Application of the adverse outcome pathway framework to genotoxic modes of action

In May 2017, the Health and Environmental Sciences Institute's Genetic Toxicology Technical Committee hosted a workshop to discuss whether mode of action (MOA) investigation is enhanced through the application of the adverse outcome pathway (AOP) framework. As AOPs are a relatively new approach in genetic toxicology, this report describes how AOPs could be harnessed to advance MOA analysis of genotoxicity pathways using five example case studies. Each of these genetic toxicology AOPs proposed for further development includes the relevant molecular initiating events, key events, and adverse outcomes (AOs), identification and/or further development of the appropriate assays to link an agent to these events, and discussion regarding the biological plausibility of the proposed AOP. A key difference between these proposed genetic toxicology AOPs versus traditional AOPs is that the AO is a genetic toxicology endpoint of potential significance in risk characterization, in contrast to an adverse state of an organism or a population. The first two detailed case studies describe provisional AOPs for aurora kinase inhibition and tubulin binding, leading to the common AO of aneuploidy. The remaining three case studies highlight provisional AOPs that lead to chromosome breakage or mutation via indirect DNA interaction (inhibition of topoisomerase II, production of cellular reactive oxygen species, and inhibition of DNA synthesis). These case studies serve as starting points for genotoxicity AOPs that could ultimately be published and utilized by the broader toxicology community and illustrate the practical considerations and evidence required to formalize such AOPs so that they may be applied to genetic toxicity evaluation schemes. Environ. Mol. Mutagen. 61:114–134, 2020. © 2019 Wiley Periodicals, Inc.     

Micronucleus test on Triturus carnifex as a tool for environmental biomonitoring

The amphibian micronucleus test has been widely used during the last 30 years to test the genotoxic properties of several chemicals and as a tool for ecogenotoxic monitoring. The vast majority of these studies were performed on peripheral blood of urodelan larvae and anuran tadpoles and to a lesser extent adults were also used. In this study, we developed protocols for measuring micronuclei in adult shed skin cells and larval gill cells of the Italian crested newt (Triturus carnifex). Amphibians were collected from ponds in two protected areas in Italy that differed in their radon content. Twenty‐three adult newts and 31 larvae were captured from the radon‐rich pond, while 20 adults and 27 larvae were taken from the radon‐free site. The animals were brought to the laboratory and the micronucleus test was performed on peripheral blood and shed skins taken from the adults and on larval gills. Samples from the radon‐rich site showed micronucleus frequencies higher than those from the radon‐free site and the difference was statistically significant in gill cells (P < 0.00001). Moreover, the larval gills seem to be more sensitive than the adult tissues. This method represents an easy (and noninvasive in the case of the shed skin) application of the micronucleus assay that can be useful for environmental studies in situ. Environ. Mol. Mutagen. 56:412–417, 2015. © 2014 Wiley Periodicals, Inc.     

Genetic toxicity assessment using liver cell models: past,present, and future

ABSTRACT

Genotoxic compounds may be detoxified to non-genotoxic metabolites while many pro-carcinogens require metabolic activation to exert their genotoxicity in vivo. Standard genotoxicity assays were developed and utilized for risk assessment for over 40 years. Most of these assays are conducted in metabolically incompetent rodent or human cell lines. Deficient in normal metabolism and relying on exogenous metabolic activation systems, the current in vitro genotoxicity assays often have yielded high false positive rates, which trigger unnecessary and costly in vivo studies. Metabolically active cells such as hepatocytes have been recognized as a promising cell model in predicting genotoxicity of carcinogens in vivo. In recent years, significant advances in tissue culture and biological technologies provided new opportunities for using hepatocytes in genetic toxicology. This review encompasses published studies (both in vitro and in vivo) using hepatocytes for genotoxicity assessment. Findings from both standard and newly developed genotoxicity assays are summarized. Various liver cell models used for genotoxicity assessment are described, including the potential application of advanced liver cell models such as 3D spheroids, organoids, and engineered hepatocytes. An integrated strategy, that includes the use of human-based cells with enhanced biological relevance and throughput, and applying the quantitative analysis of data, may provide an approach for future genotoxicity risk assessment.     

