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.     

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.     

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.     

Fluconazole induces genotoxicity in cultured human peripheral blood mononuclear cells via immunomodulation of TNF-α, IL-6, and IL-10: new challenges for safe therapeutic regimens

Context: Fluconazole (FNZ) is a drug used in antifungal therapy. However, the minimum FNZ dose to interfering with immune responses or inducing DNA damage is still unknown.

Objective: This study investigated the toxicological profile of FNZ on cultured human peripheral blood mononuclear cells (PBMCs) treated with different concentrations of this azole.

Materials and methods: Cultured PBMCs were exposed to FNZ (6, 12, 30, 60 and 120?μg/mL) and the toxicological profile was assessed by the following parameters: cytotoxic and nuclear division index (necrotic, apoptotic and viable cells), DNA damage (alkaline comet test), mutagenic potential (micronucleus test), cytokine modulation (IL-1, IL-6, IL-10, TNF-α, IFN-γ), and predictive toxicity (Osiris^® and LAZAR^® programs).

Results: Our results demonstrated that FNZ induced cellular DNA damage and mutagenicity at concentrations above the plasma peak (>30?μg/mL) and 6?μg/mL, respectively, which was associated with increased TNF-α, and decrease IL-6 and IL-10 concentrations. These effects may be related to increased apoptosis and cytotoxic nuclear division index in the cultured PBMCs. In silico results indicated potential mutagenic, tumorigenic, irritant, and carcinogenic effects, which were partially confirmed by the above assays.

Discussion and conclusions: Together, these findings suggest the need to rationalize the use of FNZ, especially if it is used for long periods or with concomitant pathologies requiring azole therapy that may increase FNZ's plasma concentration.     

Use of a high-throughput umu-microplate test system for rapid detection of genotoxicity produced by mutagenic carcinogens and airborne particulate matter

In the present study, we developed a rapid umu-microplate test system that uses the nitroreductase- and O-acetyltransferase-overproducing Salmonella typhimurium strain NM3009 and the O-acetyltransferase-overproducing S. typhimurium strain NM2009 to detect genotoxic activity in small volume samples. The assay was used to test the genotoxicity of several standard mutagens and environmental samples. Exponentially growing cultures of NM3009, NM2009, and the parental strain TA1535/pSK1002 were incubated in 96-well microplates with test chemicals both in the presence and in the absence of rat liver S9. The relative beta-galactosidase activities were then determined colorimetrically using either chlorophenol red-beta-D-galactopyranoside (CPRG) or O-nitrophenyl-beta-D-galactopyranoside (ONPG) as a measure of umuC gene induction activity. The sensitivities of NM3009 without S9 mix and NM2009 with S9 mix to nitroarenes and aromatic amines were up to 24- to 75-fold higher than those of the parent strain. Induction of umuC gene expression was detected more readily with CPRG than ONPG. The umu-microplate assay also detected genotoxicity in organic extracts of particulate matter from air samples collected in Osaka City, Japan. The pattern of the responses suggested that the genotoxic activity of the particulate extract was due primarily to nitrated polycyclic aromatic hydrocarbons. Our results indicate that the umu-microplate assay may be a useful way of carrying out rapid screens for genotoxicity in small-volume environmental samples.     

藿香正气片增强镇静催眠药的镇静与催眠作用的研究

目的研究藿香正气片增强镇静催眠药对小鼠的镇静催眠作用.为临床增添了一个中西药结合的镇静催眠合剂提供药理依据.方法应用抖笼换能器法观察藿香正气片对小鼠的自发活动的影响.由此观察藿香正气片与镇静催眠药的协同作用.测定藿香正气片与镇静催眠药对小鼠的入睡时间及睡眠持续时间的作用.两个实验均分成四组,生理水组、藿香正气片组、安定组、安定+藿香正气片组.结果1.对小鼠自发活动的影响.(1)藿香正气片组与生理盐水组比较,具有极显著性差异(P＜0.01).(2)安定组与安定+藿香正气片组比较具有显著性差异(P＜0.05).2.藿香正气片增强镇静催眠药对小鼠的睡眠作用.(1)藿香正气片组与生理盐水组比较具有显著性差异(P＜0.05).(2)藿香正气片+安定组与安定组比较具有极显著性差异(P＜0.01).结论藿香正气片与安定合用对镇静及催眠确有协同作用.