Mahsa Karbaschi

Inter-laboratory variation in DNA damage using a standard comet assay protocol

There are substantial inter-laboratory variations in the levels of DNA damage measured by the comet assay. The aim of this study was to investigate whether adherence to a standard comet assay protocol would reduce inter-laboratory variation in reported values of DNA damage. Fourteen laboratories determined the baseline level of DNA strand breaks (SBs)/alkaline labile sites and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites in coded samples of mononuclear blood cells (MNBCs) from healthy volunteers. There were technical problems in seven laboratories in adopting the standard protocol, which were not related to the level of experience. Therefore, the inter-laboratory variation in DNA damage was only analysed using the results from laboratories that had obtained complete data with the standard comet assay protocol. This analysis showed that the differences between reported levels of DNA SBs/alkaline labile sites in MNBCs were not reduced by applying the standard assay protocol as compared with the laboratorys own protocol. There was large inter-laboratory variation in FPG-sensitive sites by the laboratory-specific protocol and the variation was reduced when the samples were analysed by the standard protocol. The SBs and FPG-sensitive sites were measured in the same experiment, indicating that the large spread in the latter lesions was the main reason for the reduced inter-laboratory variation. However, it remains worrying that half of the participating laboratories obtained poor results using the standard procedure. This study indicates that future comet assay validation trials should take steps to evaluate the implementation of standard procedures in participating laboratories.

An ECVAG inter-laboratory validation study of the comet assay: inter-laboratory and intra-laboratory variations of DNA strand breaks and FPG-sensitive sites in human mononuclear cells

The alkaline comet assay is an established, sensitive method extensively used in biomonitoring studies. This method can be modified to measure a range of different types of DNA damage. However, considerable differences in the protocols used by different research groups affect the inter-laboratory comparisons of results. The aim of this study was to assess the inter-laboratory, intra-laboratory, sample and residual (unexplained) variations in DNA strand breaks and formamidopyrimidine DNA glycosylase (FPG)-sensitive sites measured by the comet assay by using a balanced Latin square design. Fourteen participating laboratories used their own comet assay protocols to measure the level of DNA strand breaks and FPG-sensitive sites in coded samples containing peripheral blood mononuclear cells (PBMC) and the level of DNA strand breaks in coded calibration curve samples (cells exposed to different doses of ionising radiation) on three different days of analysis. Eleven laboratories found dose-response relationships in the coded calibration curve samples on two or three days of analysis, whereas three laboratories had technical problems in their assay. In the coded calibration curve samples, the dose of ionising radiation, inter-laboratory variation, intra-laboratory variation and residual variation contributed to 60.9, 19.4, 0.1 and 19.5%, respectively, of the total variation. In the coded PBMC samples, the inter-laboratory variation explained the largest fraction of the overall variation of DNA strand breaks (79.2%) and the residual variation (19.9%) was much larger than the intra-laboratory (0.3%) and inter-subject (0.5%) variation. The same partitioning of the overall variation of FPG-sensitive sites in the PBMC samples indicated that the inter-laboratory variation was the strongest contributor (56.7%), whereas the residual (42.9%), intra-laboratory (0.2%) and inter-subject (0.3%) variations again contributed less to the overall variation. The results suggest that the variation in DNA damage, measured by comet assay, in PBMC from healthy subjects is assay variation rather than variation between subjects.

Novel method for the high-throughput processing of slides for the comet assay

Single cell gel electrophoresis (the comet assay), continues to gain popularity as a means of assessing DNA damage. However, the assays low sample throughput and laborious sample workup procedure are limiting factors to its application. “Scoring”, or individually determining DNA damage levels in 50 cells per treatment, is time-consuming, but with the advent of high-throughput scoring, the limitation is now the ability to process significant numbers of comet slides. We have developed a novel method by which multiple slides may be manipulated, and undergo electrophoresis, in batches of 25 rather than individually and, importantly, retains the use of standard microscope comet slides, which are the assay convention. This decreases assay time by 60%, and benefits from an electrophoresis tank with a substantially smaller footprint, and more uniform orientation of gels during electrophoresis. Our high-throughput variant of the comet assay greatly increases the number of samples analysed, decreases assay time, number of individual slide manipulations, reagent requirements and risk of damage to slides. The compact nature of the electrophoresis tank is of particular benefit to laboratories where bench space is at a premium. This novel approach is a significant advance on the current comet assay procedure.

