK in the drinking water. The effects of BITC (1 micromol/g diet), PEITC (3 micromol/g diet), and a mixture of BITC plus PEITC (1 and 3 micromol/g diet) on DNA and hemoglobin (Hb) adducts of B[a]P and NNK, and on two urinary metabolites of NNK, were examined. DNA adducts were quantified after 8 and 16 weeks of treatment. Hb adducts were quantified in blood samples withdrawn every 2 weeks. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its glucuronide NNAL-Gluc were measured in urine every 4 weeks. PEITC or BITC plus PEITC significantly reduced levels of HPB releasing DNA adducts of NNK in lung at 8 and 16 weeks, but there was no effect of BITC. There were no effects of any of the treatments on levels of HPB releasing DNA adducts of NNK in liver, or on DNA adducts of B[a]P in either lung or liver. PEITC or BITC plus PEITC significantly inhibited the formation of Hb adducts of NNK from 2-12 weeks of treatment while there were no effects on Hb adducts of B[a]P. There was a significant increase in levels of NNAL and NNAL-Gluc in the urine of the rats treated with PEITC or BITC plus PEITC. These results demonstrate that dietary PEITC, or a mixture of BITC plus PEITC, inhibit the formation of HPB releasing adducts of NNK in the rodent lung, leading to inhibition of tumorigenesis.

Benzyl isothiocyanate: an effective inhibitor of polycyclic aromatic hydrocarbon tumorigenesis in A/J mouse lung.

Polycyclic aromatic hydrocarbons (PAH) are an important group of carcinogens that are likely to be involved as one of the causes of lung cancer in smokers and occupationally exposed individuals. Previous studies have shown that benzyl isothiocyanate (BITC), administered by gavage, is a good inhibitor of lung tumorigenesis in A/J mice induced by benzo[a]pyrene (BaP), a typical PAH carcinogen. In this study, we evaluated the effects of BITC on lung tumor induction in A/J mice by two other carcinogenic PAH in cigarette smoke - 5-methylchrysene (5-MeC) and dibenz[a,h]anthracene (DBahA). We also compared the effects of BITC with two other well known chemopreventive agents - butylated hydroxyanisole (BHA) and sulforaphane. In experiment 1, groups of A/J mice were treated by gavage once weekly for 8 weeks with BaP (3 micromol) or 5-MeC (2 micromol) or DBahA (1 micromol) in 0.1 ml cottonseed oil. Fifteen minutes before each treatment, the mice were gavaged with 0.1 ml cottonseed oil or 0.1 ml cottonseed oil containing 13.4 micromol or 6.7 micromol of BITC. The experiment was terminated 19 weeks after the final carcinogen treatment. BITC significantly reduced lung tumor multiplicity in all PAH-treated groups by 63.5-90.6%. In experiment 2, groups of A/J mice were treated with BaP or BITC and BaP as in experiment 1, or with BHA or sulforaphane at doses equimolar to those of BITC. BITC was significantly more effective as an inhibitor of lung tumor induction than either BHA or sulforaphane. These results firmly establish gavaged BITC as a strong inhibitor of lung tumorigenesis induced in A/J mice by PAH, and support its further development for chemoprevention of smoking-induced lung cancer.

Effects of benzyl isothiocyanate and phenethyl isothiocyanate on DNA adduct formation by a mixture of benzo[a]pyrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in A/J mouse lung.

Dietary phenethyl isothiocyanate (PEITC) and a mixture of dietary PEITC and benzyl isothiocyanate (BITC) inhibit lung tumorigenesis in A/J mice induced by a mixture of the tobacco smoke carcinogens benzo[a]pyrene (B[a]P) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In this study, we tested the hypothesis that inhibition of tumorigenesis by these isothiocyanates was due to inhibition of DNA adduct formation. We quantified the following pulmonary DNA adducts: N2-[7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-10-yl]deoxyguanosine (BPDE-N2-dG) from B[a]P; and O(6)-methylguanine (O(6)-mG) and 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing adducts from NNK. Initial experiments demonstrated that there were no effects of B[a]P on NNK-DNA adduct formation, or vice versa, and established by way of a time course study the appropriate sacrifice intervals for the main experiment. Dietary PEITC, or dietary BITC plus PEITC, inhibited the formation of HPB-releasing DNA adducts of NNK at several of the time points examined. There were no effects of dietary isothiocyanates on levels of O(6)-mG or BPDE-N2-dG. These results, which are consistent with previous studies in rats and with tumor inhibition data in mice, support a role for inhibition of HPB-releasing DNA adducts of NNK as a mechanism of inhibition of tumorigenesis by dietary PEITC and BITC plus PEITC. However, the observed inhibition was modest, suggesting that other effects of isothiocyanates are also involved in chemoprevention in this model.

Inhibition of lung tumorigenesis in A/J mice by N-acetyl-S-(N-2-phenethylthiocarbamoyl)-L-cysteine and myo-inositol, individually and in combination.

Isothiocyanates, their N-acetylcysteine conjugates, and myo-inositol (MI) are inhibitors of lung tumorigenesis in A/J mice. However, chemoprevention by combinations of these compounds in different temporal sequences has not been examined. This is important for developing practical approaches to lung cancer chemoprevention in smokers and ex-smokers. We used a tumor model in which A/J mice are treated with 8 weekly doses of benzo[a]pyrene (B[a]P) plus 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and killed 19 weeks after the final treatment. In Experiment 1, isothiocyanates or their N-acetylcysteine conjugates were added to the diet (1 or 3 micro mol/g) from 1 week before until 1 week after carcinogen treatment. The compounds were 2-phenethyl isothiocyanate (PEITC), 3-phenylpropyl isothiocyanate (PPITC), N-acetyl-S-(N-benzyl-thiocarbamoyl)-L-cysteine (BITC-NAC), N-acetyl-S-(N-2-phenethylthiocarbamoyl)-L-cysteine (PEITC-NAC), and N-acetyl-S-(N-3-phenylpropylthiocarbamoyl)-L-cysteine (PPITC-NAC). Significant reductions in lung tumor multiplicity were observed in mice treated with PEITC, PEITC-NAC, PPITC and PPITC-NAC. PEITC-NAC was chosen for combination studies with MI (Experiment 2). Mice were treated with B[a]P plus NNK without or with PEITC-NAC (3 micro mol/g diet), MI (55.5 micro mol/g diet), or PEITC-NAC plus MI (3 micro mol plus 55.5 micro mol/g diet). Different temporal sequences of dietary additions were investigated: carcinogen treatment phase; post-carcinogen treatment phase; entire experiment; 50% of carcinogen treatment phase until termination; and 75% of carcinogen treatment phase until termination. All treatments reduced lung tumor multiplicity except PEITC-NAC post-carcinogen or from 75% of the carcinogen treatment phase. Reduction of lung tumor multiplicity by PEITC-NAC plus MI was greater than that in the mice treated with the agents alone in all temporal sequences. When all results were combined, PEITC-NAC plus MI was significantly more effective than the agents alone. There was a significant trend for reduction in lung tumor multiplicity with increased duration of treatment by the chemopreventive agents. These results provide a basis for further development of mixtures of PEITC-NAC and MI for chemoprevention of lung cancer.