耐高温亚硝酸盐降解菌的分离、鉴定及降低雪茄烟叶亚硝胺的应用

【背景】烟草特有亚硝胺(tobacco-specific nitrosamines, TSNAs)是烟草于调制和发酵阶段产生的一类致癌物质,由烟草生物碱与氮氧化物发生亚硝化反应生成,生物碱和亚硝酸盐是TSNAs的直接前体物质。【目的】发掘适用雪茄高温发酵且显著降低TSNAs形成与积累的微生物。【方法】以TSNAs前体物质亚硝酸盐的高效降解为目标,对从雪茄烟叶分离得到的烟草源微生物菌株进行高温培养、亚硝酸盐降解及亚硝酸盐耐受能力研究,得到可于50℃高效降解亚硝酸盐及耐受高浓度亚硝酸盐的微生物菌株,将菌株应用于雪茄烟叶高温发酵35 d,对发酵前后亚硝酸盐、TSNAs、常规化学成分和中性香味成分含量进行测定,分析菌株在雪茄烟叶发酵中对TSNAs含量及烟叶品质的影响。【结果】获得了3株于50℃高效降解亚硝酸盐的菌株NY7、NY8和NY9,分别鉴定为莫海威芽孢杆菌(Bacillus mojavensis) NY7、耐盐芽孢杆菌(Bacillus halotolerans) NY8和枯草芽孢杆菌(Bacillus subtilis) NY9,其中B. halotolerans NY8亚硝酸盐降解能...     

Identifying human cells capable of metabolizing various classes of carcinogens

Human cells that appear capable of metabolizing various classes of carcinogens have been identified using one of two methods: metabolism of tritiated benzo(a)pyrene to aqueous-acetone soluble forms or inhibition of cellular DNA synthesis. Each of the assay systems was optimized and the results on 15 human epithelial cell lines were compared. One or more cell lines were found to activate each of four classes of carcinogens examined: polycyclic hydrocarbons, aromatic amines, heterocyclic hydrocarbons, and nitrosamines. Cells that appeared capable of metabolizing polycyclic hydrocarbons or aromatic amines by these methods were also found to produce metabolites which were cytotoxic to cocultivated human xeroderma pigmentosum fibroblasts after a 48-hr exposure to the carcinogen.     

Molecules involved in cell–cell adhesion during development

A peculiarity of nitrosamines is the high degree of cell and organ specificity in inducing tumors. There is substantial evidence that the initiation of the carcinogenesis process by carcinogens of this group is linked to the metabolic competence of the target tissue or cell to convert these carcinogens into mutagenic metabolites and to the binding of those metabolites to cellular DNA. Alkylation occurs in the DNA at the N-1, N-3, and N-7 positions of adenine; the N-3, N-7, and O⁶ of guanine; the N-3, and O² of cytosine; and the N-3, O⁴, and O² of thymine; and the phosphate groups. The initial proportion of each DNA adduct depends upon the alkylating agent used. The various DNA adducts are lost to a variable extent from DNA in vivo by spontaneous release of bases and Or by specific DNA repair processes. Studies conducted in vitro and in vivo indicate that alkylation at the oxygen atoms of DNA bases is more critical than alkylation at other positions in the mutagenesis and carcinogenesis induced by N-nitroso compounds. In particular, tissues in which tumors occur more frequently after a pulse dose of nitrosamine are those in which O⁶-alkylguanine persists longest in DNA, presumably resulting in an increased probability that a miscoding event (mutation) will take place during DNA synthesis. The more rapid removal of O⁶-methylguanine from the DNA of liver (as compared with cxtrahepatic tissues) of rats has been associated with the absence of tumor production in this organ by a single dose of dimethylnitrosamine; however, a significant incidence of liver tumors is observed if the same dose is given 24 hr after partial hepatectomy, and tumors arc induced by such a dose of dimethyl-nitrosamine in the liver of hamsters, which has a low capacity to remove O⁶-methylguanine from its DNA. These data also indicate that the rate of disappearance of 7-methylguanine from the liver or cxtrahepatic tissues is independent of the dose of dimethylnitrosamine; whereas O⁶-methylguanine is lost from DNA more rapidly after a low dose of this nitrosamine. It has been shown that in liver the removal of O⁶-methylguanine, but not of other DNA adducts, from DNA can be affected by pretreating the animals with N-nitroso compounds. The modulation of DNA repair processes observed after a single dose and after chronic treatment with nitrosamines is discussed in relation to the tissue-specific carcinogenic effect of this group of carcinogens.     

