Occurrence of mutations in the epidermal growth factor receptor gene in X-ray-induced rat lung tumors

Epidermal growth factor receptor ( EGFR ) gene alterations have been found in human lung cancers. However, there is no information on the factors inducing EGFR mutations. In rodents, K- ras mutations are frequently found in many lung carcinogenesis models, but hitherto, Egfr mutations have not been reported. Their presence was therefore investigated in representative lung carcinogenesis models with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N -nitrosobis(2-hydroxypropyl)amine (BHP), 2-amino-3,8-dimethylimidazo[4,5- f ]quinoxaline (MeIQx) and ethyl carbamate (urethane), as well as X-ray irradiation. With the chemical carcinogenesis models, no mutations were detected in Egfr , which is in clear contrast to the high rates observed in either codon 12 or 61 of K- ras (21/23 of the lung tumors induced with NNK, 4/5 with MeIQx, 1/4 with urethane and 7/18 with BHP). However, in the X-ray-induced lung tumors, Egfr mutations with amino acid substitution were observed in exons 18 and 21 (4/12, 33%), but no activating mutation of K- ras was detected. In addition, one and four silent mutations were identified in K- ras (exon 1) and Egfr (exons 18, 20 and 21), respectively. Most mutations in both Egfr and K- ras were G/C→A/T transitions (7/8, 88% and 31/34, 91%, respectively). Although, the mutational patterns in equivalent human lesions were not completely coincident, this first report of Egfr mutations in an experimental lung tumor model suggests that X-rays or other factors producing oxygen radicals could cause EGFR mutations in some proportion of lung cancers in humans. ( Cancer Sci 2008; 99: 241–245)     

Role of the alveolar type II cell in the development and progression of pulmonary tumors induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in the A/J mouse.

The role of the type II cell in the development of pulmonary tumors induced in the adult A/J mouse (6 weeks of age) by treatment with a single dose (100 mg/kg, i.p.) of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was investigated. Twenty-four h following treatment with NNK, the concentration of O6-methylguanine was similar in Clara and type II cells. However, hyperplasias were detected only along the alveolar septa in lungs 14 weeks after carcinogen treatment. Examination of the ultrastructure of several hyperplasias revealed that the proliferating cells resembled type II pneumocytes. The proliferating cells were cuboidal in shape, with centrally localized ovoid nuclei characterized by minor indentations. Lamellar bodies, one of the major hallmarks of the type II cell, were present in the cytoplasm. The progression of pulmonary lesions was followed by sacrificing mice at 4-week intervals from 14 to 54 weeks after treatment with NNK. From 34 to 42 weeks after treatment, progression to neoplasia was demonstrated by a decline in the frequency of hyperplasias and an increase in the frequency of adenomas. Approximately 50% of the adenomas were observed arising within hyperplasias. Carcinomas appeared to increase in frequency 34 weeks after carcinogen treatment and comprised greater than 50% of the pulmonary lesions by 54 weeks. Approximately 30% of the carcinomas were observed arising within adenomas. The growth pattern of carcinomas began to change from solid to mixed (solid and papillary) 42 weeks after NNK. Moreover, electron micrographic analysis demonstrated that, within a hyperplasia, proliferating type II cells could change from cuboidal to columnar in shape and could also exhibit nuclear indentations, both characteristics displayed by the Clara cell. Thus, this divergence of the type II cell from its well characterized morphological features indicates that the selective growth advantage which these initiated cells possess can result in changes to the normal ultrastructure of this cell as it progresses toward malignancy. DNA was isolated from 20 hyperplasias and screened for the presence of an activated K-ras gene. This gene was activated in 17 of 20 lesions, with 85% of the mutations involving a GC to AT transition within codon 12 (GGT to GAT), a mutation consistent with base mispairing produced by the formation of the O6-methylguanine adduct. This specificity for activation of the K-ras gene was identical to that observed previously in adenocarcinomas induced by NNK.(ABSTRACT TRUNCATED AT 400 WORDS)     

Frequent k- ras -2 mutations and p16(INK4A)methylation in hepatocellular carcinomas in workers exposed to vinyl chloride

