Kim L. O'Neill

The comet assay: a comprehensive review

The comet assay is a sensitive and rapid method for DNA strand break detection in individual cells. Its use has increased significantly in the past few years. This paper is a review of the studies published to date that have made use of the comet assay. The principles of strand break detection using both the alkaline and neutral versions of the technique are discussed, and a basic methodology with currently used variations is presented. Applications in different fields are reviewed and possible future directions of the technique are briefly explored.

Pestacin: a 1,3-dihydro isobenzofuran from Pestalotiopsis microspora possessing antioxidant and antimycotic activities

Abstract Pestalotiopsis microspora, an endophytic fungus native to the rainforest of Papua New Guinea, produces a 1,3-dihydro isobenzofuran. This product, pestacin, is 1,5,7-trisubstituted and exhibits moderate antifungal properties and antioxidant activity 11 times greater than the vitamin E derivative trolox. Antioxidant activity is proposed to arise primarily via cleavage of an unusually reactive C–H bond and, to a lesser extent, through O–H abstraction. Isolation of pestacin was achieved by extraction of culture fluid with methylene chloride followed by silica gel chromatography. Structure was established by X-ray diffraction and 13C and 1H NMR. The X-ray data demonstrate that pestacin occurs naturally as a racemic mixture. A mechanism for post-biosynthetic racemization is proposed.

DNA damage induced by micellar-delivered doxorubicin and ultrasound: comet assay study

To minimize adverse side effects of chemotherapy, we have developed a micellar drug carrier that retains hydrophobic drugs, and then releases the drug by ultrasonic stimulation. This study investigated the DNA damage induced by doxorubicin (DOX) delivered to human leukemia (HL-60) cells from pluronic P-105 micelles with and without the application of ultrasound. The comet assay was used to quantify the amount of DNA damage. No significant DNA damage was observed when the cells were treated with 0.1, 1 and 10 wt% P-105 with or without ultrasound (70 kHz, 1.3 W/cm(2)) for 1 h or for up to 3 h in 10 wt% P-105. However, when cells were incubated with 10 microg/ml free DOX for up to 9 h, DNA damage increased with incubation time (P=0.0011). Exposure of cells to the same concentration of DOX in the presence of 10-wt% P-105 showed no significant DNA damage for up to 9 h of incubation. However, when ultrasound was applied, a rapid and significant increase in DNA damage was observed (P=0.0001). The application of ultrasound causes the release of DOX from micelles or causes the HL-60 cells to take up the micelle encapsulated DOX. Our experiments indicated that the combination of DOX, ultrasound and pluronic P105 causes the largest DNA damage to HL-60 cells. We believe that this technique can be used for controlled drug delivery.

Induction of apoptotic cell DNA fragmentation in human cells after treatment with hyperthermia

The biological significance of apoptosis is becoming increasingly clear. Its relevance in tumor response to treatment as well as recent evidence for its important function as a regulating mechanism in tumorigenesis has also been demonstrated. One of the most prominent biological features of apoptosis is nucleosomal DNA fragmentation. In this communication, we present a study of DNA fragmentation in Raji cells which have been subjected to hyperthermia treatment to induce apoptosis. We found that the induction and onset of fragmentation is swift, and consistent with previous reports that fragmentation must be a rapid event.

Critical parameters influencing hyperthermia-induced apoptosis in human lymphoid cell lines

Brief mild hyperthermia is sufficient to induce apoptosis (programmed cell death) in many cell lines. Here we describe the effects of a number of factors modulating heat shock induced apoptosis outcomes. We report the effects of cell type, heat load, recovery times, cellular growth phase, and protein synthesis on the levels of apoptoses seen in heat stressed cell populations. We observe that a number of cell lines are competent to undergo heat stress induced apoptosis using both the comet assay and cellular and nuclear morphologies. Of the cell lines tested we saw a wide spectrum of sensitivities, ranging from resistant (less than 1% apoptotic after 12 h) to exquisitely sensitive (>95%). By incrementally increasing the heat load from 37–49°C, we observed a gradual increase in apoptosis with a significant change from apoptotic to necrotic death at temperatures beyond 45°C. The kinetics of the apoptotic response to heat shock were also examined. A time dependent increase in apoptotic cell death was seen after initial hyperthermic treatment with most cell types reaching a ‘plateau’ at 18 h. In addition to these parameters we report that growth phase has a strong influence on the number of apoptoses induced as a result of heat stress. Cultured cells, grown to a plateau, undergo apoptosis at a much higher level than similarly treated cells taken during an exponential phase of growth. Finally, we determined the necessity of protein synthesis for apoptotic competency.

