nnk是烟草中特有的一种物质

nnk是烟草中特有的一种物质
nnk是烟草中特有的一种物质，可以诱导肺癌的发生。

卷烟燃烧可产生4000多种化学物质，其中40余种有明确的诱变/致癌性。

主要的致癌物有烟草特有亚硝胺（TSNA）、苯并（a）芘、多环芳烃（PAH），芳香胺、苯、二嗯英、儿茶酚及致癌的醌、肼类等。

TSNA 是尼古丁被亚硝化的产物，4-（甲基亚硝氨基）-3-吡啶-1-丁酮（NNK）是已知7种TSNA中最强的致癌源。

NNK在主流、侧流烟气及不燃烧的烟草中均大量存在。

NNK是卷烟致癌的主要标志物，尼古丁是吸烟成瘾的主要原因。

Efficient Bioelectronic Actuation of the Natural Catalytic Pathway of
Human Metabolic Cytochrome P450s
Sadagopan Krishnan,?Dhanuka Wasalathanthri,?Linlin Zhao,?John B. Schenkman,?and James F. Rusling*,?,?
Herein, we describe fabrication of LbL films made by combining pure cyt P450s with CPR microsomes on electrodes to achieve a large ratio of cyt P450 to CPR (Figure 1), as in the human liver.1,2,40 Electrons are injected into the film from the electrode to accurately mimic the natural cyt P450 catalytic cycle at high catalytic turnover.
We provide unambiguous evidence for electron transfer from electrode to CPR to cyt P450 from measured redox potentials, electron transfer rates, enzyme turnover rates, and carbon monoxide (CO) binding. Results suggest dynamic participation of a CPR-cyt P450 complex in a key equilibrium redox process facilitating efficient catalytic turnover of the excess cyt P450s. In addition, the electrode-driven turnover rate for a model oxidation reaction was as good as or better than when NADPH was utilized.
这里我们主要叙述了层层膜结构通过将纯的P450酶和P450还原酶连接到电极上来实现像人体中从P450酶到P450还原酶一个较大差异，电子从电极中被送到膜中，准确模拟再高催化转化情况下的p450的催化过程。

我们从对测量的氧化还原电势，电子转移速率，酶转化速率和CO 键合中为电子从电极传递到到CRP再到P450还原酶提供了有力的证据。

结果显示，在一个关键的氧化还原过程中CPR-cyt P450的动态参与大大的促进了大量P450酶的有效催化转化。

除此之外，电极驱动模型氧化反应的转化速率和使用NADPH差不多甚至更好！
有资料表明,吸烟者每天吸入NNK的量约为28 nmol ,40年内吸入NNK 的量约为85 mg ( 1. 1 mg/kg)
烟草特有亚硝胺NNK与肺癌的关系
3.NNK诱导的肺癌基因突变:在NNK诱导的动物肺癌中均发现了基因的突变,在NNK诱导的小鼠肺癌中发现有Kras 基因的12 位密码子GGT →GAT 的转变[4 ] 。

在人的肺腺癌中Kras 基因12 位密码子的突变约24 %～50 % ,而在其他类型的肺癌中这种突变很少见[12 ,13 ] 。

这种突变在吸烟者和被动吸烟者比在不吸烟者中更常见。

最常见的突变为:GGT→TGT ( 60 %) , 其次为GGT→GAT ( 20 %) 和GGT→GTT(15 %)
PEITC在F2344 鼠和A P J鼠内能抑制NNK诱导肺肿瘤,而异硫氰酸苯
甲酯(BITC) 在A P J鼠能抑制苯并芘(BaP) 诱导的肺肿瘤,非毒性剂量的PEITC 和BITC 能有效抑制NNK和苯并芘(BaP) 在鼠体内的代谢活化和其致癌性。

如果联合PEITC和BITC ,能在人体内抑制NNK和B (a) P 诱导的肺肿瘤,将会为吸烟导致肺癌的预防带来新的防治措施
吲哚232甲醇( I3C) 也是人类食物中的一种成分,它在一些十字花科蔬菜中以
结合形式存在,它主要通过增加肝脏清除NNK的能力来阻断NNK 诱导的实验鼠的肺肿瘤,同样能增加吸烟者肝脏代谢NNK的能力和尿排泄NNAL 和NNAL2Gluc
的能力。

还有研究提示一些药物如维生素C、维生素E、阿司匹林及黄绿色蔬菜水果等均能减少和预防动物肺肿瘤的发生[4 ] 。

维生素E 是通过调节多胺的代谢,而阿司匹林是通过抑制环氧酯酶的活性来实现其抑制NNK的致癌性的
NNK和NNAL 在体内的代谢:NNK在体内的半衰期极短,很快代谢转化为NNAL、NNAL2Gluc 和其他产物。

