Persistent binding of a new reaction product of the carcinogen N-hydroxy-N-2-acetylaminofluorene with guanine in rat liver DNA in vivo.
Persistent binding of a new reaction product of the carcinogen N-hydroxy-N-2-acetylaminofluorene with guanine in rat liver DNA in vivo.
复制标题
致癌物 N-羟基-N-2-乙酰氨基芴的新反应产物与大鼠肝脏 DNA 中的鸟嘌呤在体内持续结合。
作者：
E. Kriek
本文研究了N -羟基- N-2-乙酰氨基芴-2 ′-3H后不同时间内2-乙酰氨基芴残基（-AAF）与大鼠肝DNA的结合。在pH 6下用脾磷酸二酯酶和麦胚酸性磷酸酶的混合物水解肝DNA。
用SephadexLH-20柱层析对酶解产物进行了分析。单次注射N -羟基- N-2-乙酰氨基芴-2 ′-3H后16至18 h，在肝脏DNA中发现最大结合放射性水平。结合放射性的主要部分（80%）被鉴定为N -（脱氧鸟苷-8-基）-2-乙酰氨基芴（dGuo-AAF），其从DNA中迅速消失，生物半衰期约为7天。然而，第二种产物（占结合放射性的20%）在注射后长达8周的时间内仍与DNA结合。与dGuo-AAF不同，在75 ℃下用0.1nNaOH或0.1nHCl作用2小时，其次要产物不发生脱乙酰化，在大鼠肝rRNA和tRNA中未检测到持久性-AAF残留。
从体外与N -乙酰氧基- N-2-乙酰氨基芴-2 ′-3H反应的~（14）C标记DNA中分离出一种微量产物，其化学和色谱性质与体内持久的-AAF部分相似。该产物的3 H：14 C比率与理论3 H：14 C比率相同，该理论3 H：14 C比率是针对反应产物dGuo-AAF计算的，因此表明DNA中的持久-AAF部分也与鸟嘌呤结合。
单次注射2-乙酰氨基芴-9-14 C-2′-3H后，还观察到放射性与大鼠肝脏DNA的体内持续结合。证实了先前的报道，即在24小时时与DNA结合的总致癌物的70%已经失去了N -乙酰基。注射后4周，24小时约20%的结合AAF（总致癌物的5%）仍与DNA结合。4周时DNA的3 H：14 C比率与分离的反应产物dGuo-AAF的3 H：14 C比率相同，表明不存在持久的2-氨基芴残基。给予2-乙酰氨基芴-9-14 C-2′-3 H导致53%的N -乙酰基交换，但注射N -羟基- N-2-乙酰氨基芴-9-14 C-2′-3 H没有导致N -乙酰基的显著交换。
The binding of 2-acetylaminofluorenyl residues (-AAF) to rat liver DNA in vivo was studied at different periods of time after administration of N -hydroxy- N -2-acetylaminofluorene-2′-3H. Liver DNA was hydrolyzed at pH 6 with a mixture of spleen phosphodiesterase and wheat germ acid phosphatase.
The enzymatic digests were analyzed by Sephadex LH-20 column chromatography. Maximum levels of bound radioactivity were found in liver DNA at 16 to 18 hr following a single injection of N -hydroxy- N -2-acetylaminofluorene-2′-3H. The major part (80%) of the bound radioactivity was identified as N -(deoxyguanosin-8-yl)-2-acetylaminofluorene (dGuo-AAF), which disappeared rapidly from DNA with a biological half-life of approximately 7 days. A second product, however, constituting 20% of the bound radioactivity, remained associated with DNA for periods of up to 8 weeks after injection. Unlike dGuo-AAF, the minor product was not deacetylated by the action of 0.1 n NaOH or 0.1 n HCl at 75° for 2 hr. The persistent -AAF residue was not detected in rRNA and tRNA from rat liver.
A minor product with chemical and chromatographic properties similar to those of the persistent -AAF moiety in vivo was isolated from 14C-labeled DNA, which had been reacted with N -acetoxy- N -2-acetylaminofluorene-2′-3H in vitro . The 3H:14C ratio of this product was identical to the theoretical 3H:14C ratio, which was calculated for the reaction product dGuo-AAF, thus indicating that the persistent -AAF moiety in DNA is also bound to guanine.
Persistent binding of radioactivity to rat liver DNA in vivo was also observed following a single injection of 2-acetylaminofluorene-9-14C-2′-3H. Previous reports that 70% of the total carcinogen bound to DNA at 24 hr had lost the N -acetyl group were confirmed. Approximately 20% of bound -AAF at 24 hr (5% of total carcinogen) remained associated with DNA at 4 weeks after injection. The 3H:14C ratio of DNA at 4 weeks was identical to the 3H:14C ratio of the isolated reaction product dGuo-AAF, indicating that there are no persistent 2-aminofluorene residues. Administration of 2-acetylaminofluorene-9-14C-2′-3H resulted in 53% exchange of N -acetyl groups, but injection of N -hydroxy- N -2-acetylaminofluorene-9-14C-2′-3H did not result in significant exchange of the N -acetyl group.