DEVELOPMENT OF THE RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) TECHNIQUE TO MEASURE THE EFFECTS OF GENOTOXINSIN AQUATIC ORGANISMS

Studies were undertaken to evaluate the potential of random amplified polymorphic DNA (RAPD) to detect DNA effects (including DNA damage and mutations) in aquatic invertebrates, following their exposure to a variety of environmental contaminants under laboratory conditions. After rigorous optimisation of the RAPD method, the protocol, which used a high annealing temperature (50"C for 10-mer primers), was found to generate good-quality DNA profiles from groups of organisms belonging to the bacterial, plant and animal kingdoms. The RAPD method was initially used to detect benzo(a)pyrene [B(a)P] and copper-induced DNA effects in the water flea Daphnia magna and ultraviolet-mediated DNA effects in the marine alga Palmaria palmata. The results clearly showed that changes occurred in RAPD profiles obtained from the exposed populations when compared to controls. In these studies, the effect of the genotoxins at higher levels of biological organisation (e.g. Darwinian parameters and/or fitness parameters) were also investigated and were compared with genomic DNA template stability (GTS), a qualitative index representing clear changes in panems compared to control RAPD profiles. The results from these experiments revealed that GTS could be more sensitive than growth parameters and showed at least equal or even greater sensitivity than other measures of fitness. Changes in RAPD profiles were believed to be the result of DNA effects, namely adduct formation, DNA breakage, oxidative damage and mutations and possibly other effects (e.g. variation in gene expression). Nevertheless, the nature and amount of DNA effects could only be speculated because diverse events may induce the same category of changes (i.e. variation in band intensity, appearance of bands, and disappearance of amplicons) in RAPD patterns. Further studies confirmed that RAPD had the potential to qualitatively detect oestrogen and xeno-oestrogen -induced DNA effects in barnacles. Additional experiments emphasised that oxygen radicals and variation in gene expression may induce significant changes in RAPD profiles. To further understand the effects of DNA lesions and mutations on RAPD patterns, individual types of DNA damage were created in vitro. The results clearly indicated that BaP DNA adducts, DNA photoproducts. and DNA breakages had significant effects on RAPD profiles but that diverse types of DNA damage may induce the same category of changes in RAPD patterns which render the interpretation of the results difficult. It was also concluded that mutations could be detected provided they do not arise in a random fashion. Finally, an attempt was made to determine the kinetics of DNA damage and DNA repair and whether changes in patterns obtained from B(a)P exposed Daphnia magna could be transmitted to successive generations. This strategy was developed to distinguish between mutations and DNA damage. The results showed that some bands obtained from the exposed populations were transmitted to the first and/or second generation but not to the third. It was concluded that the transmission of modified genetic material to the offspring was more likely to be the result of large genomic rearrangements and/or base methylation (epigenetic processes) rather than point mutations. In conclusion, the results presented in this research project show the potential of the RAPD assay as a useful method for the qualitative assessment of DNA effects including genotoxicity and changes in gene expression. The main advantage of this technique is that it can be applied to any species without requiring any information about the nucleotide sequence. In the field of ecogenotoxicology, its main advantage lies in its sensitivity and speed to detect a wide range of DNA damage including DNA breakage, DNA adducts, oxidative damage as well as mutations (including point mutations and large rearrangements). On the other hand, RAPD only allows a qualitative assessment of the DNA effects and the nature of the changes occurring in profiles can only be speculated. Finally, a great deal of further experimentation and validation are required in order to assess the applicability of the technique to a variety of other species and pollutants, particularly under field conditions

A novel statistical signal processing method to estimate effects of compounds on contractility of cardiomyocytes using impedance assays

International audienceLabel free methods such as cell impedance assays are in vitro tests increasingly used in drug development and producing large and high-content data files. Since the current commercial software are not suited for fully automated analysis , there is a need to develop validated and rapid solutions to extract relevant information for biologists. This need is particularly obvious in the case of impedance signals analysis from cardiomyocytes. The proposed solution is based on three main steps. The first one consists in calculating five indices informing about the time variations of frequency (F), amplitude (A), shape (S) of beatings, trends (T) of the cardiomyocyte dependent on spreading, viability and attachment as well as irregularity (I) of the contractility. In a second phase, two summary statistics are proposed to test the concentration effect of drugs on the five FASTI indices. Results of the statistical tests are finally aggregated in a cardio-effect grade to compare the tested molecules in a cardio-impact scale graduated from 0 (no influence) to 10 (highly disturbed effects in cardiomy-ocytes). This innovative approach was tested using in vitro data obtained from cell impedance analysis of three known molecules (2 cardiotoxic and 1 non-cardiotoxic compounds). Results have clearly shown the ability of the proposed approach to identify significant effects on the contractility of cardiomyocytes. This solution speeds up the analysis of cardiomyocyte impedance data, takes into account all the kinetic data generated and is now available for biologists on a web-platform: i-Cardio TM developed by CYBERnano TM

