NNK-induced Lung Carcinogenesis Research Articles

Suppressing the activation of protein kinase A as a DNA damage-independent mechanistic lead for dihydromethysticin prophylaxis of NNK-induced lung carcinogenesis.

Our earlier work demonstrated varying potency of dihydromethysticin (DHM) as the active kava phytochemical for prophylaxis of tobacco carcinogen nicotine-derived nitrosamine ketone (NNK)-induced mouse lung carcinogenesis. Efficacy was dependent on timing of DHM gavage ahead of NNK insult. In addition to DNA adducts in the lung tissues mitigated by DHM in a time-dependent manner, our in vivo data strongly implicated the existence of DNA damage-independent mechanism(s) in NNK-induced lung carcinogenesis targeted by DHM to fully exert its anti-initiation efficacy. In the present work, RNA seq transcriptomic profiling of NNK-exposed (2 h) lung tissues with/without a DHM (8 h) pretreatment revealed a snap shot of canonical acute phase tissue damage and stress response signaling pathways as well as an activation of protein kinase A (PKA) pathway induced by NNK and the restraining effects of DHM. The activation of the PKA pathway by NNK active metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) at a concentration incapable of promoting DNA adduct was confirmed in a lung cancer cell culture model, potentially through NNAL binding to and activation of the β-adrenergic receptor. Our in vitro and in vivo data overall support the hypothesis that DHM suppresses PKA activation as a key DNA damage-independent mechanistic lead, contributing to its effective prophylaxis of NNK-induced lung carcinogenesis. Systems biology approaches with a detailed temporal dissection of timing of DHM intake versus NNK exposure are warranted to fill the knowledge gaps concerning the DNA damage-driven mechanisms and DNA damage-independent mechanisms to optimize the implementation strategy for DHM to achieve maximal lung cancer chemoprevention.

In vivo Structure-Activity Relationship of Dihydromethysticin in Reducing Nicotine-Derived Nitrosamine Ketone (NNK)-Induced Lung DNA Damage against Lung Carcinogenesis in A/J Mice.

Lung cancer is the leading cause of cancer-related deaths and chemoprevention should be developed. We recently identified dihydromethysticin (DHM) as a promising candidate to prevent NNK-induced lung tumorigenesis. To probe its mechanisms and facilitate its future translation, we investigated the structure-activity relationship of DHM on NNK-induced DNA damage in A/J mice. Twenty DHM analogs were designed and synthesized. Their activity in reducing NNK-induced DNA damage in the target lung tissues was evaluated. The unnatural enantiomer of DHM was identified to be more potent than the natural enantiomer. The methylenedioxy functional moiety did not tolerate modifications while the other functional groups (the lactone ring and the ethyl linker) accommodated various modifications. Importantly, analogs of high structural similarity to DHM with distinct efficacy in reducing NNK-induced DNA damage have been identified. They will serve as chemical probes to elucidate the mechanisms of DHM in blocking NNK-induced lung carcinogenesis.

Characterization of adductomic totality of NNK, (R)-NNAL and (S)-NNAL in A/J mice, and their correlations with distinct lung carcinogenicity.

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 8h 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.

Effects of tobacco compound 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on the expression of epigenetically regulated genes in lung carcinogenesis

ABSTRACT Cigarette smoking is a major cause of lung cancer. Although tobacco smoking-induced genotoxicity has been well established, there is apparent lack of abundance functional epigenetic effects reported On cigarette smoke-induced lung carcinogenesis. The aim of this study was to determine effects of intratracheal administration of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) utilizing target gene expression DNA methylation patterns in lung tissues of mice following twice weekly for 8 weeks treatment. An unbiased approach where genomic regions was undertaken to assess early methylation changes within mouse pulmonary tissues. A methylated-CpG island recovery assay (MIRA) was performed to map the DNA methylome in lung tissues, with the position of methylated DNA determined using a Genome Analyzer (MIRA-SEQ). Alterations in epigenetic-regulated target genes were confirmed with quantitative reverse transcription-PCR, which revealed 35 differentially hypermethylated genes including Cdkn1C, Hsf4, Hnf1a, Cdx1, and Hoxa5 and 30 differentially hypomethylated genes including Ddx4, Piwi1, Mdm2, and Pce1 in NNK-exposed lung tissue compared with controls. The main pathway of these genes for mediating biological information was analyzed using the Kyoto Encyclopedia of Genes and Genomes database. Among them, Rssf1 and Mdm2 were closely associated with NNK-induced lung carcinogenesis. Taken together, our data provide valuable resources for detecting cigarette smoke-induced lung carcinogenesis.