克百威及其代谢产物的小鼠骨髓红细胞微核试验研究

[目的]利用微核试验研究克百威及其4种代谢产物的遗传毒性。[方法]分别以0．1、0．2、0．4mg/kg3个不同剂量水平的克百威及其代谢产物3-羟基呋喃丹、3-酮基呋喃丹、呋喃酚和亚硝基呋喃丹腹腔注射染毒健康小鼠，24h后以同样剂量再次注射染毒，6h后脱颈椎处死动物，制片，观察并计数嗜多染红细胞的微核率，进行统计分析。[结果]克百威、呋喃酚及3-酮基呋喃丹小鼠胸骨骨髓嗜多染红细胞微核实验为阴性结果，高剂量处理组微核率分别为1．3‰、3．25‰和3．62‰，与阴性对照组(1．83‰)差异无显著性；而高剂量组3-羟基呋喃丹(7．00‰)和三个剂量组的亚硝基呋喃丹(微核率分别为3．62‰、5．00‰、7．85‰)均有明显微核效应。[结论]克百威、呋喃酚和3-酮基呋喃丹在受试剂量下微核试验阴性，3-羟基呋哺丹和亚硝基呋喃丹微核试验阳性，提示可能对染色体具有突变效应。     

煤焦沥青烟雾提取物的细胞毒性和遗传毒性研究

采用人外周血淋巴细胞非程序DNA合成(UDS)试验和人胚肺成纤维细胞转化试验,测试了煤焦沥青烟雾提取物(ECTPF)对人体细胞的细胞毒性和遗传毒性。UDS试验结果表明,ECTPF可使淋巴细胞UDS值明显增加,并有剂量一反应关系。引起半数淋巴细胞死亡的浓度(LC50)为33.8μg/ml。细胞转化试验表明,ECTPF能诱发人胚肺成纤维细胞明显的形态学转化,且转化细胞具有部分恶性转化细胞的特性。引起半数人胚肺成纤维细胞生长抑制的浓度为41.3μg/ml。实验结果提示,ECTPF是一种具有细胞毒性和遗传毒性的物质。     

Assessment of oxidative DNA damage in the oxyR-deficient SOS chromotest strain Escherichia coli PQ300.

The SOS chromotest is a simple short-term genotoxicity assay measuring the induction of gene sfiA in Escherichia coli K-12. The recent availability of SOS tester strains with additional mutations in DNA repair or protection systems allows testing of DNA damaging compounds for genotoxic specificity. E. coli PQ300 differs from the standard SOS tester strain PQ37 in that it contains an additional mutation in gene oxyR that renders it more sensitive to oxidative genotoxins. The generation of reactive oxygen intermediates (ROI) by hydroperoxides (H2O2, t-butyl hydroperoxide, cumene hydroperoxide), gamma-radiation, glucose oxidase, and xanthine oxidase resulted in a more vigorous SOS response in strain PQ300 compared to strain PQ37. PQ300 was also more sensitive than PQ37 for the detection of reducing agents such as ascorbic acid, cysteine, and glutathione, which also alter the redox status of the bacterial cells. However, intercalating agents (adriamycin, bleomycin, and mitomycin C) and the UV- and radiomimetic compound 4-nitroquinoline-1-oxide whose DNA damaging potential are known also to involve ROI did not show significant differences between strains PQ37 and PQ300. It is concluded that the oxyR-deficient strain PQ300 is useful for detecting certain classes of genotoxins that change the oxidative/antioxidative balance of tester bacteria in the SOS chromotest.     

Selective aflatoxin B1-induced sister chromatid exchanges and cytotoxicity in differentiating B and T lymphocytes in vivo

The purpose of this study was to assess the genotoxic and cytotoxic effects of the fungal metabolite aflatoxin B1 (AfB1) on the developing immune system of the chick embryo, a model in vivo system. Of particular interest was the assessment of AfB1 -mediated selective toxicity toward developing B lymphocytes as compared to T lymphocytes. In vivo bromodeoxyuridine (BrdU) labelling of DNA was used to detect the induction of sister chromatid exchanges (SCE) in lymphocytes and to assess the progression of these cells through successive cell cycles. Cytotoxicity was also assessed by studying the entrance and maintenance of cells in mitosis (mitotic index). Graded doses of AfB1 (1.09–17.4 μ/g embryo) were applied to chick embryos of 18 days of incubation (Dl). Embryos also received two doses of BrdU at 3 mg/200 μ (3 hr apart) to provide continuous labelling of B and T lymphocyte replicating DNA. B and T lymphocytes were harvested 20 hr post-AfB1/BrdU exposure from the bursa and thymus, respectively, and were processed for cytogenetic analyses. AfB1 induced dose-related increases in SCE in B lymphocytes; this induction was 6- to 8-fold that of controls at the higher doses tested, AfB1 -mediated induction of SCE in T cells was just 2-fold that of controls at the highest dose tested. AfB1 reduced the progression of B cells and to a lesser extent T ceels through successive rounds of replication. Furthermore, AfB1 dramatically reduced the mitotic index of B cells but not of T cells. These data indicate both selective genotoxicity and cytotoxicity of AfB1 toward B cells in the late stage embryo. © 1993 Wiley-Liss, Inc.     

