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2-Phenethyl isothiocyanate (PEITC), a natural product found as a conjugate in watercress and other cruciferous vegetables, is an inhibitor of the metabolic activation and lung carcinogenicity of the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in F344 rats and A/J mice. We carried out a clinical trial to determine whether PEITC also inhibits the metabolic activation of NNK in smokers. Cigarette smokers were recruited and asked to smoke cigarettes containing deuterium-labeled [pyridine-D4]NNK for an acclimation period of at least 1 week. Then subjects were randomly assigned to one of two arms: PEITC followed by placebo, or placebo followed by PEITC. During the 1-week treatment period, each subject took PEITC (10 mg in 1 mL of olive oil, 4 times per day). There was a 1-week washout period between the PEITC and placebo periods. The NNK metabolic activation ratio [pyridine-D4]hydroxy acid/total [pyridine-D4]NNAL was measured in urine samples to test the hypothesis that PEITC treatment modified NNK metabolism. Eighty-two smokers completed the study and were included in the analysis. Overall, the NNK metabolic activation ratio was reduced by 7.7% with PEITC treatment (P = 0.023). The results of this trial, while modest in effect size, provide a basis for further investigation of PEITC as an inhibitor of lung carcinogenesis by NNK in smokers. Cancer Prev Res; 9(5); 396–405. ©2016 AACR.

We previously reported the chemopreventive potential of kava against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)- and benzo(a)pyrene (BaP)–induced lung tumorigenesis in A/J mice during the initiation and postinitiation stages. In this study, we investigated the tumorigenesis-stage specificity of kava, the potential active compounds, and the underlying mechanisms in NNK-induced lung tumorigenesis in A/J mice. In the first experiment, NNK-treated mice were given diets containing kava at a dose of 5 mg/g of diet during different periods. Kava treatments covering the initiation stage reduced the multiplicity of lung adenomas by approximately 99%. A minimum effective dose is yet to be defined because kava at two lower dosages (2.5 and 1.25 mg/g of diet) were equally effective as 5 mg/g of diet in completely inhibiting lung adenoma formation. Daily gavage of kava (one before, during, and after NNK treatment) completely blocked lung adenoma formation as well. Kavalactone-enriched fraction B fully recapitulated kava's chemopreventive efficacy, whereas kavalactone-free fractions A and C were much less effective. Mechanistically, kava and fraction B reduced NNK-induced DNA damage in lung tissues with a unique and preferential reduction in O6-methylguanine (O6-mG), the highly tumorigenic DNA damage by NNK, correlating and predictive of efficacy on blocking lung adenoma formation. Taken together, these results demonstrate the outstanding efficacy of kava in preventing NNK-induced lung tumorigenesis in A/J mice with high selectivity for the initiation stage in association with the reduction of O6-mG adduct in DNA. They also establish the knowledge basis for the identification of the active compound(s) in kava. Cancer Prev Res; 7(1); 86–96. ©2013 AACR.

The quantity of each vegetable in the salads and the corresponding concentration of glucobrassicin is shown.

Tobacco smoking is the primary risk factor for lung cancer, driven by the addictive nature of nicotine and the indisputable carcinogenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as well as other compounds. The integration of lung cancer chemoprevention with smoking cessation is one potential approach to reduce this risk and mitigate lung cancer mortality. Experimental data from our group suggest that kava, commonly consumed in the South Pacific Islands as a beverage to promote relaxation, may reduce lung cancer risk by enhancing NNK detoxification and reducing NNK-derived DNA damage. Building upon these observations, we conducted a pilot clinical trial to evaluate the effects of a 7-day course of kava on NNK metabolism in active smokers. The primary objective was to compare urinary total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL plus its glucuronides, major metabolites of NNK) before and after kava administration as an indicator of NNK detoxification. Secondary objectives included determining kava's safety, its effects on DNA damage, tobacco use, and cortisol (a biomarker of stress). Kava increased urinary excretion of total NNAL and reduced urinary 3-methyladenine in participants, suggestive of its ability to reduce the carcinogenicity of NNK. Kava also reduced urinary total nicotine equivalents, indicative of its potential to facilitate tobacco cessation. Plasma cortisol and urinary total cortisol equivalents were reduced upon kava use, which may contribute to reductions in tobacco use. These results demonstrate the potential of kava intake to reduce lung cancer risk among smokers.

Glucobrassicin in Brassica vegetables gives rise to indole-3-carbinol (I3C), a compound with potent anticancer effects in preclinical models. We previously showed that the urinary metabolite 3,3′-diindolylmethane (DIM) could discriminate between volunteers fed high and low doses of Brassica vegetables. However, the quantitative relationship between glucobrassicin exposure and urinary DIM level is unclear. We conducted a clinical trial to examine the hypotheses that a range of glucobrassicin exposure from Brassica vegetables is reflected in urinary DIM and that this effect plateaus. Forty-five subjects consumed vegetables, a mixture of brussels sprouts and/or cabbage, at one of seven discrete dose levels of glucobrassicin ranging from 25 to 500 μmol, once daily for 2 consecutive days. All urine was collected for 24 hours after each vegetable-eating session. Urinary DIM was measured using our published liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (LC/ESI-MS/MS-SRM) method. Urinary DIM excretion increased predictably with increasing glucobrassicin dose and plateaued between 200 and 300 μmol of glucobrassicin. The association between glucobrassicin dose and urinary DIM was strong and positive (R2 = 0.68). The majority of DIM was excreted in the first 12 hours after vegetable consumption. We conclude that urinary DIM is a reliable biomarker of glucobrassicin exposure and I3C uptake and that feeding glucobrassicin beyond 200 μmol did not consistently lead to more urinary DIM, suggesting a plateau in potential chemopreventive benefit. Cancer Prev Res; 9(10); 788–93. ©2016 AACR.

Table S1. Number of [pyridine-D4]NNK cigarettes smoked per day by study participants by treatment sequence groups and treatment period, The PEITC Intervention Study 2008-2013 Table S2. Spearman correlation coefficients between number of cigarettes per day and urinary metabolites of nicotine, NNK, [pyridine-D4]NNK, and PGEM and 8-iso-PGF2α at the end of smoking adaption period (visit 2), The PEITC Intervention Study 2008-2013 Table S3. Urinary Levels of Total ITC and PEITC-NAC by treatment sequence groups and treatment period, The PEITC Intervention Study 2008-2013 Table S4. Geometric means of urinary [pyridine-D4]NNK metabolites by study period and treatment sequence assignment, The PEITC Intervention Study 2008-2013 Table S5. Mean level of urinary levels of PEITC-NAC and total ITC during PEITC treatment by glutathione-S-transferase (GST) genotypes, The PEITC Intervention Study 2008-2013 Table S6. Urinary levels of NNK and nicotine metabolites by PEITC treatment, The PEITC Intervention Study 2008-2013 Figure S1. Overview of the metabolism of [pyridine-D4]NNK and [pyridine-D4]NNAL leading to DNA adducts and urinary metabolites. Figure S2. Overview of the study design for the PEITC trial.

PDF file - 338K, Supplementary Figure 1. Study design for evaluating the chemopreventive efficacy of different kava treatment regimens against NNK-induced lung adenoma formation in A/J mice. Supplementary Figure 2. Representative photomicrographs H&E-stained sections of lungs (n = 4 in each group) from negative control mice (A), mice with NNK alone (B), and mice with NNK plus kava at a dose of 5 mg/g of diet (C). Supplementary Figure 3. 1H-NMR spectra of different kava fractions and the mass balance of each fraction. I. Kava; II. Fraction A; III. Reconstituted kava from Fractions A, B, and C; IV. Fraction B; V, Comparison between Kava (Blue) and reconstituted kava (Green) from Fractions A, B, and C; VI. Fraction C; VII. Mass balance of each fraction. Supplementary Figure 4. HPLC traces of traditional kava, kava from Gaia Herbs, reconstituted kava, and Fractions A, B, and C. Supplementary Figure 5. Chemicals isolated from kava and their natural abundance.

