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Objectives: To compare the genotoxic effects of desflurane and propofol using comet assay in patients undergoing elective discectomy surgery. Methods: This was a randomized controlled study. Patients who underwent elective lumbar discectomy under general anesthesia with propofol or desflurane were included in the study. Venous blood samples were obtained at 4 different time points: 5 minutes before anesthesia induction (T1), 2 hours after the start of anesthesia (T2), the first day after surgery (T3), and the fifth day following surgery (T4). Deoxyribonucleic acid damage in lymphocytes was assessed via the comet assay. Results: A total of 30 patients, 15 in each group, were included in the analysis. The groups were similar in terms of age and gender distribution. There were no significant differences in demographics, duration of surgery, total remifentanil consumption, and total rocuronium bromide consumption. The comet assay revealed that head length, head intensity, tail intensity, tail moment at T1 were similar in the desflurane and propofol groups. Head length, tail length and tail moment measured in the desflurane group at T4 were significantly higher compared to the propofol group. Tail lengths of the desflurane group at T1, T2 and T3 were significantly higher than the corresponding values in the propofol group. Conclusion: Propofol and desflurane do not appear to induce DNA damage in lymphocytes. However, when the quantitative data were compared, it was determined that propofol had relatively lower genotoxic potential than desflurane. ClinicalTrials.gov Reg. No.: NCT05185167 Keywords: genotoxicity, propofol, desflurane, comet assay, lymphocyte doi: 10.15537/smj.2024.45.5.20240077 [phrase omitted]

The comet assay is a widely used test for the detection of DNA damage and repair activity. However, there are interlaboratory differences in reported levels of baseline and induced damage in the same experimental systems. These differences may be attributed to protocol differences, although it is difficult to identify the relevant conditions because detailed comet assay procedures are not always published. Here, we present a Consensus Statement for the Minimum Information for Reporting Comet Assay (MIRCA) providing recommendations for describing comet assay conditions and results. These recommendations differentiate between ‘desirable’ and ‘essential’ information: ‘essential’ information refers to the precise details that are necessary to assess the quality of the experimental work, whereas ‘desirable’ information relates to technical issues that might be encountered when repeating the experiments. Adherence to MIRCA recommendations should ensure that comet assay results can be easily interpreted and independently verified by other researchers.

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.

Apoptosis has been recognized as a type of programmed cell death connected with characteristic morphological and biochemical changes in cells. This programmed cell death plays an important role in the genesis of a number of physiological and pathological processes. Thus, it can be very important to detect the signs of apoptosis in a study of cellular metabolism. The present paper provides an overview of methods often being used for detecting DNA fragmentation as one of the most specific findings in apoptosis. To date, three routine assays have been developed for detecting DNA fragmentation: DNA ladder assay, TUNEL assay, and comet assay. All these methods differ in their principles for detecting DNA fragmentation. DNA ladder assay detects the characteristic “DNA ladder” pattern formed during internucleosomal cleavage of DNA. Terminal deoxynUcleotidyl transferase Nick-End Labeling (TUNEL) assay detects DNA strand breaks using terminal deoxynucleotidyl transferase catalyzing attachment of modified deoxynucleotides on the DNA strand breaks. Comet assay can be used for detecting nucleus breakdown producing single/double-strand DNA breaks. The aim of this review is to describe the present knowledge on these three methods, including optimized approaches, techniques, and limitations.

Reports on the effects of carotenoids are conflicting. The present paper examines similarities and differences from contiguous studies in vitro and in vivo. Single-cell gel electrophoresis was used to measure the frequency of single-strand breaks (SSB) in the cell line MOLT-17 (as a model system) and human peripheral blood lymphocytes (PBL). MOLT-17 cells were supplemented with β-carotene, lutein or lycopene at a range of concentrations (0·00–8·00 μmol/l) using a liposome delivery method. Uptake was dose-dependent. β-Carotene concentration in the media had no effect on SSB in control cells, but incubation with lycopene or lutein (>2·00 μmol/l) increased the numbers of SSB in control cells. MOLT-17 DNA was less susceptible to oxidative damage (100 μmol H2O2/l, 5 min, 4 °C) following incubation with carotenoids between 0·50 and 1·00 μmol/l; at >1·00 μmol/l the effects were ambiguous. Apparently healthy male volunteers supplemented their habitual diets with lutein, β-carotene or lycopene (natural isolate capsules, 15 mg/d, 4 weeks) in three independent studies, raising plasma concentrations to different extents. Lycopene and lutein had no effect on SSB in control PBL or following oxidative challenge. However, increased plasma β-carotene was associated with more SSB in control cells whilst PBL DNA resistance to oxidative damage ex vivo was unaffected. These results suggest that the carotenoids are capable of exerting two overlapping but distinct effects: antioxidant protection by scavenging DNA-damaging free radicals and modulation of DNA repair mechanisms.

