4-(Methylnitrosamino)-1-(3-pyridyl) -1-butanone induces circulating microRNA deregulation in early lung carcinogenesis / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To study the alteration of circulating microRNAs in 4-(methylnitrosamino)-1-(3-pyridyl) -1-butanone (NNK)-induced early stage lung carcinogenesis.</p><p><b>METHODS</b>A lung cancer model of male F344 rats was induced with systemic NNK and levels of 8 lung cancer-associated miRNAs in whole blood and serum of rats were measured by quantitative RT-PCR of each at weeks 1, 5, 10, and 20 following NNK treatment.</p><p><b>RESULTS</b>No lung cancer was detected in control group and NNK treatment group at week 20 following NNK treatment. The levels of some circulating miRNAs were significantly higher in NNK treatment group than in control group. The miR-210 was down-regulated and the miR-206 was up-regulated in NNK treatment group. The expression level of circulating miRNAs changed from week 1 to week 20 following NNK treatment.</p><p><b>CONCLUSION</b>The expression level of circulating miRNAs is related to NNK-induced early stage lung carcinogenesis in rats and can therefore serve as its potential indicator.</p>

B6C3F1 mice exposed to ozone with 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone and/or dibutyl phthalate showed toxicities through alterations of NF-kappaB, AP-1, Nrf2, and osteopontin

Toxic effects of ozone, 4-(N-methyl-N-nitrosamino)-1-(3- pyridyl)-1-butanone (NNK), and/or dibutyl phthalate (DBP) were examined through NF-kappaB, AP-1, Nrf2, and osteopontin (OPN) in lungs and livers of B6C3F1 mice. Electrophoretic mobility shift assay (EMSA) indicated that mice treated with combination of toxicants induced high NF-kappaB activities. Expression levels of p105, p65, and p50 proteins increased in all treated mice, whereas IkB activity was inhibited in NNK-, DBP-, and combination-treated ones. All treated mice except ozone-treated one showed high AP-1 binding activities. Expression levels of c-fos, c-jun, junB, jun D, Nrf2, and OPN proteins increased in all treated mice. Additive interactions were frequently noted from two-toxicant combination mice compared to ozone-treated one. These results indicate treatment of mixture of toxicants increased toxicity through NF-kappaB, AP-1, Nrf2, and OPN. Our data could be applied to the elucidation of mechanism as well as the risk assessment of mixture-induced toxicity.

Molecular analysis of hprt mutation in B6C3F1 mice exposed to ozone alone and combined treatment of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone and/or dibutyl phthalate for 32 and 52 weeks

Potential toxicological interactions of 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and/or dibuthyl phthalate (DBP) on ozone were investigated after 32- and 52-wk exposures using hprt mutation assay. Male and female B6C3F1 mice exposed to ozone (0.5 ppm), NNK (1.0 mg/kg), DBP (5,000 ppm), and two or three combinations of these toxicants 6 h per day for 32- and 52-wk showed increases in the frequencies of TG rlymphocytes compared to the control groups. Additive interactions were noted from two combination groups compared to the ozone alone in both sexes of 32- and 52-wk studies. The most common specific mutation type in the hprt genes of test materials-treated male and female mice was transversion with very few transition. The results indicate that such dominant transversion may be responsible for toxicity and combined exposure to ozone, NNK, and DBP induces additive genotoxicities compared to ozone alone.

Cap-independent protein translation is initially responsible for 4-(N-methylnitrosamino)-1-(3-pyridyl)-butanone (NNK)-induced apoptosis in normal human bronchial pithelial cells

Evidences show that eukaryotic mRNAs can perform protein translation through internal ribosome entry sites (IRES). 5'-Untranslated region of the mRNA encoding apoptotic protease-activating factor 1 (Apaf-1) contains IRES, and, thus, can be translated in a cap-independent manner. Effects of changes in protein translation pattern through rapamycin pretreatment on 4-(methylnitrosamino)-1-(3-pyridyl)-butanone(NNK, tobacco-specific lung carcinogen)-induced apoptosis in human bronchial epithelial cells were examined by caspase assay, FACS analysis, Western blotting, and transient transfection. Results showed that NNK induced apoptosis in concentration- and time-dependent manners. NNK-induced apoptosis occurred initially through cap-independent protein translation, which during later stage was replaced by cap-dependent protein translation. Our data may be pplicable as the mechanical basis of lung cancer treatment.

