Besaratinia A, Maas L M, Van Breda S G, Curfs D M, Kleinjans J C, Wouters E F, Van Schooten F J
Department of Health Risk Analysis and Toxicology, Maastricht University, The Netherlands.
Cancer Epidemiol Biomarkers Prev. 2000 Apr;9(4):367-72.
The lung is a major target organ for smoking-associated cancer. We examined the applicability of induced sputum for molecular dosimetry of exposure to tobacco smoke-related carcinogens. Sputum induction was performed by inhalation of 4.5% saline delivered from an ultrasonic nebulizer for a period of up to 21 min in a group of smoking (n = 20) and nonsmoking (n = 24) healthy individuals. Samples were analyzed for total and differential cell counts and cell viability. Subsequently, DNA contents of the samples were isolated, and measurement of lipophilic DNA adducts was done by the 32P-postlabeling assay using nuclease P1 (NP1) and butanol enrichment methods. All subjects tolerated the induction procedure without experiencing any troublesome symptoms, and 90% of smokers (18 of 20) and 88% of nonsmokers (21 of 24) succeeded in producing sufficient amounts of sputum. Total cell counts and percentages of viable cells in smokers were higher than those in nonsmokers (6.7+/-6.0 versus 4.7+/-6.0 x 10(6), P = 0.40 and 80+/-15 versus 63+/-17, P = 0.01, respectively). In cell differentials, smokers had lower percentages of bronchoalveolar macrophages and higher percentages of neutrophils (69+/-24 versus 92+/-5, P = 0.002 and 26+/-26 versus 4+/-4, P = 0.008, respectively). Using the NP1 digestion method, all smokers and only one nonsmoker showed a diagonal radioactive zone in their adduct maps; adduct levels in smokers were higher than those in nonsmokers (3.1+/-1.4 versus 0.6+/-0.8/10(8) nucleotides; P = 0.0007), and also, adduct levels were significantly related to smoking indices. Applying the butanol extraction method, however, only half of the smokers and three nonsmokers showed the diagonal radioactive zone in their adduct maps; adduct levels in smokers were higher than those in nonsmokers (4.6+/-3.7 versus 1.0+/-1.9/10(8) nucleotides; P = 0.02), and the levels of adducts were significantly related to the smoking indices. There was a correlation between the levels of adducts determined by the two enrichment methods (r = 0.7; P = 0.02). Paired comparison showed no differences between the levels of adducts measured by the two methods (P = 0.55). We conclude that induced sputum can serve for molecular dosimetry of inhalatory exposure to carcinogens and that the NP1 version of the 32P-postlabeling assay is a choice of preference for studying smoking-induced DNA adducts in the lower respiratory tract.
肺是吸烟相关癌症的主要靶器官。我们研究了诱导痰用于烟草烟雾相关致癌物暴露分子剂量测定的适用性。在一组吸烟（n = 20）和不吸烟（n = 24）的健康个体中，通过吸入超声雾化器产生的4.5%盐水进行痰液诱导，持续时间最长为21分钟。对样本进行总细胞计数、分类细胞计数和细胞活力分析。随后，分离样本的DNA含量，并使用核酸酶P1（NP1）和丁醇富集方法通过32P后标记法测定亲脂性DNA加合物。所有受试者均耐受诱导过程，未出现任何不适症状，90%的吸烟者（20人中的18人）和88%的不吸烟者（24人中的21人）成功咳出足够量的痰液。吸烟者的总细胞计数和活细胞百分比高于不吸烟者（分别为6.7±6.0对4.7±6.0×10⁶，P = 0.40；80±15对63±17，P = 0.01）。在细胞分类中，吸烟者的支气管肺泡巨噬细胞百分比更低，中性粒细胞百分比更高（分别为69±24对92±5，P = 0.002；26±26对4±4，P = 0.008）。使用NP1消化方法，所有吸烟者和仅一名不吸烟者在其加合物图谱中显示出对角线放射性区域；吸烟者的加合物水平高于不吸烟者（3.1±1.4对0.6±0.8/10⁸核苷酸；P = 0.0007），并且加合物水平也与吸烟指数显著相关。然而，应用丁醇提取方法时，仅一半的吸烟者和三名不吸烟者在其加合物图谱中显示出对角线放射性区域；吸烟者的加合物水平高于不吸烟者（4.6±3.7对1.0±1.9/10⁸核苷酸；P = 0.02），加合物水平与吸烟指数显著相关。两种富集方法测定的加合物水平之间存在相关性（r = 0.7；P = 0.02）。配对比较显示两种方法测定的加合物水平之间无差异（P = 0.55）。我们得出结论，诱导痰可用于吸入致癌物暴露的分子剂量测定，并且32P后标记法的NP1版本是研究下呼吸道吸烟诱导的DNA加合物的首选方法。