NNK在动物体内主要有三条代谢途径:碳基还原反应、吡啶氮氧化反应和α2羟化反应
1.NNK和NNAL 在体内的代谢:NNK在体内的半衰期极短,很快代谢转化为NNAL、NNAL2Gluc 和其他产物。

NNK在动物体内主要有三条代谢途径:碳基还原反应、吡啶氮氧化反应和α2羟化反应NNK requires metabolic activation to exert its carcinogenic effects (3). Metabolic activation of NNK occurs
largely via cytochrome P450-catalyzed hydroxylation of the carbon atoms adjacent to the nitroso moiety (i.e.,R-hydroxylation)
NNK requires metabolic activation to exert its carcinogeniceffects . Metabolic activation of NNK occurs largely via cytochrome P450-catalyzed hydroxylation of the carbon atoms adjacent to the nitroso moiety (i.e.,α-hydroxylation) . As can be seen in Scheme 1,hydroxylation of the α-methylene carbon of NNK generates an unstable α-hydroxynitrosamine that spontaneously decomposes to methanediazohydroxide and OPB. Methanediazohydroxide , or the methyldiazonium ion, reacts with DNA bases to form methyl adducts such as O6-mG and 7-mG . Hy droxylation of the α-methyl carbon of NNK generates the unstable metabolite 4, which spontaneously breaks down to 5 and formaldehyde (Scheme 1). The diazonium ion 6 formed from this
pathway is a pyridyloxobutylating agent; it reacts with DNA bases to form
pyridyloxobutyl adducts or with water to form HPB
a preponderance of experimental data in A/J mice implicates α-methylene hydroxylation and the subsequent formation of O6-mG as crucial factors for NNK tumorigenicity
使用A/J老鼠收集实验数据很大优势是可以得出α-methylene hydroxylation（α-亚甲基羟基化）和随后产生的O6-mG（6-甲氧基鸟嘌呤）是NNK致癌的关键！
The initial P450-mediated metabolic activation of NNK is clearly an important event in the generation of a methylating species, the formation of a promutagenic base, and, ultimately, the development of A/J mouse lung tumors.
NNK的代谢最初以P450为媒介是甲基化的重要步骤，是导致A/J 小鼠产生肺癌肿瘤的基础和最终原因！
在!KTR&"T人类原发性肺腺癌中存在<,0U 基因密码子F! 的突变，但其他类型肺肿瘤中几乎未发现过=F>，!K，!&?。

这些突变在吸烟者和被动吸烟者中比在不吸烟者中更常见，表明这些突变可能是由烟草烟气中的某种成分引起在!KTR&"T人类原发性肺腺癌中存在<,0U 基因密码子F! 的突变，但其他类型肺肿瘤中几乎未发现过=F>，!K，!&?。

这些突变在吸烟者和被动吸烟者中比在不吸烟者中更常见，表明这些突变可能是由烟草烟气中的某种成分引起的=!#?。

最常观察到的突变是CCV!VCV，非常典型，占密码子F! 突变的#"T，其次是CCV!C@V （!"T）和CCV!CVV（F&T）。

C!V 突变的普遍性导致这样一种推测，这些突变是起因于苯并芘，后者可通过二醇氧化物代谢活化途径诱发这些突变=!#?。

然而，C!V 突变也可由;;<o@e（;;< bdsfid="94" p="" 实施"b甲基羟基化后与醋酸形成的酯）诱发<=""></o@e（;;<> 研究表明，异硫氰酸盐抑制SK&"U 酶是其抑制;;< 诱发小鼠肺肿瘤的主要机制=!$G%F?。

