Fast Halo Assay Research Articles

Genotoxic and Anti-Migratory Effects of Camptothecin Combined with Celastrol or Resveratrol in Metastatic and Stem-like Cells of Colon Cancer.

Background: Colorectal cancer is one of the leading and most lethal neoplasms. Standard chemotherapy is ineffective, especially in metastatic cancer, and does not target cancer stem cells. A promising approach to improve cancer treatment is the combination therapy of standard cytostatic drugs with natural compounds. Several plant-derived compounds have been proven to possess anticancer properties, including the induction of apoptosis and inhibition of cancer invasion. This study was focused on investigating in vitro the combination of camptothecin (CPT) with celastrol (CEL) or resveratrol (RSV) as a potential strategy to target metastatic (LOVO) and stem-like (LOVO/DX) colon cancer cells. Methods: The genotoxic effects that drive cancer cells into death-inducing pathways and the ability to inhibit the migratory properties of cancer cells were evaluated. The γH2AX+ assay and Fast-Halo Assay (FHA) were used to evaluate genotoxic effects, the annexin-V apoptosis assay to rate the level of apoptosis, and the scratch test to assess antimigratory capacity. Results: The results showed that both combinations CPT-CEL and CPT-RSV improve general genotoxicity of CPT alone on metastatic cells and CSCs. However, the assessment of specific double-stranded breaks (DSBs) indicated a better efficacy of the CPT-CEL combination on LOVO cells and CPT-RSV in LOVO/DX cells. Interestingly, the combinations CPT-CEL and CPT-RSV did not improve the pro-apoptotic effect of CPT alone, with both LOVO and LOVO/DX cells suggesting activation of different cell death mechanisms. Furthermore, it was found that the combinations of CPT-CEL and CPT-RSV improve the inhibitory effect of camptothecin on cell migration. Conclusions: These findings suggest the potential utility of combining camptothecin with celastrol or resveratrol in the treatment of colon cancer, including more aggressive forms of the disease. So far, no studies evaluating the effects of combinations of these compounds have been published in the available medical databases.

FACT inhibitor CBL0137, administered in an optimized schedule, potentiates radiation therapy for glioblastoma by suppressing DNA damage repair.

Standard-of-care for glioblastoma remains surgical debulking followed by temozolomide and radiation. However, many tumors become radio-resistant while radiation damages surrounding brain tissue. Novel therapies are needed to increase the effectiveness of radiation and reduce the required radiation dose. Drug candidate CBL0137 is efficacious against glioblastoma by inhibiting histone chaperone FACT, known to be involved in DNA damage repair. We investigated the combination of CBL0137 and radiation on glioblastoma. In vitro, we combined CBL0137 with radiation on U87MG and A1207 glioblastoma cells using the clonogenic assay to evaluate the response to several treatment regimens, and the Fast Halo Assay to examine DNA repair. In vivo, we used the optimum combination treatment regimen to evaluate the response of orthotopic tumors in nude mice. In vitro, the combination of CBL0137 and radiation is superior to either alone and administering CBL0137 two hours prior to radiation, having the drug present during and for a prolonged period post-radiation, is an optimal schedule. CBL0137 inhibits DNA damage repair following radiation and affects the subcellular distribution of histone chaperone ATRX, a molecule involved in DNA repair. In vivo, one dose of CBL0137 is efficacious and the combination of CBL0137 with radiation increases median survival over either monotherapy. CBL0137 is most effective with radiation for glioblastoma when present at the time of radiation, immediately after and for a prolonged period post-radiation, by inhibiting DNA repair caused by radiation. The combination leads to increased survival making it attractive as a dual therapy.

FACT inhibitor CBL0137, administered in an optimized schedule, potentiates radiation therapy for glioblastoma by suppressing DNA damage repair.

