Interpreting In Vitro Micronucleus Positive Results: Simple Biomarker Matrix Discriminates Clastogens, Aneugens, and Misleading Positive Agents

被引:44
作者
Bryce, Steven M. [1 ]
Bemis, Jeffrey C. [1 ]
Mereness, Jared A. [1 ]
Spellman, Richard A. [2 ]
Moss, Jocelyn [2 ]
Dickinson, Donna [2 ]
Schuler, Maik J. [2 ]
Dertinger, Stephen D. [1 ]
机构
[1] Litron Labs, Rochester, NY 14623 USA
[2] Pfizer Worldwide Res & Dev, Genet Toxicol Ctr Emphasis, Groton, CT USA
关键词
micronuclei; genotoxicity; TK6; cells; specificity; sensitivity; flow cytometry; FLOW-CYTOMETRIC ANALYSIS; MOUSE PERIPHERAL-BLOOD; INDUCED ER STRESS; A GENE MUTATION; CHROMOSOME-ABERRATIONS; CYTOSINE-ARABINOSIDE; GENOTOXICITY TESTS; CELL-PROLIFERATION; HUMAN-LYMPHOCYTES; HISTONE H2AX;
D O I
10.1002/em.21868
中图分类号
X [环境科学、安全科学];
学科分类号
08 ; 0830 ;
摘要
The specificity of in vitro mammalian cell genotoxicity assays is low, as they yield a high incidence of positive results that are not observed in animal genotoxicity and carcinogenicity tests, that is, "misleading" or "irrelevant" positives. We set out to develop a rapid and effective follow-up testing strategy that would predict whether apparent in vitro micronucleus-inducing effects are due to a clastogenic, aneugenic, or secondary irrelevant mode(s) of action. Priority was given to biomarkers that could be multiplexed onto flow cytometric acquisition of micronucleus frequencies, or that could be accomplished in parallel using a homogeneous-type assay. A training set of 30 chemicals comprised of clastogens, aneugens, and misleading positive chemicals was studied. These experiments were conducted with human TK6 cells over a range of closely spaced concentrations in a continuous exposure design. In addition to micronucleus frequency, the following endpoints were investigated, most often at time of harvest: cleaved Parp-positive chromatin, cleaved caspase 3-positive chromatin, ethidium monoazide bromide-positive chromatin, polyploid nuclei, phospho-histone H3-positive (metaphase) cells, tetramethylrhodamine ethyl ester-negative cells, cellular ATP levels, cell cycle perturbation, and shift in gamma-H2AX fluorescence relative to solvent control. Logistic regression was used to identify endpoints that effectively predict chemicals' a priori classification. Cross validation using a leave-one-out approach indicated that a promising base model includes gamma-H2AX shift and change in phospho-histone H3-positive events (25/30 correct calls). Improvements were realized when one or two additional endpoints were included (26-30/30 correct calls). These models were further evaluated with a test set of 10 chemicals, and also by evaluating 3 chemicals at a collaborating laboratory. The resulting data support the hypothesis that a matrix of high throughput-compatible biomarkers can effectively delineate two important modes of genotoxic action as well as identify cytotoxicity that can lead to irrelevant positive results. (C) 2014 Wiley Periodicals, Inc.
引用
收藏
页码:542 / 555
页数:14
相关论文
共 72 条
[1]   MOUSE MICRONUCLEUS TESTS WITH KNOWN AND SUSPECT SPINDLE POISONS - RESULTS FROM 2 LABORATORIES [J].
ADLER, ID ;
KLIESCH, U ;
VANHUMMELEN, P ;
KIRSCHVOLDERS, M .
MUTAGENESIS, 1991, 6 (01) :47-53
[2]   Molecular cytogenetic evaluation of the mechanism of genotoxic potential of amsacrine and nocodazole in mouse bone marrow cells [J].
Attia, Sabry M. .
JOURNAL OF APPLIED TOXICOLOGY, 2013, 33 (06) :426-433
[3]   Molecular Cytogenetic Evaluation of the Mechanism of Micronuclei Formation Induced by Camptothecin, Topotecan, and Irinotecan [J].
Attia, Sabry M. ;
Aleisa, Abdulaziz M. ;
Bakheet, Saleh A. ;
Al-Yahya, Abdulaziz A. ;
Al-Rejaie, Salim S. ;
Ashour, Abdelkader E. ;
Al-Shabanah, Othman A. .
ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, 2009, 50 (02) :145-151
[4]   Use of the γH2AX assay for assessing the genotoxicity of polycyclic aromatic hydrocarbons in human cell lines [J].
Audebert, M. ;
Riu, A. ;
Jacques, C. ;
Hillenweck, A. ;
Jamin, E. L. ;
Zalko, D. ;
Cravedi, J. -P. .
TOXICOLOGY LETTERS, 2010, 199 (02) :182-192
[5]   In vitro micronucleus scoring by flow cytometry: Differential staining of micronuclei versus apoptotic and necrotic chromatin enhances assay reliability [J].
Avlasevich, SL ;
Bryce, SM ;
Cairns, SE ;
Dertinger, SD .
ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, 2006, 47 (01) :56-66
[6]   Flow cytometric analysis of micronuclei in mammalian cell cultures: past, present and future [J].
Avlasevich, Svetlana ;
Bryce, Steven ;
De Boeck, Marlies ;
Elhajouji, Azeddine ;
Van Goethem, Freddy ;
Lynch, Anthony ;
Nicolette, John ;
Shi, Jing ;
Dertinger, Stephen .
MUTAGENESIS, 2011, 26 (01) :147-152
[7]   RELATIONSHIPS AMONG CYTOTOXICITY, LYSOSOMAL BREAKDOWN, CHROMOSOME-ABERRATIONS, AND DNA DOUBLE-STRAND BREAKS [J].
BRADLEY, MO ;
TAYLOR, VI ;
ARMSTRONG, MJ ;
GALLOWAY, SM .
MUTATION RESEARCH, 1987, 189 (01) :69-79
[8]   Flow cytometric 96-well microplate-based in vitro micronucleus assay with human TK6 cells: Protocol optimization and transferability assessment [J].
Bryce, Steven M. ;
Avlasevich, Svetlana L. ;
Bemis, Jeffrey C. ;
Tate, Matthew ;
Walmsley, Richard M. ;
Saad, Frederic ;
Van Dijck, Kris ;
De Boeck, Marlies ;
Van Goethem, Freddy ;
Lukamowicz-Rajska, Magdalena ;
Elhajouji, Azeddine ;
Dertinger, Stephen D. .
ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, 2013, 54 (03) :180-194
[9]   Miniaturized flow cytometric in vitro micronucleus assay represents an efficient tool for comprehensively characterizing genotoxicity dose-response relationships [J].
Bryce, Steven M. ;
Avlasevich, Svetlana L. ;
Bemis, Jeffrey C. ;
Phonethepswath, Souk ;
Dertinger, Stephen D. .
MUTATION RESEARCH-GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS, 2010, 703 (02) :191-199
[10]   A Simple High-Content Cell Cycle Assay Reveals Frequent Discrepancies between Cell Number and ATP and MTS Proliferation Assays [J].
Chan, Grace Ka Yan ;
Kleinheinz, Tracy L. ;
Peterson, David ;
Moffat, John G. .
PLOS ONE, 2013, 8 (05)