A data-driven modeling method to analyze cardiomyocyte impedance data

Présentation PosterInternational audienceOne goal of the Comprehensive in vitro ProArrhythmia Assay initiative is to predict more accurately potentially torsadogenic compounds in an earlier stage of drug development. To that aim one of the CiPA component is to assess capabilities of label-free in vitro assays (impedance and extracellular field potential signals) applied to human stem cell-derived cardiomyocytes

Investigative safety strategies to improve success in drug development

Understanding and reducing attrition rate remains a key challenge in drug development. Preclinical and clinical safety issues still represent about 40% of drug discontinuation, of which cardiac and liver toxicities are the leading reasons. Reducing attrition rate can be achieved by various means, starting with a comprehensive evaluation of the potential safety issues associated to the primary target followed by an evaluation of undesirable secondary targets. To address these risks, a risk mitigation plan should be built at very early development stages, using a panel of in silico, in vitro, and in vivo models. While most pharmaceutical companies have developed robust safety strategies to de-risk genotoxicity and cardiotoxicity issues, partly driven by regulatory requirements; safety issues affecting other organs or systems, such as the central nervous system, liver, kidney, or gastro-intestinal system are less commonly addressed during early drug development. This paper proposes some de-risking strategies that can be applied to these target organ systems, including the use of novel biomarkers that can be easily integrated in both preclinical and clinical studies. Experiments to understand the mechanisms’ underlying toxicity are also important. Two examples are provided to demonstrate how such mechanistic studies can impact drug development. Novel trends in investigative safety are reviewed, such as computational modeling, mitochondrial toxicity assessment, and imaging technologies. Ultimately, understanding the predictive value of non-clinical safety testing and its translatability to humans will enable to optimize assays in order to address the key objectives of the drug discovery process, i.e., hazard identification, risk assessment, and mitigation

Evaluation of Impedance-Based Label-Free Technology as a Tool for Pharmacology and Toxicology Investigations

The use of label-free technologies based on electrical impedance is becoming more and more popular in drug discovery. Indeed, such a methodology allows the continuous monitoring of diverse cellular processes, including proliferation, migration, cytotoxicity and receptor-mediated signaling. The objective of the present study was to further assess the usefulness of the real-time cell analyzer (RTCA) and, in particular, the xCELLigence platform, in the context of early drug development for pharmacology and toxicology investigations. In the present manuscript, four cellular models were exposed to 50 compounds to compare the cell index generated by RTCA and cell viability measured with a traditional viability assay. The data revealed an acceptable correlation (ca. 80%) for both cell lines (i.e., HepG2 and HepaRG), but a lack of correlation (ca. 55%) for the primary human and rat hepatocytes. In addition, specific RTCA profiles (signatures) were generated when HepG2 and HepaRG cells were exposed to calcium modulators, antimitotics, DNA damaging and nuclear receptor agents, with a percentage of prediction close to 80% for both cellular models. In a subsequent experiment, HepG2 cells were exposed to 81 proprietary UCB compounds known to be genotoxic or not. Based on the DNA damaging signatures, the RTCA technology allowed the detection of ca. 50% of the genotoxic compounds (n = 29) and nearly 100% of the non-genotoxic compounds (n = 52). Overall, despite some limitations, the xCELLigence platform is a powerful and reliable tool that can be used in drug discovery for toxicity and pharmacology studies

Current trends in in silico , in vitro toxicology, and safety biomarkers in early drug development

International audienc

Marqueurs de génotoxicité et effets in situ, individuels et populationnels

2de éditionInternational audienc

Evaluation of Impedance-Based Label-Free Technology as a Tool for Pharmacology and Toxicology Investigations

The use of label-free technologies based on electrical impedance is becoming more and more popular in drug discovery. Indeed, such a methodology allows the continuous monitoring of diverse cellular processes, including proliferation, migration, cytotoxicity and receptor-mediated signaling. The objective of the present study was to further assess the usefulness of the real-time cell analyzer (RTCA) and, in particular, the xCELLigence platform, in the context of early drug development for pharmacology and toxicology investigations. In the present manuscript, four cellular models were exposed to 50 compounds to compare the cell index generated by RTCA and cell viability measured with a traditional viability assay. The data revealed an acceptable correlation (ca. 80%) for both cell lines (i.e., HepG2 and HepaRG), but a lack of correlation (ca. 55%) for the primary human and rat hepatocytes. In addition, specific RTCA profiles (signatures) were generated when HepG2 and HepaRG cells were exposed to calcium modulators, antimitotics, DNA damaging and nuclear receptor agents, with a percentage of prediction close to 80% for both cellular models. In a subsequent experiment, HepG2 cells were exposed to 81 proprietary UCB compounds known to be genotoxic or not. Based on the DNA damaging signatures, the RTCA technology allowed the detection of ca. 50% of the genotoxic compounds (n = 29) and nearly 100% of the non-genotoxic compounds (n = 52). Overall, despite some limitations, the xCELLigence platform is a powerful and reliable tool that can be used in drug discovery for toxicity and pharmacology studies