Standard-of-care for glioblastoma remains surgical debulking followed by temozolomide and radiation. However, many tumors become radio-resistant while radiation damages surrounding brain tissue. Novel therapies are needed to increase the effectiveness of radiation and reduce the required radiation dose. Drug candidate CBL0137 is efficacious against glioblastoma by inhibiting histone chaperone FACT, known to be involved in DNA damage repair. We investigated the combination of CBL0137 and radiation on glioblastoma. In vitro, we combined CBL0137 with radiation on U87MG and A1207 glioblastoma cells using the clonogenic assay to evaluate the response to several treatment regimens, and the Fast Halo Assay to examine DNA repair. In vivo, we used the optimum combination treatment regimen to evaluate the response of orthotopic tumors in nude mice. In vitro, the combination of CBL0137 and radiation is superior to either alone and administering CBL0137 two hours prior to radiation, having the drug present during and for a prolonged period post-radiation, is an optimal schedule. CBL0137 inhibits DNA damage repair following radiation and affects the subcellular distribution of histone chaperone ATRX, a molecule involved in DNA repair. In vivo, one dose of CBL0137 is efficacious and the combination of CBL0137 with radiation increases median survival over either monotherapy. CBL0137 is most effective with radiation for glioblastoma when present at the time of radiation, immediately after and for a prolonged period post-radiation, by inhibiting DNA repair caused by radiation. The combination leads to increased survival making it attractive as a dual therapy.

In vitro cytotoxic and genotoxic effects of Phyllanthus niruri extract loaded chitosan nanoparticles in TM4 cells and their influence on spermatogenesis

Purpose: This paper introduces a complete in vitro investigation of cytotoxic and genotoxic effects of Phyllanthus niruri extract loaded chitosan nanoparticles (PNNP) on mouse Sertoli cell line (TM4), as well as their impact on spermatogenesis. Methods: Chitosan nanoparticles (ChNP) and PNNP were prepared using an ionic gelation process, while their cytotoxicity on TM4 cells was assessed using the Cell Counting Kit-8 (CCK-8) assay. Comet and fast halo assays were used to quantify single-strand DNA breaks in TM4 cells. To detect changes in cell morphology during apoptosis, nuclear staining with Hoechst 33342 was performed. An immunofluorescence assay was employed to examine the expression level of proteins connexin 43 and Claudin 11 in TM4 cells after exposure to PNNP concentration of 125 µg/mL. Results: The synthesized PNNP had a size of 170.6 nm, a polydispersity index of 0.269, a zeta potential of +37.8 mV, and a good entrapment efficiency of 71.0%. Encapsulation of Phyllanthus niruri into ChNP induced DNA damage in TM4 cells as determined by alkaline comet and fast halo assay (FHA). Additionally, it stimulated apoptosis, as determined by changes in cell morphology by Hoechst 33342 staining. There was significant down regulation of blood-testis barrier (BTB) proteins in TM4 cells after exposure to PNNP which could compromise the integrity of BTB and subsequently disrupt spermatogenesis process in male. Conclusion: Our investigation confirms the cytotoxic and genotoxic effects of PNNP in TM4 cells, which could lead to spermatogenesis disruption and male infertility.

Detection of DNA Damage in Hematopoietic Stem Cells.

Single-cell gel electrophoresis (SCGE or Comet assay) and the Fast Halo assay, also known as the Halo assay, are powerful tools to generate DNA damage measurements with single-cell resolution. Though these techniques are prone to have variability, they can be robust tools for quantifying DNA damage when planned and executed carefully. Here, we present both assays and highlight each technique's advantages and challenges in measuring DNA damage in cells with limiting cell number, such as hematopoietic stem cells (HSCs). The Comet assay is highly sensitive at the cost of increased variability. The Halo assay attenuates some of the effects of variability present in the Comet assay but does not eliminate them entirely and is less sensitive. Overall, the Comet and Halo assays are powerful means of directly measuring DNA damage. We recommend the below methods for detecting damage in hematopoietic stem cells, but the methods can easily be adjusted for measuring damage in any type of single cells in suspension.