Rescue of cells from apoptosis increases DNA repair in UVB exposed cells: implications for the DNA damage response

Classically, the nucleotide excision repair (NER) of cyclobutane pyrimidine dimers (CPD) is a lengthy process (t1/2 > 48 h). Using the T4 endonuclease V-modified comet assay, we uniquely found a far more rapid repair of UVA-induced CPD (t1/2 = 4.5 h) in human skin keratinocytes. The repair of UVB-induced CPD began to slow within 1 h of irradiation, causing damage to persist for over 36 h. A similar trend was noted for the repair of oxidatively-modified purine nucleobases. Supportive of this differential repair, we noted an up-regulation of key genes associated with NER in UVA-irradiated cells, whereas the same genes were down regulated in UVB-irradiated cells. There were no significant differences in cell viability between the two treatments over the first 6 h post-irradiation, but after 24 h apoptosis had increased significantly in the UVB-irradiated cells. The role of apoptosis was confirmed using a pan-caspase inhibitor, which increased CPD repair, similar to that seen with UVA. These data indicate that the cellular ‘decision’ for apoptosis/DNA repair occurs far earlier than previously understood, and that the induction of apoptosis leads to lesion persistence, and not vice versa. This also highlights a new, potential increased carcinogenic risk from UVA-induced DNA damage as, rather than undergoing apoptosis, high levels of damage are tolerated and repaired, with the attendant risk of mutation.

Vitamin E inhibits the UVAI induction of “light” and “dark” cyclobutane pyrimidine dimers, and oxidatively generated DNA damage, in keratinocytes

Solar ultraviolet radiation (UVR)-induced DNA damage has acute, and long-term adverse effects in the skin. This damage arises directly by absorption of UVR, and indirectly via photosensitization reactions. The aim of the present study was to assess the effects of vitamin E on UVAI-induced DNA damage in keratinocytes in vitro. Incubation with vitamin E before UVAI exposure decreased the formation of oxidized purines (with a decrease in intracellular oxidizing species), and cyclobutane pyrimidine dimers (CPD). A possible sunscreening effect was excluded when similar results were obtained following vitamin E addition after UVAI exposure. Our data showed that DNA damage by UVA-induced photosensitization reactions can be inhibited by the introduction of vitamin E either pre- or post-irradiation, for both oxidized purines and CPD (including so-called “dark” CPDs). These data validate the evidence that some CPD are induced by UVAI initially via photosensitization, and some via chemoexcitation, and support the evidence that vitamin E can intervene in this pathway to prevent CPD formation in keratinocytes. We propose the inclusion of similar agents into topical sunscreens and aftersun preparations which, for the latter in particular, represents a means to mitigate on-going DNA damage formation, even after sun exposure has ended.

Immuno-Slot Blot Assay for Detection of UVR-Mediated DNA Damage

Solar ultraviolet radiation (UVR), through the formation of DNA photolesions, is the primary cause of most skin cancers. A better understanding of the mechanisms of UVR-induced DNA damage may help prevent skin cancer and this may be achieved using methods to quantify DNA damage. The immuno-slot blot (ISB) method is routinely used for detection and quantification of any heat- and alkali-stable DNA adducts for which a sufficiently specific monoclonal antibody is available. The main steps in ISB are fragmentation and denaturation of the DNA, immobilization of DNA to a nitrocellulose filter, incubation with primary antibody against a specific DNA adduct, incubation with an enzyme-linked secondary antibody and finally chemiluminescence detection and quantification of the DNA adducts.

Light-based methods for whole blood bacterial inactivation enabled by a recirculating flow system

Light of certain wavelengths can be used to inactivate pathogens. Whole blood is opaque; thus, the penetration of light is reduced. Here, we overcame this limitation using a thin transparent tube that is illuminated from all angles. Three light‐based techniques were evaluated: photodynamic therapy (PDT) using a 660‐nm light and antibody‐photosensitizer conjugates, ultraviolet, and violet light. We observed a reduction of 55–71% of Staphylococcus aureus after 5 h of exposure (starting concentration 107 CFU mL−1) and an 88–97% reduction in methicillin‐resistant Staphylococcus aureus (MRSA) (starting 104 CFU mL−1). An 83–92% decrease for S. aureus and 98–99.9% decrease for MRSA were observed when combined with an immunocapture approach. Complete blood count with differential analysis did not reveal any significant changes in the blood cell numbers. Genotoxicity studies showed that violet and ultraviolet did not induce any significant level of single strand breaks and alkali labile sites in the peripheral blood mononuclear cells (PBMC). In contrast, ultraviolet did induce a very low level of cyclobutane pyrimidine dimers, a UV damage indicator. PDT generated a significant level of single strand breaks and 8‐oxoGua in these cells. The approaches showed promise for whole blood pathogen inactivation with minimal collateral damage to PBMC.