这导致O#B7C 的形成和肿瘤被抑制。

当被加至小鼠肺微粒中孵化时，S(WVX 通过竞争和非竞争机制抑制;;< 氧化
In order for NNK to exert its carcinogenicity, it must be metabolically activated. The metabolic activation of NNK involves a-hydroxylation of the methyl or methylene carbon, leading to the formation of electrophiles, which can pyridyloxobutylate and methylate DNA, respectively
NNK的代谢活化被认为是诱发癌变的首要条件
[22]
DNA adduct formation from tobacco-specific N-nitrosamines Stephen S. Hecht )
Uniíersity of Minnesota Cancer Center Box 806, Mayo, 420 Delaware Street SE Minneapolis, MN 55455, USA
This paper will describe DNA adduct formation from tobacco-specific nitrosamines. Almost all studies to date have been carried out with NNK, NNAL, and NNN. Although some of the adducts are highly specific and can be derived only from tobacco-specific nitrosamines,others have a
variety of sources.
In this paper, metabolic activation and DNA adduct formation by NNK, NNAL, and NNN will be summarized. An extensive review of the literature on this topic has recently been completed
Similarly, CNPB is a precursor to diazohydroxide 3 Diazohydroxide 3 yields diazonium ion 4 which has three fates: reaction with nucleophiles Y:. producing 5, formation of the cyclic oxonium ion 6, or loss of N2 and H＋yielding the α, β-unsaturated ketone 7. The latter two react with nucleophiles to form products 8 and 9. Extensive studies conclusively demonstrate that the major DNA adducts. formed by this
pathway in vitro and in vivo, accounting for at least 50% of the DNA bind-ing, releases the
keto alcohol HPB Fig. 2. upon acid or neutral thermal, but not base hydrolysis. This adducts. is produced via intermediates—3, 4 and/or 6—but not by HPB itself . The HPB releasing adducts. have differing stabilities in DNA, being released in a triphasic manner
NNK羰基还原反应的主要催化剂不是细胞色素P450，而是11β-羟基类固醇脱氢酶，它是一种微粒体酶，主要作用是将活泼的11-羟基糖皮质激素转化为不活泼的11-羰基式
1.2.1.3 吡啶-N-氧化反应
吡啶-N-氧化反应只在体外实验中被发现，根据实验动物种类和组织的不同，NNK产生N-氧化产物也不同。

NNK在大鼠和小鼠肺微粒体中主要代谢生成NNKN-oxide，而在未经预处理的大鼠肝微粒体、小鼠肝微粒体和大鼠鼻粘膜微粒体中则仅为次要反应或不能被检测到。

吡啶-N-氧化反应依赖细胞色素P450的催化。

在动物组织体外实验中，CYP450 2B1主要表现出催化NNK形成NNK N-oxide的活性还有实验表明CYP450 3A4在人肝微粒体中有催化作用
POB-DNA加合物还会影响O6-烷基鸟嘌呤-DNA烷基转移酶(AGT)对DNA的修复作用。

AGT是一种重要的DNA修复酶，它可通过复原DNA链上烷化剂导致的鸟嘌呤O6位烷基化而达到修复DNA 的目的
however, evidence has accumulated that 1,4-phenylenebis (methylene) selenocyanate (p-XSC) is able to prevent the initiation phase of carcinogenesis caused by DMBA and NNK by inhibiting the formation of DNA adducts.
(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) Cardnogenesis vol.17 no.4 pp.749-753, 1996
While Sohn et al. have demonstrated that p-XSC (苯二甲硒氰 ) pretreatment has no effect on phase I enzymes such as P450 1A1, 2B1, 2E1 and 3A4 in the liver of male F344 rats and of female Sprague-Dawley rats, the effects of p-XSC on the induction of such enzymes in extrahepatic tissues, such as the lung, have not been studied(通过膳食补充）Both doses of p-XSC inhibited the formation of (AmGua and 7-mGua in mouse liver. The highest inhibition of O6- mGua was observed with p-XSC at the 15 p.p.m. Se dose. In hepatic DNA, dietary supplementation of p-XSC at this level resulted in 69.5% inhibition of this lesion after 4 h and 73.8% after 96 h (Figure 1)
Suppressive oligodeoxynucleotides reduce lung cancer susceptibility in mice with silicosis
Carcinogenesis vol.00 no.00 p.1 of 6, 2014
doi:10.1093/carcin/bgu005 Advance Access publication January 8, 2014
Kinetics of O6?Pyridyloxobutyl-2′-deoxyguanosine Repair by Human O6?alkylguanine DNA Alkyltransferase
Biochemistry 2013, 52, 4075?4088
We found that HBEC cells were capable of removing O6-POB-dG lesions, and the repair rates were significantly reduced in the presence of an AGT inhibitor (O6-benzylguanine).
O6-Alkylguanine-DNA alkyltransferase (AGT) protein can directly remove the O6-alkyl group from O6-alkylguanines in DNA, restoring normal guanine. AGT protein binds to the minor groove of DNA via the helix-turn-helix motif, inducing flipping of O6-Alk-dG out of the DNA helix to enter the protein active
site.Cysteine-145 thiol within the active site of the AGT protein is deprotonated via interactions with other active
site residues, and the resulting Cys-145 thiolate anion undergoes nucleo philic a ttack at the α-carbon of the O6-alkyl group, leading to its transfer from DNA to the protein.
6甲氧基鸟嘌呤DNA甲基转移酶(AGT)蛋白能够直接将6甲氧基基团从6甲氧基鸟嘌呤移除，使鸟嘌呤恢复正常，AGT蛋白根据DNA 的双螺旋结构直接键合在小沟中，诱导O6-Alk-dG从DNA螺旋中翻转出去，从而进入蛋白活性点，半胱氨酸的巯基在AGT蛋白的活性点中通过与其他残基活性点相互作用去质子化，具有亲核作用的半胱氨酸巯基阴离子在攻击6甲氧基基团上的α碳原子是6甲氧基基团从DNA上转移到蛋白质中。