PARP Inhibition with Talazoparib Enhances DNA Damage and Anti-Leukemic Activity of Venetoclax in Preclinical Human Acute Myeloid Leukemia (AML) Models

Background: BCL-2 is an anti-apoptotic protein associated with chemotherapy resistance and survival of acute myeloid leukemia (AML) cells following cell stress or DNA damage. Venetoclax (Ven) is a BH3-mimetic that competitively binds to BCL-2, allowing BH3 proteins to bind to pro-apoptotic proteins (BAX, BAK) and induce cell death. The combination of Ven with Azacitidine (Aza), is now standard of care therapy for older patients with AML. Poly (ADP-ribose) polymerase (PARP) inhibitors prevent the repair of single stranded DNA breaks by blocking the nicotinamide adenine dinucleotide (NAD) catalytic domain of the PARP protein and preventing dissociation of PARP from the DNA (termed PARP trapping). The PARP inhibitor, talazoparib (Tala) displays 10,000-fold increased PARP trapping as compared to other clinical PARP inhibitor agents in tumor models. We hypothesized that Tala could enhance the anti-leukemic activity of Ven on AML cells specifically by increasing DNA damage.Methods: Human AML (Molm13) cells were continuously exposed to increasing doses of Ven (1nM - 10mM), Tala (1nM - 10mM), and Aza (0.1-5mM) alone and in combination. Synergy reports were generated using CompuSyn software. Cell viability was measured using a WST colorimetric assay. Apoptosis (annexin /PI), DNA damage (pH2AX), and cell cycle arrest (propidium iodide) were quantified by flow cytometric analysis. DNA damage was also evaluated using the Fast-Halo assay. In vivo efficacy was assessed in NSG mice systematically engrafted with stably luciferase transfected Molm13 BLIV cells via tail vein injection. Cohorts of mice (5-10) were treated with vehicle, Ven (100 mg/kg given 5 days/week for 3 weeks), Tala (0.33 mg/kg for 5 days/week over 3 weeks), Ven + Tala (same doses), and Ven+ Aza (1.25 or 2.5 mg/kg daily for 7 days). Treatment effects on leukemia burden, toxicity, and overall survival were determined using weekly whole animal bioluminescent imaging, total animal weights, and time to morbidity, respectively.Results: Human AML cells treated with Ven displayed dose and time-dependent induction of DNA damage, apoptosis, and cell death. Similarly, treatment with single agent Tala resulted in dose-dependent anti-proliferative effects and PARP trapping. Combination in vitro therapy with Ven (0.1 - 1 μM) + Tala (10- 100 nM) or Ven (same doses) + Aza (1 μM) resulted in significant and synergistic anti-leukemic effects improving upon monotherapy with Ven, Tala, or Aza alone. However, in contrast to Ven+Aza, increased levels of DNA damage (as determined by phosphorylated H2AX and Fast Halo assays) were detected as well as induction of apoptosis, cell cycle arrest, decreased viability, and increased cell death vs vehicle and single agent therapy.We then evaluated the effects of in vivo treatment with vehicle, Ven, Tala, Ven + Tala, and Ven +Aza in NSG mice systemically engrafted with human luciferase-tagged AML cells (Molm13 BLIV). Single agent Ven or Tala therapy decreased systemic AML burden but did not prolong overall survival over vehicle treated mice. Combination Ven + Tala significantly prolonged overall survival of leukemia xenografted mice over vehicle, Ven, or Tala monotherapy. Of note, Ven + Tala treatment appeared to result in similar disease burden reduction and prolonged overall survival as Ven + Aza treated animals (Figure 1). Preliminary experiments of iv vivo triple therapy with Ven + Aza + Tala in AML xenografted mice resulted in further reduction in AML disease burden but was associated with marked weight loss leading to early morbidity and shortened overall survival (data not shown). Additional doses and schedules of this triplet approach are under investigation.Conclusions: Our results demonstrate that addition of the potent PARP inhibitor, talazoparib, to the BCL-2 inhibitor, venetoclax, enhances DNA damage and results in potent in vitro and in vivo activity in preclinical human AML models. Further evaluation of this combination in primary AML samples with varying genetic backgrounds as well as in confirmatory primary xenograft models is underway. These data strongly support future clinical investigation of PARP inhibitors as a novel class of agents with the potential to significantly enhance the efficacy of novel targeted therapies for acute leukemia. [Display omitted] DisclosuresWang: BMS/Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Other: Advisory Board, Speakers Bureau; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy, Honoraria, Other: Advisory Board; Mana Therapeutics: Consultancy, Honoraria; DAVA Oncology: Consultancy, Speakers Bureau; Takeda: Consultancy, Honoraria, Other: Advisory board; Stemline Therapeutics: Consultancy, Honoraria, Other: Advisory board, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Kite Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Kura Oncology: Consultancy, Honoraria, Other: Advisory board, steering committee, Speakers Bureau; Novartis: Consultancy, Honoraria, Other: Advisory Board; Rafael Pharmaceuticals: Other: Data safety monitoring committee; Gilead: Consultancy, Honoraria, Other: Advisory board; Daiichi Sankyo: Consultancy, Honoraria, Other: Advisory board; PTC Therapeutics: Consultancy, Honoraria, Other: Advisory board; Genentech: Consultancy; MacroGenics: Consultancy.

