Mitsubishi Chemical Medience Corporation, Kamisu-shi, Ibaraki, Japan.
Mutat Res. 2012 Sep 18;747(2):234-9. doi: 10.1016/j.mrgentox.2012.05.012. Epub 2012 Jun 5.
Various liver micronucleus assay methods, such as those involving partial hepatectomy, treatment with mitogens, and the use of juvenile animals, have been developed. These assays have been proven to be of high sensitivity and specificity to predict hepatocarcinogenicity of compounds that cannot be detected by bone marrow micronucleus assays. On the contrary, the existing assays have only been evaluated for their use in detecting micronucleus induction in the settings of relatively short-term cell proliferation. However, the integration of in vivo genotoxicity endpoints into routine toxicity studies is increasingly desired from the viewpoint of animal welfare to reduce the number of animals used. In the present study, the rodent hepatocarcinogens diethylnitrosamine (DEN) and 2,4-diaminotoluene (2,4-DAT) were repeatedly administered orally to male Crl:CD (SD) rats (6 weeks old at the beginning of administration) for 5, 14, and 28 days, and changes in the frequency of hepatocytes with micronuclei in liver tissues that had undergone no artificial treatment to accelerate cell proliferation were evaluated. At the same time, a new method of hepatocyte isolation involving the treatment of a portion of the liver with collagenase in a centrifuge tube, without the use of in situ perfusion, was established. The induction of micronucleated hepatocytes was achieved after the repeated administration of DEN for 5 days or longer and of 2,4-DAT for 14 days or longer. Micronucleus frequencies were increased depending on the number of administrations, indicating that micronucleated hepatocytes had possibly remained for a long period of time and accumulated additively. It therefore appears that even in adult rat liver with low mitotic activity, a repeated-dose of a chemical substance for 14 days or longer enables the detection of micronucleus induction. In addition, the establishment of a method to isolate hepatocytes without perfusion using only a part of the liver enables the integration of liver micronucleus assays into general toxicity studies.
已经开发出了各种肝微核试验方法，如部分肝切除术、有丝分裂原处理和使用幼年动物等。这些试验方法已被证明对预测骨髓微核试验无法检测到的化合物的致癌性具有很高的敏感性和特异性。相反，现有的试验方法仅在相对短期细胞增殖的背景下评估了其用于检测微核诱导的能力。然而，从动物福利的角度出发，越来越希望将体内遗传毒性终点纳入常规毒性研究，以减少使用的动物数量。在本研究中，反复给雄性 Crl:CD（SD）大鼠（开始给药时 6 周龄）口服给予二乙基亚硝胺（DEN）和 2,4-二氨基甲苯（2,4-DAT）作为啮齿动物肝致癌物，连续给药 5、14 和 28 天，评估未进行任何人工处理以加速细胞增殖的肝组织中具有微核的肝细胞的频率变化。同时，建立了一种新的肝细胞分离方法，该方法涉及在离心管中用胶原酶处理部分肝脏，而无需原位灌流。在 DEN 重复给药 5 天或更长时间，2,4-DAT 重复给药 14 天或更长时间后，诱导了微核化的肝细胞。微核频率随给药次数的增加而增加，表明微核化的肝细胞可能已经长时间存在并进行了累加。因此，即使在具有低有丝分裂活性的成年大鼠肝脏中，重复给予化学物质 14 天或更长时间也能够检测到微核诱导。此外，仅使用一部分肝脏建立无需灌流即可分离肝细胞的方法，使肝微核试验能够纳入一般毒性研究。