The comet assay (single cell gel electrophoresis) is the most common method for measuring DNA damage in eukaryotic cells or disaggregated tissues. The assay depends on the relaxation of supercoiled DNA in agarose-embedded nucleoids (the residual bodies remaining after lysis of cells with detergent and high salt), which allows the DNA to be drawn out towards the anode under electrophoresis, forming comet-like images as seen under fluorescence microscopy. The relative amount of DNA in the comet tail indicates DNA break frequency. The assay has been modified to detect various base alterations, by including digestion of nucleoids with a lesion-specific endonuclease. We describe here recent technical developments, theoretical aspects, limitations as well as advantages of the assay, and modifications to measure cellular antioxidant status and different types of DNA repair. We briefly describe the applications of this method in genotoxicity testing, human biomonitoring, and ecogenotoxicology.

Methods for quantifying DNA damage, as well as repair of that damage, in a high-throughput format are lacking. Single cell gel electrophoresis (SCGE; comet assay) is a widely-used method due to its technical simplicity and sensitivity, but the standard comet assay has limitations in reproducibility and throughput. We have advanced the SCGE assay by creating a 96-well hardware platform coupled with dedicated data processing software (CometChip Platform). Based on the original cometchip approach, the CometChip Platform increases capacity ~200 times over the traditional slide-based SCGE protocol, with excellent reproducibility. We tested this platform in several applications, demonstrating a broad range of potential uses including the routine identification of DNA damaging agents, using a 74-compound library provided by the National Toxicology Program. Additionally, we demonstrated how this tool can be used to evaluate human populations by analysis of peripheral blood mononuclear cells to characterize susceptibility to genotoxic exposures, with implications for epidemiological studies. In summary, we demonstrated a high level of reproducibility and quantitative capacity for the CometChip Platform, making it suitable for high-throughput screening to identify and characterize genotoxic agents in large compound libraries, as well as for human epidemiological studies of genetic diversity relating to DNA damage and repair.

Context: This study was carried out on the assumption that oral mucosal cells might show DNA damage in oral squamous cell carcinoma (OSCC). Aims: To evaluate the extent of DNA damage in oral smears of patients with OSCC and determine correlation if any of the extent of DNA damage to TNM staging of oral cancer. Settings and design: A randomized controlled study at a regional cancer centre was designed for this project. Smears were taken from lesion proper of 30 patients with OSCC and from the buccal mucosa of 30 normal healthy volunteers. Materials and methods: Collected cells were centrifuged and single-cell gel electrophoresis (SCGE) assay was performed. DNA damage was visualized under a fluorescent microscope. Statistical analysis used : Mean DNA damage levels of both the groups were measured and statistically analyzed with students' test. The extent of DNA damage was correlated with the TNM stages by employing the one way ANOVA 'F' technique. Results: High statistical significance (P < 0.0001) was found in DNA damage levels between control and study groups. A stepwise increase in DNA damage levels with high statistical significance (P < 0.005) was also found between all the TNM stages. Conclusions: Statistically significant increased DNA damage levels in OSCC patients and their correlation to clinical staging suggest that comet assay may be used effectively to assess the prognosis of OSCC.

Nanoscale particles can have impressive and useful characteristics, but the same properties may be problematic for human health. From this perspective it is critical to assess the ability of nanoparticles to cause DNA damage. This review focuses on the use of the comet assay in nanotoxicology research. In the alkaline version of the assay, DNA strand breaks and alkali-labile sites are detected and oxidatively damaged DNA can be analyzed using the enzyme formamidopyrimidine glycosylase. The article reviews studies that have used the comet assay to investigate the toxicity of manufactured nanoparticles. It is shown that at least 46 cellular in vitro studies and several in vivo studies have used the comet assay and that the majority of the nanoparticles tested cause DNA strand breaks or oxidative DNA lesions. This is not surprising considering the sensitivity of the method and the reactivity of many nanomaterials. Interactions between the particles and the assay cannot be totally excluded and need further consideration. It is concluded that studies including several particle types, to enable the assessment of their relative potency, are valuable as are studies focusing both on comet assay end points and mutagenicity. Finally, the article discusses the potential future use of the comet assay in human biomonitoring studies, which could provide valuable information for hazard identification of nanoparticles.

With a direct link to cancer, aging, and heritable diseases as well as a critical role in cancer treatment, the importance of DNA damage is well-established. The intense interest in DNA damage in applications ranging from epidemiology to drug development drives an urgent need for robust, high throughput, and inexpensive tools for objective, quantitative DNA damage analysis. We have developed a simple method for high throughput DNA damage measurements that provides information on multiple lesions and pathways. Our method utilizes single cells captured by gravity into a microwell array with DNA damage revealed morphologically by gel electrophoresis. Spatial encoding enables simultaneous assays of multiple experimental conditions performed in parallel with fully automated analysis. This method also enables novel functionalities, including multiplexed labeling for parallel single cell assays, as well as DNA damage measurement in cell aggregates. We have also developed 24- and 96-well versions, which are applicable to high throughput screening. Using this platform, we have quantified DNA repair capacities of individuals with different genetic backgrounds, and compared the efficacy of potential cancer chemotherapeutics as inhibitors of a critical DNA repair enzyme, human AP endonuclease. This platform enables high throughput assessment of multiple DNA repair pathways and subpathways in parallel, thus enabling new strategies for drug discovery, genotoxicity testing, and environmental health.