Effect of Novel Pyrrolo[3,4-d]pyridazinone Derivatives on Lipopolysaccharide-Induced Neuroinflammation.

Neuroinflammation is considered to be one of the potential causes for the development of neurodegenerative diseases, including Alzheimer’s disease. In this study, we evaluated the effect of four newly synthesized pyrrolo[3,4-d]pyridazinone derivatives on the neuron-like PC12 cells under simulated inflammation conditions by preincubation with lipopolysaccharide (LPS). Our novel derivatives are selective cyclooxygenase-2 (COX-2) inhibitors and have similar effects to nonsteroidal anti-inflammatory drugs (NSAIDs). We assessed viability (LDH assay), metabolic activity (MTT assay), DNA damage (number of double-strand breaks measured by fast halo assay), and the neuronal features of cells (average neurite length and neurite outgrowth measured spectrofluorimetrically). DCF-DA and Griess assays were also performed, which allowed determining the impact of the tested compounds on the level of oxygen free radicals and nitrites. LPS administration significantly negatively affected the results in all tests performed, and treatment with the tested derivatives in most cases significantly reduced this negative impact. Multiple-criteria decision analysis indicated that overall, the best results were observed for compounds 2a and 2b at a concentration of 10 µM. The new derivatives showed intense activity against free oxygen radicals and nitrites. Reduced reactive oxygen species level also correlated with a decrease in the number of DNA damage. The compounds improved neuronal features, such as neurite length and outgrowth, and they also increased cell viability and mitochondrial activity. Our results suggest that derivatives 2a and 2b may also act additionally on mechanisms other than 3a and 3b.

Phytochemical composition, antioxidant and antiproliferative activities and effects on nuclear DNA of ethanolic extract from an Italian mycelial isolate of Ganoderma lucidum