Abstract 2596: Stage-specific inhibition of NNK-induced lung carcinogenesis by 1,4-phenylenebis(methylene)seleno-aspirin (p-XS-Asp)

Abstract Chemopreventive efficacy of p-XSC is well documented but it failed due to systemic toxicity issues. This toxicity could partially be due to the release of poisonous hydrogen cyanide generated after p-XSC metabolizes to form active bis-selenol (p-XSeH). To address the said concern, we recently designed and developed p-XS-Asp, with the rationale that it would cleave in vivo to release the active p-XSeH and aspirin instead of undesired HCN, thus making the compound less toxic and possibly more potent than p-XSC. Indeed, we have shown previously that p-XS-Asp inhibits NNK-induced lung tumorigenesis in A/J mice more effectively than p-XSC in a complete A/J mouse model, and is better tolerable. The complete model, however, does not reveal at what stage of carcinogenesis does p-XS-Asp acts. In the current study, we evaluated the stage-specificity of chemopreventive efficacy of p-XS-Asp in NNK-induced lung carcinogenesis in A/J mice models. A/J mice were divided into 7 distinct groups (n=30 per group, half male, half female; except group 1 and 2 where n=10). The mice were fed AIN-93M diet (control) or p-XS-Asp diet until they reached termination end point of 26 weeks (adenomas) and 40 weeks (adenocarcinomas). Two weeks after the experiment started, all the groups, except for group 1 and 2, were given a single IP injection of 10 µmol (100 mg/kg) of NNK. Group 3 and 4 were on complete control AIN93M and p-XS-Asp diets, respectively. Group 5 (peri-initiation) was on p-XS-Asp for the first 3 weeks and then on the control diet till the end of the experiment. On the other hand, group 6 (post-initiation) was on control diet for 3 weeks and then was changed to the p-XS-Asp diet. In the progression group 7, mice were on control diet for 14 weeks and subsequently changed to p-XS-Asp diet. The Groups 4 and 5 mice showed a remarkable decrease in the tumor multiplicity (TM) and incidence (TI) as compared to the mice on control diet at both 26 and 40 week time-points. The TM in male mice for groups 1, 2, 3, 4, 5, 6 and 7 at 26 weeks was 0.6, 0, 5.7, 1, 1.54, 4.27, and 6.27, respectively, while at 40 weeks, it was 0.2, 0, 7.7, 1, 1.8, 4.1, and 4.9, respectively. A similar trend was also observed in female mice. These data clearly demonstrated that robust inhibition at peri-initiation stage is mainly responsible for the activity observed in the complete model. The inhibition of both O6-methylguanine and pyridoxobutyl mutagenic DNA adducts by p-XS-Asp further compliments that the compound acts at initial stages of carcinogenesis. Body weights comparison and the blood and tissue analyses showed no systemic toxicity for the p-XS-Asp fed groups. RNA-seq data of the tumor tissues showed numerous signaling pathways to be affected in p-XS-Asp treated mice. The exact mechanism of action is still under investigation. In summary, our results show that p-XS-Asp may be promising candidate for future clinical evaluation as a lung cancer preventive agent. Citation Format: Asif Raza, Amandeep Singh, Cesar Aliaga, Daniel Plano, Shantu Amin, Arun K. Sharma. Stage-specific inhibition of NNK-induced lung carcinogenesis by 1,4-phenylenebis(methylene)seleno-aspirin (p-XS-Asp) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2596.

