We are evaluating the use of metabolically competent HepaRG™ cells combined with CometChip® for DNA damage and the micronucleus (MN) assay as a New Approach Methodology (NAM) alternative to animals for follow up genotoxicity assessment to in vitro positive genotoxic response. Naphthalene is genotoxic in human TK6 cells inducing a nonlinear dose-response for the induction of micronuclei in the presence of rat liver S9. of naphthalene. In HepaRG™ cells, naphthalene genotoxicity was assessed using either 6 (CometChip™) or 12 concentrations of naphthalene (MN assay) with the top dose used for assessment of genotoxicity for the Comet and MN assay was 1.25 and 1.74 mM respectively, corresponding to approximately 45% cell survival. In contrast to human TK6 cell with S9, naphthalene was not genotoxic in either the HepaRG™ MN assay or the Comet assay using CometChip®. The lack of genotoxicity in both the MN and comet assays in HepaRG™ cells is likely due to Phase II enzymes removing phenols preventing further bioactivation to quinones and efficient detoxication of naphthalene quinones or epoxides by glutathione conjugation. In contrast to CYP450 mediated metabolism, these Phase II enzymes are inactive in rat liver S9 due to lack of appropriate cofactors causing a positive genotoxic response. Rat liver S9-derived BMD10 over-predicts naphthalene genotoxicity when compared to the negative genotoxic response observed in HepaRG™ cells. Metabolically competent hepatocyte models like HepaRG™ cells should be considered as human-relevant NAMs for use genotoxicity assessments to reduce reliance on rodents.

OBJECTIVE: Uniparental disomy (UPD) is one of the common causes of imprinting disorders, which can have an impact on gene expression according to the origin of the parental chromosome. Paternal UPD14 leads to Kagami-Ogata syndrome (KOS), which has a more severe phenotype than maternal UPD14, also called Temple syndrome. Small supernumerary marker chromosomes (SSMCs) are defined as structural chromosomal abnormalities that may be inherited or come from micronucleus-mediated chromothripsis. The association of UPD and SSMC is very rare but not fortuitous and several mechanisms can explain this phenomenon.
METHODS: We report the first prenatal case of paternal isodisomy for chromosome 14 associated with a de novo SSMC originating from chromosome 15 and revealed by KOS. The mechanism could be a chromothripsis mediated by trisomy rescue.
CONCLUSIONS: Regarding this case, in relation to a de novo SSMC, it could be important to extend the research of UPD to other acrocentric chromosomes if ultrasound signs are evocative.

The contamination of freshwaters by heavy metals represents a great problem, posing a threat for human and environmental health. Cadmium is classified as carcinogen to humans and its mechanism of carcinogenicity includes genotoxic events. In this study a recently developed eco-friendly cellulose-based nanosponge (CNS) was investigated as a candidate in freshwater nano-remediation process. For this purpose, CdCl2 (0.05 mg L-1) contaminated artificial freshwater (AFW) was treated with CNS (1.25 g L-1 for 2 h), and cellular responses were analyzed before and after CNS treatment in Dreissena polymorpha hemocytes. A control group (AFW) and a negative control group (CNS in AFW) were also tested. DNA primary damage was evaluated by Comet assay while chromosomal damage and cell proliferation were assessed by Cytome assay. AFW exposed to CNS did not cause any genotoxic effect in zebra mussel hemocytes. Moreover, DNA damage and cell proliferation induced by Cd(II) turned down to control level after 2 days when CNS were used. A reduction of Cd(II)-induced micronuclei and nuclear abnormalities was also observed. CNS was thus found to be a safe and effective candidate in cadmium remediation process being efficient in metal sequestering, restoring cellular damage exerted by Cd(II) exposure, without altering cellular physiological activity.

Patients with end-stage renal disease (ESRD) require hemodialysis. However, dialysis therapy may cause genomic damage due to increased oxidative stress. Non-invasive assessment of genotoxicity may be helpful for developing management strategies. We applied the buccal micronucleus cytome (BMCyt) assay to ESRD patients on dialysis. Patients (n=35, age 52±2 year) on dialysis therapy (20.9±0.8months) had low glomerular filtration rates (GFR=5.00±0.36ml/min/1.73m2); controls (n=21, age 51±2 year) were healthy adults with no known recent illnesses or exposures. Patients had significantly increased chromosome damage: clastogenic/aneugenic events (frequency of cells with MN), cellproliferation (basal cells), cytokinesis defects (binucleated cells), and celldeath (pyknotic cells); Repair Index was lower in the patient group. Receiver Operator Characteristic (ROC) curve analysis showed that cells with MN were the best predictor for discriminating between patients and controls. Other predictivebiomarkers were the frequencies of basal, binucleated,and pyknotic.

In most lineages, diversity among gene family members results from gene duplication followed by sequence divergence. Because of the genome rearrangements during the development of somatic nuclei, gene family evolution in ciliates involves more complex processes. Previous work on the ciliate Chilodonella uncinata revealed that macronuclear β-tubulin gene family members are generated by alternative processing, in which germline regions are alternatively used in multiple macronuclear chromosomes. To further study genome evolution in this ciliate, we analyzed its transcriptome and found that (1) alternative processing is extensive among gene families; and (2) such gene families are likely to be C. uncinata specific. We characterized additional macronuclear and micronuclear copies of one candidate alternatively processed gene family-a protein kinase domain containing protein (PKc)-from two C. uncinata strains. Analysis of the PKc sequences reveals that (1) multiple PKc gene family members in the macronucleus share some identical regions flanked by divergent regions; and (2) the shared identical regions are processed from a single micronuclear chromosome. We discuss analogous processes in lineages across the eukaryotic tree of life to provide further insights on the impact of genome structure on gene family evolution in eukaryotes.

The currently used regulatory in vitro mutagenicity/genotoxicity test battery has a high sensitivity for detecting genotoxicants, but it suffers from a large number of irrelevant positive results (i.e. low specificity) thereby imposing the need for additional follow-up by in vitro and/or in vivo genotoxicity tests. This could have a major impact on the cosmetic industry in Europe, seen the imposed animal testing and marketing bans on cosmetics and their ingredients. Afflicted, but safe substances could therefore be lost. Using the example of triclosan, a cosmetic preservative, we describe here the potential applicability of a human toxicogenomics-based in vitro assay as a potential mechanistically based follow-up test for positive in vitro genotoxicity results. Triclosan shows a positive in vitro chromosomal aberration test, but is negative during in vivo follow-up tests. Toxicogenomics analysis unequivocally shows that triclosan is identified as a compound acting through non-DNA reactive mechanisms. This proof-of-principle study illustrates the potential of genome-wide transcriptomics data in combination with in vitro experimentation as a possible weight-of-evidence follow-up approach for de-risking a positive outcome in a standard mutagenicity/genotoxicity battery. As such a substantial number of cosmetic compounds wrongly identified as genotoxicants could be saved for the future.