一株源于醇化烟叶表面高效降解TSNA 菌株AS97 的分离筛选、鉴定及应用

摘要:【目的】烟草特有亚硝胺(Tobacco-specific nitrosamines,简称TSNA)是烟叶中的主要致癌物质。本研究从筛选建立的特有菌库中发现了1 株可有效降解TSNA 的菌株AS97,并对其进行了鉴定及初步应用研究。【方法】采用富集驯化及选择培养基筛选得到硝酸盐与亚硝酸盐转化能力最强的菌株AS97;根据菌株的形态特征、生理生化特性及16S rRNA 基因序列分析对其进行鉴定;并将AS97 自制发酵液喷施于烟丝表面,确定适宜接种量和发酵条件,采用LC-MS /MS(液相色谱串联质谱)方法检测TSNA 中四种主要成分的含量。【结果】菌株AS97 源于云南玉溪烤烟样品表面,经分析确定其为荧光假单胞菌(Pseudomonas fluorescens,GenBank 登录号:JF449445)。将AS97 自制发酵液以5% 的接种量喷施于烟丝,30℃(相对湿度是60%)条件下发酵10 d 检测烟叶中硝酸盐、亚硝酸盐、TSNA、4-(N-甲基-亚硝基) -1-(3-吡啶基)-1-丁酮(NNK)、N-亚硝基去甲基烟碱(NNN)、N-亚硝基新烟草碱(NAT) 及N-亚硝基假木贼碱(NAB) 的转化率分别达到68. 77%、45. 57%、45. 47%、59. 08%、38. 79%、21. 41% 及11. 76%。相关性分析结果表明烤烟中硝酸盐、亚硝酸盐与TSNA 的含量均呈极显著相关(P＞0.01),进一步证实硝酸盐与亚硝酸盐是TSNA 的主要前体物质。【结论】醇化烟叶表面的荧光假单胞菌AS97 能够显著降低TSNA 的含量。本文首次报道了源于醇化烤烟表面对TSNA 有良好转化能力的Pseudomonas fluorescens。可将其开发成新型微生物制剂,应用于低害卷烟制品的生 产实践中。     

RNA interference (RNAi)-induced suppression of nicotine demethylase activity reduces levels of a key carcinogen in cured tobacco leaves

Technologies for reducing the levels of tobacco product constituents that may contribute to unwanted health effects are desired. Target compounds include tobacco-specific nitrosamines (TSNAs), a class of compounds generated through the nitrosation of pyridine alkaloids during the curing and processing of tobacco. Studies have reported the TSNA N '-nitrosonornicotine (NNN) to be carcinogenic in laboratory animals. NNN is formed via the nitrosation of nornicotine, a secondary alkaloid produced through enzymatic N -demethylation of nicotine. Strategies to lower nornicotine levels in tobacco ( Nicotiana tabacum L.) could lead to a corresponding decrease in NNN accumulation in cured leaves. The major nicotine demethylase gene of tobacco has recently been isolated. In this study, a large-scale field trial was conducted to evaluate transgenic lines of burley tobacco carrying an RNA interference (RNAi) construct designed to inhibit the expression of this gene. Selected transgenic lines exhibited a six-fold decrease in nornicotine content relative to untransformed controls. Analysis of cured leaves revealed a commensurate decrease in NNN and total TSNAs. The inhibition of nicotine demethylase activity is an effective means of decreasing significantly the level of a key defined animal carcinogen present in tobacco products.     

Repair of DNA alkylation adducts in mammalian cells

Carcinogenic alkylating agents, including nitrosamines, are able to alkylate DNA at various sites. This review presents evidence of the high degree of specificity in the type of DNA damage induced by various N-nitroso compounds and in the DNA repair processes among tissues or cells of different species. The O6-alkylguanine DNA alkyltransferase activity in various human and rodent tissues is discussed as well as the detection of O6-methylguanine in human DNA, using monoclonal antibodies and radioimmunoassay. The relevance of these findings to the mechanisms of cancer induction by nitrosamines is discussed.     

Gene expression profiles in HPV-immortalized human cervical cells treated with the nicotine-derived carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

Human papillomavirus (HPV) infection is an established etiological factor for cervical cancer. Epidemiological studies suggest that smoking in combination with HPV infection plays a significant role in the etiology of this disease. We have previously shown that the tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is present in human cervical mucus. Here, we hypothesized that treatment of HPV-16-immortalized human ectocervical cells (Ecto1/E6E7) with NNK would alter the expression of genes involved in cellular transformation. Ecto1/E6E7 cells were treated with water (vehicle control) alone or with 1 μM, 10 μM, and 100 μM of NNK in water for 12 weeks. The colony-forming efficiency increased following NNK treatment; the maximum effect was observed after 12 weeks with 100 μM NNK. Microarray analysis revealed that, independent of the dose of NNK, expression of 30 genes was significantly altered; 22 of these genes showed a dose-response pattern. Genes identified are categorized as immune response (LTB4R), RNA surveillance pathway (SMG1), metabolism (ALDH7A1), genes frequently expressed in later stages of neoplastic development (MT1F), DNA binding (HIST3H3 and CHD1L), and protein biosynthesis (UBA52). Selected genes were confirmed by qRT-PCR. Western blot analysis indicates that phosphorylation of histone 3 at serine 10, a marker of cellular transformation, was up-regulated in cells treated with NNK. This is the first study showing that NNK can alter gene expression that may, in part, account for transformation of HPV-immortalized human cervical cells. The results support previous epidemiological observations that, in addition to HPV, tobacco smoking also plays an important role in the development of cervical cancer.     