Perspective on this Article from Chemopreventive Effect of Kava on 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone plus Benzo[a]pyrene–Induced Lung Tumorigenesis in A/J Mice

Supplemental Tables 1-7 and Supplemental Figures 1-5

Lung cancer is the leading cause of cancer-related deaths. While tobacco use is the main cause, only 10–20% of smokers eventually develop clinical lung cancer. Thus, the ability of lung cancer risk prediction among smokers could transform lung cancer management with early preventive interventions. Given that DNA damage by tobacco carcinogens is the potential root cause of lung carcinogenesis, we characterized the adductomic totality of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (a potent lung carcinogen in tobacco, commonly known as NNK) in the target lung tissues, the liver tissues and the peripheral serum samples in a single-dose NNK-induced lung carcinogenesis A/J mouse model. We also characterized these adductomic totalities from the two enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, the major in vivo metabolite of NNK) given their distinct carcinogenicity in A/J mice. With these adductomic data, we demonstrated that tissue protein adductomics have the highest abundance. We also identified that the adductomic levels at the 8 h time point after carcinogen exposure were among the highest. More importantly, the relationships among these adductomics were characterized with overall strong positive linear correlations, demonstrating the potential of using peripheral serum protein adductomics to reflect DNA adductomics in the target lung tissues. Lastly, we explored the relationships of these adductomics with lung tumor status in A/J mice, providing preliminary but promising evidence of the feasibility of lung cancer risk prediction using peripheral adductomic profiling.

The International Agency for Research on Cancer has classified the tobacco-specific nitrosamines N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as "carcinogenic to humans" (Group 1). To exert its carcinogenicity, NNN requires metabolic activation to form reactive intermediates which alkylate DNA. Previous studies have identified cytochrome P450-catalyzed 2'-hydroxylation and 5'-hydroxylation of NNN as major metabolic pathways, with preferential activation through the 5'-hydroxylation pathway in some cultured human tissues and patas monkeys. So far, the only DNA adducts identified from NNN 5'-hydroxylation in rat tissues are 2-[2-(3-pyridyl)-N-pyrrolidinyl]-2'-deoxyinosine (Py-Py-dI), 6-[2-(3-pyridyl)-N-pyrrolidinyl]-2'-deoxynebularine (Py-Py-dN), and N6-[4-hydroxy-1-(pyridine-3-yl)butyl]-2'-deoxyadenosine (N6-HPB-dAdo) after reduction. To expand the DNA adduct panel formed by NNN 5'-hydroxylation and identify possible activation biomarkers of NNN metabolism, we investigated the formation of dAdo-derived adducts using a new highly sensitive and specific liquid chromatography-nanoelectrospray ionization-high-resolution tandem mass spectrometry method. Two types of NNN-specific dAdo-derived adducts, N6-[5-(3-pyridyl)tetrahydrofuran-2-yl]-2'-deoxyadenosine (N6-Py-THF-dAdo) and 6-[2-(3-pyridyl)-N-pyrrolidinyl-5-hydroxy]-2'-deoxynebularine (Py-Py(OH)-dN), were observed for the first time in calf thymus DNA incubated with 5'-acetoxyNNN. More importantly, Py-Py(OH)-dN was also observed in relatively high abundance in the liver and lung DNA of rats treated with racemic NNN in the drinking water for 3 weeks. These new adducts were characterized using authentic synthesized standards. Both NMR and MS data agreed well with the proposed structures of N6-Py-THF-dAdo and Py-Py(OH)-dN. Reduction of Py-Py(OH)-dN by NaBH3CN led to the formation of Py-Py-dN both in vitro and in vivo, which was confirmed by its isotopically labeled internal standard [pyridine-d4]Py-Py-dN. The NNN-specific dAdo adducts Py-THF-dAdo and Py-Py(OH)-dN formed by NNN 5'-hydroxylation provide a more comprehensive understanding of the mechanism of DNA adduct formation by NNN.

Tobacco smoke is a complex mixture of chemicals, many of which are toxic and carcinogenic. Hazard assessments of tobacco smoke exposure have predominantly focused on either single chemical exposures or the more complex mixtures of tobacco smoke or its fractions. There are fewer studies exploring interactions between specific tobacco smoke chemicals. Aldehydes such as formaldehyde and acetaldehyde were hypothesized to enhance the carcinogenic properties of the human carcinogen, 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) through a variety of mechanisms. This hypothesis was tested in the established NNK-induced A/J mouse lung tumor model. A/J mice were exposed to NNK (intraperitoneal injection, 0, 2.5, or 7.5 μmol in saline) in the presence or absence of acetaldehyde (0 or 360 ppmv) or formaldehyde (0 or 17 ppmv) for 3 h in a nose-only inhalation chamber, and lung tumors were counted 16 weeks later. Neither aldehyde by itself induced lung tumors. However, mice receiving both NNK and acetaldehyde or formaldehyde had more adenomas with dysplasia or progression than those receiving only NNK, suggesting that aldehydes may increase the severity of NNK-induced lung adenomas. The aldehyde coexposure did not affect the levels of NNK-derived DNA adduct levels. Similar studies tested the ability of a 3 h nose-only carbon dioxide (0, 5, 10, or 15%) coexposure to influence lung adenoma formation by NNK. While carbon dioxide alone was not carcinogenic, it significantly increased the number of NNK-derived lung adenomas without affecting NNK-derived DNA damage. These studies indicate that the chemicals in tobacco smoke work together to form a potent lung carcinogenic mixture.

Metabolic activation of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N’-nitrosonornicotine (NNN) results in formation of reactive electrophiles that modify DNA to produce a variety of products including methyl, 4-(3-pyridyl)-4-oxobutyl (POB)-, and 4-(3-pyridyl)-4-hydroxybutyl adducts. Among these are adducts such as 7-POB-deoxyguanosine (N(7)POBdG) which can lead to apurinic/apyrimidinic (AP) sites by facile hydrolysis of the base-deoxyribonucleoside bond. In this study, we used a recently developed highly sensitive mass spectrometric method to quantitate AP sites by derivatization with O-(pyridin-3-yl-methyl)hydroxylamine (PMOA) (detection limit, 2 AP sites per 10(8) nucleotides). AP sites were quantified in DNA isolated from tissues of rats treated with NNN and NNK and from human lung tissue and leukocytes of cigarette smokers and non-smokers. Rats treated with 5 or 21 mg/kg bw NNK for four days by s.c. injection had 2–6 and 2–17 times more AP sites than controls in liver and lung DNA (p

Our early studies demonstrated an impressive chemopreventive efficacy of dihydromethysticin (DHM), unique in kava, against tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis in A/J mice, in which DHM was supplemented in the diet. The current work was carried out to validate the efficacy, optimize the dosing schedule and further elucidate the mechanisms using oral bolus dosing of DHM. The results demonstrated a dose-dependent chemopreventive efficacy of DHM (orally administered 1 h before each of the two NNK intraperitoneal injections, 1 week apart) against NNK-induced lung adenoma formation. Temporally, DHM at 0.8 mg per dose (~32 mg per kg body weight) exhibited 100% lung adenoma inhibition when given 3 and 8 h before each NNK injection and attained >93% inhibition when dosed at either 1 h or 16 h before each NNK injection. The simultaneous treatment (0h) or 40 h pretreatment (−40h) decreased lung adenoma burden by 49.8% and 52.1%, respectively. However, post-NNK administration of DHM (1 h to 8 h after each NNK injection) was ineffective against lung tumor formation. In short-term experiments for mechanistic exploration, DHM treatment reduced the formation of NNK-induced O(6)-methylguanine (O(6)-mG, a carcinogenic DNA adduct in A/J mice) in the target lung tissue and increased the urinary excretion of NNK detoxification metabolites as judged by the ratio of urinary NNAL-O-gluc to free NNAL, generally in synchrony with the tumor prevention efficacy outcomes in the dose scheduling time-course experiment. Overall, these results suggest DHM as a potential chemopreventive agent against lung tumorigenesis in smokers, with O(6)-mG and NNAL detoxification as possible surrogate biomarkers.

