Dunn B P, Black J J, Maccubbin A
Environmental Carcinogenesis Unit, British Columbia Cancer Research Centre, Vancouver, Canada.
Cancer Res. 1987 Dec 15;47(24 Pt 1):6543-8.
Brown bullheads (Ictalurus nebulosus) were sampled from sites in the Buffalo and Detroit Rivers where fish are exposed to high levels of sediment bound polycyclic aromatic hydrocarbons, and suffer from an elevated frequency of liver cancer. DNA was isolated from the livers of these wild fish and from control specimens which were raised in clean aquariums. DNA was enzymatically digested to normal and adducted nucleotides, and hydrophobic/bulky adducts were enriched in the digests either by preparative reverse-phase high-pressure liquid chromatography, or selective nuclease P1 dephosphorylation of normal nucleotides. Aromatic DNA-carcinogen adducts were then quantitated using 32P-postlabeling analysis. Using both adduct enrichment procedures, chromatograms derived from DNA of fish from polluted areas showed a diffuse diagonal radioactive zone not present in DNA from aquarium raised fish. The diagonal zone appeared to consist at least in part of multiple overlapping discrete adduct spots which could be partially separated by gradient high-pressure liquid chromatography prior to 32P-postlabeling analysis, and most of which were more strongly retained on a reverse-phase column than the major benzo(a)pyrene-DNA adduct. The behavior of the adducts in the diagonal radioactive zone and of their unlabeled precursors is consistent with their identification as nucleotide adducts of a variety of bulky hydrophobic aromatic environmental compounds. Total pollution-related adduct levels as analyzed by HPLC adduct enrichment and 32P-postlabeling were 70.1 +/- 29 (SD) nmol/mol normal nucleotide in fish from the Buffalo River, and 52 and 56 nmol/mol for two specimens from the Detroit River.
棕色牛头鲇（Ictalurus nebulosus）取自布法罗河和底特律河的一些地点，那里的鱼类接触到高浓度的与沉积物结合的多环芳烃，且肝癌发病率较高。从这些野生鱼类的肝脏以及在清洁水族箱中饲养的对照样本中提取DNA。DNA经酶消化为正常核苷酸和加合物核苷酸，通过制备型反相高压液相色谱法或正常核苷酸的选择性核酸酶P1去磷酸化作用，使消化物中的疏水/大分子加合物得以富集。然后使用32P后标记分析法对芳香族DNA - 致癌物加合物进行定量。使用这两种加合物富集方法，来自污染区域鱼类DNA的色谱图显示出一个弥散的对角放射性区域，而水族箱饲养鱼类的DNA中不存在该区域。对角区域似乎至少部分由多个重叠的离散加合物斑点组成，在进行32P后标记分析之前，可通过梯度高压液相色谱法将其部分分离，其中大多数在反相柱上的保留比主要的苯并(a)芘 - DNA加合物更强。对角放射性区域中加合物及其未标记前体的行为与它们被鉴定为多种大分子疏水芳香族环境化合物的核苷酸加合物一致。通过HPLC加合物富集和32P后标记分析的与污染相关的总加合物水平，在布法罗河鱼类中为70.1 +/- 29（标准差）nmol/mol正常核苷酸，在底特律河的两个样本中分别为52和56 nmol/mol。