Abstract 2349: Modulation of inflammation in the tumor microenvironment by ER and PPARγ in an NNK-induced lung carcinogenesis model

Abstract Lung cancer is the number one cause of cancer-related fatalities, and to date, there are no FDA-approved chemoprevention options for lung cancer prevention. NNK, the principal carcinogen in cigarette smoke can induce mutations in oncogenes and can also act as an inflammatory mediator of the tumor microenvironment (TME) by promoting macrophage infiltration into the lungs. Macrophages play a key role in regulation of the lung TME. Pioglitazone, a synthetic peroxisome proliferator-activated receptor gamma (PPARγ) agonist belonging to the thiazolidinediones (TZDs) drug class has been used in preclinical studies to mitigate lung tumorigenesis, progression, and metastasis. A retrospective analysis found that diabetics using TZDs experienced a 33% reduction in lung cancer incidence. Cross-talk between PPARγ and estrogen receptor (ER) signaling has been previously reported, and ER has already been implicated as a driver of lung cancer severity. Both ER and PPARγ are also known to be functional in macrophages. Agents that block ER signaling such as fulvestrant, a complete ER antagonist, have shown activity against lung cancer. We therefore tested the ability of pioglitazone and fulvestrant in combination to reduce lung tumor formation and to modulate the TME. Preliminary results from a co-culture simulation of the TME in vitro using human macrophages and lung adenocarcinoma cells show pioglitazone and fulvestrant together can significantly suppress inflammatory modulators such as IL-1β, IL-10, Amphiregulin (AREG), and VEGF compared to single treatments. Similar effects were observed in a mouse model system of the TME. This drug combination also reduced number of tumor cell colonies in a soft agar assay, suggesting a direct effects on tumor cells. In vivo use of combined pioglitazone and fulvestrant in an NNK female mouse model of former smokers showed a significant reduction in tumor size by 40% compared to placebo (p= 0.0010), 13% by pioglitazone alone and 5% by fulvestrant alone. Inflammatory cytokine array analysis of the bronchio-alveolar lavage fluid (BALF) revealed maximum down-modulation of pro-tumor inflammatory mediators with combination treatment compared to single treatments. Fulvestrant alone did not suppress tumor-promoting inflammatory pathways, suggesting possible activation of compensatory signaling pathways. Further analysis of the cell cultures and the BALF confirmed EGFR compensatory activation through release of AREG with fulvestrant single treatment, that is suppressed with the addition of pioglitazone. Thus, the strong combination effect of pioglitazone and fulvestrant on lung tumor growth and macrophage function, and the ability of the combination to suppress compensatory signaling pathways, supports the hypothesis of ER/PPARγ cross-talk in lung cancer and provides a rationale for further investigation of chemopreventive effects of this combination. Citation Format: Erika Louiselle, Oshin Miranda, Huiyu Li, MiKayla Boeder, Zhijie Li, Jill M. Siegfried, Mariya Farooqui. Modulation of inflammation in the tumor microenvironment by ER and PPARγ in an NNK-induced lung carcinogenesis model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2349.

Dose-Related Modulatory Effects of Polymeric Black Tea Polyphenols (PBPs) on Initiation and Promotion Events in B(a)P and NNK-Induced Lung Carcinogenesis

Our understanding of dose-related effects of polymeric black tea polyphenols (PBPs), the most abundant polyphenols in black tea, is limited. In the present study, the effect of various doses of black tea (0.75, 1.5, and 3%)-derived PBP-rich extract on biochemical parameters and lung carcinogenicity in A/J mice was investigated. Pretreatment with PBPs showed the dose-related decrease in B(a)P-induced expression and activity of CYP1A1 in the liver while CYP1A2 expression and activity in the lung. Dose-dependent significant increase in PBP-mediated over-expression and activity of GSTs (alpha in the liver while pi in the lung) were observed in polyphenol-treated groups. Significant dose-related decrease in number and intensity of BPDE-DNA adducts were observed in liver and lung. Black tea (1.5%, 3%)-derived PBPs showed dose-mediated decrease in lung tumor incidence and multiplicity which was further correlated with different molecular markers like cell proliferation and apoptosis in B(a)P and NNK model. In conclusion, dose-dependent chemopreventive effects of PBPs, both anti-initiating (induction of phase II and inhibition of carcinogen-induced phase-I enzymes leading to decrease in BPDE-DNA adducts) and anti-promoting (decreased cell proliferation and increased apoptosis lowering incidence and/or multiplicity of lung lesions), were observed in A/J mice without significant toxicity.