Nicotine,through upregulating pro‐survival signaling,cooperates with NNK to promote transformation

Cigarette smoking is a mixture of thousands of compounds, many of which are carcinogens, such as NNK [4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone]. Nicotine, as an addictive substance in cigarette, has been shown to promote growth of non‐neuronal cells. It is unclear how nicotine cooperates with tobacco‐related carcinogens during tumorigenesis. Here, by concurrent treatment of nicotine and NNK, we investigate the effect of the cooperation of these two compounds on cell growth and apoptosis in various different lung epithelial (RLE) or cancer (LKR) cells. We demonstrated that short‐term nicotine exposure moderately activated mitogenic signaling pathways (such as PKC, ERK, and Akt) and a mediocre protection against cisplatin‐mediated apoptosis. In contrast, NNK strongly stimulated mitogenic signaling and rendered the cells a high resistance to cisplatin. The pre‐ligation of nAChR by nicotine interfered with NNK‐mediated mitogenic signaling and resistance to cisplatin, the magnitude of which was similar as that exposed to nicotine alone. Interestingly, a week after the exposure to nicotine or nicotine plus NNK, Bcl‐2 expression was augmented, accompanied with the increased resistance to cisplatin‐induced apoptosis. In comparison, long‐term NNK treatment provided very little protection of the cells from cisplatin. We also showed that the combination treatment promoted more cells to grow in an anchorage‐independent fashion than NNK exposure alone. Thus, the data suggest that through occupying nAChR, nicotine appears to modulate NNK‐mediated signaling and persistently sustain pro‐survival activities to promote transformation. J. Cell. Biochem. 109: 152–161, 2010. © 2009 Wiley‐Liss, Inc.     

Formation of genotoxic products from N-nitrosoheptamethyleneimine (NHMI), 4-(Methylnitrosamino)-1-(3-Pyridyl)-1-Butanone (NNK) and N′-Nitrosonornicotine (NNN) by isolated rabbit lung cells

The genotoxic potentials of N-nitrosoheptamethyleneimine (NHMI), 4-(methylnitrosamino)-1-(3 pyridyl)-1-butanone (NNK) and N-nitrosonornicotine (NNN) were studied in fresh preparations of Clara cells and type II cells isolated by centrifugal elutriation and density gradient centrifugation, and macrophages from rabbit lung. The activation of the compounds to bacterial mutagens was assayed in the Salmonella mutagenicity test using strains of TA 100 and TA 1530 preincubated with test chemicals and cells placed in chambers with nucleopore membranes to separate cells and bacteria. Unscheduled DNA synthesis was measured by incorporation of [³H]-thymidine in the cells after exposure to the compounds. NHMI, NNK and NNN were not activated to bacterial mutagens by Clara cells, type II cells or macrophages, presumably because the reactive metabolites generated were not released into the incubation medium. However, NHMI and NNK increased unscheduled DNA synthesis in Clara cells, and the highest repair activity was found after incubation with NNK. The effect of NNN was only marginal. This indicates that NHHI and NNK are genotoxic in the rabbit lung and that the Clara cells are involved in the metabolic activation of these compounds.Abbreviations NHMI N-nitrosoheptamethyleneimine - NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone - NNN N-nitrosonornicotine Supported by a grant and a fellowship (R.B.) from the Royal Norwegian Council for Scientific and Industrial Research.     

Expression of a constitutively active nitrate reductase variant in tobacco reduces tobacco‐specific nitrosamine accumulation in cured leaves and cigarette smoke

Burley tobaccos (Nicotiana tabacum) display a nitrogen‐use‐deficiency phenotype that is associated with the accumulation of high levels of nitrate within the leaf, a trait correlated with production of a class of compounds referred to as tobacco‐specific nitrosamines (TSNAs). Two TSNA species, 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) and N‐nitrosonornicotine (NNN), have been shown to be strong carcinogens in numerous animal studies. We investigated the potential of molecular genetic strategies to lower nitrate levels in burley tobaccos by overexpressing genes encoding key enzymes of the nitrogen‐assimilation pathway. Of the various constructs tested, only the expression of a constitutively active nitrate reductase (NR) dramatically decreased free nitrate levels in the leaves. Field‐grown tobacco plants expressing this NR variant exhibited greatly reduced levels of TSNAs in both cured leaves and mainstream smoke of cigarettes made from these materials. Decreasing leaf nitrate levels via expression of a constitutively active NR enzyme represents an exceptionally promising means for reducing the production of NNN and NNK, two of the most well‐documented animal carcinogens found in tobacco products.