Vinyl chloride (VC) is a know animal and human carcinogen associated with liver angiosarcomas (LAS) and hepatocellular carcinomas (HCC). In VC-associated LAS mutations of the K- ras -2 gene have been reported; however, no data about the prevalence of such mutations in VC associated HCCs are available. Recent data indicate K- ras -2 mutations induce P16 methylation accompanied by inactivation of the p16 gene. The presence of K- ras -2 mutations was analysed in tissue from 18 patients with VC associated HCCs. As a control group, 20 patients with hepatocellular carcinoma due to hepatitis B (n = 7), hepatitis C (n = 5) and alcoholic liver cirrhosis (n = 8) was used. The specific mutations were determined by direct sequencing of codon 12 and 13 of the K- ras -2 gene in carcinomatous and adjacent non-neoplastic liver tissue after microdissection. The status of p16 was evaluated by methylation-specific PCR (MSP), microsatellite analysis, DNA sequencing and immunohistochemical staining. All patients had a documented chronic quantitated exposure to VC (average 8883 ppmy, average duration: 245 months). K- ras -2 mutations were found in 6 of 18 (33%) examined VC-associated HCCs and in 3 cases of adjacent non-neoplastic liver tissue. There were 3 G --> A point mutations in the tumour tissue. All 3 mutations found in non-neoplastic liver from VC-exposed patients were also G --> A point mutations (codon 12- and codon 13-aspartate mutations). Hypermethylation of the 5' CpG island of the p16 gene was found in 13 of 18 examined carcinomas (72%). Of 6 cancers with K- ras -2 mutations, 5 specimens also showed methylated p16. Within the control group, K- ras -2 mutation were found in 3 of 20 (15%) examined HCC. p16 methylation occurred in 11 out of 20 (55%) patients. K- ras -2 mutations and p16 methylation are frequent events in VC associated HCCs. We observed a K- ras -2 mutation pattern characteristic of chloroethylene oxide, a carcinogenic metabolite of VC. Our results strongly suggest that K- ras -2 mutations play an important role in the pathogenesis of VC-associated HCC.     

Development of ferret as a human lung cancer model by injecting 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

Objective

Development of new animal lung cancer models that are relevant to human lung carcinogenesis is important for lung cancer research. Previously we have shown the induction of lung tumor in ferrets (Mustela putorius furo) exposed to both tobacco smoke and a tobacco carcinogen (4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone, NNK). In the present study, we investigated whether NNK treatment alone induces both preneoplastic and neoplastic lesions in the lungs of ferrets.

Methods

We exposed ferrets to NNK by i.p. injection of NNK (50 mg/kg BW) once a month for four consecutive months and then followed up for 24, 26 and 32 weeks. The incidences of pulmonary preneoplastic and neoplastic lesions were assessed by histopathological examination. The expressions of α7 nicotinic acetylcholine receptor (α7 nAChR, which has been shown to promote lung carcinogenesis) and its related molecular biomarkers in lungs were examined by immunohistochemistry and/or Western blotting analysis.

Results

Ferrets exposed to NNK alone developed both preneoplastic lesions (squamous metaplasia, dysplasia and atypical adenomatous hyperplasia) and tumors (squamous cell carcinoma, adenocarcinoma and adenosquamous carcinoma), which are commonly seen in humans. The incidence of tumor induced by NNK was time-dependent in the ferrets (16.7%, 40.0% and 66.7% for 24, 26 and 32 weeks, respectively). α7 nAChR is highly expressed in the ferret bronchial/bronchiolar epithelial cells, and alveolar macrophages in ferrets exposed to NNK, and in both squamous cell carcinoma and adenocarcinoma of the ferrets. In addition, we observed the tendency for an increase in phospho-ERK and cyclin D1 protein levels (p = 0.081 and 0.080, respectively) in the lungs of ferrets exposed to NNK.

Conclusion

The development of both preneoplastic and neoplastic lesions in ferret lungs by injecting NNK alone provides a simple and highly relevant non-rodent model for studying biomarkers/molecular targets for the prevention, detection and treatment of lung carcinogenesis in humans.     