P3.01-052 DNA Adductomics to Identify the Role of Inflammation in NNK-Induced Lung Carcinogenesis: Topic: Models of Lung Cancer

The association between pulmonary inflammation and lung cancer is well established. Smokers with chronic obstructive pulmonary disease (COPD) have higher risk of developing lung cancer than smokers without this condition. However, the molecular events underlying the association between inflammation and cancer in the lung are poorly understood. To better understand this association, an A/J mouse model was recently developed which combines exposure to the tobacco specific lung carcinogen NNK and the pro-inflammatory agent LPS. Using an innovative mass spectrometry based DNA adductomic approach, we plan to measure the DNA damage resulting from these exposures in this model. Both NNK and LPS can induce DNA modifications (DNA adducts), if not eliminated or repaired these adducts can result in miscoding events that can lead to misregulation of normal cellular growth control mechanisms and ultimately may result in cancer formation. Traditionally, the standard LC-MS methodology used for DNA adduct measurement focuses on the investigation of small numbers of anticipated DNA adducts based on a priori assumptions regarding their formation from specific exposures or chemicals. This approach does not account for the complexity of in vivo DNA adduct formation resulting from endogenous sources such as oxidative stress, lipid peroxidation or aberrant metabolism, or as a result of exposure to complex mixtures of chemicals which cannot be completely anticipated or predicted. To address this limitation, we have developed a high resolution/accurate mass data dependent-constant neutral loss-MS3 methodology for DNA adductomics using ion trap-orbital trap technology to screen for all DNA modifications simultaneously. We have successfully tested our method on mixtures of standards and applied it to lung DNA samples collected from mice exposed to NNK and to LPS. Our method allowed for the detection of the expected DNA adducts resulting from NNK as well as a number of endogenous DNA adducts resulting from lipid peroxidation, oxidative stress and aberrant metabolism resulting from the LPS induced inflammatory process. These results confirm the ability of our DNA adductomic approach to characterize the DNA damage deriving from these exposures. Our comprehensive DNA adductomic approach contributes to the development of new tools needed to investigate lung carcinogenesis, to elucidate its mechanisms and dissect the molecular pathways involved in inflammation-driven lung cancer, with the ultimate goal of identifying preventive and therapeutic strategies.

Gender-dependent effects of gonadectomy on lung carcinogenesis by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in female and male A/J mice

The present study was conducted to investigate the effects of gonadectomy on lung carcinogenesis in female and male mice, and to determine an association between sex hormone and lung carcinogenesis. Female and male A/J mice were divided into gonadectomized and unoperated control groups and all animals were treated intraperitoneally with 1 or 2 injections of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) at the dose of 2 mg/mouse. The mice were sacrificed 18 or 56 weeks after surgery. Serum levels of estradiol in females and testosterone in males were confirmed to be decreased by gonadectomy. Lung white nodules were detected in all mice of all groups. In the control groups of 18- and 56-week studies, the multiplicities of lung nodules in females were significantly greater than in males. In males in the 56-week study, the multiplicity of macroscopical lung nodules, bronchiolo-alveolar hyperplasias, adenomas and tumors (adenomas and adenocarcinomas) showed significant increase with castration. In females in the 18-week study, the multiplicity of adenomas decreased significantly by ovariectomy. Based on the results of the present study, female A/J mice were confirmed to be more susceptible to NNK-induced lung carcinogenesis than males. Furthermore, it was suggested that the process is inhibited by testosterone and accelerated by estradiol. These findings indicate the possibility that sex hormones play important roles in determining sex differences in lung carcinogenesis in the A/J mice initiated by NNK.

