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Multiple-endpoint in vitro carcinogenicity test in human cell line TK6 distinguishes carcinogens from non-carcinogens and highlights mechanisms of action.

ABSTRACT: Current in vitro genotoxicity tests can produce misleading positive results, indicating an inability to effectively predict a compound's subsequent carcinogenic potential in vivo. Such oversensitivity can incur unnecessary in vivo tests to further investigate positive in vitro results, supporting the need to improve in vitro tests to better inform risk assessment. It is increasingly acknowledged that more informative in vitro tests using multiple endpoints may support the correct identification of carcinogenic potential. The present study, therefore, employed a holistic, multiple-endpoint approach using low doses of selected carcinogens and non-carcinogens (0.001-770 µM) to assess whether these chemicals caused perturbations in molecular and cellular endpoints relating to the Hallmarks of Cancer. Endpoints included micronucleus induction, alterations in gene expression, cell cycle dynamics, cell morphology and bioenergetics in the human lymphoblastoid cell line TK6. Carcinogens ochratoxin A and oestradiol produced greater Integrated Signature of Carcinogenicity scores for the combined endpoints than the "misleading" in vitro positive compounds, quercetin, 2,4-dichlorophenol and quinacrine dihydrochloride and toxic non-carcinogens, caffeine, cycloheximide and phenformin HCl. This study provides compelling evidence that carcinogens can successfully be distinguished from non-carcinogens using a holistic in vitro test system. Avoidance of misleading in vitro outcomes could lead to the reduction and replacement of animals in carcinogenicity testing.

SUBMITTER: Chapman KE

PROVIDER: S-EPMC7811515 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Multiple-endpoint in vitro carcinogenicity test in human cell line TK6 distinguishes carcinogens from non-carcinogens and highlights mechanisms of action.

Chapman Katherine E KE Wilde Eleanor C EC Chapman Fiona M FM Verma Jatin R JR Shah Ume-Kulsoom UK Stannard Leanne M LM Seager Anna L AL Tonkin James A JA Brown M Rowan MR Doherty Ann T AT Johnson George E GE Doak Shareen H SH Jenkins Gareth J S GJS

Archives of toxicology 20200910 1

Current in vitro genotoxicity tests can produce misleading positive results, indicating an inability to effectively predict a compound's subsequent carcinogenic potential in vivo. Such oversensitivity can incur unnecessary in vivo tests to further investigate positive in vitro results, supporting the need to improve in vitro tests to better inform risk assessment. It is increasingly acknowledged that more informative in vitro tests using multiple endpoints may support the correct identification ...[more]

PMID: 32910239

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Project description:Background: Information on the carcinogenic potential of chemicals is only availably for High Production Volume products. There is however, a pressing need for alternative methods allowing for the chronic toxicity of substances, including carcinogenicity, to be detected earlier and more reliably. Here we applied advanced genomics to a cellular transformation assay to identify gene signatures useful for the prediction of risk for carcinogenicity. Methods: Genome wide gene expression analysis and qRT-PCR were applied to untransformed and transformed Balb/c 3T3 cells that exposed to 2, 4-diaminotoluene (DAT), benzo(a)pyrene (BaP), 2-Acetylaminoflourene (AAF) and 3-methycholanthrene (MCA) for 24h and 120h, at different concentrations, respectively. Furthermore, various bioinformatics tools were used to identify gene signatures predicting for the carcinogenic risk. Results: Bioinformatics analysis revealed distinct datasets for the individual chemicals tested while the number of significantly regulated genes increased with ascending treatment concentration of the cell cultures. Filtering of the data revealed a common gene signature that comprised of 13 genes whose regulation in cancer tissue has already been established. Strikingly, this gene signature was already identified prior to cell transformation therefore confirming the predictive power of this gene signature in identifying carcinogenic risks of chemicals. Comparison of fold changes determined by microarray analysis and qRT-PCR were in good agreement. Conclusion: Our data describes selective and commonly regulated carcinogenic pathways observed in an easy to use in vitro carcinogenicity assay. Here we defined a set of genes which can serve as a simply assay to predict the risk for carcinogenicity by use of an alternative in vitro testing strategy. Balb/c 3T3 cells were seeded at 200 cells in each 60 x 15 mm culture dish with 4 ml M10F, using six culture dishes for every treatment. When cells reached a confluence of 60-65%, the culture medium was removed and replaced with fresh medium containing all tested chemicals at a specific concentration and two time points (24 and 120h). First we treated the cells both 24h and 120h with concentrations reported in the literature (0.5ÂµM BaP, 50ÂµM DAT, 25ÂµM AAF and 2ÂµM MCA). In a second approach IC20 concentrations were investigated for each chemical at both time points. The concentrations determined for IC20 ranged from 1.5 ÂµM BaP to 700 ÂµM DAT for 24h of treatment, and from 0.1 ÂµM BaP or MCA to 10ÂµM AAF for 120h of treatment. Balb/c 3T3 cells treated with 0.75% DMSO alone were kept as controls. Each experiment was run in triplicate.

Dataset Information

Multiple-endpoint in vitro carcinogenicity test in human cell line TK6 distinguishes carcinogens from non-carcinogens and highlights mechanisms of action.

Publications

Multiple-endpoint in vitro carcinogenicity test in human cell line TK6 distinguishes carcinogens from non-carcinogens and highlights mechanisms of action.

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