Department of Entomology, CSK Himachal Pradesh Agricultural University, Palampur, Himachal Pradesh, India.
Department of Entomology, PG College of Agriculture, Dr. Rajendra Prasad Central Agricultural University, Pusa, Bihar, India.
Sci Rep. 2023 Jul 27;13(1):12151. doi: 10.1038/s41598-023-37656-2.
Nicotine is a highly addictive alkaloid and a neurostimulator found in tobacco that causes addiction in humans and makes tobacco a high-demand commercial product. It is popularly used for recreational purposes and is a harmful substance (Oral LD value for rat is 50 mg/kg) and causes addiction. The metabolites of nicotine such as the Tobacco-specific Nitrosamines (TSNAs) are hazardous substances whose metabolites are highly electrophilic and form DNA adducts, which will initiate the process of carcinogenesis. TSNAs are formed during curing, storage and fermentation due to the nitrosation of nicotine and other tobacco alkaloids. TSNAs are used as biomarkers for cancer risk assessment in humans exposed to tobacco and its products. To determine the occasional formation of TSNAs in tobacco-feeding insects, 5th instar larvae of Spodoptera litura and their faeces were analyzed for the presence of N'-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) along with the stored tobacco leaves (PT-76) using an Agilent 6470B LC-MS/MS system following ISO/DIS 19290:2015 protocol. The larvae are extracted in a buffered acetonitrile-water extraction and the amount of TSNAs are quantified in multiple reaction monitoring (MRM) mode. 20 [Formula: see text]l of each extracted and cleaned up sample was injected into the LC-MS/MS system for quantification. The Limit of Detection (LOD) and Limit of Quantification (LOQ) were 0.001 mg/kg and 0.005 mg/kg for all the tested nitrosamines. NNN was found to be 0.361 mg/kg, 0.340 mg/kg, and 5.66 mg/kg in insect whole-body samples, faeces, and tobacco leaves, respectively. NNK was found to be 0.060 mg/kg, 0.035 mg/kg and 0.93 mg/kg in insect whole body samples, faeces and tobacco leaves, respectively. However, NNAL was not detected in both the insect's whole body and faeces. Recoveries ranged between 95 and 98% for all compounds when spiked at LOD and LOQ. The presence of TSNAs is a biomarker for cancer risk and their presence in insects would point to cancer risk assessment in tobacco feeding insects and any possible TSNA-detoxifying pathways in insects that might prevent mutagenesis caused these compounds.
尼古丁是一种高度成瘾的生物碱和神经刺激物，存在于烟草中，会导致人类上瘾，并使烟草成为高需求的商业产品。它通常被用于娱乐目的，是一种有害物质（大鼠口服 LD 值为 50mg/kg），会导致上瘾。尼古丁的代谢物，如烟草特异性亚硝胺（TSNAs），是危险物质，其代谢物具有高度亲电性，并形成 DNA 加合物，从而引发致癌过程。TSNAs 是在烟草的加工、储存和发酵过程中由于尼古丁和其他烟草生物碱的亚硝化而形成的。TSNAs 被用作评估接触烟草及其制品的人类癌症风险的生物标志物。为了确定在以烟草为食的昆虫中偶尔形成 TSNAs，使用安捷伦 6470B LC-MS/MS 系统，按照 ISO/DIS 19290:2015 协议，分析了 5 龄期斜纹夜蛾幼虫及其粪便中 N'-亚硝基降烟碱（NNN）、4-（甲基亚硝氨基）-1-（3-吡啶基）-1-丁酮（NNK）和 4-（甲基亚硝氨基）-1-（3-吡啶基）-1-丁醇（NNAL）的存在情况，以及使用经过缓冲的乙腈-水提取法提取的储存烟草叶片（PT-76）。在多重反应监测（MRM）模式下定量测定 TSNAs 的含量。对每个提取和净化后的样品进行 20μl 注入 LC-MS/MS 系统进行定量。所有测试亚硝胺的检测限（LOD）和定量限（LOQ）均为 0.001mg/kg 和 0.005mg/kg。在昆虫全身体样本、粪便和烟草叶片中分别发现 NNN 为 0.361mg/kg、0.340mg/kg 和 5.66mg/kg。在昆虫全身体样本、粪便和烟草叶片中分别发现 NNK 为 0.060mg/kg、0.035mg/kg 和 0.93mg/kg。然而，在昆虫的全身体和粪便中均未检测到 NNAL。当在 LOD 和 LOQ 处添加时，所有化合物的回收率在 95%至 98%之间。TSNAs 的存在是癌症风险的生物标志物，它们在昆虫中的存在表明在以烟草为食的昆虫中存在癌症风险评估，以及昆虫体内可能存在的任何 TSNA 解毒途径，这些途径可能防止这些化合物引起的突变。