Tobacco smoking is the primary risk factor for lung cancer, driven by the addictive nature of nicotine and the indisputable carcinogenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) as well as other compounds. The integration of lung cancer chemoprevention with smoking cessation is one potential approach to reduce this risk and mitigate lung cancer mortality. Experimental data from our group suggest that kava, commonly consumed in the South Pacific Islands as a beverage to promote relaxation, may reduce lung cancer risk by enhancing NNK detoxification and reducing NNK-derived DNA damage. Building upon these observations, we conducted a pilot clinical trial to evaluate the effects of a 7-day course of kava on NNK metabolism in active smokers. The primary objective was to compare urinary total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL plus its glucuronides, major metabolites of NNK) before and after kava administration as an indicator of NNK detoxification. Secondary objectives included determining kava's safety, its effects on DNA damage, tobacco use, and cortisol (a biomarker of stress). Kava increased urinary excretion of total NNAL and reduced urinary 3-methyladenine in participants, suggestive of its ability to reduce the carcinogenicity of NNK. Kava also reduced urinary total nicotine equivalents, indicative of its potential to facilitate tobacco cessation. Plasma cortisol and urinary total cortisol equivalents were reduced upon kava use, which may contribute to reductions in tobacco use. These results demonstrate the potential of kava intake to reduce lung cancer risk among smokers.

Aim: A hypoxia-activated combi-nitrosourea prodrug, N-(2-chloroethyl)-N′-2-(2-(4-nitrobenzylcarbamate)-O6-benzyl-9-guanine)ethyl-N-nitrosourea (NBGNU), was synthesized and evaluated for its hypoxic selectivity and anticancer activity in vitro. Results: The prodrug was designed as a tripartite molecule consisting of a chloroethylnitrosourea pharmacophore to induce DNA interstrand crosslinks (ICLs) and an O6-benzylguanine analog moiety masked by a 4-nitrobenzylcarbamate group to induce hypoxia-activated inhibition of O6-alkylguanine-DNA alkyltransferase. NBGNU was tested for hypoxic selectivity, cytotoxicity and DNA ICLs ability. The reduction product amounts, cell death rates and DNA ICL levels induced by NBGNU under hypoxic conditions were all significantly higher than those induced by NBGNU under normoxic conditions. Conclusion: The tripartite combi-nitrosourea prodrug exhibits desirable tumor-hypoxia targeting ability and abolished chemoresistance compared with the conventional chloroethylnitrosoureas.

The International Agency for Research on Cancer has classified the tobacco-specific nitrosamines

Although members of the hyaluronan (HA)-CD44/HA-mediated motility receptor (RHAMM) signaling pathway have been shown to be overexpressed in lung cancer, their role in lung tumorigenesis is unclear. In the present study, we first determined levels of HA and its receptors CD44 and RHAMM in human non-small cell lung cancer (NSCLC) cells and stromal cells as well as mouse lung tumors. Subsequently, we examined the role of HA-CD44/RHAMM signaling pathway in mediating the proliferation and survival of NSCLC cells and the cross-talk between NSCLC cells and normal human lung fibroblasts (NHLFs)/lung cancer-associated fibroblasts (LCAFs). The highest levels of HA and CD44 were observed in NHLFs/LCAFs followed by NSCLC cells, whereas THP-1 monocytes/macrophages showed negligible levels of both HA and CD44. Simultaneous silencing of HA synthase 2 (HAS2) and HAS3 or CD44 and RHAMM suppressed cell proliferation and survival as well as the EGFR/AKT/ERK signaling pathway. Exogenous HA partially rescued the defect in cell proliferation and survival. Moreover, conditioned media (CM) generated by NHLFs/LCAFs enhanced the proliferation of NSCLC cells in a HA-dependent manner as treatment of NHLFs and LCAFs with HAS2 siRNA, 4-methylumbelliferone, an inhibitor of HASs, LY2228820, an inhibitor of p38MAPK, or treatment of A549 cells with CD44 blocking antibody suppressed the effects of the CM. Upon incubation in CM generated by A549 cells or THP-1 macrophages, NHLFs/LCAFs secreted higher concentrations of HA. Overall, our findings indicate that targeting the HA-CD44/RHAMM signaling pathway could be a promising approach for the prevention and therapy of lung cancer.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) are carcinogenic in animal models and are believed to play an important role in human lung carcinogenesis in cigarette smokers. Cytochrome P450-mediated metabolism of these tobacco-specific nitrosamines produces reactive species that alkylate DNA in the form of pyridyloxobutyl (POB)- or pyridylhydroxybutyl (PHB)-DNA adducts. Understanding the formation mechanism and overall levels of these adducts can potentially enhance cancer prevention methods through the identification of particularly susceptible smokers. Previous studies have identified and measured a panel of POB- and PHB-DNA base adducts of dGuo, dCyd, and Thd; however, dAdo adducts have yet to be determined. In this study, we complete this DNA adduct panel by identifying and quantifying levels of NNK- and NNAL-derived dAdo adducts in vitro and in vivo. To accomplish this, we synthesized standards for expected dAdo-derived DNA adducts and used isotope-dilution LC-ESI+-MS/MS to identify POB adducts formed in vitro from the reaction of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc) with calf thymus DNA. Adduct levels were then quantified in lung and liver DNA of rats chronically treated with NNK or NNAL for 50 weeks using similar LC-MS detection methods. The in vitro studies identified N6-POB-dAdo and N1-POB-dIno as products of the reaction of NNKOAc with DNA, which supports our proposed mechanism of formation. Though both N6-dAdo and N1-dIno adducts were found in vitro, only N6-dAdo adducts were found in vivo, implying possible intervention by DNA repair mechanisms. Analogous to previous studies, levels of N6-POB-dAdo and N6-PHB-dAdo varied both with tissue and treatment type. Despite the adduct levels being relatively modest compared to most other POB- and PHB-DNA adducts, they may play a biological role and could be used in future studies as NNK- and NNAL-specific DNA damage biomarkers.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a powerful lung carcinogen in animal models and is considered a causative factor for lung cancer in tobacco users. NNK is stereoselectively and reversibly metabolized to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which is also a lung carcinogen. Both NNK and NNAL undergo metabolic activation by α-hydroxylation on their methyl groups to form pyridyloxobutyl and pyridylhydroxybutyl DNA base and phosphate adducts, respectively. α-Hydroxylation also occurs on the α-methylene carbons of NNK and NNAL to produce methane diazohydroxide, which reacts with DNA to form methyl DNA base adducts. DNA adducts of NNK and NNAL are important in their mechanisms of carcinogenesis. In this study, we characterized and quantified methyl DNA phosphate adducts in the lung of rats treated with 5 ppm of NNK, (S)-NNAL, or (R)-NNAL in drinking water for 10, 30, 50, and 70 weeks, by using a novel liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry method. A total of 23, 21, and 22 out of 32 possible methyl DNA phosphate adducts were detected in the lung tissues of rats treated with NNK, (S)-NNAL, and (R)-NNAL, respectively. Levels of the methyl DNA phosphate adducts were 2290–4510, 872–1120, and 763–1430 fmol/mg DNA, accounting for 15–38%, 8%, and 5–9% of the total measured DNA adducts in rats treated with NNK, (S)-NNAL, and (R)-NNAL, respectively. The methyl DNA phosphate adducts characterized in this study further enriched the diversity of DNA adducts formed by NNK and NNAL. These results provide important new data regarding NNK- and NNAL-derived DNA damage and new insights pertinent to future mechanistic and biomonitoring studies of NNK, NNAL, and other chemical methylating agents.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a powerful lung carcinogen in animal models and is considered a causative factor for lung cancer in people who use tobacco products. NNK undergoes metabolic activation-a critical step in its mechanism of carcinogenesis-to an intermediate which reacts with DNA to form pyridyloxobutyl DNA base and phosphate adducts. Another important metabolic pathway of NNK is its conversion to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which similarly forms pyridylhydroxybutyl DNA base adducts that have been characterized previously. In this study, we investigated the potential formation of pyridylhydroxybutyl DNA phosphate adducts. We report the characterization and quantitation of 107 structurally unique pyridylhydroxybutyl DNA phosphate adducts in the lungs of rats treated chronically with a carcinogenic dose of 5 ppm of NNK in their drinking water for up to 70 weeks, by using a novel liquid chromatography-nanoelectrospray ionization-high-resolution tandem mass spectrometry method. Our findings demonstrate that pyridylhydroxybutyl phosphate adducts account for 38-55 and 34-40% of all the measured pyridine-containing DNA adducts in rat lung and liver, respectively, upon treatment with NNK. Some of the pyridylhydroxybutyl DNA phosphate adducts persisted in both tissues for over 70 weeks, suggesting that they could be potential biomarkers of chronic exposure to NNK and NNAL. This study provides comprehensive characterization and relative quantitation of a panel of NNK/NNAL-derived DNA phosphate adducts, thus identifying NNK as the source of the most structurally diverse set of DNA adducts identified to date from any carcinogen.