SHORT COMMUNICATION: G to A transitions and G to T transversions in codon 12 of the Ki-ras oncogene isolated from mouse lung tumors induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and relati DNA methylating and pyridyloxobutylating agents

Lung tumors were induced in A/J mice by the tobacco-specificnitrosamine 4Kmethylnitrosaniino)-1-(3-pyridyI)-1-butanone (NNK)and the related compounds acetoxymethylmethyl-nitrosamine (AMMN)and 4-acetoxymethylnitrosamino)-1-(3-pyridyI)-1-butanone (NNKOAc).NNK both methylates and pyridyloxobutylates DNA while AMMN andNNKOAc only methylate or pyridyloxobutylate DNA, respectively.The lung tumors were analyzed for mutations in the Ki-ras oncogeneby PCR amplification followed by either restriction fragmentlength polymorphism, hybridization, or sequencing procedures.NNK induced GGT to GAT mutations in codon 12 (26 of 28 samplesanalyzed). AMMN induced GGT to GAT mutations in 18 of 18 samples.In contrast, NNKOAc induced a variety of changes including GGTto GAT (8/21), GGT to TGT (5/21) and GGT to GTT (4/21) mutations.These results demonstrate that DNA methylation causes mainlyG to A transitions in the Ki-ras gene of A/J mouse lung tumors,consistent with previous results and a role for O⁶-methyl-guanine,while DNA pyridyloxobutylation induces G to A transitions aswell as G to T transversions, perhaps due to the steric bulkof the adducts which are formed. The results are discussed withrespect to mutations observed in rodent and human lung tumors.     

Inhibition of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis by dietary olive oil and squalene

Epidemiological studies have suggested that frequent olive oil consumption may be a protective factor against lung cancer formation. Squalene, a characteristic compound in olive oil, is an inhibitor of 3- hydroxy-3-methylglutaryl coenzyme A reductase activity and has been proposed to inhibit the farnesylation of ras oncoproteins. The present study investigated the effect of dietary olive oil and squalene in a mouse lung tumorigenesis model. Female A/J mice were fed AIN-76A diets containing 5% corn oil (control), 19.6% olive oil, or 2% squalene starting at 3 weeks before a single dose of 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone (NNK) (103 mg/kg, i.p.). Animals were maintained on their respective diets throughout the study. At 16 weeks after NNK administration, 100% of the mice in the control group had lung tumors with a tumor multiplicity of 16 tumors per mouse. The olive oil and squalene diets significantly (P < 0.05) decreased the lung tumor multiplicity by 46 and 58%, respectively. The squalene diet significantly (P < 0.05) decreased lung hyperplasia by 70%. In mice fed a diet containing 2% squalene for 3 weeks, the activation of NNK was increased by 1.4- and 2.0-fold in lung and liver microsomes, respectively, but its relationship to the inhibition of carcinogenesis is not clear. These results demonstrate that dietary olive oil and squalene can effectively inhibit NNK-induced lung tumorigenesis.      

Role of ras protooncogene activation in the formation of spontaneous and nitrosamine-induced lung tumors in the resistant C3H mouse

