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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

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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Correspondence to Gamze Ates.

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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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Keywords

  • Genotoxicity
  • Mutagenicity
  • qPCR array
  • Toxicogenomics
  • HepaRG
  • In vitro screening