(+)-Dihydromethysticin was recently identified as a promising lung cancer chemopreventive agent, while (+)-dihydrokavain was completely ineffective. A pilot in vivo structure–activity relationship (SAR) was explored, evaluating the efficacy of its analogs in blocking 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced short-term O6-methylguanine and long-term adenoma formation in the lung tissues in A/J mice. Both results revealed cohesive SARs, demonstrating that the methylenedioxy functional group in DHM is essential while the lactone functional group tolerates modifications.

Two indole compounds, indole-3-carbinol (I3C) and its acid condensation product, 3,3'-diindolymethane (DIM), have been shown to suppress the expression of flavin-containing monooxygenases (FMO) and to induce some hepatic cytochrome P450s (CYPs) in rats. In liver microsomes prepared from rats fed I3C or DIM, FMO-mediated nicotine N-oxygenation was decreased, whereas CYP-mediated nicotine metabolism to nicotine iminium and subsequently to cotinine was unchanged. Therefore, it was hypothesized that in mice DIM would also suppress nicotine N-oxygenation without affecting CYP-mediated nicotine metabolism. Liver microsomes were produced from male and female C57BL/6 J and CD1 mice fed 2500 parts per million (ppm) DIM for 14 days. In liver microsomes from DIM-fed mice, FMO-mediated nicotine N-oxygenation did not differ from the controls, but CYP-mediated nicotine metabolism was significantly increased, with results varying by sex and strain. To confirm the effects of DIM in vivo, control and DIM-fed CD1 male mice were injected subcutaneously with nicotine, and the plasma concentrations of nicotine, cotinine and nicotine-N-oxide were measured over 30 minutes. The DIM-fed mice showed greater cotinine concentrations compared with the controls 10 minutes following injection. It is concluded that the effects of DIM on nicotine metabolism in vitro and in vivo differ between mice and rats and between mouse strains, and that DIM is an effective inducer of CYP-mediated nicotine metabolism in commonly studied mouse strains.

Glucobrassicin in Brassica vegetables gives rise to indole-3-carbinol (I3C), a compound with potent anticancer effects in preclinical models. We previously showed that the urinary metabolite 3,3′-diindolylmethane (DIM) could discriminate between volunteers fed high and low doses of Brassica vegetables. However, the quantitative relationship between glucobrassicin exposure and urinary DIM level is unclear. We conducted a clinical trial to examine the hypotheses that a range of glucobrassicin exposure from Brassica vegetables is reflected in urinary DIM and that this effect plateaus. Forty-five subjects consumed vegetables, a mixture of brussels sprouts and/or cabbage, at one of seven discrete dose levels of glucobrassicin ranging from 25 to 500 μmol, once daily for 2 consecutive days. All urine was collected for 24 hours after each vegetable-eating session. Urinary DIM was measured using our published liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (LC/ESI-MS/MS-SRM) method. Urinary DIM excretion increased predictably with increasing glucobrassicin dose and plateaued between 200 and 300 μmol of glucobrassicin. The association between glucobrassicin dose and urinary DIM was strong and positive (R2 = 0.68). The majority of DIM was excreted in the first 12 hours after vegetable consumption. We conclude that urinary DIM is a reliable biomarker of glucobrassicin exposure and I3C uptake and that feeding glucobrassicin beyond 200 μmol did not consistently lead to more urinary DIM, suggesting a plateau in potential chemopreventive benefit. Cancer Prev Res; 9(10); 788–93. ©2016 AACR.

// Jung Min Song 1 , Arunkumar Anandharaj 1 , Pramod Upadhyaya 1 , Ameya R. Kirtane 2 , Jong-Hyuk Kim 1, 4 , Kwon Ho Hong 3 , Jayanth Panyam 1, 2 , Fekadu Kassie 1, 4 1 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA 2 Department of Pharmaceutics, University of Minnesota, Minneapolis, MN 55455, USA 3 Institute for Therapeutics Discovery and Development, University of Minnesota, Minneapolis, MN 55414, USA 4 Department of Veterinary Clinical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN 55108, USA Correspondence to: Fekadu Kassie, email: kassi012@umn.edu Keywords: chemoprevention, honokiol, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, lung tumor, EGFR Received: April 26, 2016 Accepted: July 10, 2016 Published: July 21, 2016 ABSTRACT Since epidermal growth factor receptor (EGFR) is commonly deregulated in pre-malignant lung epithelium, targeting EGFR may arrest the development of lung cancer. Here, we showed that honokiol (2.5–7.5 μM), a bioactive compound of Magnolia officinalis , differentially suppressed proliferation (up to 93%) and induced apoptosis (up to 61%) of EGFR overexpressing tumorigenic bronchial cells and these effects were paralleled by downregulation of phospho-EGFR, phospho-Akt, phospho-STAT3 and cell cycle-related proteins as early as 6–12 h post-treatment. Autocrine secretion of EGF sensitized 1170 cells to the effects of honokiol. Molecular docking studies indicated that honokiol binds to the tyrosine kinase domain of EGFR although it was less efficient than erlotinib. However, the anti-proliferative and pro-apoptotic activities of honokiol were stronger than those of erlotinib. Upon combinatory treatment, honokiol sensitized bronchial cells and erlotinib resistant H1650 and H1975 cells to erlotinib. Furthermore, in a mouse lung tumor bioassay, intranasal instillation of liposomal honokiol (5 mg/kg) for 14 weeks reduced the size and multiplicity (49%) of lung tumors and the level of total- and phospho-EGFR, phospho-Akt and phospho-STAT3. Overall, our results indicate that honokiol is a promising candidate to suppress the development and even progression of lung tumors driven by EGFR deregulation.

Lee W. Wattenberg, who spent his entire career at the University of Minnesota, was a true pioneer in the field of chemoprevention. This paper is a tribute to his groundbreaking research which uncovered the cancer prevention properties of many dietary compounds, including those discussed here in some detail-indole-3-carbinol and diindolylmethane. These compounds occur as glucosinolate conjugates in cruciferous vegetables and are released when one chews or otherwise macerates the vegetable. They have numerous beneficial effects including the ability to prevent cancer in laboratory animals treated with carcinogens. We review some of the early work on indole-3-carbinol and diindolylmethane which spurred subsequent studies on their efficacy and molecular mechanisms of prevention. We also present unique data on field conditions that affect levels of their glucosinolate precursors in vegetables and on the release of diindolylmethane in people who consume cruciferous vegetables.

2-Phenethyl isothiocyanate (PEITC), a natural product found as a conjugate in watercress and other cruciferous vegetables, is an inhibitor of the metabolic activation and lung carcinogenicity of the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in F344 rats and A/J mice. We carried out a clinical trial to determine whether PEITC also inhibits the metabolic activation of NNK in smokers. Cigarette smokers were recruited and asked to smoke cigarettes containing deuterium-labeled [pyridine-D4]NNK for an acclimation period of at least 1 week. Then subjects were randomly assigned to one of two arms: PEITC followed by placebo, or placebo followed by PEITC. During the 1-week treatment period, each subject took PEITC (10 mg in 1 mL of olive oil, 4 times per day). There was a 1-week washout period between the PEITC and placebo periods. The NNK metabolic activation ratio [pyridine-D4]hydroxy acid/total [pyridine-D4]NNAL was measured in urine samples to test the hypothesis that PEITC treatment modified NNK metabolism. Eighty-two smokers completed the study and were included in the analysis. Overall, the NNK metabolic activation ratio was reduced by 7.7% with PEITC treatment (P = 0.023). The results of this trial, while modest in effect size, provide a basis for further investigation of PEITC as an inhibitor of lung carcinogenesis by NNK in smokers. Cancer Prev Res; 9(5); 396–405. ©2016 AACR.