The role of ras activation in the formation of spontaneous and chemically induced tumors was evaluated in the C3H mouse, a strain that has a low incidence of spontaneous lung tumors. Lung tumors were induced in C3H mice by treatment with 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 50 mg/kg, or nitrosodimethylamine (NDMA), 3 mg/kg for 7 weeks (3 times/week, i.p.). Eleven tumors from each treatment group were evaluated for activated ras genes by direct sequencing and oligonucleotide hybridization to slot blots of amplified DNA from these tumors. An activated K-ras gene was detected in 100% of NDMA- and NNK-induced lung tumors, and the activating mutation detected in all samples was a GC to AT transition (GGT to GAT) in codon 12. In contrast, only 40% of the seven spontaneous lung tumors analyzed contained an activated K-ras gene and the mutations identified were not localized to either a specific base or codon. Both NNK and NDMA can be activated via alpha-hydroxylation to methylating agents. The GC to AT mutation observed in codon 12 in the nitrosamine-induced tumors is consistent with the formation of an O6-methylguanine (O6MG) adduct. Similar concentrations (13-15 pmoles/mumol deoxyguanosine) of this promutagenic adduct were detected in lungs during treatment with either NNK or NDMA. Thus, both these nitrosamines appear to activate the K-ras gene in lung through a direct genotoxic mechanism involving the formation of the O6MG adduct. The frequency of K-ras activation was similar in chemically induced lung tumors from the sensitive A/J strain and the C3H mouse, indicating that susceptibility for neoplasia in these stains is not related to the ability to activate this gene. Although tumors were induced in lung from 100% of C3H mice following chronic carcinogen exposure, both the size and the multiplicity was significantly less, while latency was longer than that observed in the A/J mouse. These differences could not be attributed to an altered propensity for DNA damage, but rather suggest that genetic loci which regulate clonal expansion and growth of initiated cells play a major role in the susceptibility of pulmonary neoplasia.     

Effects of deuterium substitution on the tumorigenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in A/J mice

Bioassays and DNA-binding studies of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its analogs with deuterium substitution at the positions alpha to the nitrosamino group ([4,4-D2]NNK and [CD3]NNK) were carried out in A/J mice in order to assess the potential importance of DNA methylation or pyridyloxobutylation in lung tumor induction. The tumorigenic activities of the major NNK metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its analog with deuterium at the carbinol carbon ([1-D]NNAL) were also determined. Groups of A/J mice were given single i.p. injections of either 10 or 5 mumol of NNK, [4,4-D2]NNK, [CD3]NNK, NNAL and [1-D]NNAL, and were killed 16 weeks later. Lung tumor multiplicities were as follows in mice treated with 10 mumol: NNK, 7.3 +/- 3.5; [4,4-D2]NNK, 1.4 +/- 1.6; [CD3]NNK, 11.7 +/- 5.4; NNAL, 3.2 +/- 2.0; [1-D]NNAL, 3.2 +/- 2.0. Similar relative tumorigenic activities were observed in mice treated with 5 mumol of these compounds. These results demonstrated that [4,4-D2]NNK was less tumorigenic than NNK and [CD3]NNK was more tumorigenic than NNK. NNAL was less tumorigenic than NNK; substitution of deuterium at the carbinol carbon did not affect its activity. Levels of O6-methylguanine (O6-mG) were measured in pulmonary DNA of A/J mice treated with 10 mumol of NNK, [4,4-D2]NNK or [CD3]NNK, and killed 2 or 24 h later. O6-mG levels were lower in mice treated with [4,4-D2]NNK than in those treated with NNK; no difference in O6-mG levels was observed between those treated with NNK and [CD3]NNK. The results of this study support the hypothesis that O6-mG formation in pulmonary DNA is the key step in lung tumor induction by NNK in A/J mice.     

Evaluation of the transplacental tumorigenicity of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in mice

