Project 6 (NILU)
Project 6: Assessment of genotoxic potential of nanoparticles, oxidative DNA damage and repair
Fellow: Anna Huk (ESR6), 36 months
Tutor: Dr. Maria Dusinska (NILU)
NP may induce DNA damage and could be potentially mutagenic. A positive effect on DNA damage measured by the comet assay was found in 14 out of 19 studies. However, many conflicting results have been published and confounding factors are being identified to influence the effect. The comet assay in basic form detects DNA damage (strand breaks). We have modified this assay with the lesion specific enzyme FPG for detection of oxidized purines and EndoIII for detection of oxidized pyrimidines. In a previous project (FP5 FIBRETOX) with nanomaterial – asbestos and mineral fibres – we found elevated levels of oxidative DNA damage measured by the Comet assay in human lymphocytes of exposed subjects. Within the FP6 COMICS and FP6 NewGeneris the comet assay was developed towards high throughput with automated screening. Although previous studies have proposed a role for ROS in NP-induced toxicity the downstream pathways through which NP signal in human cells and induce DNA damage are unclear. Here we are focusing on development of alternative testing strategies for the assessment of toxic potential of NP used in medical diagnostics with a focus on crucial toxicological endpoints including cytotoxicity and genotoxicity.
An ESR will perform studies to further standardize the comet assay with human OGG1 (detection of 8-oxoguanin as most mutagenic lesion), and UVRABC (detection of bulk DNA adducts) repair enzymes to detect more specific DNA lesions which may be induced by NP and on the application of high throughput assays in nanogenotoxicity. An ESR will focus on identifying the most appropriate reference standards relevant to nanotoxicology, especially the nano-related positive and negative controls, and will develop SOP for using the comet assay in nanotoxicity testing. As the mechanisms of action of NP at the genome level are not known, the focus will be on the investigation of interaction of NP with DNA either directly by detecting different DNA lesions using the modified comet assay or indirectly through DNA repair system. The ESR will investigate if NP can compromise DNA repair, both cellular and in vitro nucleotide or base excision repair. This research will provide information on mechanisms of action of metal NP at the level of DNA, and specifically on involvement of NP induced ROS in DNA damage and repair and will pre-validate the measurement of specific DNA lesions as biomarkers for nanogenotoxicity testing. Main skills acquired are in molecular and cell biology, in vitro toxicology assays, genotoxicology tests like comet assay and other cytogenetic methods. Secondments to partners 1 (NLR and gene expression assays), 2 (inflammatory reactions vs. genotoxicity), 3 (chip-based miniaturization), 4 (assay validation and standardisation), 5 (comparing health and ecotox data), 7 (LVOCs, ROS production and link to genotoxicity), 10 (airborne NP delivery to cultured cells), and 11 (optical NP-cell interaction analysis).
In WP1 (task 1.4), WP2 (tasks 2.1 and 2.2) and WP4 (tasks 4.2, 4.4 and 4.5).