| 1. |
- Bivehed, Erik, et al.
(författare)
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DNA integrity under alkaline conditions : An investigation of factors affecting the comet assay
- 2023
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Ingår i: Mutation research. Genetic toxicology and environmental mutagenesis. - : Elsevier. - 1383-5718 .- 1879-3592. ; 891
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Tidskriftsartikel (refereegranskat)abstract
- The effect of pH on DNA integrity was assessed using a three-step approach. The comet assay was used on a whole genome level, with three different protocols: neutral (no alkaline unwinding), flash (pH 12.5 with 2.5 min unwinding), and the conventional alkaline protocol (pH>13 with 40 min unwinding). Real-time quantitative PCR (RT-qPCR) was then used to study the isolated DNA, revealing that gene amplification decreased with increasing pH, indicating DNA degradation. Specially designed molecular beacons were used to examine DNA at the molecular level, with or without alkali-labile site (ALS) insertions. At pH 12.5, fluorescence in the hairpins with ALS started to increase after 30 min, while at pH> 13, this increase was already observed after 5 min, indicating a significant increase in DNA strand breaks. Liquid chromatography analysis was also used, demonstrating that the hairpins remained intact up to pH 10, even after 1 h exposure, whereas, at pH 12.5, partial conversion into strand breaks occurred after 30 min. At pH> 13, the hairpins were almost completely degraded after 30 min. The flash protocol effectively detects DNA single- and double-strand breaks and identified these damages after 2.5 min of alkaline treatment at pH 12.5. When the hairpins were exposed to pH 12.5 for 60 min, ALS were converted to strand breaks, demonstrating the sensitivity of this approach to detect changes in DNA structure. These findings indicate that pH poses a substantial risk to DNA integrity, leading to significantly higher background levels of DNA damage compared to conditions closer to neutrality. Our study demonstrates the importance of understanding the influence of pH on DNA stability and provides insights into risks associated with alkaline environments, especially at pH> 13.
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| 2. |
- Bivehed, Erik, et al.
(författare)
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Evaluation of Potential DNA-Damaging Effects of Nitenpyram and Imidacloprid in Human U937-Cells Using a New Statistical Approach to Analyse Comet Data
- 2020
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Ingår i: Exposure and Health. - : Springer Nature. - 2451-9766 .- 2451-9685. ; 12:3, s. 547-554
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Tidskriftsartikel (refereegranskat)abstract
- Even if the two neonicotinoids nitenpyram and imidacloprid have been considered safe for humans, their potential genotoxicity still remains a matter of discussion. The DNA-damaging effects of these two compounds were therefore evaluated in a lymphoma cell line of human origin (U-937) using the comet assay after 3-h exposure to up to 50 mu M, with or without metabolic activation using S9 from human liver. The comet data were analysed using a traditional one-way ANOVA after pooling the data on cellular level, and a new alternative approach we have called Uppsala Comet Data Analysis Strategy (UCDAS). UCDAS is a proportional odds model tailored to continuous outcomes, taking the number of pooled cultures, slides and cells into consideration in the same analysis. To the best of our knowledge, the UCDAS approach when analysing comet data has never been presented before. Without metabolic activation, no increase in DNA damage was observed in the neonicotinoide-exposed cells. Nitenpyram was also without DNA-damaging effects when S9 was added. However, in the presence of S9, imidacloprid was found to increase the level of DNA damage. Whereas the ANOVA showed an increase (P<0.001) both at 5 and 50 mu M, UCDAS showed an increase only at the lowest concentration (P<0.001). Based on these findings, the two neonicotinoids seem to be of little concern when it comes to their potential genotoxicity. However, since the U-937 cells were rather resistant to our positive controls, they may not be the best cells to use when evaluating potential genotoxicity of chemicals.