Ethnopharmacological relevanceGanoderma lucidum (Curtis) P. Karst. (also known as Linghzhi and Reishi) is the most appreciated and revered medicinal mushroom across many Asian countries, but its properties have also attracted interest in Western countries. Indeed, in the West, it is now commercially available as a dietary supplement in preparations mainly made from spores, fruiting bodies and mycelia. It is employed in both nutraceutical and pharmacological formulations either for its immuno-modulating anti-inflammatory properties or as an effective adjuvant therapy in the treatment of several chronic diseases as well as in cancer treatment. Aim of the studyThe aim of this investigation was to show the phytochemical composition and antioxidant and antiproliferative activities of an ethanolic extract from an Italian mycelial isolate of Ganoderma lucidum and to assess its effects on nuclear DNA. Materials and methodsLC/ESI-MS and tandem mass spectrometry MSMS were used to obtain structural identification of ethanolic G. lucidum extract constituents. Antioxidant activities were determined by the DPPH method, chelating effect on Fe2+ and lipoxygenase inhibition while cytotoxic activities using the MTT assay. Effects on nuclear DNA were evaluated using the DNA nicking assay in a cell-free system and the fast halo assay performed on oxidatively injured human U937 cells; apoptosis induction was investigated using the non-denaturing fast halo assay and DNA laddering detection. ResultsThis extract was rich in several bioactive compounds, mainly phenolic and triterpenic acids. It showed antioxidant activity and protective effects in oxidatively injured DNA in cell-free analyses and antiproliferative, genotoxic, and proapoptotic effects in the cell model. ConclusionsItalian G. lucidum mycelium isolate appears to be a source of various natural compounds that may have applications as chemopreventive agents or functional foods.

Citrus peels prevent cancer

Background: Citrus comprises the largest fruit sector worldwide, and its fruit peels are the dominant ‘residue’ of the industry. Though not profitable, Citrus peels are industrially used for making some byproducts (cattle feed, molasses, ethanol, fiber) and for the extraction of bioactives (flavonoids, essential oils, d-limonene). Still huge amounts of peels are wasted by Citrus industries, juice and other vending sectors.Purpose: The biological potentials of these unutilized or ‘wasted’ Citrus peels are least exploited. Here we tested the anticancer potentials of Citrus medica (2 morphotypes), C. sinensis, C. maxima, C. limon and C. reticulata peels by in vitro assays and in vivo cancer models.Methods: Chemical profiles of Citrus peel oils and peel extracts were analyzed by gas chromatographic techniques (GC-FID, GC–MS) and HPTLC-densitometry, respectively. Anticancer potentials of Citrus peels (Citrus medica 2 morphotypes, C. sinensis, C. maxima, C. limon and C. reticulata) were evaluated by various in vitro assays (MTT assay, morphological observations, fast halo assay, flow cytometric analysis) and in vivo cancer models.Results: C. reticulata peels (extracts, essential oils) showed significant activity against DLA cell line in MTT assay. We found C. reticulata peel water extract inducing cell cycle arrest of DLA in G0/G1 phase followed by nuclear condensation, membrane blebbing, formation of apoptotic bodies and DNA damage leading to apoptosis. In in vivo experiments, C. reticulata peel extract pre-treated mice were significantly (50%) protected from DLA compared to post-treated mice (33%), without any conspicuous toxic symptoms. Citrus peels have volatiles (essential oils, limonoids) and non-volatiles (mainly polymethoxy flavones) as their bioactive/anticancer constituents.Conclusion: Our results encourage the use of Citrus peels, which is wasted in huge amounts, as cancer preventive food additives and as anticancer agents.

Protective Effect of Juglans regia L. Walnut Extract Against Oxidative DNA Damage.

Walnuts (Juglans regia L.) are relevant components of the Mediterranean diet providing important macronutrients, micronutrients and other bioactive constituents including unsaturated fatty acids, proteins, fiber, vitamins, minerals, phytosterols and polyphenols. Although the walnut beneficial effects in human health are widely recognized by a lot of epidemiologic studies very little is known regarding its effect on damaged DNA. The aim of the present study was to investigate the effect of Juglans regia L. ethanolic extract from kernel on the induction of DNA strand breaks by thiol/Fe3+/O2 mixed function oxidase, tert-butyl hydroperoxide or UVC radiations in acellular and cellular models. Plasmid DNA cleavage and fast Halo assay were used to monitor oxidative damage to DNA. Both approaches showed protection of oxidatively injured DNA. These results agree with a lot of scientific proofs which recommend walnut as dietary adjunct in health promotion and prevention as well as in treatment of lifestyle-related oxidative diseases.

The Fast-Halo Assay for the Detection of DNA Damage.

