Liu Zi, Kullman Seth W, Bencic David C, Torten Michael, Hinton David E
Department of Anatomy, Physiology and Cell Biology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA.
Mutat Res. 2003 Aug 5;539(1-2):43-53. doi: 10.1016/s1383-5718(03)00133-5.
Medaka fish are an established non-mammalian research model for the study of liver carcinogenesis and exposure to environmental pollutants. Studies have emphasized the development of hepatic neoplasms in medaka following exposure to model carcinogens. To date however, little information is known regarding the mechanisms underlying initiation of hepatic tumors in this species. The aim of this study was to relate our understanding of diethylnitrosamine (DEN)-induced tumor formation to ras gene activation in hepatic neoplasms of exposed medaka. Initial studies were conducted to identify medaka ras exons 1 and 2 by reverse transcriptase polymerase chain reaction (RT-PCR). Amplification of ras exons 1 and 2 from untreated medaka liver resulted in the identification of three polymorphic ras sequence variants exhibiting a high degree of homology to other teleost and mammalian ras genes. Exposure of medaka to 159 ppm of DEN resulted in a wide range of hepatic neoplasms including: hepatocellular adenomas, hepatocellular carcinomas, cholangiomas, and mixed hepatocholangiocellular carcinomas. Individual liver tumors were examined for oncogenically activating ras mutations by probing genomic DNA with probes specific for activating point mutations or by direct cloning and sequencing of ras transcripts using RT-PCR. Using allele-specific oligonucleotide (ASO) analysis, a single point mutation was detected in codon 12 position two in 8/25 (32%) tumors examined. Mutated ras alleles were additionally detected in 12 of 39 (30%) medaka liver tumors by sequence analysis. Ten of the 12 mutations identified contained a single point mutation at codon 12 resulting in a Gly to Asp amino acid substitution. Two unique mutations were identified at codon 16 resulting in either Lys to Asn or Lys to Thr amino acid substitutions. Our results show that ras mutations are induced by DEN and are present in over 30% of the fish that developed tumors. A ras mutation incidence of 30% is similar to that reported in mammalian species exposed to DEN. While mutations at codon 12 have previously been reported, the present study is the first in vivo report of ras point mutations at codon 16.
青鳉鱼是一种成熟的非哺乳动物研究模型，用于研究肝癌发生和环境污染物暴露。研究强调了青鳉鱼暴露于模型致癌物后肝脏肿瘤的发展。然而，迄今为止，关于该物种肝脏肿瘤起始的潜在机制知之甚少。本研究的目的是将我们对二乙基亚硝胺（DEN）诱导的肿瘤形成的理解与暴露的青鳉鱼肝脏肿瘤中的ras基因激活联系起来。最初的研究通过逆转录聚合酶链反应（RT-PCR）来鉴定青鳉鱼ras外显子1和2。从未经处理的青鳉鱼肝脏中扩增ras外显子1和2，结果鉴定出三个多态性ras序列变体，它们与其他硬骨鱼和哺乳动物的ras基因具有高度同源性。将青鳉鱼暴露于159 ppm的DEN会导致多种肝脏肿瘤，包括：肝细胞腺瘤、肝细胞癌、胆管瘤和混合性肝内胆管癌。通过用特异性针对激活点突变的探针探测基因组DNA，或使用RT-PCR对ras转录本进行直接克隆和测序，来检查单个肝脏肿瘤中致癌性激活的ras突变。使用等位基因特异性寡核苷酸（ASO）分析，在检测的25个肿瘤中的8个（32%）中，密码子12的第2位检测到一个单点突变。通过序列分析，在39个青鳉鱼肝脏肿瘤中的12个（30%）中还检测到了突变的ras等位基因。鉴定出的12个突变中有10个在密码子12处含有一个单点突变，导致甘氨酸到天冬氨酸的氨基酸替换。在密码子16处鉴定出两个独特的突变，分别导致赖氨酸到天冬酰胺或赖氨酸到苏氨酸的氨基酸替换。我们的结果表明，ras突变是由DEN诱导的，并且在超过30%的发生肿瘤的鱼中存在。30%的ras突变发生率与暴露于DEN的哺乳动物物种中报道的发生率相似。虽然之前已经报道过密码子12处的突变，但本研究是首次在体内报道密码子16处的ras点突变。
