卷烟烟气有害成分联合作用的细胞毒性

为评价卷烟烟气有害成分的细胞毒性及其联合作用,以人支气管上皮细胞(BEASe-2B细胞)为靶细胞,分别用卷烟烟气中5种有害成分4-甲基亚硝基-1-(3-吡啶基)-1-丁酮(NNK)、氨、苯并[a]芘、苯酚和巴豆醛单独及两两联合染毒,用MTT法测定染毒24 h后的细胞存活率,计算细胞的平均半数致死剂量(LC50),用效应图解法对联合作用进行评价.结果显示:①NNK、氨、苯并[a]芘、苯酚和巴豆醛的LC50分别为87.2、395.5、5.4、61.9和6.9 mg/L;②NNK与氨、NNK与苯并[a]芘、NNK与苯酚、NNK与巴豆醛、氨与苯并[a]芘、氨与苯酚、氨与巴豆醛、苯并[a]芘与苯酚、苯并[a]芘与巴豆醛、苯酚与巴豆醛的LC50分别为46.2、10.9、14.1、30.4、76.4、86.1、39.0、6.1、1.1和22.1 mg/L;③(NNK与氨、NNK与苯并[a]芘、NNK与苯酚、苯并[a]芘与苯酚、苯并[a]芘与巴豆醛、氨与巴豆醛存在明显的协同作用,而NNK与巴豆醛、苯并[a]芘与氨、苯酚与氨、苯酚与巴豆醛之间无协同作用.因此,卷烟烟气有害成分的细胞毒性存在明显差异,其联合作用的细胞毒性效应各不相同.     

高效液相色谱-大气压化学电离串联质谱法研究4-(甲基亚硝胺基)-1-(3-吡啶基)-1-丁酮体外模拟代谢作用

烟草特异亚硝胺4-(甲基亚硝胺基)-1-(3-吡啶基)-1-丁酮(NNK)是一种存在于烟草烟气及烟草制品中的强致癌物,其致癌机理是通过细胞色素P450酶的代谢活化生成活泼亲电试剂,导致DNA损伤,因此,建立NNK体外模拟代谢产物的定量分析方法对于研究烟草致癌物具有重要意义。本实验利用高效液相色谱-大气压化学电离串联质谱法（HPLC-APCI-MS/MS）对NNK代谢产物4-羟基-1-(3-吡啶基)-1-丁酮(HPB)进行定量分析。使用选择反应扫描（SRM）模式监测了HPB和内标[3,3,4,4-D₄]HPB,分析方法在0.2~400 nmol/L范围内线性关系良好,线性相关系数R²=0.999 9,检出限(LOD)为0.025 nmol/L (S/N=3),定量限(LOQ)为0.05 nmol/L(S/N=10)。方法的日内和日间准确度为96.6%~101.8%,回收率为98.1%~102.6%。所建立的NNK体外模拟代谢模型可用于烟草特异亚硝胺代谢活化分子机制的研究,为定量分析吸烟致癌相关生物标志物奠定基础。     

NNK和B[a]P在卷烟烟气复杂基质中的联合遗传毒性

为对卷烟进行安全性评价,以肯塔基参比卷烟3R4F为研究对象,以B[a]P、NNK为目标化合物,采用检测哺乳动物细胞致突变性的微核试验,结合三者对人体支气管上皮细胞的毒性作用,分析了B[a]P、NNK在卷烟烟气复杂基质中的联合作用.结果表明:(1)B[a]P、NNK与CSC均具有体外细胞遗传毒性;(2)两两联合作用诱发细胞微核存在一定的拮抗效应;(3)三者联合作用呈现一定的协同作用.     

硝酸盐、亚硝酸盐和烟碱对卷烟主流烟气中烟草特有N-亚硝胺的影响

研究了卷烟主流烟气中烟草特有N亚硝胺及其前体的相互关系,通过在卷烟烟支中分别加入硝酸盐、亚硝酸盐、烟碱,采用标准方法对烟气总粒相物进行捕集,捕集后的样品进行前处理,采用内标法对烟草特有N亚硝胺(TSNAs)进行气相热能分析仪(GCTEA)定量分析,分别研究了4种烟草特有N亚硝胺(NNN、NAT、NAB、NNK)及其总量与硝酸盐、亚硝酸盐和烟碱的相互关系。研究结果表明:硝酸盐、亚硝酸盐、烟碱均是烟草特有N亚硝胺的重要前体物质,随着这些前体物质含量的增加,卷烟主流烟气中的烟草特有N亚硝胺含量也增加,表现出显著的相关关系。      

