Safety Assessment, AstraZeneca R&D, Alderley Park, Macclesfield, Cheshire SK104TG, UK.
Toxicology. 2010 Jan 12;267(1-3):45-53. doi: 10.1016/j.tox.2009.10.012. Epub 2009 Oct 22.
Potassium bromate (KBrO3) is a well-established rodent kidney carcinogen and its oxidising activity is considered to be a significant factor in its mechanism of action. Although it has also been shown to be clearly genotoxic in a range of in vivo and in vitro test systems, surprisingly, it is not readily detected in several cell lines using the standard alkaline Comet assay. However, previous results from this laboratory demonstrated huge increases in tail intensity by modifying the method to include incubation with either human 8-oxodeoxyguanosine DNA glycosylase-1 (hOGG1) or bacterial formamidopyrimidine DNA glycosylase (FPG) indicating that, as expected, significant amounts of 8-oxodeoxyguanosine (8-OHdG) were induced. The purpose of this work, therefore, was to investigate why KBrO3, in contrast to other oxidising agents, gives a relatively poor response in the standard Comet assay. Results confirmed that it is a potent genotoxin in mouse lymphoma L5178Y cells inducing micronuclei and mutation at the tk and hprt loci at relatively non-cytotoxic concentrations. Subsequent time-course studies demonstrated that substantial amounts of 8-OHdG appear to remain in cells 24h after treatment with KBrO3 but result in no increase in frank stand breaks (FSB) even though phosphorylated histone H2AX (gamma-H2AX) antibody labelling confirmed the presence of double-strand breaks. Using bromodeoxyuracil (BrdU) incorporation together with measured increases in cell numbers, L5178Y cells also appeared to go through the cell cycle with unrepaired hOGG1-recognisable damage. Since unrepaired 8-OHdG can give rise to point mutations through G:C-->T:A transversions, it was also surprising that mutation could not be detected at the Na+/K+ATPase locus as determined by ouabain resistance. Some increases in strand breakage could be seen in the Comet assay by increasing the unwinding time, but only at highly toxic concentrations and to a much smaller extent than would be expected from the magnitude of the other genotoxic responses. It was considered unlikely that these anomalous observations were due to the inability of L5178Y cells to recognise 8-OHdG because these cells were shown to express mOGG1 and have functional cleavage activity at the adducted site. It appears that the responses of L5178Y cells to KBrO3 are complex and differ from those induced by other oxidising agents.
溴酸钾（KBrO3）是一种公认的啮齿动物肾脏致癌物质，其氧化活性被认为是其作用机制的重要因素。尽管它在一系列体内和体外测试系统中也被证明具有明显的遗传毒性，但令人惊讶的是，使用标准碱性彗星试验在几种细胞系中却不易检测到它。然而，本实验室的先前结果表明，通过修改方法，包括与人 8-氧脱氧鸟苷 DNA 糖基化酶-1（hOGG1）或细菌形式嘧啶 DNA 糖基化酶（FPG）孵育，大大增加了尾部强度，表明如预期的那样，大量 8-氧脱氧鸟苷（8-OHdG）被诱导。因此，这项工作的目的是研究为什么溴酸钾与其他氧化剂相比，在标准彗星试验中反应相对较差。结果证实，它在相对非细胞毒性浓度下，在小鼠淋巴瘤 L5178Y 细胞中是一种有效的遗传毒素，可诱导微核和 tk 和 hprt 基因座的突变。随后的时间过程研究表明，在用 KBrO3 处理 24 小时后，大量的 8-OHdG 似乎仍然存在于细胞中，但即使磷酸化组蛋白 H2AX（γ-H2AX）抗体标记证实存在双链断裂，也不会导致明显的单链断裂（FSB）增加。使用溴脱氧尿苷（BrdU）掺入，并结合细胞数量的测量增加，L5178Y 细胞似乎也能在未修复的 hOGG1 可识别的损伤下通过细胞周期。由于未修复的 8-OHdG 可以通过 G:C-->T:A 颠换导致点突变，因此令人惊讶的是，在 Na+/K+ATPase 基因座上也无法检测到突变，这是通过哇巴因抗性确定的。通过增加解旋时间，可以在彗星试验中看到一些链断裂增加，但仅在高毒性浓度下，而且程度远小于其他遗传毒性反应的预期程度。由于这些异常观察结果不太可能归因于 L5178Y 细胞无法识别 8-OHdG 的能力，因为这些细胞被证明表达 mOGG1 并且在加合物位点具有功能性切割活性，因此不太可能。L5178Y 细胞对 KBrO3 的反应似乎很复杂，与其他氧化剂诱导的反应不同。