N'-Nitrosonornicotine (NNN) is carcinogenic in multiple animal models and has been evaluated as a human carcinogen. NNN can be metabolized by cytochrome P450s through two activation pathways: 2'-hydroxylation and 5'-hydroxylation. While most previous studies have focused on 2'-hydroxylation in target tissues of rats, available evidence suggests that 5'-hydroxylation is a major activation pathway in human enzyme systems, in nonhuman primates, and in target tissues of some other rodent carcinogenicity models. In the study reported here, we investigated DNA damage resulting from NNN 5'-hydroxylation by quantifying the adduct 2-(2-(3-pyridyl)-N-pyrrolidinyl)-2'-deoxyinosine (py-py-dI). In rats treated with NNN in the drinking water (7-500 ppm), py-py-dI was the major DNA adduct resulting from 5'-hydroxylation of NNN in vivo. Levels of py-py-dI in the lung and nasal cavity were the highest, consistent with the tissue distribution of CYP2A3. In rats treated with (S)-NNN or (R)-NNN, the ratios of formation of (R)-py-py-dI to (S)-py-py-dI were not the expected mirror image, suggesting that there may be a carrier for one of the unstable intermediates formed upon 5'-hydroxylation of NNN. Rat hepatocytes treated with (S)- or (R)-NNN or (2'S)- or (2'R)-5'-acetoxyNNN exhibited a pattern of adduct formation similar to that of live rats. In vitro studies with human liver S9 fraction or human hepatocytes incubated with NNN (2-500 μM) demonstrated that py-py-dI formation was greater than the formation of pyridyloxobutyl-DNA adducts resulting from 2'-hydroxylation of NNN. (S)-NNN formed more total py-py-dI adducts than (R)-NNN in human liver enzyme systems, which is consistent with the critical role of CYP2A6 in the 5'-hydroxylation of NNN in human liver. The results of this study demonstrate that the major DNA adduct resulting from NNN metabolism by human enzymes is py-py-dI and provide potentially important new insights into the metabolic activation of NNN in rodents and humans.

Recently, we have shown that (S)-N'-Nitrosonornicotine [(S)-NNN], the major form of NNN in tobacco products, is a potent oral cavity and esophageal carcinogen in rats. To determine the early molecular alterations induced by (S)-NNN in the oral and esophageal mucosa, we administered the carcinogen to rats in the drinking water for 10 wk and global gene expression alterations were analyzed by RNA sequencing. At a false discovery rate P-value

Tobacco is a complex chemical mixture, containing many toxicants and carcinogens. Most rodent risk assessment studies have focused on single chemicals or the complicated mixtures of tobacco smoke or its fractions. There are few studies evaluating how specific chemicals interact with one another to form the potent carcinogenic mixture of tobacco smoke. We hypothesized that tobacco smoke aldehydes like formaldehyde and acetaldehyde could enhance the carcinogenic properties of the tobacco-specific nitrosamines, N’-nitrosonornicotine (NNN) and 4-methylnitrosamine-1-(3-pyridyl)-1-butanone (NNK), through a variety of mechanisms. This hypothesis was tested in two established rodent tumor models, the NNN-induced rat esophageal tumor model and the NNK-induced A/J mouse lung tumor model. In the first model, rats were exposed to 0, 4, or 8 ppm NNN in the drinking water in the presence or absence of 3000 ppm acetaldehyde for up to 100 weeks. The number of esophageal papillomas per rat was doubled in animals receiving both acetaldehyde and 8 ppm NNN (0.5 versus 1.15 tumors/rat, respectively). Acetaldehyde alone did not cause esophageal tumors. DNA adduct levels were not affected by the combination of the two chemicals. In the second model, A/J mice were exposed to NNK (i.p, 0, 2.5, or 7.5 μmol in saline) in the presence or absence of acetaldehyde (0 or 360 ppmv) or formaldehyde (0 or 15 ppmv) for 3 hours in a nose-only inhalation chamber. Lung tumors were counted 16 weeks later. Neither aldehyde by itself induced lung tumors. However, mice receiving both NNK and acetaldehyde or formaldehyde had an increased number of adenomas with dysplasia or progression than those receiving only NNK, suggesting that aldehydes may increase dysplasia in tumors initiated by NNK. As in the rat study, DNA adduct levels were not affected by the coexposure. In a separate experiment, we tested the hypothesis that the elevated levels of carbon dioxide in tobacco smoke could affect the carcinogenic properties of NNK in the A/J mouse; tobacco smoke contains 12.5% carbon dioxide. Mice received a 3 h nose-only carbon dioxide (0, 5, 10, or 15%) coexposure of mice receiving NNK (i.p. in saline, 0, 2.5, or 7.5 μmol). The coexposure to carbon dioxide more than doubled the number of lung adenomas induced by 2.5 μmol NNK, with the maximal effect observed with 10% carbon dioxide (0%: 1.8 ± 1.9; 5%: 3.9 ± 2.8; 10%: 7.1 ± 3.5; 15%; 5.5 ± 2.9 lung adenomas/mouse). Lung adenomas were also significantly increased in mice receiving 7.5 μmol NNK, although to a lesser extent (0%: 11 ± 6.2; 5%: 13 ± 7.9; 10%: 18 ± 6.0; 15%; 12 ± 5.3 lung adenomas/mouse). This additive and synergistic effect of carbon dioxide was highly significant (p value = 1.0 × 10-14). The mechanism of this interaction is under investigation. Collectively, these studies support the hypothesis that the aldehydes and carbon dioxide present in the tobacco mixture interact to enhance the carcinogenic potency of the tobacco specific nitrosamines. (Funded by CA-184987.) Citation Format: Lisa A. Peterson, Marissa K. Oram, Donna E. Seabloom, William E. Smith, Alessia Stornetta, Karin R. Vevang, Monica Flavin, Alexandru F. Tabaran, Ingrid Cornax, M. Gerard O’Sullivan, Pramod Upadhyaya, Lin Zhang, Stephen S. Hecht, Silvia Balbo, Timothy S. Wiedmann. Interactions between tobacco smoke chemicals in rodent tumor models [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A19.

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a potent lung carcinogen and exerts its carcinogen effects upon metabolic activation. The identification and quantitation of NNK metabolites could identify potential biomarkers of bio-activation and detoxification of this potent carcinogen and may be used to predict lung cancer susceptibility among smokers. Here, we have used in vivo isotope labeling and high-resolution mass spectrometry-based methods for the comprehensive profiling of all known and unknown NNK metabolites. The sample enrichment, LC/MS, and data analysis workflow, including the custom script for automated d(0)-d(4) m/z pair peak detection, enabled unbiased identification of numerous NNK metabolites. The structures of the metabolites were confirmed using targeted LC-MS(2) with retention time (t(R)) and MS(2) fragmentation comparison to standards when possible. Eleven known metabolites and the unchanged NNK were identified simultaneously. More importantly, our workflow revealed new and novel NNK metabolites including: the 1,3-Diol (13); α-OH-methyl-NNAL-Gluc (14); nitro-NK-N-oxide (15); nitro-NAL-N-oxide (16); γ-OH NNAL (17); and three N-acetylcysteine (NAC) metabolites (18a-c). We measured the differences in the relative distribution of a panel of nitroso-containing NNK-specific metabolites in rats before and after phenobarbital (PB) treatment, and this serves as a demonstration of a general strategy for the detection of metabolic differences in animal and cell systems. Lastly, we have generated a d(4)-labeled NNK metabolite mixture to be used as internal standards (d(4)-rat urine) for relative quantitation of NNK metabolites in humans, and this new strategy will be used to assess carcinogen exposure and ultimately to evaluate lung cancer risk and susceptibility in smokers.