Chromosome Aberration During Malignant Transformation of Human Bronchial Epithelial Cells Induced by NNK / 重庆医科大学学报

Objective To investigate chromosome aberration during malignant transformation of immortalized human bronchial epithelial cells ( BEAS - 2B) induced by tobacco specific nitrosamine ( NNK) , and explore those significance of early - warning and mass screening of lung cancer in high - risk smoking population. Methods Immortalized human bronchial epithelial cells ( BEAS - 2B) were induced to malignant transformation cells ( BEAS - 2BNNK ) by NNK, and chromosome aberration were detected in the different passage of BEAS - 2BNNK and BEAS - 2B cells by analysis of metaphase chromosome. Results 1. Model of malignant transformation of BEAS -2B cells induced by NNK was created (1)The serum resistance was significantly increased in the 5th passage BEAS - 2BNNK cells.(2) The anchorage independent growth was appeared in the 15th passage. (3)The ultrastructure of the 20th passage BEAS - 2BNNK cells shown obvious heteromorphy characterization. (4)The 25th passage BEAS -2BNNK cells formed tumors in nude mice, and tumors were overdifferentiation squamous cell carcinoma confirmed by histopathological examination. 2. Chromosome aberration (1) BEAS -2B cells hold relatively stable diploid karyotype which ratio was 91%-97% up to the 25th passage. Different passage BEAS -2BNNK cells gradually lost the normal diploid karyotype and the ratio of diploid cells decreased from 83% to 54% , the proportion of polyploid and aneu-ploid cells increased in process of passing generation. (2)The ratio of structure aberration neared to 2% ~ 4% in BEAS - 2BNNK and BE-AS -2B cells but the 25th passage of BEAS -2BNNK cells was 11%. Conclusion The model of malignant transformation of BEAS - 2B cells induced by NNK could be created successfully and provided for investigating the molecular biological mechanism of lung cancer, especially smoking - related cases. The polyploid and aneuploid chromosomes could be the early event in the process of malignant transformation of BEAS - 2B cells induced by NNK. In a word, the chromosome aberration might be useful markers for the early warning and mass screening of lung cancer, especially in high - risk smoking population.

The Effects of Nicotine and NNK on gingival fibroblast / 대한치주과학회지

In order to observe the effects of Nicotine and NNK on cultured human gingival fibroblast, several factors were examined including mutagenicity, the number of cells attached culture plate surface through MTT test, the abundance of collagen & collagenase in mRNA level and collagenolytic activity in extracellular matrix. The results were as follows; 1. Regardless of the co-existence of S9, Nicotine did not show the mutagenicity by itself and NNK by itself showd the same result; However, dose related mutagenicity was shown in NNK with S9. 2. The number of fibroblasts attached cultured plate surface was measured by MTT procedure. The number of cells in Non-smokers increased at all time periods as compared to those of smoker. 3. Non-smoker's fibroblast treated by NNK or Nicotine was dose-dependently decreased in the number of cells when compared to untreated control. In higher dose, Nicotine showed the cellular toxicity , but NNK did not. 4. No change in the abundance of mRNA for proalpha1 and proalpha2 was shown in Nicotine treated group but in gingival fibroblasts following treatment with NNK, the abundance of mRNA for proalpha1, but not proalpha2 collagen was decreased. 5. The abundance of mRNA for collagenase was decreased when NNK was treated but no change occurred in Nicotine treated group. 6. The effect of NNK and Nicotine in collagenolytic activity showed that ,collagenase activity exclusively react to type I collagen, was increased in both group, but gelatinase exclusively react to type IV collagen was not influenced at all. Collagenase activity of smoker's fibroblast was also increased as much as Nicotine and NNK group. The findings suggest that both of Nicotine and NNK lead gingival fibroblast to decrease in the abundance of collagen. And it seems to be that Nicotine and NNK have independent pathway toward the gingival fibroblast.