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| 3. |
- Bivehed, Erik, 1990-
(författare)
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Evolving the Methodology for Detection of Primary DNA Damage : Development, adaptation and assessment of the single cell gel electrophoresis (comet) assay
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Deoxyribonucleic acid (DNA) is one of the most important molecules in nature. It is the fundamental carrier of evolutionary information and constitutes the genetic blueprint of all living organisms. Being the sole source of information, it is vital for the cell to transmit the correct genetic information from generation to generation. DNA damage is a critical precursor to cancer development, highlighting the need for tests to predict genotoxicity and mutagenicity of various agents, including pharmaceuticals and environmental factors. This thesis focuses on enhancing the single cell gel electrophoresis (comet assay) for assessing primary DNA damage.The work was concentrated around several fundamental aspects of the methodology where a novel statistical approach, Uppsala Comet Data Analysis Strategy (UCDAS), was developed for data evaluation. A proportional odds model tailored to continuous outcomes was used, accommodating the experimental design's hierarchy, large zero values, and avoiding data transformation. A revisit of the formulation of the electrophoresis medium led to the introduction of a low conductive lithium hydroxide-based solution, enabling higher field strengths, significantly reducing runtimes and increasing sensitivity compared to the conventional comet assay. A lot of work was done on the investigation of the pH's impact on DNA integrity, revealing elevated background DNA damage at higher pH levels. Extended unwinding at pH >13, typical of the most commonly used versions of alkaline comet assays, jeopardizes the integrity of DNA, resulting in greater background DNA damage than at lower pH values. The study underscores pH's significance for DNA stability, highlighting risks associated with extremely alkaline conditions.A new method was developed, the Polymerase Assisted DNA Damage Assay (PADDA), to label and quantify single- and double-strand DNA breaks selectively in comet heads and tails after exposure to established DNA-damaging agents. This approach also allowed detection of DNA damage inside comet heads, an ability lacking in traditional comet assays.In conclusion, this research enhances DNA damage assessment methodologies, introducing new statistical innovations, novel electrophoresis mediums, and a novel technique for the selective detection and quantification of single- and double-strand breaks. These advancements deepen our understanding of DNA damage's complexities and underscore the crucial role of pH in influencing DNA stability and its implications for genotoxicity assessment.
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| 4. |
- Bivehed, Erik, et al.
(författare)
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Flash-comet : Significantly improved speed and sensitivity of the comet assay through the introduction of lithium-based solutions and a more gentle lysis
- 2020
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Ingår i: Mutation research. Genetic toxicology and environmental mutagenesis. - : ELSEVIER. - 1383-5718 .- 1879-3592. ; 858
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Tidskriftsartikel (refereegranskat)abstract
- Evaluation of primary DNA-damage is one way to identify potential genotoxic agents and for this purpose the Comet assay has, for the last decades, been used to monitor DNA single strand and double strand breaks in individual cells. Various attempts have been made to modify the different steps in the in vitro protocol for the Comet assay in order to improve its sensitivity. However, to the best of our knowledge, nobody has tried to replace the traditionally used NaOH-based electrophoresis solution (pH > 13), with another type of solution. In the present paper, using TK-6 cells exposed to different concentrations of H2O2 or ionizing radiation, we present evidence clearly showing that a low-conductive LiOH-based electrophoresis solution at pH 12.5, and a more gentle lysis procedure, significantly improved both the speed and sensitivity of the assay. The new approach, which we call the Flash-comet, is based on a lysis buffer at pH 8.5, an unwinding time of 2.5 min in a LiOH solution without EDTA at pH 12.5, and an electrophoresis time of 1 min at 150 V (5 V/cm) using the same solution.
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| 5. |
- Bivehed, Erik, et al.
(författare)
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Flash-comet assay
- 2020
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Ingår i: MethodsX. - : ELSEVIER. - 1258-780X .- 2215-0161. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- In the present paper, we present a substantially revised protocol of the widely used SCGE assay performed under alkaline conditions. In our updated version of the comet assay, which we call the Flash-comet, LiOH is used instead of NaOH during the unwinding and electrophoresis. This allows a higher voltage during the electrophoresis (5 V/cm instead of 0.7 V/cm), making it possible to reduce the unwinding time from 20 to 40 to 2.5 min, and the electrophoresis time from 10 to 20 to 1 min. Still, the Flash-comet was found to detect DNA strand breaks and alkali-labile sites with a higher degree of sensitivity than the conventional protocol in cells that had been exposed to H2O2 or ionizing radiation. In order to prevent alkaline hydrolysis of DNA, the wash and lysis solutions have been modified in the Flash-comet protocol. By using an alkaline LiOH-based medium, the Flash-comet allows for much shorter times for both unwinding and electrophoresis than the conventional comet assay without compromising the sensitivity. The reduced run-times of the unwinding and electrophoresis steps in the Flash-comet should also reduce the risk of laboratory-induced alkaline hydrolysis of DNA when evaluating the potential DNA-damaging effects of different types of xenobiotics.