(S)-N'-Nitrosonornicotine [(S)-NNN] and racemic NNN are powerful oral and esophageal carcinogens in the F344 rat, whereas (R)-NNN has only weak activity. Tumor formation in these tissues of rats treated with racemic NNN was far greater than the sum of the activities of the individual enantiomers. We hypothesized that metabolites of (R)-NNN enhanced levels of DNA adducts produced by (S)-NNN. A test of that hypothesis necessitated the development of a novel liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry method for the analysis of O(6)-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (O(6)-POB-dGuo), a highly mutagenic DNA adduct not previously quantified in rats treated with NNN. The new method, with a limit of detection of 6.5 amol for diluted standard and 100 amol for DNA samples, was applied in this study. Groups of nine F344 rats were treated with doses as follows: 7 ppm (R)-NNN, 7 ppm (S)-NNN, and 14 ppm racemic NNN; 14 ppm (R)-NNN, 14 ppm (S)-NNN, and 28 ppm racemic NNN; or 28 ppm (R)-NNN, 28 ppm (S)-NNN, and 56 ppm racemic NNN for 5 weeks, and tissues were analyzed for DNA adducts. We found statistically significant, but modest, synergistic enhancement of levels of O(6)-POB-dGuo in the esophagus but not the oral cavity of rats treated with racemic NNN (low and median doses only) compared to the sum of the amounts formed in these tissues of rats treated with (S)-NNN or (R)-NNN. There was no synergy in the formation of other POB-DNA adducts of NNN in oral cavity and esophagus, nor was there any evidence for synergy in nasal respiratory and olfactory epithelium, lung, or liver. Our results provide the first quantitation of O(6)-POB-dGuo in DNA from tissues of rats treated with NNN and evidence for synergy in DNA adduct formation as one possible mechanism by which (R)-NNN enhances the carcinogenicity of (S)-NNN in rats.

Although regular aspirin use has been shown to lower the risk of colorectal cancer, its efficacy against lung cancer is weak or inconsistent. Moreover, aspirin use increases the risk of ulcers and stomach bleeding. In this study, we determined the efficacy of nitric oxide-donating aspirin (NO-Aspirin), a safer form of aspirin in which the parent drug is linked to a nitric oxide-releasing moiety through a spacer, to suppress lung tumorigenesis. Under in vitro conditions, NO-Aspirin significantly reduced the proliferation and survival of tumorigenic bronchial cell line (1170) and non-small cell lung cancer (NSCLC) cell lines (A549, H1650, H1975 and HCC827) and colony formation by NSCLC cells at sub- or low micromolar concentrations (≤1 µM for 1170 cells and ≤6 µM for NSCLC cells) in a COX-2 independent manner. These effects were paralleled by suppression of phospho-epidermal growth factor receptor (EGFR), -STAT3, -Akt and -ERK and enhanced caspase 3 and PARP cleavage. Among NSCLC cells, EGFR mutant cells (H1650, H1975 and HCC827) were more sensitive than cells expressing wild-type EGFR (A549) and H1650 cells were the most sensitive. Moreover, NO-Aspirin sensitized H1650 and H1975 cells to the antiproliferative effects of erlotinib, a tyrosine kinase inhibitor. In in vivo studies using 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) + lipopolysaccharide (LPS)-induced model of lung tumorigenesis, NO-Aspirin significantly reduced the number and size of lung tumors, expression of phospho-EGFR and -Akt as well as the pro-inflammatory molecules TNF-α and interferon-gamma. Overall, these results indicate the potential of NO-Aspirin for the chemoprevention of lung cancer in high risk populations.

Glucobrassicin, a quantitatively significant constituent of Brassica vegetables, gives rise to indole-3-carbinol (I3C) and its dimer di-indolylmethane (DIM) when the vegetables are chewed. I3C and DIM have been extensively studied with respect to their anti-carcinogenic properties. However, the presumed intermediate isothiocyanate in their formation, indole-3-methyl isothiocyanate (IMITC), has to our knowledge never been observed, despite the fact that isothiocyanates derived from cruciferous vegetables are known to have anti-carcinogenic properties. Therefore, we investigated the formation and presence in human urine of IMITC by analyzing for its N-acetylcysteine conjugate, IMITC-NAC, in order to gain a more complete understanding of the biochemical pathways leading to formation of I3C and DIM upon consumption of vegetables rich in glucobrassicin. Standard IMITC-NAC was synthesized and its structure confirmed by NMR and MS. IMITC-NAC was identified in extracts of Brussels sprouts chopped in the presence of N-acetylcysteine. An LC-ESI-MS/MS-SRM method for analysis of IMITC-NAC, with [13C,15N]IMITC-NAC as internal standard, was developed and validated. Then, ten subjects (7 females) consumed a salad of Brussels sprouts and cabbage (containing 100 to 500 μmol glucobrassicin) once daily for 3 days. Urine was collected at intervals up to 24 h after vegetable consumption. Levels of IMITC-NAC in the urine of these 10 subjects ranged from 0.2 to 30.2 pmol/mL urine. These results provide the first evidence for the presumed intermediacy of IMITC in the formation of I3C and DIM in humans who consumed Brussels sprouts and cabbage as a source of glucobrassicin.

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted mechanism of metabolic activation involves formation of α-hydroxynitrosamines that spontaneously decompose to DNA alkylating agents. Accumulation of DNA damage and the resulting mutations can ultimately lead to cancer. New evidence indicates that α-hydroxynitrosamines can be further oxidized to nitrosamides processively by cytochrome P450s. Because nitrosamides are generally more stable than α-hydroxynitrosamines and can also alkylate DNA, nitrosamides may play a role in carcinogenesis. In this report, we describe a general protocol for evaluating nitrosamide production from in vitro cytochrome P450-catalyzed metabolism of nitrosamines. This protocol utilizes a general approach to the synthesis of the relevant nitrosamides and an in vitro cytochrome P450 metabolism assay using liquid chromatography-nanospray ionization-high resolution tandem mass spectrometry for detection. This method detected N′-nitrosonorcotinine as a minor metabolite of N′-nitrosonornicotine in the example study. The method has high sensitivity and selectively due to accurate mass detection. Application of this method to a wide variety of nitrosamine-cytochrome P450 systems will help determine the generality of this transformation. Because cytochrome P450s are polymorphic and vary in activity, a better understanding of nitrosamide formation could aid in individual cancer risk assessment.

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted mechanism of metabolic activation involves formation of α-hydroxynitrosamines that spontaneously decompose to DNA alkylating agents. Accumulation of DNA damage and the resulting mutations can ultimately lead to cancer. New evidence indicates that α-hydroxynitrosamines can be further oxidized to nitrosamides processively by cytochrome P450s. Because nitrosamides are generally more stable than α-hydroxynitrosamines and can also alkylate DNA, nitrosamides may play a role in carcinogenesis. In this report, we describe a general protocol for evaluating nitrosamide production from in vitro cytochrome P450-catalyzed metabolism of nitrosamines. This protocol utilizes a general approach to the synthesis of the relevant nitrosamides and an in vitro cytochrome P450 metabolism assay using liquid chromatography-nanospray ionization-high resolution tandem mass spectrometry for detection. This method detected N'-nitrosonorcotinine as a minor metabolite of N'-nitrosonornicotine in the example study. The method has high sensitivity and selectively due to accurate mass detection. Application of this method to a wide variety of nitrosamine-cytochrome P450 systems will help determine the generality of this transformation. Because cytochrome P450s are polymorphic and vary in activity, a better understanding of nitrosamide formation could aid in individual cancer risk assessment.

(+)-Dihydromethysticin was recently identified as a promising lung cancer chemopreventive agent while (+)-dihydrokavain was completely ineffective. A pilot in vivo structure-activity relationship (SAR) was explored, evaluating the efficacy of its analogs in blocking 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced short-term O6-methylguanine and long-term adenoma formation in the lung tissues in A/J mice. Both results revealed cohesive SARs, demonstrating that the methylenedioxy functional group in DHM is essential while the lactone functional group tolerates modifications.