In vitro genotoxicity of dyes present in colored smoke munitions

Genetic toxicology studies were conducted on organic dyes and mixtures used in colored smoke munitions. The dyes studied included Solvent Red 1; two different batches (Lot 1 and Lot 2) of Disperse Red 11; terephthalic acid; and a mixture of 25 parts Solvent Red 1, 5 parts Disperse Red 11, and 16 parts terephthalic acid. The dyes were evaluated for their ability to produce mutations in Salmonella bacterial strains and in Chinese hamster ovary (CHO) cells. The dyes were also tested in CHO cells to determine cytotoxicity and the induction of sister chromatid exchanges and chromosome aberration. None of the dyes were genotoxic in the standard Ames assay using bacterial strain TA1535 or TA100 with or without the addition of S-9 or in TA98 and TA1538 without S-9. With S-9, Disperse Red 11 (Lot 2) showed significant mutagenic activity in TA98 and TA1538 which increased as a function of S-9 concentration. However, the maximum level of mutagenic activity detected was low (3.8 revertants/micrograms). The azo dye Solvent Red 1 was also negative in a pre-incubation assay designed to reduce azo compounds to free amines. Solvent Red 1 was cytotoxic to mammalian cells, caused a significant increase in SCE, but was not mutagenic or clastogenic. Disperse Red 11 (Lot 1 and Lot 2) were not cytotoxic or clastogenic but produced an increase in cell cycle time and SCE frequency. Only Disperse Red 11 (Lot 2) increased mutations in the CHO/hypoxanthine-guanine phosphoribosyltransferase (HGPRT) assay. The mutagenic activity of the dye mixture was not significant, suggesting no synergistic interaction between the dyes. These studies demonstrated that none of the dyes was clastogenic and that a contaminant in Disperse Red 11 (Lot 2) may be responsible for the weak mutagenic activity in both mammalian and bacterial cell systems.     

Activity of the human carcinogen MeCCNU in the mouse bone marrow micronucleus assay

The nitrosourea mustard MeCCNU is the most recent organic chemical to be classified as a human carcinogen by IARC. MeCCNU gave a strong positive response when tested in the mouse bone marrow micronucleus assay. Activity was evident using either ip injection or oral gavage of the test chemical. These results further support the correlation between human carcinogens and their genotoxicity.     

Evaluation of mutagenic activity in an extract of pepper tree stem bark (Schinus terebinthifolius Raddi)

An extract (decoction) from pepper tree stem bark (Schinus terebinthifolius Raddi) is widely used in Brazil as a topical antiinflammatory agent and to cicatrize wounds. The extract contains catechin, tannins, terpenes, flavonoids, and saponins; of these components, both mutagenic potential and antioxidant properties have been ascribed to flavonoids. The mutagenicity of some flavonoids is believed to be associated with the formation of reactive oxygen species and seems to depend on the number and position of hydroxyl groups. In the present study, we evaluated an extract of S. terebinthifolius in a series of cell-free and bacterial assays in order to determine its genotoxic potential. The extract was negative in a cell-free plasmid DNA test, indicating that it did not directly break DNA. Positive results, however, were obtained in the SOS chromotest, in a forward mutagenesis assay employing CC104 and CC104mutMmutY strains of Escherichia coli, and in the Salmonella reversion assay, using strains TA97, TA98, TA100, and TA102. All the bacterial tests were performed without exogenous metabolic activation due to the topical use of this preparation. The results indicate that pepper tree stem bark extract produces DNA damage and mutation in bacteria, and that oxidative damage may be responsible for the genotoxicity.