The transplacental tumorigenicity of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was assessed in three strains of mice: A/J; C3H/He x C57BL/6 F1 (hereafter called C3B6F1); and Swiss outbred [Cr:NIH(S)]. NNK (100 mg/kg) was administered i.p. on Days 14, 16, and 18 of gestation to A/J and C3H/He mice and on Days 15, 17 and 19 of gestation to the Swiss mice. The effects of postnatal treatment with tumor-promoting agents, including 0.05% sodium barbital in the drinking water until death or a single dose of Aroclor 1254 (a mixture of polychlorinated biphenyls, PCB) given on Postnatal Day 8 or 56, were also examined. Progeny were sacrificed at age 24 wk (A/J) or 72 wk (C3B6F1 and Swiss). Significant incidences of tumors occurred in the lungs of strain A/J progeny and in the livers of male C3B6F1 and Swiss progeny. Lung tumor incidence was 8 of 34 (24%) in the female offspring of the A/J mice treated with NNK, compared with 1 of 39 (3%) in controls (P less than 0.05). A 2-fold difference in lung tumor incidence in male offspring of NNK-treated (4 of 23, 13%) versus control (3 of 48, 6%) A/J mice was not of statistical significance. However, the incidence of lung tumors in NNK-exposed progeny A/J mice in both sexes combined (12 of 66, 18%) was also significantly greater than in controls (4 of 87, 5%). The incidence of liver tumors in the male C3B6F1 mice exposed transplacentally to NNK was 12 of 30 (40%) compared to 8 of 46 (17%) in controls (P less than 0.05). No effects of postnatal sodium barbital or PCB were observed on transplacental NNK tumorigenicity in C3B6F1 mice. The combined incidence of liver carcinoma in male mice in all NNK-treated groups (13 of 141, 9%) was significantly greater (P less than 0.05) than in controls (5 of 144, 3%). In male Swiss mice exposed transplacentally to NNK, the incidence of liver tumors was 3 of 57 (5%) compared to 0 of 35 controls, and postnatal treatment with PCB on Day 56 caused a significant increase (5 of 26, 19%) (P less than 0.05) in the incidence of NNK-induced liver tumors. The combined incidence of liver tumors in the male offspring of the Swiss mice treated with NNK, with or without PCB, was 8 of 83 (10%) which was significantly greater (P less than 0.05) than in controls (0 of 66).(ABSTRACT TRUNCATED AT 400 WORDS)     

Point mutation of K-ras gene in cisplatin-induced lung tumours in A/J mice

The risks of secondary lung cancer in patients with early stage non-small and small cell lung cancers are estimated to be 1-2% and 2-10% per patient per year, respectively. Surprisingly, the incidence of second primary cancer in locally advanced non-small cell lung cancer at 10 years, following cisplatin-based chemotherapy with concurrent radiotherapy, increases to 61%. Those patients, on the road to being cured, cannot overlook the possibility of developing a second primary cancer. We developed a second primary lung cancer model using cisplatin as a carcinogen in A/J mice to screen for chemopreventive agents for a second malignancy. In the primary lung tumour model, 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), benzo(a)pyrene (BaP), urethane induces specific K-ras mutations in codon 12, codon 12, and codon 61, respectively, in the A/J mice. In this study, we investigated the mechanisms of carcinogenicity by cisplatin in the A/J mice. In the cisplatin-induced tumours, we found no K-ras codon 12 mutation, which is the major mutation induced by NNK or BaP. K-ras gene mutations in codon 13 and codon 61 were found in one tumour (4%) and five tumours (17.8%), respectively. These findings suggest that cisplatin is partially related to K-ras codon 61 mutations, and that the mechanism of carcinogenicity by cisplatin is different from that by NNK or BaP.     

Inhibitory effects of cinnamaldehyde on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung carcinogenesis in rasH2 mice.

Previously we reported a lack of modification by cinnamaldehyde (CNMA) of development of lung proliferative lesions induced by urethane in CB6F1-TgHras2 (rasH2) mice. In the present study, we re-evaluated CNMA effects using the same rasH2 strain and non-transgenic littermates initiated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Sixteen mice/strain/sex received intraperitoneal NNK injections at a dose of 3 mg/mouse once a week for 2 weeks followed by free feeding of commercial diet containing 5000 ppm CNMA for 26 weeks. Additional groups were maintained without NNK injection and/or CNMA feeding for 28 weeks. Lung tumors were induced by NNK in both rasH2 and non-Tg males and females at incidence ranging from 63 to 100%. CNMA treatment significantly reduced the combined incidence of adenomas and carcinomas from 86 to 31% in rasH2 males (P<0.05), but no significant influence was evident in females. The multiplicity of NNK-induced lung tumors was also significantly reduced in rasH2 males given CNMA (P<0.01). Similar effects were also observed in non-Tg females given CNMA after NNK initiation. The results of our study strongly indicate that CNMA is capable of inhibiting development of NNK-initiated pulmonary tumorigenesis in rasH2 and non-Tg mice.     

Inhibitory effects of diallyl sulfide on the metabolism and tumorigenicity of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mouse lung.

