Multi-endpoint analysis of cadmium chloride-induced genotoxicity shows role for reactive oxygen species and p53 activation in DNA damage induction, cell cycle irregularities, and cell size aberrations.

Cadmium chloride (CdCl2) is a known genotoxic carcinogen, with a mechanism of action thought to partly involve the generation of reactive oxygen species (ROS). We applied here a multi-endpoint approach in vitro to explore the impact of CdCl2 on both the genome and on wider cell biology pathways relevant to cancer. Multi-endpoint approaches are believed to offer greater promise in terms of understanding the holistic effects of carcinogens in vitro. This richer understanding may help better classification of carcinogens as well as allowing detailed mechanisms of action to be identified. We found that CdCl2 caused DNA damage [micronuclei (MN)] in both TK6 and NH32 cells in a dose-dependent manner after 4 h exposure (plus 23 h recovery), with lowest observable effect levels (LOELs) for MN induction of 1 µM (TK6) and 1.6 µM (NH32). This DNA damage induction in TK6 cells was ROS dependent as pretreatment with the antioxidant N-Acetyl Cysteine (1 mM), abrogated this effect. However, 2',7'-dichlorofluorescin diacetate was not capable of detecting the ROS induced by CdCl2. The use of NH32 cells allowed an investigation of the role of p53 as they are a p53 null cell line derived from TK6. NH32 showed a 10-fold increase in MN in untreated cells and a similar dose-dependent effect after CdCl2 treatment. In TK6 cells, CdCl2 also caused activation of p53 (accumulation of total and phosphorylated p53), imposition of cell cycle checkpoints (G2/M) and intriguingly the production of smaller and more eccentric (elongated) cells. Overall, this multi-endpoint study suggests a carcinogenic mechanism of CdCl2 involving ROS generation, oxidative DNA damage and p53 activation, leading to cell cycle abnormalities and impacts of cell size and shape. This study shows how the integration of multiple cell biology endpoints studied in parallel in vitro can help mechanistic understanding of how carcinogens disrupt normal cell biology.

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

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.

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

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.