Osman Sozeri

Variation in the measurement of DNA damage by comet assay measured by the ECVAG inter-laboratory validation trial

The comet assay has become a popular method for the assessment of DNA damage in biomonitoring studies and genetic toxicology. However, few studies have addressed the issue of the noted inter-laboratory variability of DNA damage measured by the comet assay. In this study, 12 laboratories analysed the level of DNA damage in monocyte-derived THP-1 cells by either visual classification or computer-aided image analysis of pre-made slides, coded cryopreserved samples of cells and reference standard cells (calibration curve samples). The reference standard samples were irradiated with ionizing radiation (0-10 Gy) and used to construct a calibration curve to calculate the number of lesions per 10(6) base pair. All laboratories detected dose-response relationships in the coded samples irradiated with ionizing radiation (1.5-7 Gy), but there were overt differences in the level of DNA damage reported by the different laboratories as evidenced by an inter-laboratory coefficient of variation (CV) of 47%. Adjustment of the primary comet assay end points by a calibration curve prepared in each laboratory reduced the CV to 28%, a statistically significant reduction (P < 0.05, Levenes test). A large fraction of the inter-laboratory variation originated from differences in image analysis, whereas the intra-laboratory variation was considerably smaller than the variation between laboratories. In summary, adjustment of primary comet assay results by reference standards reduces inter-laboratory variation in the level of DNA damage measured by the alkaline version of the comet assay.

An ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay

The increasing use of single cell gel electrophoresis (the comet assay) highlights its popularity as a method for detecting DNA damage, including the use of enzymes for assessment of oxidatively damaged DNA. However, comparison of DNA damage levels between laboratories can be difficult due to differences in assay protocols (e.g. lysis conditions, enzyme treatment, the duration of the alkaline treatment and electrophoresis) and in the end points used for reporting results (e.g. %DNA in tail, arbitrary units, tail moment and tail length). One way to facilitate comparisons is to convert primary comet assay end points to number of lesions/106 bp by calibration with ionizing radiation. The aim of this study was to investigate the inter-laboratory variation in assessment of oxidatively damaged DNA by the comet assay in terms of oxidized purines converted to strand breaks with formamidopyrimidine DNA glycosylase (FPG). Coded samples with DNA oxidation damage induced by treatment with different concentrations of photosensitizer (Ro 19-8022) plus light and calibration samples irradiated with ionizing radiation were distributed to the 10 participating laboratories to measure DNA damage using their own comet assay protocols. Nine of 10 laboratories reported the same ranking of the level of damage in the coded samples. The variation in assessment of oxidatively damaged DNA was largely due to differences in protocols. After conversion of the data to lesions/106 bp using laboratory-specific calibration curves, the variation between the laboratories was reduced. The contribution of the concentration of photosensitizer to the variation in net FPG-sensitive sites increased from 49 to 73%, whereas the inter-laboratory variation decreased. The participating laboratories were successful in finding a dose–response of oxidatively damaged DNA in coded samples, but there remains a need to standardize the protocols to enable direct comparisons between laboratories.

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.

Constitutive expression of bioactivating enzymes in normal human prostate suggests a capability to activate pro‐carcinogens to DNA‐damaging metabolites

The constitutive bioactivating capacity of human prostate may play a role in determining risk of adenocarcinoma developing in this tissue. Expression of candidate enzymes that convert exogenous and/or endogenous agents into reactive DNA‐damaging species would suggest the potential to generate initiating events in prostate cancer (CaP).

Cytotoxic and genotoxic responses of the RTgill-W1 fish cells in combination with the yeast oestrogen screen to determine the sediment quality of Lagos lagoon, Nigeria

Economic advancements in developing countries have seen an increase in urbanisation and industrialisation with a rise in the levels of discharge of effluents and municipal waste into aquatic ecosystems. Unfortunately, aquatic environmental regulations in these countries are often rudimentary and the development of environmental monitoring programmes will help identify ecological risks. As an example, the current study assesses the pollution status of 11 sampling sites in Lagos lagoon, Nigeria. The organic solvent sediment extracts were assessed for cytotoxicity and genotoxicity in rainbow trout gill-W1 cells. The induction of oestrogenic activities using the yeast oestrogen screen was also determined. The sediments were analysed for polycyclic aromatic hydrocarbons (PAHs) and other contaminants (polychlorinated biphenyls, organochlorine and organophosphate pesticides). Only sediments from three sites were cytotoxic at both 25 and 12.5mg eQsed/ml using the Alamar Blue cell viability assay. The alkaline Comet assay showed that all sites caused significant DNA damage at 7 mg eQsed/ml; the extent of the damage was site specific. The measure of oxidative damage to DNA via the formamidopyrimidine DNA-glycosylase-modified Comet assay revealed similar results. Toxicity to yeast cells was observed in extracts from six sites; of the remaining sites, only two exhibited oestrogenic activity. There was no strong consistent relationship between sediment PAH concentrations and the cell toxicity endpoints. The dynamic nature of Lagos lagoon with its tides and freshwater inputs are suggested as factors that make it difficult to link the sources of pollution observed at each site with PAH levels and toxic endpoints. The study has demonstrated that the Comet assay is a sensitive endpoint to identify sediments that possess genotoxic contaminants, and this in vitro bioassay has the potential to be incorporated into an environmental monitoring framework for Lagos lagoon.