Diallyl sulfide (DAS), a component of garlic oil, has been shown to inhibit tumorigenesis by several chemical carcinogens. Our previous work demonstrated that DAS inhibited the metabolic activation of carcinogenic nitrosamines, including the tobacco-specific 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), in rat lung and nasal mucosa microsomes. In the present study, the effects of DAS on the tumorigenicity and the metabolism of NNK in A/J mouse lung were examined. Female A/J mice at 7 weeks of age were pretreated with DAS (200 mg/kg body wt in corn oil, p.o) daily for 3 days. Two hours after the final DAS treatment, the mice were either given a single dose of NNK (2 mg/mouse, i.p.) and kept for an additional 16 weeks for determining the production of pulmonary tumors, or were killed immediately so as to measure the microsomal activity in metabolizing NNK. In comparison to the vehicle control group, DAS pretreatment significantly decreased the incidence of NNK-induced lung tumors (37.9 versus 100%) and the tumor multiplicity (0.6 versus 7.2 tumors/mouse). In pulmonary metabolism of NNK, DAS pretreatment reduced the rates of formation of keto aldehyde, keto alcohol, NNAL-N-oxide, and NNK-N-oxide by 70-90%. In addition, the formation of NNK oxidative metabolites from NNK in the liver microsomes from DAS-pretreated mice was remarkably reduced. DAS also inhibited the metabolism of NNK in mouse lung microsomes in vitro. These results demonstrate that DAS is an effective chemopreventive agent against NNK-induced lung tumorigenesis, probably by inhibiting the metabolic activation of NNK.     

Relationship between the formation of promutagenic adducts and the activation of the K-ras protooncogene in lung tumors from A/J mice treated with nitrosamines

Lung and liver tumors were induced in female A/J mice after treatment for 7 weeks (3 times/week, i.p.) with either 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) (50 mg/kg) or nitrosodimethylamine (NDMA) (3 mg/kg). Both compounds can be activated via alpha-hydroxylation to methylating agents, while NNK may also undergo hydroxylation at the N-methyl carbon to form a pyridyloxobutylated adduct. The purpose of these studies was to identify and characterize the activated oncogenes present in tumors induced by NDMA and NNK. Following transfection of high molecular weight DNA onto NIH/3T3 mouse fibroblasts, transforming genes were detected in 90% of both NNK- (10 of 11) and NDMA- (9 of 10) induced lung tumors. In contrast, transformation of NIH/3T3 fibroblasts was observed only in 40% (2 of 5) and 13% (1 of 8) of the liver tumors from NNK- and NDMA-treated mice, respectively. Southern blot analysis indicated that the transforming gene present in all lung tumors was an activated K-ras oncogene. Both rearranged bands and amplified signals were detected in the transfectants. The one transformant from the NDMA-induced liver tumor contained an activated K-ras gene. In contrast, the two liver transformants from NNK-induced tumors did not contain an activated ras or raf gene. Hybridization with oligonucleotide probes that were centered around either codon 12 or 61 of the K-ras gene were utilized to localize the mutations. Activation of this gene appeared to occur largely via a mutation in codon 12 (15 of 20 transformants) and was observed with a similar frequency in pulmonary tumors induced by either compound. The remaining mutations were found in codon 61. The specific mutation within these two codons was determined by amplifying the exon containing the base change, followed by direct sequencing. With one exception the mutation observed in codon 12 was a GC to AT transition (GGT to GAT). One transformant contained a GC to TA transversion. The activating mutation detected in codon 61 was always an AT to GC transition of the middle A (CAA to CGA). The GC to AT mutation observed in codon 12 is consistent with the formation of the O6-methylguanine adduct. Similar concentrations (23 to 32 pmol/mumol deoxyguanosine) of this promutagenic adduct were detected in lungs during treatment with either NNK or NDMA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of dietary aromatic isothiocyanates fed subsequent to the administration of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone on lung tumorigenicity in mice

