Abstract
Although the value of the regulatory accepted batteries for in vitro genotoxicity testing is recognized, they result in a high number of false positives. This has a major impact on society and industries developing novel compounds for pharmaceutical, chemical, and consumer products, as afflicted compounds have to be (prematurely) abandoned or further tested on animals. Using the metabolically competent human HepaRG™ cell line and toxicogenomics approaches, we have developed an upgraded, innovative, and proprietary gene classifier. This gene classifier is based on transcriptomic changes induced by 12 genotoxic and 12 non-genotoxic reference compounds tested at sub-cytotoxic concentrations, i.e., IC10 concentrations as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The resulting gene classifier was translated into an easy-to-handle qPCR array that, as shown by pathway analysis, covers several different cellular processes related to genotoxicity. To further assess the predictivity of the tool, a set of 5 known positive and 5 known negative test compounds for genotoxicity was evaluated. In addition, 2 compounds with debatable genotoxicity data were tested to explore how the qPCR array would classify these. With an accuracy of 100%, when equivocal results were considered positive, the results showed that combining HepaRG™ cells with a genotoxin-specific qPCR array can improve (geno)toxicological hazard assessment. In addition, the developed qPCR array was able to provide additional information on compounds for which so far debatable genotoxicity data are available. The results indicate that the new in vitro tool can improve human safety assessment of chemicals in general by basing predictions on mechanistic toxicogenomics information.
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Abbreviations
- 2NF :
-
2-nitrofluorene
- AFB :
-
Aflatoxin B1
- AMP :
-
Ampicillin trihydrate
- ANT :
-
Anthranilic acid
- B[α]P :
-
Benzo[α]pyrene
- BLE :
-
Bleomycin sulfate
- BOR :
-
Basic orange 31
- CAP :
-
Caprolactam
- CAvit :
-
In vitro chromosome aberration test
- CdCl 2 :
-
Cadmium chloride
- CHF :
-
Chloramphenicol
- CIS :
-
Cisplatin
- CLI :
-
Climbazole
- CLR :
-
4chlororesorcinol
- CND :
-
Clonidine
- CYC :
-
Cyclophosphamide
- DAT :
-
2,4-diaminotoluene
- DMN :
-
Dimethylnitrosamine
- EMS :
-
Ethyl methanesulphonate
- ENU :
-
1-ethyl-1-nitrosourea
- ETO :
-
Etoposide
- FAM :
-
6-carboxyfluorescein
- FDR :
-
False discovery rate
- HBM :
-
Hydroxybenzomorpholine
- HCA :
-
Hierarchical cluster analysis
- HKG :
-
Housekeeping genes
- MAN :
-
d-mannitol
- MAP :
-
m-aminophenol
- MELA :
-
Melamine
- MMS :
-
Methyl methanesulphonate
- MNvit :
-
In vitro micronucleus test
- MNviv :
-
In vivo micronucleus test
- MTT :
-
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
- NAC :
-
No amplification control
- NaCl :
-
Sodium chloride
- NAP :
-
1-naphthol
- NIF :
-
Nifedipine
- NNK :
-
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone
- NTC :
-
No template control
- OECD :
-
Organisation for economic co-operation and development
- PAM :
-
Prediction analysis of micorarrays
- pCA :
-
p-chloroaniline
- PCA :
-
Principal component analysis
- RIN :
-
RNA integrity number
- SCCS :
-
Scientific Committee on Consumer Safety
- SDF :
-
Sodium diclofenac
- SOR :
-
Sorbitol
- SVM :
-
Support vector machine
- TAMRA :
-
Tetramethylrhodamine
- TOL :
-
Tolbutamide
- TRI :
-
Triclosan
- VIN :
-
Vinblastine sulfate
- WHO :
-
World Health Organization
- ZID :
-
Zidovudine
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Acknowledgements
This study was supported by the Vrije Universiteit Brussel (VUB) and the Scientific Institute of Public Health (WIV-ISP), Belgium.
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Ates, G., Mertens, B., Heymans, A. et al. A novel genotoxin-specific qPCR array based on the metabolically competent human HepaRG™ cell line as a rapid and reliable tool for improved in vitro hazard assessment. Arch Toxicol 92, 1593–1608 (2018). https://doi.org/10.1007/s00204-018-2172-5
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DOI: https://doi.org/10.1007/s00204-018-2172-5