NNK和B[a]P在卷烟烟气基质中的联合致突变性

以苯并[a]芘(B[a]P)、甲基亚硝胺吡啶基丁酮(NNK)为目标化合物,设置卷烟烟气冷凝物(CSC)的浓度为0(CK),0.005,0.010,0.015,0.020 mg/mL;NNK的浓度为0(CK),0.005,0.010,0.020,0.040 mg/mL;B[a]P的浓度为0(CK),0.00025,0.00050,0.00075,0.00100 mg/mL,研究了NNK,B[a]P和CSC的联合致突变性。结果表明:①CSC,NNK和B[a]P均可诱导鼠伤寒沙门氏菌TA98,TA100发生突变,且剂量-效应明显。②B[a]P与CSC联合呈相加作用;B[a]P与NNK联合高剂量呈弱相加作用;CSC与NNK联合低剂量呈弱相加作用。③在不同情况下,三者联合作用致TA98呈弱相加作用。综上,NNK和B[a]P在卷烟烟气基质中的联合致突变性呈现量效、时效和先后作用顺序的复杂性。     

“三丝”掺兑量对卷烟主流烟气有害成分释放量的影响

为考察膨胀梗丝、膨胀烟丝或再造烟叶(简称“三丝”)掺兑量对主流烟气有害成分释放量的影响规律,在控制烟支硬度一致的条件下,制备了15种不同“三丝”掺兑量的卷烟,对其烟丝重量、吸阻、抽吸口数及主流烟气中焦油、烟碱、CO、氢氰酸、NNK、氨、BaP、苯酚、巴豆醛的释放量进行了测定,并建立了“三丝”掺兑量与上述有害成分释放量的回归方程.结果表明:①膨胀梗丝、膨胀烟丝或再造烟叶掺兑量增加10％,烟丝重量分别减少约1.8％,31％和1.3％,卷烟抽吸口数均减少约0.4口;膨胀梗丝或膨胀烟丝掺兑量增加10％,卷烟吸阻均增加约3.0％;②随着膨胀梗丝掺兑量增加,CO和巴豆醛释放量增加,其它7项有害成分释放量降低;随着膨胀烟丝掺兑量增加,巴豆醛释放量增加,焦油、烟碱、CO、BaP、氨和苯酚释放量降低;随着再造烟叶掺兑量增加,巴豆醛释放量增加,焦油、烟碱、氢氰酸、BaP、氨和苯酚释放量降低;③通过增加膨胀梗丝、膨胀烟丝或再造烟叶掺兑量,可以选择性降低卷烟主流烟气中的BaP、氨和苯酚.     

Low ratio of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol-glucuronides (NNAL-Gluc)/free NNAL increases urothelial carcinoma risk

Cigarette smoking is the most important risk factor for bladder cancer. The compound 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) are viewed as biomarkers for cigarette smoking exposure. Therefore, we wanted to explore the effects of these urinary metabolites on urothelial carcinoma (UC) risk. We recruited 127 pairs of UC cases and matched healthy participants for a hospital-based case-control study. Participants completed questionnaires of medical and social information, including smoking history, and provided 50 mL urine samples. Urine samples were analyzed for free NNAL and NNAL-Gluc using the liquid chromatography-tandem mass spectrometry method. Nonparametric analysis and multivariate logistic regression were applied to compare the differences in NNK-related metabolites between UC cases and controls, and to estimate the UC risk associated with certain risk factors. Overall, controls with higher cumulative cigarette smoking exposure had higher total NNAL, free NNAL and NNAL-Gluc. In addition, a decreased NNAL-Gluc/free NNAL ratio corresponded to a significantly increased UC risk. The association between the NNAL-Gluc/free NNAL ratio and UC risk was significant in a dose-response manner. Furthermore, cumulative cigarette smoking exposure was found to interact significantly with low NNAL-Gluc/free NNAL ratio to affect UC risk in this study. This is the first study to conclude that the metabolic products of total NNAL, free NNAL and NNAL-Gluc might be measured as biomarkers of cigarette smoking exposure. Furthermore, the NNAL-Gluc/free NNAL ratio was a better biomarker to evaluate UC risk than total NNAL.     

三重四极杆气质联用法测定卷烟主流烟气中NNK

为了测定卷烟主流烟气中4-(甲基亚硝胺基)-1-(3-吡啶基)-1-丁酮（NNK）的含量,建立了三重四极杆气质联用仪（GC-MS/MS）测定方法。按照标准条件抽吸卷烟,捕集在剑桥滤片上的卷烟烟气粒相物用二氯甲烷萃取并以氘代NNK为内标,经固相萃取净化后,用三重四极杆气质联用仪测定卷烟烟气中NNK的含量。方法的相对标准偏差为2.3%～2.7%（n=6）、回收率为98.9%～116.8%、定量下限为0.087 ng/cig,完全可以满足日常检测的要求。本方法采用商品化固相萃取柱,避免了样品浓缩、转移等操作步骤,节省试剂并且提高了工作效率。应用本方法对2R4F和其他不同类型的卷烟样品烟气中NNK进行了检测,检测数据与国家标准方法GB/T 23228 2008（GC-TEA）一致性好,验证了本方法的准确性。