Naturally-occurring aromatic isothiocyanates, benzyl isothiocyanate (BITC) and phenethyl isothiocyanate (PEITC), were tested for their post-treatment effects on lung tumorigenicity by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mice. Mice at 7 weeks of age were administered a single i.p. dose of NNK (10 mumol/mouse). One week after NNK dosing, mice were placed on AIN-76A diet containing 1 or 3 mumol/g diet of BITC or PEITC. The control group was maintained on AIN-76A diet after NNK administration. Mice were killed 16 weeks after NNK treatment and lung adenomas were counted. The results showed mice fed control diet developed 7.8 tumors/mouse. Mice fed PEITC at concentrations of 1 or 3 mumol/g diet had 8.2 or 6.1 tumors/mouse, respectively. Feeding BITC at 1 mumol/g diet resulted in a tumor yield of 8.0 tumors/mouse, whereas BITC diet at 3 mumol/g diet gave 5.2 tumors/mouse, a small but significant inhibition. However, in the high BITC dose group, a loss in weight gain due to reduced food intake was noted. The results of this study showed that post-treatment of aromatic isothiocyanates had little, if any, effect on NNK lung tumorigenicity in A/J mice. This is in contrast to our previous findings in which pretreatment with PEITC greatly inhibited lung tumor induction by NNK in A/J mice and suggests that tumor inhibition by PEITC is due to inhibition of NNK metabolic activation.     

Effects of indole-3-carbinol on lung tumorigenesis and DNA methylation induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and on the metabolism and disposition of NNK in A/J mice

The effects of indole-3-carbinol (I3C) on lung neoplasia induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were assessed in an A/J mouse pulmonary adenoma bioassay. Mice were administered corn oil or I3C (25 or 125 mumol/mouse/day) by gavage for 4 consecutive days. Two h after the final pretreatment, mice were administered a single dose of NNK (10 mumol/mouse) i.p. Pulmonary adenomas were quantitated 16 wk after NNK dosing. Mice pretreated with corn oil developed 10.7 tumors/mouse; I3C pretreatment at either dose level inhibited tumor multiplicity by approximately 40%. The effects of I3C on NNK-induced DNA methylation in the lungs and livers of A/J mice were assessed using the same dosing regimen as in the bioassay. Both dose levels of I3C inhibited pulmonary O6-methylguanine formation by at least 50%, but enhanced hepatic DNA methylation at 2 or at 6 h after NNK administration. The effects of I3C pretreatment on NNK metabolism were also investigated. Hepatic microsomes of I3C-pretreated mice showed increased formation of alpha-hydroxylation products, while no significant effect of I3C pretreatment was observed in pulmonary microsomes. The effects of I3C on [5-3H]NNK disposition were also evaluated. I3C pretreatment produced lower levels of total radioactivity in the lung when compared with controls. Additionally, lower proportions of NNK and its carcinogenic metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol were found in the lungs of I3C-pretreated mice. These results demonstrate that I3C inhibits NNK-induced lung neoplasia in A/J mice and suggest that the basis of this inhibition is the decrease in O6-methylguanine formation in A/J lung caused by I3C pretreatment. This decrease in lung DNA methylation appears to be due to the decreased bioavailability of NNK and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in the lungs of I3C-treated mice which, in turn, may be a result of increased metabolic alpha-hydroxylation of NNK by the liver.     

Topographic analysis of K- ras mutations in histologically normal lung tissues and tumours of lung cancer patients.

Mutations in the K- ras gene are very common in lung tumours and are implicated in the development of lung cancer, but the timing of their occurrence remains poorly understood. We investigated K- ras mutations in cell samples microdissected by laser capture microscopy at multiple sites from lung tissue sections representing tumour tissue and matched histologically normal tissue obtained from 48 lung cancer patients. K- ras mutations were detected in cell samples from 10 of 38 (26.3%) lung adenocarcinomas and in none of the histologically normal or tumour cell samples taken from 10 lung squamous cell carcinomas. Of the K- ras mutation-positive adenocarcinomas, in 4 cases a mutation was found in only the tumour tissue, in 1 case a mutation was found only in the histologically normal tissue, and in 5 cases mutations were found in both the tumour tissue and histologically normal tissue. Among these 5 cases, 2 had identical mutations in both the tumour tissue and histologically normal tissue, 2 had 1 mutation in the tumour tissue and 2 mutations in the histologically normal tissue, 1 of which was identical to the mutation found in the tumour, and 1 case had 2 codon 12 mutations in tumour tissue and 2 mutations, in codons 9 and 11, in histologically normal tissue. These results showed that K- ras mutations are frequent in histologically normal cells taken from outside lung adenocarcinomas and suggest that some of these mutations may represent early events which could pave the way of lung carcinogenesis.     