The need for express screening of the DNA damaging potential of chemicals has progressively increased over the past 20years due to the wide number of new synthetic molecules to be evaluated, as well as the adoption of more stringent chemical regulations such as the EU REACH and risk reduction politics. In this regard, DNA diffusion assays such as the microelectrophoretic comet assay paved the way for rapid genotoxicity testing. A more significant simplification and speeding up of the experimental processes was achieved with the fast halo assay (FHA) described in the present chapter. FHA operates at the single cell level and relies on radial dispersion of the fragments of damaged DNA from intact nuclear DNA. The fragmented DNA is separated by diffusion in an alkaline solvent and is stained, visualized, and finally quantified using computer-assisted image analysis programs. This permits the rapid assessment of the extent of DNA breakage caused by different types of DNA lesions. FHA has proven to be sensitive, reliable, and flexible. This is currently one of the simplest, cheapest, and quickest assays for studying DNA damage and repair in living cells. It does not need expensive reagents or electrophoretic equipment and requires only 40 min to prepare samples for computer-based quantification. This technique can be particularly useful in rapid genotoxicity assessments and in high-throughput genotoxicity screenings.

A simplified chromatin dispersion (nuclear halo) assay for detecting DNA breakage induced by ionizing radiation and chemical agents

Methods for visualizing DNA damage at the microscopic level are based on treatment of cell nuclei with saline or alkaline solutions. These procedures for achieving chromatin dispersion produce halos that surround the nuclear remnants. We improved the fast halo assay for visualizing DNA breakage in cultured cells to create a simplified method for detection and quantitative evaluation of DNA breakage. Nucleated erythrocytes from chicken blood were selected as a model test system to analyze the production of nuclear halos after treatment with X-rays or H2O2. After staining with ethidium bromide or Wright's methylene blue-eosin solution, nuclear halos were easily observed by fluorescence or bright-field microscopy, respectively, which permits rapid visualization of DNA breakage in damaged cells. By using image processing and analysis with the public domain ImageJ software, X-ray dose and H2O2 concentration could be correlated well with the size of nuclear halos and the halo:nucleus ratio. Our results indicate that this simplified nuclear halo assay can be used as a rapid, reliable and inexpensive procedure to detect and quantify DNA breakage induced by ionizing radiation and chemical agents. A mechanistic model to explain the differences between the formation of saline or alkaline halos also is suggested.

Assessment of DNA damage by comet assay and fast halo assay in buccal epithelial cells of Indian women chronically exposed to biomass smoke

Genotoxicity of indoor air pollution from biomass burning was evaluated in buccal epithelial cells (BECs) of 85 pre-menopausal Indian women who were engaged in cooking with biomass (wood, dung, crop residues) and 76 age-matched control women who were cooking with cleaner fuel liquefied petroleum gas (LPG). DNA damage was evaluated by comet assay and fast halo assay (FHA). The concentrations of particulate matter with aerodynamic diameters of less than 10 and 2.5 μm (PM(10) and PM(2.5), respectively) in indoor air were measured by real-time aerosol monitor. Generation of reactive oxygen species (ROS) was measured by flow cytometry and the level of superoxide dismutase (SOD) by spectrophotometry. Compared with control, BEC of biomass users illustrated 2.6-times higher comet tail % DNA (32.2 vs. 12.4, p < 0.001), 2.7-times greater comet tail length (37.8 μm vs. 14.2 μm, p < 0.001) and 2.2-times more olive tail moment (7.1 vs. 3.2, p < 0.001), suggesting marked increase in DNA damage. FHA also showed 5-times more mean nuclear diffusion factor (9.2 vs. 1.8, p < 0.0001) in BEC of biomass users, confirming sharp rise in DNA single strand breaks. Airway cells of biomass-using women showed 51% rise in ROS generation but 28% reduction in SOD, suggesting oxidative stress in the airways. Indoor air of biomass-using households had 3-times more PM(10) and PM(2.5) than LPG-using families, and DNA damage showed positive association with PM(10) and PM(2.5) levels controlling education, kitchen location and family income as potential confounders. In summary, chronic inhalation of biomass smoke elicits oxidative stress and extensive DNA damage in BEC.