Alteration of serum miR-206 and miR-133b is associated with lung carcinogenesis induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

The alteration of microRNA (miRNA) expression plays an important role in chemical carcinogenesis. Presently, few reports have been published that concern the significance of circulating miRNAs in lung carcinogenesis induced by environmental carcinogens. The purpose of this study was to identify serum miRNAs that could be associated with lung carcinogenesis induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Male F344 rats were systemically administered with NNK. The rat serum differential expression profiles of miRNAs were analyzed by small RNA solexa sequencing. Using quantitative real-time PCR, the differentially expressed serum miRNAs were identified in each individual rat. Serum miR-206 and miR-133b were selected for further identification in rat serum at different stages of lung carcinogenesis; we detected the levels of serum miR-206 and miR-133b in lung cancer tissues induced by NNK. NNK causes significant alteration of serum miRNA expression. Compared to the control group, serum miR-206 and miR-133b were significantly up-regulated in the early stage of NNK-induced lung carcinogenesis. miR-206 and miR-133b exhibited low-expression in lung cancer tissues. Our results demonstrate that lung carcinogen NNK exposure changes the expression of serum miRNAs. Serum miR-206 and miR-133b could be associated with lung carcinogenesis induced by NNK.

Deficiency of CCAAT/enhancer binding protein family DNA binding prevents malignant conversion of adenoma to carcinoma in NNK-induced lung carcinogenesis in the mouse

BackgroundThe CCAAT/enhancer binding proteins (C/EBPs) play important roles in carcinogenesis of many tumors including the lung. Since multiple C/EBPs are expressed in lung, the combinatorial expression of these C/EBPs on lung carcinogenesis is not known.MethodsA transgenic mouse line expressing a dominant negative A-C/EBP under the promoter of lung epithelial Clara cell secretory protein (CCSP) gene in doxycycline dependent fashion was subjected to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung carcinogenesis bioassay in the presence and absence of doxycycline, and the effect of abolition of DNA binding activities of C/EBPs on lung carcinogenesis was examined.ResultsA-C/EBP expression was found not to interfere with tumor development; however, it suppressed the malignant conversion of adenoma to carcinoma during NNK-induced lung carcinogenesis. The results suggested that Ki67 may be used as a marker for lung carcinomas in mouse.ConclusionsThe DNA binding of C/EBP family members can be used as a potential molecular target for lung cancer therapy.

NNK-Induced Lung Tumors: A Review of Animal Model

The incidence of lung adenocarcinoma has been remarkably increasing in recent years due to the introduction of filter cigarettes and secondary-hand smoking because the people are more exposed to higher amounts of nitrogen oxides, especially 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK), which is widely applied in animal model of lung tumors. In NNK-induced lung tumors, genetic mutation, chromosome instability, gene methylation, and activation of oncogenes have been found so as to disrupt the expression profiles of some proteins or enzymes in various cellular signal pathways. Transgenic animal with specific alteration of lung cancer-related molecules have also been introduced to clarify the molecular mechanisms of NNK in the pathogenesis and development of lung tumors. Based on these animal models, many antioxidant ingredients and antitumor chemotherapeutic agents have been proved to suppress the NNK-induced lung carcinogenesis. In the future, it is necessary to delineate the most potent biomarkers of NNK-induced lung tumorigenesis, and to develop efficient methods to fight against NNK-associated lung cancer using animal models.

