Fichtinger-Schepman A M, van Dijk-Knijnenburg H C, van der Velde-Visser S D, Berends F, Baan R A
TNO Food and Nutrition Research Institute, Rijswijk, The Netherlands.
Carcinogenesis. 1995 Oct;16(10):2447-53. doi: 10.1093/carcin/16.10.2447.
In order to determine the nature of the cytotoxic lesion(s) formed by the antitumour drugs cisplatin and carboplatin, a comparative study was made of bifunctional DNA-adduct formation by these drugs. The kinetics of bifunctional cisplatin adduct formation were studied with DNA in vitro and in cultured Chinese hamster ovary (CHO) cells. Prior to adduct measurements with AAS in in vitro platinated DNA and with ELISA in cellular DNA, the monoadducts were inactivated with thiourea (10 mM; 1 h at 37 degrees C). The data indicated that the conversion of monofunctional to bifunctional adducts, with t1/2 of approximately 2 h (37 degrees C), leads to maximum intrastrand adduct levels after approximately 4-6 h postincubation. This interval coincided with the period during which the cytotoxic effect of cisplatin could be reduced by a 1 h 10 mM thiourea post-incubation of the cells. The formation of interstrand crosslinks continued for approximately 7 h of post-incubation; then these amounted to approximately 2% of the total DNA adducts. When a DNA sample was dialysed against 0.1 M NH4HCO3 (16 h, 37 degrees C) immediately after cisplatin treatment, in order to block mono- to bifunctional adduct conversion, adduct levels were found similar to those after the 4-6 h post-incubation. From this it is clear that the high values reported earlier for bifunctional cisplatin adducts in such DNA samples are not correct. These values apparently represent the amounts of adducts that eventually would have been formed during post-incubation in DNA in vitro but also in cells in the absence of cellular repair. The cisplatin data of CHO cells were compared with those after treatment of the cells with equitoxic doses of carboplatin. The data indicate that after 12 h post-incubation, when all bifunctional adducts are formed, the total amount of the various bifunctional adducts after cisplatin treatment (37.5 +/- 4.5 fmol/micrograms DNA) was in the same range as that after carboplatin (32.8 +/- 6.3 fmol/micrograms DNA). However, because the relative occurrences of the adducts were different, it could also be concluded that if one of the diadducts were exclusively responsible for the cytotoxic effect of these platinum antitumour drugs, Pt-AG is the only likely candidate.
为了确定抗肿瘤药物顺铂和卡铂形成的细胞毒性损伤的性质，对这些药物形成双功能DNA加合物进行了一项比较研究。用体外DNA和培养的中国仓鼠卵巢（CHO）细胞研究了双功能顺铂加合物形成的动力学。在用原子吸收光谱法（AAS）测量体外铂化DNA中的加合物以及用酶联免疫吸附测定法（ELISA）测量细胞DNA中的加合物之前，先用硫脲（10 mM；37℃ 1小时）使单加合物失活。数据表明，单功能加合物向双功能加合物的转化，其半衰期约为2小时（37℃），在温育后约4 - 6小时导致链内加合物水平达到最高。这个时间段与顺铂的细胞毒性作用可通过在细胞温育后用10 mM硫脲处理1小时而降低的时间段一致。链间交联的形成在温育后持续约7小时；然后这些交联约占总DNA加合物的2%。当顺铂处理后立即将DNA样品对0.1 M碳酸氢铵进行透析（37℃ 16小时），以阻止单功能向双功能加合物的转化时，发现加合物水平与温育4 - 6小时后的相似。由此清楚地表明，早期报道的此类DNA样品中双功能顺铂加合物的高值是不正确的。这些值显然代表了在体外DNA以及在没有细胞修复的细胞中温育过程中最终会形成的加合物量。将CHO细胞的顺铂数据与用等毒性剂量卡铂处理细胞后的数据进行了比较。数据表明，温育12小时后，当所有双功能加合物形成时，顺铂处理后各种双功能加合物的总量（37.5±4.5 fmol/μg DNA）与卡铂处理后的总量（32.8±6.3 fmol/μg DNA）处于同一范围。然而，由于加合物的相对发生率不同，也可以得出结论，如果其中一种双加合物专门负责这些铂类抗肿瘤药物的细胞毒性作用，Pt - AG是唯一可能的候选物。
