In vivo flow cytometric Pig-a and micronucleus assays: highly sensitive discrimination of the carcinogen/noncarcinogen pair benzo(a)pyrene and pyrene using acute and repeated-dose designs.
In vivo flow cytometric Pig-a and micronucleus assays: highly sensitive discrimination of the carcinogen/noncarcinogen pair benzo(a)pyrene and pyrene using acute and repeated-dose designs.
复制标题
体内流式细胞术 Pig-a 和微核测定：使用急性和重复剂量设计对致癌物/非致癌物对苯并 (a) 芘和芘进行高度灵敏的辨别。
DOI：
10.1002/em.21709
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发表时间：
2012
影响因子：
2.8
通讯作者：
Dertinger,StephenD
中科院分区：
文献类型：
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作者：
Torous,DorotheaK;Phonethepswath,Souk;Avlasevich,SvetlanaL;Mereness,Jared;Bryce,StevenM;Bemis,JeffreyC;Weller,Pamela;Bell,Sara;Gleason,Carol;Custer,LauraL;MacGregor,JamesT;Dertinger,StephenD
将多个遗传毒理学终点组合到单个体内研究中，和/或将一个或多个遗传毒性测定整合到一般毒理学研究中，是有吸引力的，因为它减少了动物使用，并且能够使用来自同一动物的毒性、代谢和药代动力学信息进行全面的比较分析。该实验室开发了流式细胞术评分技术，用于监测两个血液遗传毒性终点-微核网织红细胞频率和猪的基因突变-从而使组合和整合研究切实可行。使用致癌物/非致癌物对苯并（a）芘（BP）和芘（Pyr）研究了在短期和重复给药方案中有效监测这些终点的能力。雄性Sprague-道利大鼠连续3天或28天经口灌胃给予多个剂量水平的Pyr，包括最大耐受剂量。BP暴露通过相同途径给药，但在3天和28天研究中分别以250或125 mg/kg/天的剂量水平给药。采集连续血样直至第45天，并采用双标记方法（SYTO 13联合抗CD 59-PE）分析猪突变，该方法有助于测量总红细胞和网织红细胞亚群中的突变细胞频率。使用基于免疫磁性柱分离的突变体细胞富集步骤来增加测定的统计功效。BP在第15天诱导了稳健的突变型网织红细胞应答，并且频率升高持续至研究终止。突变型红细胞应答滞后于突变型网织红细胞应答，在3天研究的第30天（增加43倍）和28天研究的第42天（增加171倍）观察到峰值发生率。Pyr无明显致突变作用。评价28天研究第4天和第29天采集的血样的微核网织红细胞频率。观察到BP的微核率显著增加，而Pyr无影响。这些结果表明，Pig-a和微核终点可区分这些结构相关的致癌剂和非致癌剂。此外，富集方案证明的高灵敏度表明Pig-a终点适用于重复给药和急性研究，允许将致突变和致染色体断裂终点整合到正在进行的毒理学研究中，并用作短期试验，提供有效的体内筛选和机制信息。Environ.摩尔变异体2012.© 2012 Wiley Periodicals，Inc.
Combining multiple genetic toxicology endpoints into a single in vivo study, and/or integrating one or more genotoxicity assays into general toxicology studies, is attractive because it reduces animal use and enables comprehensive comparative analysis using toxicity, metabolism, and pharmacokinetic information from the same animal. This laboratory has developed flow cytometric scoring techniques for monitoring two blood‐based genotoxicity endpoints—micronucleated reticulocyte frequency and gene mutation at thePig‐alocus—thereby making combination and integration studies practical. The ability to effectively monitor these endpoints in short‐term and repeated dosing schedules was investigated with the carcinogen/noncarcinogen pair benzo(a)pyrene (BP) and pyrene (Pyr). Male Sprague‐Dawley rats were treated via oral gavage for 3 or 28 consecutive days with several dose levels of Pyr, including maximum tolerated doses. BP exposure was administered by the same route but at one dose level, 250 or 125 mg/kg/day for 3‐day and 28‐day studies, respectively. Serial blood samples were collected up to Day 45, and were analyzed forPig‐amutation with a dual labeling method (SYTO 13 in combination with anti‐CD59‐PE) that facilitated mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. A mutant cell enrichment step based on immunomagnetic column separation was used to increase the statistical power of the assay. BP induced robust mutant reticulocyte responses by Day 15, and elevated frequencies persisted until study termination. Mutant erythrocyte responses lagged mutant reticulocyte responses, with peak incidences observed on Day 30 of the 3‐day study (43‐fold increase) and on Day 42 of the 28‐day study (171‐fold increase). No mutagenic effects were apparent for Pyr. Blood samples collected on Day 4, and Day 29 for the 28‐day study, were evaluated for micronucleated reticulocyte frequency. Significant increases in micronucleus frequencies were observed with BP, whereas Pyr had no effect. These results demonstrate thatPig‐aand micronucleus endpoints discriminate between these structurally related carcinogenic and noncarcinogenic agents. Furthermore, the high sensitivity demonstrated with the enrichment protocol indicates that thePig‐aendpoint is suitable for both repeated‐dose and acute studies, allowing integration of mutagenic and clastogenic endpoints into on‐going toxicology studies, and use as a short‐term assay that provides efficient screening and mechanistic information in vivo. Environ. Mol. Mutagen. 2012. © 2012 Wiley Periodicals, Inc.