One possible mechanism for the increased mutagenesis at these sites involves inefficient repair of tobacco carcinogen-induced DNA adducts such as O6-Me-dG and O6-POB-dG, leading to their accumulation at methylated CpG sequences.
Determination of DNA Melting Temperatures. DNA duplexes containing site-specific O6-POB-dG adducts (3 nmol) were dissolved in sodium phosphate buffer (10 mM, pH 7.0) containing 50 mM sodium chloride (9.7 μM DNA). DNA melting temperatures were obtained with
a Varian Cary-100 Bio UV?visible spectrophotometer using a temperature gradient between 30 and 90 °C. Temperature increments or decrements of 0.5 °C/min were used. The experiment was repeated 4?6 times to determine DNA melting temperatures using Cary WinUV Thermal software
Comparison of the Chemopreventive Efficacies of
1,4-phenylenebis(methylene)selenocyanate and Selenium-Enriched Yeast on 4-(Methylnitrosamino)-1-(3-pyridyl)-1-
butanone Induced Lung Tumorigenesis in A/J Mouse Arunangshu Das , Dhimant Desai , Brian Pittman , Shantu Amin & Karam
I n biochemical studies, p-XSC was shown to significantly inhibit formation ofO6-methylguanine (O6-MG)and 7-methylguanine (7-MG) in the lungs and livers of mice treated with NNK.
NNK was purchased from Chemsyn Science Laboratories (Lenexa, KS).
Benzylmorpholine Analogs as Selective Inhibitors of Lung Cytochrome P450 2A13 for the Chemoprevention of Lung Cancer in Tobacco Users
Received: 14 January 2013 / Accepted: 2 April 2013 / Published online: 12 June 201 Biochemical applications of ultrathin films of enzymes, polyions and
DNA\
Received (in Cambridge, UK) 15th June 2007, Accepted 3rd August 2007First published as an Advance Article on the web 30th August 2007
DOI: 10.1039/b709121b
Construction begins by adsorbing an initial layer of charged polyion from solution onto an oppositely charged solid surface. Loosely bound polyions are removed by washing with water, then a second layer of polyions of the opposite charge to the first is adsorbed.
起初通过吸附通过静电力结合与固体表面上结合的聚离子层上，用水将松散的聚离子洗掉，然后带相反电荷的第二层聚离子再吸附第一层。

Herein we present a short account of our biochemically-related applications utilizing LbL films, first to make stable films
of enzymes and polyions for biocatalysis,
and second to make films of DNA, metabolic enzymes and polyions for toxicity screening
Lvov and I specifically wished to apply the LbL method to facilitate direct electrochemical activation of metabolic enzymes on solid electrodes
A key is to place the protein in a film or on a surface that protects it from denaturing and fouling the electrode
A second, non-stereoselective pathway is likely to involve olefin oxidation by a peroxyl radical on an amino-acid residue at the protein’s surface
. Major advantages include greatly enhanced enzyme stability and the tiny amount of enzyme required
The LbL film ‘‘nanoreactor’’on the sensor synthesizes reactive metabolites in the vicinity of large concentrations of DNA in the film. The rate of DNA damage from metabolite–nucleobase adduct formation is a measure of relative genotoxicity, and can be detected by voltammetric, electrochemiluminescent, or LC-MS/MS methods Our first step in biosensor development was to evaluate methodologies to detect DNA damage. LbL films of DNA and polycations were used with no enzymes, and incubated with known DNA damaging agents including epoxides and
methylating agents.8,65
The most successful electrochemical detection employed square wave voltammetry (SWV) for DNA oxidation using soluble catalyst Ru(bpy)3 or a catalytic Ru-polyvinylpyridine polymer [Ru(bpy)2 2+ Cl–PVPor ClRu–PVP) within the film to provide ‘‘reagentless’’sensors. The signal results mainly from catalytic electrochemical oxidation of the guanines in DNA
(Scheme 4).
Detection relies on the fact that partly unfolded and ss-DNA have more accessible
guanines (G) than ds-DNA. As DNA is damaged, it partly unfolds, making the guanines in the damaged ds-DNA more accessible to the Ru catalysts and providing larger signal Genotoxicity Screening Using Biocatalyst/DNA Films and Capillary LC-MS/MS
Maricar Tarun,?Besnik Bajrami,?and James F. Rusling*,?,?
Mb in Mb/DNA film was activated by H2O2 to convert styrene to styrene oxide.Styrene oxide then reacted with DNA in the film forming covalently bound adducts
We view the biocatalyst/DNA films as an important feature of our approach, since the metabolites are formed in a thin film with large effective concentrations of DNA. This provides a high probability of reaction of the metabolites with DNA as they diffuse out of the films past the DNA。