Fish Oil Supplementation Inhibits NNK-Induced Lung Carcinogenesis in the A/J Mouse

High intake of fish oil with a low omega-6 (n-6)/omega-3 (n-3) polyunsaturated fatty acid (PUFA) ratio has been suggested to protect against many chronic diseases. However, the effect of different ratios of dietary n-6 and n-3 PUFA on lung tumorigenesis has not been investigated. In this study, we examined the effect of a 4 mo dietary supplementation with corn oil (with a high n-6/n-3 ratio) and fish oil (with a low n-6/n-3 ratio) as compared with soybean oil (isocaloric control with the same n-6/n-3 ratio as the base diet) on tumor incidence and tumor prevalence in the A/J mouse model of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung carcinogenesis. We found that dietary supplementation had no effect on overall lung tumor incidence, but fish oil supplementation was able to decrease lung tumor prevalence by 78% and 80% compared to groups receiving soybean oil and corn oil supplementation, respectively. The inhibitory effect of fish oil on lung tumor prevalence was associated with increased expressions of cell cycle inhibitor p21Cip1 and lipoxygenase isoform 15-LOX in the lungs. These data suggest that fish oil with a low ratio of n-6/n-3 PUFA could be beneficial in the prevention of lung carcinogenesis.

The pyridyloxobutyl DNA adduct, O6-[4-oxo-4-(3-pyridyl)butyl]guanine, is detected in tissues from 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-treated A/J mice.

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a potent pulmonary carcinogen. This unsymmetric nitrosamine can be metabolically activated to lung DNA methylating and pyridyloxobutylating intermediates. The methyl DNA adducts are well characterized. The pyridyloxobutyl adducts are unstable under DNA hydrolysis conditions and decompose to release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB). One of the HPB-releasing adducts,O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-pobG), has been detected in DNA reacted in vitro with the model pyridyloxobutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc). To determine whether this adduct was formed in vivo, A/J mice were treated with 10 mumol of [5-3H]NNK and sacrificed 24 h postinjection. The mutagenic O6-pobG was detected in liver but not lung DNA from these animals. Since 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a major metabolite of NNK, it is also possible that these animals are activating NNAL to a pyridylhydroxybutylating agent. Therefore, we also measured the levels of O6-[4-hydroxy-4-(3-pyridyl)butyl]guanine (O6-phbG) in these DNA samples. While radioactivity did coelute with synthetic standard for this potential NNAL adduct in one lung DNA sample, significant levels of O6-phbG were not detected in any other lung or liver DNA samples. The pyridyloxobutyl adduct, O6-pobG, was also observed in lung and liver DNA from mice treated with 4.2 mumol of [5-3H]NNKOAc in the presence but not absence of 2.5 mumol of O6-benzylguanine, a known depletor of the repair protein O6-alkylguanine-DNA alkyltransferase (AGT). These data indicate that this adduct is formed in vivo but is repaired in part by AGT. Cell-free extracts from A/J mouse lung and liver were used to determine the relative rate of O6-alkylguanine repair. O6-mG and O6-pobG were removed from DNA to the same extent in a competitive assay, suggesting that low levels of O6-pobG in lungs of NNK-treated mice did not result from preferential repair of O6-pobG by AGT. It is more likely that initial levels of O6-pobG are much lower than initial levels of O6-mG in lung DNA from NNK-treated A/J mice. These data are consistent with previous studies, which indicate that DNA methylation is the critical pathway for NNK-induced lung carcinogenesis in A/J mice.

Beta-adrenergic modulation of NNK-induced lung carcinogenesis in hamsters.

Lung cancer is the leading cause of cancer death in industrialized countries. Pulmonary adenocarcinoma (PAC) is the most common histologic type of lung cancer, and it is reproducibly induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)- 1-(3-pyridyl)-1-butanone (NNK) in laboratory rodents. We have recently shown that the growth of cell lines derived from human PACs is controlled by beta-adrenergic receptors, and that NNK is a high affinity agonist for this receptor family. In the current study, we have tested the relevance of these in in vitro findings for in vivo NNK-induced lung tumorigenesis, using a well-established hamster model of NNK-induced PAC. Our experiments demonstrate a significant increase in NNK-induced PAC multiplicity in animals chronically exposed to the beta-adrenergic agonist epinephrine or theophylline which causes intracellular accumulation of the beta-adrenergic second messenger cAMP. On the other hand, our data show that administration of the beta-adrenergic antagonist propranolol prior to each NNK injection significantly inhibited the development of PACs. Our findings support the hypothesis that the development of tobacco-associated PAC may be modulated by beta-adrenergic agents, and that the interaction of NNK with beta-adrenergic receptors contributes to the genesis of this histologic lung cancer type.

