A novel, integrated in vitro carcinogenicity test to identify genotoxic and non-genotoxic carcinogens using human lymphoblastoid cells

Abstract

Human exposure to carcinogens occurs via a plethora of environmental sources, with 70–90% of cancers caused by extrinsic factors. Aberrant phenotypes induced by such carcinogenic agents may provide universal biomarkers for cancer causation. Both current in vitro genotoxicity tests and the animal-testing paradigm in human cancer risk assessment fail to accurately represent and predict whether a chemical causes human carcinogenesis. The study aimed to establish whether the integrated analysis of multiple cellular endpoints related to the Hallmarks of Cancer could advance in vitro carcinogenicity assessment. Human lymphoblastoid cells (TK6, MCL-5) were treated for either 4 or 23 h with 8 known in vivo carcinogens, with doses up to 50% Relative Population Doubling (maximum 66.6 mM). The adverse effects of carcinogens on wide-ranging aspects of cellular health were quantified using several approaches; these included chromosome damage, cell signalling, cell morphology, cell-cycle dynamics and bioenergetic perturbations. Cell morphology and gene expression alterations proved particularly sensitive for environmental carcinogen identification. Composite scores for the carcinogens’ adverse effects revealed that this approach could identify both DNA-reactive and non-DNA reactive carcinogens in vitro. The richer datasets generated proved that the holistic evaluation of integrated phenotypic alterations is valuable for effective in vitro risk assessment, while also supporting animal test replacement. Crucially, the study offers valuable insights into the mechanisms of human carcinogenesis resulting from exposure to chemicals that humans are likely to encounter in their environment. Such an understanding of cancer induction via environmental agents is essential for cancer prevention.Electronic supplementary materialThe online version of this article (10.1007/s00204-017-2102-y) contains supplementary material, which is available to authorized users.

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

“…The parallel assessment of multiple, holistic endpoints may enable a broad range of carcinogenic mechanisms to be monitored and link to adverse outcome pathways (AOPs) (Burden et al 2015 ). Previously, we have demonstrated that integrated in vitro endpoints show promise in distinguishing genotoxic carcinogens from non-genotoxic carcinogens, with results also correlating well with in vivo data (Wilde et al 2018 ).…”

“…While there was some overlap between toxic non-carcinogens and misleading positives, toxic non-carcinogens generally ranked higher with an average ISC of 22.3, compared to 15.7 for misleading positives. There did appear to be some overlap between non-carcinogen ISCs and non-genotoxic carcinogens ISCs published previously by Wilde et al (2018); non-carcinogen cycloheximide's ISC was 28.4, whereas non-genotoxic carcinogens NiCl 2 and DEHP produced lower ISCs of 27.1 and 26.4, respectively. As previously mentioned, it is, however, possible that cycloheximide is inherently genotoxic (Fig.…”

“…A shortlist of genes for qRT-PCR analysis was generated via mRNA microarray chip technology (Illumina) to measure genome-wide transcriptome alterations, as detailed by Wilde et al ( 2018 ). qRT-PCR was completed for cyclin-dependent kinase inhibitor 1A ( CDKN1A ), choline kinase alpha ( CHKA ) and serine/threonine protein kinase ( SGK1 ).…”

“…A previously published method was followed (Brusehafer, et al 2014 ). Primer sequences are available in Wilde et al ( 2018 ).…”

“…Cell and nuclear morphology analysis was performed using the INCell Analyzer 2000 followed by a MATLAB-based script to identify cells and nuclei from captured images. The full methodology was previously outlined by Wilde et al ( 2018 ). For the toxic non-carcinogens, the CellProfiler 2.2.0 software was used to obtain equivalent data on cell and nuclear morphology.…”