An integrated in vitro carcinogenicity test that distinguishes between genotoxic carcinogens, non-genotoxic carcinogens, and non-carcinogens.

Chemical safety testing plays a crucial role in product and pharmacological development, as well as chemoprevention; however, in vitro genotoxicity safety tests do not always accurately predict the chemicals that will be in vivo carcinogens. If chemicals test positive in vitro for genotoxicity but negative in vivo, this can contribute to unnecessary testing in animals used to confirm erroneous in vitro positive results. Current in vitro tests typically evaluate only genotoxicity endpoints, which limits their potential to detect non-genotoxic carcinogens. The frequency of misleading in vitro positive results can be high, leading to a requirement for more informative in vitro tests. It is now recognized that multiple-endpoint genotoxicity testing may aid more accurate detection of carcinogens and non-carcinogens. The objective of this review was to evaluate the utility of our novel, multiple-endpoint in vitro test, which uses multiple cancer-relevant endpoints to predict carcinogenic potential. The tool assessed micronucleus frequency, p53 expression, p21 expression, mitochondrial respiration, cell cycle abnormalities and, uniquely, cell morphology changes in human lymphoblastoid cell lines, TK6 and MCL-5. The endpoints were used to observe cellular responses to 18 chemicals within the following categories: genotoxic carcinogens, non-genotoxic carcinogens, toxic non-carcinogens, and misleading in vitro positive and negative agents. The number of endpoints significantly altered for each chemical was considered, alongside the holistic Integrated Signature of Carcinogenicity score, derived from the sum of fold changes for all endpoints. Following the calculation of an overall score from these measures, carcinogens exhibited greater potency than non-carcinogens. Genotoxic carcinogens were generally more potent than non-genotoxic carcinogens. This novel approach therefore demonstrated potential for correctly predicting whether chemicals with unknown mechanism may be considered carcinogens. Overall, while further validation is recommended, the test demonstrates potential for the identification of carcinogenic compounds. Adoption of the approach could enable reduced animal use in carcinogenicity testing.

Evaluation of endpoints for the study and diagnosis of mitochondrial toxicity and disease: a narrative review.

Mitochondrial DNA mutation and toxicity have been linked to several inherited and acquired diseases; however, these are challenging to diagnose and characterize due to clinical and genetic heterogeneity. This review investigates current techniques for the analysis of mitochondrial perturbations, and novel, emerging endpoints for routine application within the clinical setting. Particular focus is given to the biochemistry of the mitochondria influencing each endpoint and the relation of these to toxicity. Current approaches such as the use of metabolic markers (e.g. lactate production), and muscle biopsies to measure mitochondrial proteins were found to lack specificity. Newly emerging identified endpoints were: fibroblast growth factor-21, glucose uptake, mitochondrial membrane potential, mitochondrial morphology, mtDNA heteroplasmy, and mutation of mtDNA and nuclear DNA. Owed to the advancement in genetic analysis techniques, it is suggested by this review that genotypic endpoints of mtDNA mutation and heteroplasmy show particular promise as indicators of mitochondrial disease. It is, however, acknowledged that any single endpoint in isolation offers limited information; therefore, it is recommended that analysis of several endpoints simultaneously will offer the greatest benefit in terms of disease diagnosis and study. It is hoped that this review further highlights the need for advancement in understanding mitochondrial disease.