Indole-3-carbinol (I3C), a constituent of commonly consumed Brassica vegetables, has been shown to have anticancer effects in a variety of preclinical models of lung cancer. However, it has shown only limited efficacy in clinical trials, likely due to its poor oral bioavailability. Intranasal administration of I3C has the potential to enhance the pulmonary accumulation of the drug, thereby improving its availability at the target site of action. In this study, we developed a liposomal formulation of I3C and evaluated its lung delivery and chemopreventive potential in tobacco smoke carcinogen [4-(methylnitro-samino)-1-(3-pyridyl)-1-butanone (NNK)]-treated mice. Intranasal administration of I3C liposomes led to a ~100-fold higher lung exposure of I3C than the oral route of administration. Further, intranasal delivery of liposomal I3C led to a significant reduction (37%; p

Introduction Based on previous metabolism studies carried out in patas monkeys, we hypothesized that urinary 3'-hydroxynorcotinine could be a specific biomarker for uptake and metabolism of the carcinogen N'-nitrosonornicotine in people who use tobacco products. Methods We developed a method for quantitation of 3'-hydroxynorcotinine in human urine. [Pyrrolidinone-(13)C4]3'-hydroxynorcotinine was added to urine as an internal standard, the samples were treated with β-glucuronidase, partially purified by solid supported liquid extraction and quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry. Results The method was accurate (average accuracy = 102%) and precise (coefficient of variation = 5.6%) in the range of measurement. 3'-Hydroxynorcotinine was detected in 48 urine samples from smokers (mean 393±287 pmol/ml urine) and 12 samples from individuals who had stopped smoking and were using the nicotine patch (mean 658±491 pmol/ml urine), but not in any of 10 samples from nonsmokers. Conclusions Since the amounts of 3'-hydroxynorcotinine found in smokers' urine were approximately 50 times greater than the anticipated daily dose of N'-nitrosonornicotine, we concluded that it is a metabolite of nicotine or one of its metabolites, comprising perhaps 1% of nicotine intake in smokers. Therefore, it would not be suitable as a specific biomarker for uptake and metabolism of N'-nitrosonornicotine. Since 3'-hydroxynorcotinine has never been previously reported as a constituent of human urine, further studies are required to determine its source and mode of formation.

We previously reported the chemopreventive potential of kava against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)- and benzo(a)pyrene (BaP)–induced lung tumorigenesis in A/J mice during the initiation and postinitiation stages. In this study, we investigated the tumorigenesis-stage specificity of kava, the potential active compounds, and the underlying mechanisms in NNK-induced lung tumorigenesis in A/J mice. In the first experiment, NNK-treated mice were given diets containing kava at a dose of 5 mg/g of diet during different periods. Kava treatments covering the initiation stage reduced the multiplicity of lung adenomas by approximately 99%. A minimum effective dose is yet to be defined because kava at two lower dosages (2.5 and 1.25 mg/g of diet) were equally effective as 5 mg/g of diet in completely inhibiting lung adenoma formation. Daily gavage of kava (one before, during, and after NNK treatment) completely blocked lung adenoma formation as well. Kavalactone-enriched fraction B fully recapitulated kava's chemopreventive efficacy, whereas kavalactone-free fractions A and C were much less effective. Mechanistically, kava and fraction B reduced NNK-induced DNA damage in lung tissues with a unique and preferential reduction in O6-methylguanine (O6-mG), the highly tumorigenic DNA damage by NNK, correlating and predictive of efficacy on blocking lung adenoma formation. Taken together, these results demonstrate the outstanding efficacy of kava in preventing NNK-induced lung tumorigenesis in A/J mice with high selectivity for the initiation stage in association with the reduction of O6-mG adduct in DNA. They also establish the knowledge basis for the identification of the active compound(s) in kava. Cancer Prev Res; 7(1); 86–96. ©2013 AACR.

We quantified DNA adducts resulting from 2'-hydroxylation of enantiomers of the tobacco-specific nitrosamine N'-nitrosonornicotine (NNN) in tissues of male F-344 rats after 10, 30, 50, and 70 weeks of treatment with 14 ppm in the drinking water. These rats were in subgroups of a carcinogenicity study in which (S)-NNN was highly tumorigenic in the oral cavity and esophagus, while (R)-NNN was relatively weakly active. DNA adducts were quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry in six tissues: oral mucosa, esophageal mucosa, nasal respiratory mucosa, nasal olfactory mucosa, liver, and lung. O²-[4-(3-Pyridyl)-4-oxobut-1-yl]thymidine (O²-POB-dThd, 7) and 7-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxyguanosine (7-POB-dGuo, 8), the latter as 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine (7-POB-Gua, 11), were detected at each time point in each tissue. In the target tissues for carcinogenicity, oral mucosa and esophageal mucosa, levels of 7-POB-Gua (11) and O²-POB-dThd (7) were similar, or 11 predominated, while in all other tissues at all time points for both enantiomers, 7 was clearly present in greater amounts than 11. Total measured DNA adduct levels in esophageal mucosa and oral mucosa were higher in rats treated with (S)-NNN than (R)-NNN. The highest adduct levels were found in the nasal respiratory mucosa. DNA adducts generally persisted in all tissues without any sign of substantial decreases throughout the 70 week time course. The results of this study suggest that inefficient repair of 7-POB-dGuo (8) in the rat oral cavity and esophagus may be important in carcinogenesis by NNN and support the development of these DNA adducts as potential biomarkers of NNN metabolic activation in people who use tobacco products.

N′-Nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are carcinogenic tobacco-specific nitrosamines believed to play a vital role in the initiation of tobacco-related cancers. To exhibit their carcinogenicity, both NNN and NNK must be metabolically activated by cytochrome P450s, specifically P450 2A6 and P450 2A13, respectively. Prior research has focused on α-hydroxylation, which leads to the formation of several DNA adducts that have been identified and quantified in vivo. However, some studies indicate that P450s can retain substrates within their active sites and perform processive oxidation. For nitrosamines, this would oxidize the highly unstable α-hydroxynitrosamines to potentially more stable nitrosamides, which could also alkylate DNA. Thus, we hypothesized that both NNN and NNK are processively oxidized in vitro to nitrosamides by P450 2A6 and P450 2A13, respectively. To test this hypothesis, we synthesized the NNN- and NNK-derived nitrosamides, determined their half-lives at pH 7.4 and 37 °C, and monitored for nitrosamide formation in an in vitro P450 system with product analysis by LC-NSI+-HRMS/MS. Half-lives of the nitrosamides were determined by HPLC-UV and ranged from 7–35 min, which is more than 40 times longer than the corresponding α-hydroxynitrosamines. Incubation of NNN in the P450 2A6 system resulted in the formation of the nitrosamide, N'-nitrosonorcotinine (NNC) at low levels. Similarly, the nitrosamide 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanedione (CH2-oxo-NNK), was detected in low amounts in the incubation of NNK with the P450 2A13 system. The other possible NNK-derived nitrosamide, 4-(nitrosoformamido)-1-(3-pyridyl)-1-butanone (CH3-oxo-NNK), was not observed in the P450 2A13 reactions. CH2-oxo-NNK readily formed O6meGua in reactions with dGuo and calf thymus DNA. These results demonstrate that NNC and CH2-oxo-NNK are novel metabolites of NNN and NNK, respectively. Though low-forming, their increased stability may allow for mutagenic DNA damage in vivo. More broadly, this study provides the first account of a cytochrome-P450 mediated conversion of nitrosamines to nitrosamides which warrants further studies to determine how general this phenomenon is in nitrosamine metabolism.