Approaches to chemoprevention of lung cancer based on carcinogens in tobacco smoke.

Chemoprevention may be one way to prevent lung cancer in smokers who are motivated to quit but cannot stop. The approach to chemoprevention of lung cancer described in this article is based on an understanding of the lung carcinogens present in tobacco smoke. The available data indicate that the compounds in cigarette smoke most likely involved in the induction of lung cancer in humans are the complex of polynuclear aromatic hydrocarbons typified by benzo[a]pyrene (B[a]P) and the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). A large number of compounds are now available that inhibit lung tumorigenesis by B[a]P or NNK in rodents. Inhibition of NNK-induced lung carcinogenesis by phenethyl isothiocyanate (PEITC) and inhibition of B[a]P-induced lung carcinogenesis by benzyl isothiocyanate (BITC) are discussed as examples. Studies with PEITC in rodents clearly demonstrate that it inhibits NNK-induced lung tumorigenesis by inhibiting the metabolic activation of NNK. Similar changes appear to occur in humans according to data generated in smokers who ate watercress, a source of PEITC. It is likely that mixtures of chemopreventive agents with activity against carcinogens in tobacco smoke, such as NNK and B[a]P, will be useful in chemoprevention of lung cancer in smokers. Furthermore, there is a need to develop suppressing agents for lung cancer that might be applicable in both smokers and ex-smokers.

Effects of dietary fat content on the metabolism of NNK and on DNA methylation induced by NNK.

The available data support the concept that high-fat diets increase cytochrome P-450 activities in the liver, leading to increased rates of carcinogen metabolism and, in some instances, DNA adduct formation. Therefore we investigated whether a high-fat diet can also influence DNA methylation by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the lungs of rats. Male F344 rats were fed a regular AIN-76A low-fat (5% corn oil) or AIN-76A high-fat (23.5% corn oil) diet. After three weeks on this dietary regimen, the animals were injected subcutaneously once daily for four days with NNK at 0.39 mmol/kg body wt. Groups of rats were sacrificed 4 and 24 hours after the last NNK administration; livers and lungs were excised for DNA isolation. We found that the high-fat diet significantly enhanced the formation of O6-methylguanine (O6-mGua) in the rat lung four hours (p < 0.01) after the last carcinogen administration. This may, in part, account for our previous finding in regard to the enhancing effect of the high-fat diet on NNK-induced lung carcinogenesis. There was no effect on O6-mGua or 7-mGua in the rat liver at either time point. To further elucidate the enhancing effect of the high-fat diet on DNA methylation by NNK in the lung, we determined its effect on the in vitro and in vivo metabolism of NNK. The in vitro data indicated that dietary fat has no measurable effect on liver and lung microsomal mixed-function oxidase in catalyzing the metabolic activation of NNK. The results of the metabolism study of NNK in vivo appear to be consistent with the in vitro finding, in that fat had no effect on the excretion pattern of NNK or on the distribution pattern of its urinary metabolites. It is apparent that the enhancing effect of the high-fat diet on O6-mGua in the lung of rats that was measured four hours after NNK injection requires future investigations.

Effects of dietary 1,4-phenylenebis(methylene)selenocyanate on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced DNA adduct formation in lung and liver of A/J mice and F344 rats.