Nuclear Tau, a Key Player in Neuronal DNA Protection

Tau, a neuronal protein involved in neurodegenerative disorders such as Alzheimer disease, which is primarily described as a microtubule-associated protein, has also been observed in the nuclei of neuronal and non-neuronal cells. However, the function of the nuclear form of Tau in neurons has not yet been elucidated. In this work, we demonstrate that acute oxidative stress and mild heat stress (HS) induce the accumulation of dephosphorylated Tau in neuronal nuclei. Using chromatin immunoprecipitation assays, we demonstrate that the capacity of endogenous Tau to interact with neuronal DNA increased following HS. Comet assays performed on both wild-type and Tau-deficient neuronal cultures showed that Tau fully protected neuronal genomic DNA against HS-induced damage. Interestingly, HS-induced DNA damage observed in Tau-deficient cells was completely rescued after the overexpression of human Tau targeted to the nucleus. These results highlight a novel role for nuclear Tau as a key player in early stress response.

Effects of a 300 mT static magnetic field on human umbilical vein endothelial cells

This study describes the effects of a static magnetic field (SMF) on cell growth and DNA integrity of human umbilical vein endothelial cells (HUVECs). Fast halo assay was used to investigate nuclear damage; quantitative polymerase chain reaction (QPCR), standard PCR, and real-time PCR were used to evaluate mitochondrial DNA integrity, content, and gene expression. HUVECs were continually exposed to a 300 mT SMF for 4, 24, 48, and 72 h. Compared to control samples (unexposed cultures) the SMF-exposed cells did not show a statistically significant change in their viability. Conversely, the static field was shown to be significant after 4 h of exposure, inducing damage on both the nuclear and mitochondrial levels, reducing mitochondrial content and increasing reactive oxygen species. Twenty-four hours of exposure increased mitochondrial DNA content as well as expression of one of the main genes related to mitochondrial biogenesis. No significant differences between exposed and sham cultures were found after 48 and 72 h of exposure. The results suggest that a 300 mT SMF does not cause permanent DNA damage in HUVECs and stimulates a transient mitochondrial biogenesis.

Sulforaphane induces DNA single strand breaks in cultured human cells

Sulforaphane (SFR), an isothiocyanate from cruciferous vegetables, possesses growth-inhibiting and apoptosis-inducing activities in cancer cell lines. Recently, SFR has been shown to promote the mitochondrial formation of reactive oxygen species (ROS) in human cancer cell lines. The present study was undertaken to see whether SFR-derived ROS might cause DNA damage in cultured human cells, namely T limphoblastoid Jurkat and human umbilical vein endothelial cells (HUVEC). 1–3 h treatments with 10–30 μM SFR elicited intracellular ROS formation (as assayed with dihydrorhodamine, DHR, oxidation) as well as DNA breakage (as assessed with fast halo assay, FHA). These effects lacked cell-type specificity, since could be observed in both Jurkat and HUVEC. Differential-pH FHA analysis of damaged DNA showed that SFR causes frank DNA single strand breaks (SSBs); no DNA double strand breaks (DSBs) were found within the considered treatment times (up to 3 h). SFR-derived ROS were formed at the mitochondrial respiratory chain (MRC) level: indeed rotenone or myxothiazol (MRC Complex I and III inhibitors, respectively) abrogated ROS formation. Furthermore ROS were not formed in Jurkat cells pharmacologically depleted of respiring mitochondria (MRC−/Jurkat). Formation of ROS was causally linked to the induction of SSBs: indeed all the experimental conditions capable of preventing ROS formation also prevented the damage of nuclear DNA from SFR-intoxicated cells. As to the toxicological relevance of SSBs, we found that their prevention slightly but significantly attenuated SFR cytotoxicity, suggesting that high-dose SFR toxicity is the result of a complex series of events among which GSH depletion seems to play a pivotal role. In conclusion, the present study identifies a novel mechanism contributing to SFR toxicity which – since DNA damage is a prominent mechanism underlying the cytotoxic activity of established antineoplastic agents – might help to exploit the therapeutic value of SFR in anticancer drug protocols.