Inhibition of tobacco-specific nitrosamine 4-(N-nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) tumorigenesis with aromatic isothiocyanates

4-(N-Nitrosomethylamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific carcinogenic nitrosamine. At low doses, it induces primarily lung tumours in mice, hamsters and rats, regardless of the route of administration. Its unique organ specificity and potency suggest its possible role in the high incidence of lung cancer in smokers. The goal of this study was to find agents that would potentially prevent NNK tumorigenesis. Previous results led us to test phenethyl isothiocyanate (PEITC) on NNK tumorigenesis in a two-year bioassay in Fischer 344 rats. The NNK-treated group developed 80% lung tumour incidence, whereas NNK-treated rats fed PEITC diets had only 40% lung tumour incidence. Incidences in other organs were not affected by this treatment. We also tested PEITC in a 16-week, short-term bioassay against NNK-induced lung adenomas in A/J mice. Pretreatment of mice with PEITC by gavage at four daily doses of 5 mumol or 25 mumol reduced the formation of NNK-induced lung adenomas by 70% or 100%, respectively. Interestingly, benzyl isothiocyanate and phenyl isothiocyanate, the lower homologues of PEITC, were inactive in this bioassay. Using a protocol similar to that used in the bioassays, PEITC was shown to decrease DNA methylation by NNK in the lungs of rats and mice and suppress the metabolism of NNK by mouse lung microsomes. These results are consistent with the previous data, suggesting that the inhibition of NNK-induced lung tumour formation by PEITC is a consequence of reduced DNA methylation caused by inhibition of NNK metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Strain-Specific Spontaneous and NNK-Mediated Tumorigenesis in Pten+/- Mice

Pten is a negative regulator of the Akt pathway, and its inactivation is believed to be an etiological factor in many tumor types. Pten+/- mice are susceptible to a variety of spontaneous tumor types, depending on strain background. Pten+/- mice, in lung tumor-sensitive and -resistant background strains, were treated with a tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), to determine whether allelic Pten deletion can cooperate with NNK in carcinogenesis in lung or other tissues. In lung tumor-resistant C57BL/6 Pten+/- or +/+ mice, NNK treatment did not lead to any lung tumors and did not increase the incidence or severity of tumors previously reported for this strain. In contrast, in a lung tumor-susceptible pseudo-A/J strain, there was a dose-dependent increase in lung tumor size in Pten+/- compared with +/+ mice, although there was no increase in multiplicity. No other tumor types were observed in pseudo-A/J Pten+/- mice regardless of NNK treatment. Lung tumors from these Pten+/- mice had K-ras mutations, retained Pten expression and had similar Akt pathway activation as lung tumors from +/+ mice. Therefore, deletion of a single copy of Pten does not substantially add to the lung tumor phenotype conferred by mutation of K-ras by NNK, and there is likely no selective advantage for loss of the second Pten allele in lung tumor initiation.     

Inhibitory effects of propolis granular A P C on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis in A/J mice

We examined the effect of propolis granular A. P. C on lung tumorigenesis in female A/J mice. Lung tumors were induced by the tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) administered in drinking water for 7 weeks in mice maintained on an AIN-76A semi-synthetic diet. Propolis granular A. P. C (100 mg/kg body wt.) was administered orally daily for 6 days/week from 1 week before NNK administration and throughout the experiment. Sixteen weeks after the NNK treatment, the mice were killed and the number of surface lung tumors was measured. The number of lung tumors in mice treated with NNK alone for 7 weeks (9.4 mg/mouse) was significantly more than in that observed in control mice. Propolis granular A. P. C significantly decreased the number of lung tumors induced by NNK. These results indicate that propolis granular A. P. C is effective in suppressing NNK-induced lung tumorigenesis in mice.