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| 6. |
- Bivehed, Erik, et al.
(författare)
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Region-specific bioconversion of dynorphin neuropeptide detected by in situ histochemistry and MALDI imaging mass spectrometry
- 2017
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Ingår i: Peptides. - : Elsevier BV. - 0196-9781 .- 1873-5169. ; 87, s. 20-27
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Tidskriftsartikel (refereegranskat)abstract
- Brain region-specific expression of proteolytic enzymes can control the biological activity of endogenous neuropeptides and has recently been targeted for the development of novel drugs, for neuropathic pain, cancer, and Parkinson's disease. Rapid and sensitive analytical methods to profile modulators of enzymatic activity are important for finding effective inhibitors with high therapeutic value. Combination of in situ enzyme histochemistry with MALDI imaging mass spectrometry allowed developing a highly sensitive method for analysis of brain-area specific neuropeptide conversion of synthetic and endogenous neuropeptides, and for selection of peptidase inhibitors that differentially target conversion enzymes at specific anatomical sites. Conversion and degradation products of Dynorphin B as model neuropeptide and effects of peptidase inhibitors applied to native brain tissue sections were analyzed at different brain locations. Synthetic dynorphin B (2 pmol) was found to be converted to the N-terminal fragments on brain sections whereas fewer C-terminal fragments were detected. N-ethylmaleimide (NEM), a non-selective inhibitor of cysteine peptidases, almost completely blocked the conversion of dynorphin B to dynorphin B(1-6; Leu-Enk-Arg), (1-9), (2-13), and (7-13). Proteinase inhibitor cocktail, and also incubation with acetic acid displayed similar results. Bioconversion of synthetic dynorphin B was region-specific producing dynorphin B(1-7) in the cortex and dynorphin B (2-13) in the striatum. Enzyme inhibitors showed region-and enzyme-specific inhibition of dynorphin bioconversion. Both phosphoramidon (inhibitor of the known dynorphin converting enzyme neprilysin) and opiorphin (inhibitor of neprilysin and aminopeptidase N) blocked cortical bioconversion to dynorphin B(1-7), wheras only opiorphin blocked striatal bioconversion to dynorphin B(2-13). This method may impact the development of novel therapies with aim to strengthen the effects of endogenous neuropeptides under pathological conditions such as chronic pain. Combining histochemistry and MALDI imaging MS is a powerful and sensitive tool for the study of inhibition of enzyme activity directly in native tissue sections. (C) 2016 The Authors. Published by Elsevier Inc.
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| 7. |
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| 8. |
- Bivehed, Erik, et al.
(författare)
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Visualizing DNA single- and double-strand breaks in the Flash comet assay by DNA polymerase-assisted end-labelling
- 2024
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 52:4
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Tidskriftsartikel (refereegranskat)abstract
- In the comet assay, tails are formed after single-cell gel electrophoresis if the cells have been exposed to genotoxic agents. These tails include a mixture of both DNA single-strand breaks (SSBs) and double-strand breaks (DSBs). However, these two types of strand breaks cannot be distinguished using comet assay protocols with conventional DNA stains. Since DSBs are more problematic for the cells, it would be useful if the SSBs and DSBs could be differentially identified in the same comet. In order to be able to distinguish between SSBs and DSBs, we designed a protocol for polymerase-assisted DNA damage analysis (PADDA) to be used in combination with the Flash comet protocol, or on fixed cells. By using DNA polymerase I to label SSBs and terminal deoxynucleotidyl transferase to label DSBs with fluorophore-labelled nucleotides. Herein, TK6-cells or HaCat cells were exposed to either hydrogen peroxide (H2O2), ionising radiation (X-rays) or DNA cutting enzymes, and then subjected to a comet protocol followed by PADDA. PADDA offers a wider detection range, unveiling previously undetected DNA strand breaks. Graphical Abstract
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