Measuring DNA modifications with the comet assay: a compendium of protocols.

The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.

Hazard Assessment of Benchmark Metal-Based Nanomaterials Through a Set of In Vitro Genotoxicity Assays.

For safety assessment of nanomaterials (NMs), in vitro genotoxicity data based on well-designed experiments is required. Metal-based NMs are amongst the most used in consumer products. In this chapter, we report results for three metal-based NMs, titanium dioxide (NM-100), cerium dioxide (NM-212) and silver (NM-302) in V79 cells, using a set of in vitro genotoxicity assays covering different endpoints: the medium-throughput comet assay and its modified version (with the enzyme formamidopyrimidine DNA glycosylase, Fpg), measuring DNA strand beaks (SBs) and oxidized purines, respectively; the micronucleus (MN) assay, assessing chromosomal damage; and the Hprt gene mutation test. The results generated by this test battery showed that all NMs displayed genotoxic potential. NM-100 induced DNA breaks, DNA oxidation damage and point mutations but not chromosome instability. NM-212 increased the level of DNA oxidation damage, point mutations and increased the MN frequency at the highest concentration tested. NM-302 was moderately cytotoxic and induced gene mutations, but not DNA or chromosome damage. In conclusion, the presented in vitro genotoxicity testing strategy allowed the identification of genotoxic effects caused by three different metal-based NMs, raising concern as to their impact on human health. The results support the use of this in vitro test battery for the genotoxicity assessment of NMs, reducing the use of more expensive, time-consuming and ethically demanding in vivo assays, in compliance with the 3 R's.

Epigenetics in Breast Cancer Therapy-New Strategies and Future Nanomedicine Perspectives.

Epigenetic dysregulation has been recognized as a critical factor contributing to the development of resistance against standard chemotherapy and to breast cancer progression via epithelial-to-mesenchymal transition. Although the efficacy of the first-generation epigenetic drugs (epi-drugs) in solid tumor management has been disappointing, there is an increasing body of evidence showing that epigenome modulation, in synergy with other therapeutic approaches, could play an important role in cancer treatment, reversing acquired therapy resistance. However, the epigenetic therapy of solid malignancies is not straightforward. The emergence of nanotechnologies applied to medicine has brought new opportunities to advance the targeted delivery of epi-drugs while improving their stability and solubility, and minimizing off-target effects. Furthermore, the omics technologies, as powerful molecular epidemiology screening tools, enable new diagnostic and prognostic epigenetic biomarker identification, allowing for patient stratification and tailored management. In combination with new-generation epi-drugs, nanomedicine can help to overcome low therapeutic efficacy in treatment-resistant tumors. This review provides an overview of ongoing clinical trials focusing on combination therapies employing epi-drugs for breast cancer treatment and summarizes the latest nano-based targeted delivery approaches for epi-drugs. Moreover, it highlights the current limitations and obstacles associated with applying these experimental strategies in the clinics.

DNA damage in blood cells in relation to chemotherapy and nutritional status in colorectal cancer patients-A pilot study.

DNA damage can be considered as a biomarker for toxicity and response to chemotherapy. It is not known whether the chemotherapy-induced genotoxicity is associated with malnutrition. In this pilot study, we assess genotoxicity by means of DNA damage in patients with lymph-node positive colorectal cancer (CRC) and explore associations with chemotherapy treatment and nutritional status. DNA damage was compared between patients receiving chemotherapy (n = 24) and those not receiving chemotherapy (n = 20). DNA damage was measured in frozen whole blood by the comet assay. Associations between DNA damage and various indicators of malnutrition were also explored, including Patient-Generated Subjective Global Assessment (PG-SGA), bioelectrical impedance analysis (BIA) and anthropometric measurements, using multiple linear regression models. Patients on chemotherapy have higher levels of DNA damage in blood cells than patients not receiving chemotherapy (median of 16.9 and 7.9% tail DNA respectively, p = 0.001). The moderately malnourished patients (PG-SGA category B), representing 41% of the patients, have higher levels of cellular DNA damage than patients with good nutritional status (mean difference of 7.5% tail DNA, p = 0.033). In conclusion, adjuvant chemotherapy and malnutrition are both associated with increased levels of DNA damage in blood cells of CRC patients. Carefully controlled longitudinal studies or randomized controlled trials should be performed to determine whether good nutritional status may protect against chemotherapy-induced genotoxicity and enhance compliance to therapy in CRC patients.

Coffee and oxidative stress: a human intervention study.

PURPOSE: Coffee is known to contain phytochemicals with antioxidant potential. The aim of this study was to investigate possible antioxidant effects of coffee in healthy human volunteers. METHODS: A placebo-controlled intervention trial was carried out on 160 healthy human subjects, randomised into three groups, receiving 3 or 5 cups of study coffee or water per day, for 8 weeks. Blood samples were taken before, during, and after the intervention. Serum was used for analysis of blood lipids and standard clinical chemistry analytes. Peripheral blood mononuclear cells were isolated, and DNA damage (strand breaks and oxidised bases) was measured with the comet assay. The lipid oxidation product isoprostane 8-iso-PGF2α was assayed in urine samples by LC-MS/MS. RESULTS: There was no significant effect of coffee consumption on the markers of oxidation of DNA and lipids. Creatinine (in serum) increased by a few per cent in all groups, and the liver enzyme γ-glutamyl transaminase was significantly elevated in serum in the 5 cups/day group. Other clinical markers (including glucose and insulin), cholesterol, triacylglycerides, and inflammatory markers were unchanged. There was no effect of coffee on blood pressure. CONCLUSION: In a carefully controlled clinical trial with healthy subjects, up to 5 cups of coffee per day had no detectable effect, either beneficial or harmful, on human health.

High throughput toxicity screening and intracellular detection of nanomaterials.

With the growing numbers of nanomaterials (NMs), there is a great demand for rapid and reliable ways of testing NM safety-preferably using in vitro approaches, to avoid the ethical dilemmas associated with animal research. Data are needed for developing intelligent testing strategies for risk assessment of NMs, based on grouping and read-across approaches. The adoption of high throughput screening (HTS) and high content analysis (HCA) for NM toxicity testing allows the testing of numerous materials at different concentrations and on different types of cells, reduces the effect of inter-experimental variation, and makes substantial savings in time and cost. HTS/HCA approaches facilitate the classification of key biological indicators of NM-cell interactions. Validation of in vitro HTS tests is required, taking account of relevance to in vivo results. HTS/HCA approaches are needed to assess dose- and time-dependent toxicity, allowing prediction of in vivo adverse effects. Several HTS/HCA methods are being validated and applied for NM testing in the FP7 project NANoREG, including Label-free cellular screening of NM uptake, HCA, High throughput flow cytometry, Impedance-based monitoring, Multiplex analysis of secreted products, and genotoxicity methods-namely High throughput comet assay, High throughput in vitro micronucleus assay, and γH2AX assay. There are several technical challenges with HTS/HCA for NM testing, as toxicity screening needs to be coupled with characterization of NMs in exposure medium prior to the test; possible interference of NMs with HTS/HCA techniques is another concern. Advantages and challenges of HTS/HCA approaches in NM safety are discussed. WIREs Nanomed Nanobiotechnol 2017, 9:e1413. doi: 10.1002/wnan.1413 For further resources related to this article, please visit the WIREs website.