1,4-Phenylenebis(methylene)selenocyanate (p-XSC) was tested for its ability to inhibit DNA adduct formation induced by the tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the liver and lung of A/J mice and F344 rats. Dietary p-XSC, providing a dose of 5 p.p.m. selenium, significantly inhibited the formation of 7-methylguanine (7-mGua) induced by a single i.p. injection of 10 mumol of NNK(12.8% inhibition at 4 h and 19.9% at 96 h) and O6-methylguanine (O6-mGua) (16.5% at 4 h and 34.8% at 96 h) in the liver of A/J mice. Dietary supplements of p-XSC providing 15 p.p.m. of selenium reduced the levels of 7-mGua by 17.3% (4 h) and 33.6% (96 h). The formation of O6-mGua was inhibited by 69.5% (4th) and 73.8 (96h). In A/J mouse lung DNA the most significant reduction was observed in levels of O6-mGua. Dietary p-XSC at 5 p.p.m. as selenium inhibited the formation of this adduct by 73.1% (4 h). Ninety-six hours after NNK injection, and at both time points with p-XSC providing 15 p.p.m. selenium, O6-mGua was not detected. Although levels of 7- mGua in mouse lung DNA were also reduced, this was significant only 4 h after carcinogen administration. In general, selenite at a5 p.p.m. as selenium had no significant effect on the levels of these lesions; however, it inhibited O6-mGua in the liver only 4 h after NNK administration. These effects may explain why there is chemopreventive activity for p-XSC, but not for selenite, in NNK-induced lung carcinogenesis in A/J mice. Moreover, these findings raised our interest in determining the potential chemopreventive activity of p-XSC against NNK-induced lung adenocarcinomas in male F344 rats by first determining its effects on NNK-induced DNA methylation in the lungs of rats. Diet supplemented with 10 p.p.m. selenium as p-XSC did indeed inhibit the formation of adducts in pulmonary DNA of F344 rats treated with four consecutive injections of 81 mg/kg of NNK. Statistically significant inhibition of O6-mGua formation was observed 4 h after carcinogen treatment in both pulmonary (49.1% inhibition) and hepatic (39.8%) DNA. Statistically significant inhibition of 7-mGua formation was also measured in lung DNA isolated 24 h after the last NNK injection (45.0%) and in liver DNA 4 h after carcinogen treatment (31.8%). These results suggest that p-XSC would also inhibit induction of lung adenocarcinoma in male F344 rats by NNK.

Matabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by cytochrome P450IIB1 in a reconstituted system

Several previous studies have suggested that cytochrome P450IIB1 is involved in the bioactivation of the tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), in rats as well as in mouse lung microsomes. The present investigation was undertaken to study the metabolism of NNK by purified cytochrome P450IIB1 in a reconstituted system. The metabolites 4-hydroxy-4-(3-pyridyl) butyric acid (hydroxy acid), 4-oxo-4-(3-pyridyl) butyric acid (keto acid), 4-oxo-4-(3-pyridyl) butanol (keto aldehyde), 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone (NNK-N-oxide) and 4-oxo-4-(3-pyridyl)-1-butanol (keto alcohol) were quantitated by HPLC. The results showed that, in addition to alpha-hydroxylations, cytochrome P450IIB1 also catalyzed the formation of NNK-N-oxide efficiently, and to a certain extent, the conversion of NNK primary hydroxylation metabolites (keto aldehyde and keto alcohol) to secondary metabolites (keto acid and hydroxy acid). Cytochrome b5 at a ratio of 1:1 or 2:1 to P450IIB1 had no significant effect on the metabolic activities and profiles of NNK. The apparent Km values for the formation of keto aldehyde, NNK-N-oxide and keto alcohol were respectively 191.2, 131.4 and 318.0 microM with corresponding apparent Vmax values of 89.7, 295.5 and 333.3 pmol/min/nmol P450, indicating that hydroxylation at the alpha-methyl position is preferred over the alpha-methylene position. Measurement of formaldehyde, a product derived from the alpha-methyl hydroxylation, was developed as a convenient method to study NNK metabolism. Thiourea activated cytochrome P450IIB1-catalyzed NNK metabolism significantly. Phenethyl isothiocyanate, an inhibitor of NNK-induced lung carcinogenesis, inhibited P450IIB1-catalyzed NNK demethylation in a concentration-dependent manner. This work demonstrates that purified P450IIB1 can catalyze the conversion of NNK to most of its oxidative metabolites.