Could a strong alkali deproteinization replace the standard lysis step in alkaline single cell gel electrophoresis (comet) assay (pH > 13)?

The alkaline version of single cell gel electrophoresis (comet) assay is widely used for evaluating DNA damage at the individual cell level. The standard alkaline method of the comet assay involves deproteinization of cells embedded in agarose gel using a high salt–detergent lysis buffer, followed by denaturation of DNA and electrophoresis using a strong alkali at pH > 13 [N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell. Res. 175 (1988) 184–191]. However, a recent report showed that a strong alkali treatment results in simultaneous deproteinization of cells and denaturation of genomic DNA [P. Sestili, C. Martinelli, V. Stocchi, The fast halo assay: an improved method to quantify genomic DNA strand breakage at the single cell-level, Mutat. Res. 607 (2006) 205–214]. This study was carried out to test whether the strong alkali deproteinization of cells could replace the high salt–detergent lysis step used in the standard method of the alkaline comet assay. Peripheral blood lymphocytes from 3 healthy individuals were irradiated with gamma rays at doses varying between 0 and 10 Gy. Following irradiation, the comet assay was performed according to the standard alkaline method (pH > 13) and a modified method. In the modified method, agarose embedded cells were treated with a strong alkali (0.3 M NaOH, 0.02 M Trizma and 1 mM EDTA, pH > 13) for 20 min to allow deproteinization of cells and denaturation of DNA. This was followed by electrophoresis using the same alkali solution to obtain comets. DNA damage expressed in terms of comet tail length, percentage of DNA in comet tail and tail moment obtained by the standard alkaline method and the modified method were compared. In both methods, DNA damage showed a good correlation with the dose of gamma ray. The results indicate a satisfactory sensitivity of the modified method in detecting radiation-induced DNA damage in human peripheral blood lymphocytes.

The fast halo assay: An improved method to quantify genomic DNA strand breakage at the single-cell level

The present study describes the improvement of a technique, the alkaline-halo assay (AHA), for the assessment of DNA single-strand breakage at the single-cell level. AHA involves a series of sequential steps in which cells are embedded in melted agarose and spread onto microscope slides, incubated in a high-salt alkaline lysis solution, then in a hypotonic alkaline solution and, finally, stained with ethidium bromide (EB). Under these conditions, single-stranded DNA fragments diffuse radially from the nuclear cage and generate a fluorescent image that resembles a halo concentric to the nuclear remnants: the area of the halo is a direct function of the extent of DNA strand scission. These phenomena can be conveniently monitored with a fluorescence microscope and quantified by image-processing analysis. The behaviour of single-stranded DNA fragments under the conditions of the modified assay, called fast halo assay (FHA), is essentially the same as in AHA. The modifications consist in the simplification of the lysis, denaturation and staining procedures, and allow, as compared with AHA, the preparation of samples within 15 min, with a two-third reduction in total processing time, using only two reagents to promote DNA extraction and staining: NaOH and EB. A variation of the FHA operating at non-denaturing conditions to discriminate apoptotic cells from non-apoptotic cells bearing DNA single-strand breaks is also illustrated. To benchmark FHA sensitivity and reliability, the DNA single-strand breaks (SSBs) resulting either from exposure of cultured mammalian cells to different DNA-damaging agents or from secondary apoptotic DNA cleavage, have been quantified and results compared with the outcomes of reference techniques run in parallel, namely AHA, comet assay and Hoechst 33342 staining. The results indicate that FHA has the same reliability and sensitivity of the reference assays, but presents the additional advantages of being inexpensive, more rapid and strikingly simple.