DNA-repair measurements by use of the modified comet assay: an inter-laboratory comparison within the European Comet Assay Validation Group (ECVAG)

The measurement of DNA-repair activity by extracts from cells or tissues by means of the single-cell gel electrophoresis (comet) assay has a high potential to become widely used in biomonitoring studies. We assessed the inter-laboratory variation in reported values of DNA-repair activity on substrate cells that had been incubated with Ro19-8022 plus light to generate oxidatively damaged DNA. Eight laboratories assessed the DNA-repair activity of three cell lines (i.e. one epithelial and two fibroblast cell lines), starting with cell pellets or with cell extracts provided by the coordinating laboratory. There was a large inter-laboratory variation, as evidenced by the range in the mean level of repair incisions between the laboratory with the lowest (0.002incisions/10(6)bp) and highest (0.988incisions/10(6)bp) incision activity. Nevertheless, six out of eight laboratories reported the same cell line as having the highest level of DNA-repair activity. The two laboratories that reported discordant results (with another cell line having the highest level of DNA-repair activity) were those that reported to have little experience with the modified comet assay to assess DNA repair. The laboratories were also less consistent in ordering the repair activity of the other two cell lines, probably because the DNA-repair activity by extracts from these cell lines were very similar (on average approximately 60-65% of the cell line with the highest repair capacity). A significant correlation was observed between the repair activity found in the provided and the self-made cell extracts (r=0.71, P<0.001), which indicates that the predominant source for inter-laboratory variation is derived from the incubation of the extract with substrate cells embedded in the gel. Overall, we conclude that the incubation step of cell extracts with the substrate cells can be identified as a major source of inter-laboratory variation in the modified comet assay for base-excision repair.

Refinement of a Methodology for Untargeted Detection of Serum Albumin Adducts in Human Populations

Covalently modified blood proteins (e.g., serum albumin adducts) are increasingly being viewed as potential biomarkers via which the environmental causes of human diseases may be understood. The notion that some (perhaps many) modifications have yet to be discovered has led to the development of untargeted adductomics methods, which attempt to capture entire populations of adducts. One such method is fixed-step selected reaction monitoring (FS-SRM), which analyses distributions of serum albumin adducts via shifts in the mass of a tryptic peptide [Li et al. (2011) Mol. Cell. Proteomics 10, M110.004606]. Working on the basis that FS-SRM might be able to detect biological variation due to environmental factors, we aimed to scale the methodology for use in an epidemiological setting. Development of sample preparation methods led to a batch workflow with increased throughput and provision for quality control. Challenges posed by technical and biological variation were addressed in the processing and interpretation of the data. A pilot study of 20 smokers and 20 never-smokers provided evidence of an effect of smoking on levels of putative serum albumin adducts. Differences between smokers and never-smokers were most apparent in putative adducts with net gains in mass between 105 and 114 Da (relative to unmodified albumin). The findings suggest that our implementation of FS-SRM could be useful for studying other environmental factors with relevance to human health.

The impact of chemotherapeutic drugs on the CYP1A1-catalysed metabolism of the environmental carcinogen benzo[a]pyrene: Effects in human colorectal HCT116 TP53(+/+), TP53(+/−) and TP53(−/−) cells

Polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) can induce cytochrome P450 1A1 (CYP1A1) via a p53-dependent mechanism. The effect of different p53-activating chemotherapeutic drugs on CYP1A1 expression, and the resultant effect on BaP metabolism, was investigated in a panel of isogenic human colorectal HCT116 cells with differing TP53 status. Cells that were TP53(+/+), TP53(+/–) or TP53(–/–) were treated for up to 48 h with 60 μM cisplatin, 50 μM etoposide or 5 μM ellipticine, each of which caused high p53 induction at moderate cytotoxicity (60–80% cell viability). We found that etoposide and ellipticine induced CYP1A1 in TP53(+/+) cells but not in TP53(–/–) cells, demonstrating that the mechanism of CYP1A1 induction is p53-dependent; cisplatin had no such effect. Co-incubation experiments with the drugs and 2.5 μM BaP showed that: (i) etoposide increased CYP1A1 expression in TP53(+/+) cells, and to a lesser extent in TP53(–/–) cells, compared to cells treated with BaP alone; (ii) ellipticine decreased CYP1A1 expression in TP53(+/+) cells in BaP co-incubations; and (iii) cisplatin did not affect BaP-mediated CYP1A1 expression. Further, whereas cisplatin and etoposide had virtually no influence on CYP1A1-catalysed BaP metabolism, ellipticine treatment strongly inhibited BaP bioactivation. Our results indicate that the underlying mechanisms whereby etoposide and ellipticine regulate CYP1A1 expression must be different and may not be linked to p53 activation alone. These results could be relevant for smokers, who are exposed to increased levels of BaP, when prescribing chemotherapeutic drugs. Beside gene-environment interactions, more considerations should be given to potential drug-environment interactions during chemotherapy.