DNA adducts are a measure of internal exposure to genotoxicants and an important biomarker for human risk assessment. However, the employment of DNA adducts as biomarkers in human studies is often restricted because fresh-frozen tissues are not available. In contrast, formalin-fixed paraffin-embedded (FFPE) tissues with clinical diagnosis are readily accessible. Recently, our laboratory reported that DNA adducts of aristolochic acid, a carcinogenic component of Aristolochia herbs used in traditional Chinese medicines worldwide, can be recovered quantitatively from FFPE tissues. In this study, we have evaluated the efficacy of our method for retrieval of DNA adducts from archived tissue by measuring DNA adducts derived from four other classes of human carcinogens: polycyclic aromatic hydrocarbons (PAHs), aromatic amines, heterocyclic aromatic amines (HAAs), and N-nitroso compounds (NOCs). Deoxyguanosine (dG) adducts of the PAH benzo[a]pyrene (B[a]P), 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (dG-N(2)-B[a]PDE); the aromatic amine 4-aminobiphenyl (4-ABP), N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP); the HAA 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), N-(deoxyguanosin-8-yl)-PhIP (dG-C8-PhIP); and the dG adducts of the NOC 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), O(6)-methyl-dG (O(6)-Me-dG) and O(6)-pyridyloxobutyl-dG (O(6)-POB-dG), formed in liver, lung, bladder, pancreas, or colon were recovered in comparable yields from fresh-frozen and FFPE preserved tissues of rodents treated with the procarcinogens. Quantification was achieved by ultraperformance liquid chromatography coupled with electrospray ionization ion-trap multistage mass spectrometry (UPLC/ESI-IT-MS(3)). These advancements in the technology of DNA adduct retrieval from FFPE tissue clear the way for use of archived pathology samples in molecular epidemiology studies designed to assess the causal role of exposure to hazardous chemicals with cancer risk.

of chemotherapeutic agents (3–10). Also, in murine models of early phase tumorigenesis, DIM has been shown to inhibit tumor formation in the prostate (11), cervix (12) and lung (13). Despite the promising anticancer effects of DIM, most of the preclinical studies in murine models have been carried out at dose levels that are at least one order of magnitude higher than the amount of DIM tolerated by humans. Another issue is the poor bioavailability of DIM. Administration of DIM to human volunteers at the maximum tolerated dose level (14) led to a plasma concentration that was two orders of magnitude less than the concentrations of the agent required to induce apoptotic and antiproliferative effects in cell line models (3). In lung chemoprevention studies, one promising strategy to overcome the poor bioavailability and potential systemic toxicities of DIM is administration of the agent via the intranasal route, a non-invasive drug administration method, which enables direct delivery of the agent to the lung, thereby imparting optimal DIM concentration at the target organ but decreased exposure of non-target normal tissues. In this study, we examined the efficacy of intranasally administered bio-response DIM (BRD), absorption-enhanced DIM formulation, against 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)induced lung tumorigenesis in mice. Moreover, we assessed, using an in vitro model of lung tumorigenesis consisting of bronchial cells at different stages of transformation, the antiproliferative and apoptotic activities of BRD and the mechanisms through which these effects are mediated. Our findings indicate that intranasal administration of BRD, at dose levels equivalent to those found to be well tolerated in humans, significantly inhibited NNK-induced lung tumorigenesis in mice. Also, BRD differentially suppressed the growth and survival of premalignant and malignant bronchial cells with minimal effects in parental immortalized cells through, at least partly, inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway.

We studied the chemopreventive efficacy of indole-3-carbinol (I3C), a phytochemical found in cruciferous vegetables, to inhibit tobacco carcinogen-induced lung adenocarcinoma in A/J mice when given following post-initiation or progression protocol. Moreover, we assessed the potential mechanisms responsible for the anticancer effects of I3C. Post-initiation administration of I3C decreased the multiplicity of surface tumors as well as all forms of histopathological lesions, including adenocarcinoma, whereas administration of the compound during tumor progression failed to decrease the multiplicity of surface tumors and early forms of microscopic lesions but reduced the frequency of adenocarcinoma. Mechanistic studies in A549 lung adenocarcinoma cells indicated that the lung cancer preventive effects of I3C are mediated, at least in part, via modulation of the receptor tyrosine kinase/PI3K/Akt signaling pathway.

Polycyclic aromatic hydrocarbons (PAHs) are believed to be among the causative agents for lung cancer in smokers. PAHs require metabolic activation for carcinogenicity. One pathway produces diol epoxides that react with DNA, causing mutations. Because diol epoxides are converted to tetraols, quantitation of tetraols can potentially be used to identify smokers who may be at higher risk for lung cancer. Our approach uses [D(10)]phenanthrene, a labeled version of phenanthrene, a noncarcinogenic PAH structurally analogous to carcinogenic PAH. Although smokers are exposed to PAH by inhalation, oral dosing would be more practical for phenotyping studies. Therefore, we investigated [D(10)]phenanthrene metabolism in smokers after administration by inhalation in cigarette smoke or orally. Sixteen smokers received 10 μg of [D(10)]phenanthrene in a cigarette or orally. Plasma and urine samples were analyzed for [D(10)]r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene ([D(10)]PheT), the major end product of the diol epoxide pathway, by gas chromatography-negative ion chemical ionization-tandem mass spectrometry. The ratios of [D(10)]PheT (oral dosing/inhalation) in 15 smokers were 1.03 ± 0.32 and 1.02 ± 0.35, based on plasma area under the concentration-time curve (0-∞) and total 48-h urinary excretion, respectively. Overall, there was no significant difference in the extent of [D(10)]PheT formation after the two different routes of exposure in smokers. A large interindividual variation in [D(10)]PheT formation was observed. These results demonstrate that the level of [D(10)]PheT in urine after oral dosing of [D(10)]phenanthrene can be used to assess individual capacity of PAH metabolism by the diol epoxide pathway.

This perspective reviews our work on the identification of DNA adducts of N-nitrosopyrrolidine and some related aldehydes. The research began as a focused project to investigate mechanisms of cyclic nitrosamine carcinogenesis but expanded into other areas, as aldehyde metabolites of NPYR were shown to have their own diverse DNA adduct chemistry. A total of 69 structurally distinct DNA adducts were identified, and some of these, found in human tissues, have provided intriguing leads for investigating carcinogenesis mechanisms in humans due to exposure to both endogenous and exogenous agents.

In earlier studies, we demonstrated the efficacy of indole-3-carbinol (I3C) against lung adenocarcinoma in A/J mice. However, these effects were accompanied by reductions in body weight gain. We therefore assessed if combinations of low doses of I3C with silibinin could inhibit lung tumorigenesis without causing undesirable side effects. In in vitro assays with A549 and H460 lung cancer cells, exposure of the cells to a mixture of low concentrations of I3C (50 μM) plus silibinin (50 μM) for 72 h caused inhibition of cell growth and extracellular signal-regulated kinase (ERK) and Akt activation and induction of apoptosis, whereas the individual agents did not have any effect. In mice pretreated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and given I3C (10 μmol/g diet) plus silibinin (7 μmol/g diet), multiplicities of tumors on the surface of the lung and adenocarcinoma were reduced by 60 and 95%, respectively. The individual effects of I3C and silibinin were relatively weaker: 43 and 36% reductions, respectively, in the multiplicity of tumors on the surface of the lung and 83 and 50% reductions, respectively, in the number of adenocarcinoma. Also, the expression of phospho-Akt, phospho-ERK and cyclin D1 and poly (ADP-ribose) polymerase cleavage were strongly modulated by I3C plus silibinin than by I3C or silibinin alone, suggesting that the chemopreventive activities of the mixture could be mediated, at least partly, via modulation of the level of these proteins. Taken together, our findings showed that mixtures of I3C and silibinin are more potent than the individual compounds for the chemoprevention of lung cancer in A/J mice.

Polycyclic aromatic hydrocarbons (PAH) are among the likely major causative agents for lung cancer in smokers. PAH require metabolic activation to exert their carcinogenic effects, and one important pathway proceeds through a three-step sequence resulting in the formation of diol epoxides, which react with DNA to produce adducts that can cause mutations and initiate the carcinogenic process. However, no previous published studies have examined this critical pathway in humans specifically exposed to PAH by inhalation of cigarette smoke. This study used a unique approach employing a stable isotope derivative of phenanthrene, the simplest PAH with a bay region, a feature closely associated with PAH carcinogenicity. Twelve subjects each smoked a cigarette to which [D(10)]phenanthrene had been added. Plasma was analyzed for [D(10)]r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene ([D(10)]PheT), the major end product of the diol epoxide metabolism pathway of phenanthrene. The analysis was performed by gas chromatography--negative ion chemical ionization--tandem mass spectrometry, using [(13)C(6)]PheT as internal standard. The results demonstrated that the three-step pathway resulting in the formation of diol epoxides, as monitored by [D(10)]PheT, occurred with remarkable rapidity. Levels of [D(10)]PheT in plasma of all subjects were maximal at the earliest time points examined, 15-30 min after smoking the cigarette containing [D(10)]phenanthrene, and decreased thereafter. These results demonstrate that the formation of a PAH diol epoxide occurs rapidly in smokers. Because PAH diol epoxides are mutagenic and carcinogenic, the results clearly demonstrate immediate negative health consequences of smoking, which should serve as a major warning to anyone contemplating initiating tobacco use.