Ward E J, Stewart B W
Biochemistry. 1987 Mar 24;26(6):1709-17. doi: 10.1021/bi00380a033.
Analysis, by benzoylated DEAE-cellulose chromatography, has been made of structural change in eu- and heterochromatic DNA from rat liver following administration of the carcinogen N-nitrosodimethylamine (10 mg/kg body weight). Either hepatic DNA was prelabeled with [3H]thymidine administered 2-3 weeks before injection of the carcinogen or the labeled precursor was given during regenerative hyperplasia in rats treated earlier with N-nitrosodimethylamine. Following phenol extraction of either whole liver homogenate or nuclease-fractionated eu- and heterochromatin, carcinogen-modified DNA was examined by stepwise or caffeine gradient elution from benzoylated DEAE-cellulose. In whole DNA, nitrosamine-induced single-stranded character was maximal 4-24 h after treatment, declining rapidly thereafter; gradient elution of these DNA preparations also provided short-term evidence of structural change. Following incubation of purified nuclei with micrococcal nuclease, 10-12% of labeled DNA was solubilized (eu-chromatin) by 1.0 unit of micrococcal nuclease (5 mg of DNA)-1 mL-1 after 9 min. In prelabeled animals, administration of N-nitrosodimethylamine caused a marked fall in the specific radioactivity of solubilized DNA, while that of sedimenting DNA was not affected. Caffeine gradient chromatography suggested short-term nitrosamine-induced structural change in euchromatic DNA, while increased binding of heterochromatic DNA was evident for up to 3 months after carcinogen treatment. Preparations of newly synthesized heterochromatic DNA from animals subjected to hepatectomy up to 2 months after carcinogen treatment provided evidence of heritable structural damage. Carcinogen-induced binding of heterochromatic DNA to benzoylated DEAE-cellulose was indicative of specific structural lesions whose affinity equalled that of single-stranded DNA up to 1.0 kilobase in length.(ABSTRACT TRUNCATED AT 250 WORDS)
通过苯甲酰化二乙氨基乙基纤维素色谱法，对给予致癌物N-亚硝基二甲胺（10毫克/千克体重）后大鼠肝脏常染色质和异染色质DNA的结构变化进行了分析。在注射致癌物前2 - 3周，用[3H]胸腺嘧啶对肝脏DNA进行预标记，或者在早期用N-亚硝基二甲胺处理的大鼠再生性增生期间给予标记前体。在对全肝匀浆或经核酸酶分级分离的常染色质和异染色质进行酚提取后，通过从苯甲酰化二乙氨基乙基纤维素上逐步洗脱或咖啡因梯度洗脱来检测致癌物修饰的DNA。在全DNA中，亚硝胺诱导的单链特征在处理后4 - 24小时达到最大，此后迅速下降；这些DNA制剂的梯度洗脱也提供了结构变化的短期证据。用微球菌核酸酶孵育纯化的细胞核后，9分钟后，1.0单位微球菌核酸酶（5毫克DNA）-1毫升-1可使10 - 12%的标记DNA溶解（常染色质）。在预标记的动物中，给予N-亚硝基二甲胺导致溶解DNA的比放射性显著下降，而沉淀DNA的比放射性不受影响。咖啡因梯度色谱法表明常染色质DNA在短期内有亚硝胺诱导的结构变化，而异染色质DNA在致癌物处理后长达3个月的时间里结合增加明显。在致癌物处理后长达2个月接受肝切除术的动物中新合成的异染色质DNA制剂提供了可遗传结构损伤的证据。致癌物诱导的异染色质DNA与苯甲酰化二乙氨基乙基纤维素的结合表明存在特定的结构损伤，其亲和力与长度达1.0千碱基的单链DNA相当。（摘要截于250字）