Supplementation with fruit and vegetable extracts may decrease DNA damage in the peripheral lymphocytes of an elderly population

Abstract Fruit and vegetable consumption has been heralded for its ability to decrease the overall risk of developing cancer and other diseases. Mounting evidence supports the beneficial nature of antioxidants, carotenoids, and other phytonutrients found in fruits and vegetables. One proposed mechanism of antioxidant protection is the shielding of cellular DNA from oxidative damage and therefore mutations. This may be especially helpful in older populations. We tested the concept that a daily regimen of supplementation with fruit and vegetable extracts (JuicePlus™) would reduce the amount of DNA damage in the peripheral lymphocytes of the elderly. In a blind study, a group of twenty elderly volunteers (mean age=68) were given supplements twice daily for 80 days with blood samples drawn before and after intervention. These samples were compared using the comet assay, a technique that quantifies DNA damage to individual nuclei. Each sample was tested in triplicate, and tail moment data was collected from over 200 comets per sample. Paired t-test analysis revealed a highly significant (p

A comparative evaluation of aflatoxin B1 genotoxicity in fish models using the Comet assay

Aflatoxin B1 (AFB1) is classified as a Group I hepatocarcinogen in humans by the International Agency for Research on Cancer (IARC). The alkaline Comet assay is a simple and rapid method by which DNA damage can be demonstrated as a function of tail moment. The present work is the first to evaluate the genotoxicity of AFB1 in fish using the Comet assay. Two different species of fish were selected as models due to previously established sensitivity to AFB1: rainbow trout (sensitive) and channel catfish (resistant). Fish were i.p. injected with 0.5 mg AFB1/1 ml DMSO/1 kg body weight. The Comet assay was performed after 4 and 24 h on whole blood, liver, and kidney cells of both species. Trout blood and kidney tissue tested displayed significant (p < 0.05) and extensive DNA damage (shown by increased tail moment) after 4 h which then decreased by 24 h. In liver cells, damage progressively increased over time. Conversely, similarly treated catfish showed no elevation in DNA damage over controls at the same doses. These results suggest that the Comet assay is a useful tool for monitoring the genotoxicity of mycotoxins such as AFB1 and for evaluating organ specific effects of these agents in different species.

Thymidine kinase: diagnostic and prognostic potential

Thymidine kinase is a cell cycle-dependent marker that can be detected in the serum of patients diagnosed with many different types of cancer. Serum levels of thymidine kinase have also been shown to reflect the progression of cancer as well as an indication of the efficacy of chemotherapeutic intervention. A new monoclonal antibody assay for thymidine kinase has been developed, which is capable of detecting thymidine kinase in both serum and tumor tissue. Thymidine kinase assay kits should be available at low cost and could serve as an effective low cost test for the detection and progression of many types of human cancer.

Mechanics behind Breast Cancer Prevention - Focus on Obesity, Exercise and Dietary Fat

Cancer prevention is rapidly emerging as a major strategy to reduce cancer mortality. In the field of breast cancer, significant strides have recently been made in the understanding of underlying preventive mechanisms. Currently, three major strategies have been linked to an increase in breast cancer risk: obesity, lack of physical exercise, and high levels of saturated dietary fat. As a result, prevention strategies for breast cancer are usually centered on these lifestyle factors. Unfortunately, there remains controversy regarding epidemiological studies that seek to determine the benefit of these lifestyle changes. We have identified crucial mechanisms that may help clarify these conflicting studies. For example, recent reports with olive oil have demonstrated that it may influence crucial transcription factors and reduce breast tumor aggressiveness by targeting HER2. Similarly, physical exercise reduces sex hormone levels, which may help protect against breast cancer. Obesity promotes tumor cell growth and cell survival through upregulation of leptin and insulin-like growth factors. This review seeks to discuss these underlying mechanisms, and more behind the three major prevention strategies, as a means of understanding how breast cancer can be prevented.

Analysis of nucleotide pools in human lymphoma cells by capillary electrophoresis.

Capillary electrophoresis is applied to determine nucleotide pool levels in human Burkitt lymphoma cells. The analysis was performed on a 65 cm x 50 microns I.D. Ucon-coated column with on-column UV detector. The method requires only nanoliters of sample and a simple sample preparation procedure. Over 12 nucleotides were separated and quantitated with high resolution and reproducibility. The whole capillary electrophoretic separation time was only 35 min. These results demonstrate that capillary electrophoresis provides a useful and easy way to analyze nucleotide pools in cells.