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.

Sri Lanka is a biodiversity hotspot and one of the richest geographical locations of marine sponges in the Indian ocean. However, the most extensive taxonomical study on Sri Lankan sponge biodiversity dates back ~100 years and only a limited number of studies have been conducted on sponge natural products. In the current study, 35 marine sponge specimens (collected from 16 sponge habitats around Sri Lanka) were identified, microfractionated and evaluated for antibacterial and anticancer assays. In total, 30 species were characterized, of which 19 species gave extracts with antibacterial and/or cytotoxic activities. Microfractionated organic extract of Aciculites orientalis gave the most potent antibacterial activity against Staphylococcus aureus and strongest lymphoma cell toxicity was exhibited by the organic extract of Acanthella sp. Guided by the molecular ion peaks in the bioactive fractions, large-scale extraction of Stylissa massa led to the isolation of three bromopyrrole alkaloids, sceptrin, hymenin and manzacidin A/C. Of these, sceptrin exhibited broad spectrum antibacterial activity against both Escherichia coli and S. aureus (MIC of 62.5 μM against both species). Based on natural product literature, seven promising species were identified as understudied. Their further exploration may lead to the discovery of structurally novel compounds.

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.

The authors regret that in our paper it is stated that 2.5 mM NaCl (lysis buffer solution) and 0.3 mM NaOH (electrophoresis buffer solutions) were used, it should of course be 2.5 M NaCl and 0.3 M NaOH. Further it is stated that the S9-mix was prepared by only adding 2 mM NADPH, it should of course be 2 mM NADPH and 2 mM NADH.

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.

Physiological Glucocorticoids are important regulators of the immune system. Pharmacological GCs are in widespread use to treat inflammatory diseases. Adrenalectomy (ADX) has been shown to exacerbate renal injury through inflammation and oxidative stress that results in renal impairment due to depletion of GCs. In this study, the effect of myrcene to attenuate renal inflammation and oxidative stress was evaluated in the adrenalectomized rat model. Rats were adrenalectomized bilaterally or the adrenals were not removed after surgery (sham). Myrcene (50 mg/kg body weight, orally) was administered post ADX. Myrcene treatment resulted in significant downregulation of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) compared to untreated ADX rats. In addition, myrcene resulted in significant downregulation of immunomodulatory factors (IFNγ and NF-κB) and anti-inflammatory markers (IL-4 and IL-10) in treated ADX compared to untreated ADX. Myrcene significantly increased the antioxidant molecules (CAT, GSH, and SOD) and decreased MDA levels in treated ADX compared to untreated. Moreover, myrcene treatment reduced the expression of COX-2, iNOS, KIM-1, and kidney functional molecules (UREA, LDH, total protein, and creatinine) in ADX treated compared to ADX untreated. These results suggest that myrcene could be further developed as a therapeutic drug for treatment of kidney inflammation and injury.

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.

The presence of chemical pollutants in sources of drinking water is a key environmental problem threatening public health. Efficient removal of pollutants in drinking water treatment plants (DWTPs) is needed as well as methods for assessment of the total impact of all present chemicals on water quality. In the present study we have analyzed the bioactivity of water samples from source to tap, including effects of various water treatments in a DWTP, using a battery of cell-based bioassays, covering health-relevant endpoints. Reporter gene assays were used to analyze receptor activity of the aryl hydrocarbon receptor (AhR), estrogen receptor (ER), androgen receptor (AR), peroxisome proliferator-activated receptor alpha (PPARα) and induction of oxidative stress by the nuclear factor erythroid 2-related factor 2 (Nrf2). DNA damage was determined by Comet assay. Grab water samples were concentrated by HLB or ENV solid phase extraction and the water samples assayed at a relative enrichment factor of 50. The enrichment procedure did not induce any bioactivity. No bioactivity was detected in Milli-Q water or drinking water control samples. Induction of AhR, ER and Nrf2 activities was revealed in source to tap water samples. No cytotoxicity, PPARα or AR antagonist activity, or DNA damage were observed in any of the water samples. A low AR agonist activity was detected in a few samples of surface water, but not in the samples from the DWTP. The treatment steps at the DWTP, coagulation, granulated activated carbon filtration, UV disinfection and NH2Cl dosing had little or no effect on the AhR, Nrf2 and ER bioactivity. However, nanofiltration and passage through the distribution network drastically decreased AhR activity, while the effect on Nrf2 activity was more modest and no apparent effect was observed on ER activity. The present results suggest that bioassays are useful tools for evaluation of the efficiency of different treatment steps in DWTPs in reducing toxic activities. Bioassays of AhR and Nrf2 are useful for screening of effects of a broad range of chemicals in drinking water and ER activity can be monitored with a high sensitivity.

Background: Most of herbal medicines are used without any standard safety and toxicological trials although common assumption is that these products are nontoxic. However, this assumption is incorrect and dangerous, so toxicological studies should be done for herbal drugs. Although Pterolobium stellatum, Otostegia integrifolia and Vernonia amygdalina root extracts are frequently used in Ethiopian traditional medicine, there are no evidences of their active toxic compounds. Therefore, we made an effort to assess probable genotoxic effect of these plant extracts on DNA of human hematoma (HepG(2)) cells using alkaline comet assay. Methods: Genotoxic effects of extracts were evaluated using single cell gel electrophoresis (SCGE) method on HepG(2) cell. Regarding comet data, the average mean tail intensities (TI) from each individual experiment and treatment (usually at least 3 cultures/treatment) were pooled and the average mean TI was used as an indicator of DNA damage and the standard error of mean (SEM) as the measure of variance. Results: DNA damage in the form of comet tail has been observed for 1 and 0.5 mg/ml P. stellatum chloroform and 80% methanol extracts on HepG(2) cells, respectively. The chloroform extract of P. stellatum showed increased tail DNA percentage in a concentration dependent manner. Comet tail length in the chloroform P. stellatum extract treated cells (1 mg/ml) was significantly higher by 89% (p < 0.05) compared to vehicle treated controls. The rest of test extracts seemed to be without genotoxic effect up to a concentration of 0.5 mg/ml. Conclusions: Our findings show that two extracts from one plant evaluated have a genotoxic potential in vitro which calls for a more thorough safety evaluation. Such evaluation should include other end-points of genotoxicity apart from DNA damage, and possibly also pure compounds.

An extract of Glinus lotoides, a medicinal plant used in Africa and Asia for various therapeutic purposes, was recently shown to cause DNA damage in vitro. To further explore the potential genotoxicity of this plant, fractionation of the crude extract was performed using reverse phase solid‐phase extraction and a stepwise gradient elution of methanol in water. Four fractions were collected and subsequently analysed for their DNA damaging effects in mouse lymphoma cells using an alkaline version of the comet assay. To identify potential genotoxic and non‐genotoxic principles, each fraction was then subjected to liquid chromatography coupled to mass spectrometry, LC‐MS/MS. 1D and 2D nuclear magnetic resonance analyses were used to confirm the identity of some saponins. Although fractions containing a mixture of flavonoids and oleanane‐type saponins or oleanane‐type saponins alone produced no DNA damage, those containing hopane‐type saponins exhibited a pronounced DNA damaging effect without affecting the viability of the cells. To conclude, even if this study presents evidence that hopane‐type of saponins are endowed with a DNA damaging ability, further studies are needed before individual saponins can be cited as a culprit for the previously reported genotoxicity of the crude extract of G. lotoides. Copyright © 2012 John Wiley & Sons, Ltd.

Prochloraz is widely used in horticulture and agriculture, e.g. as a post-harvest anti-mold treatment. Prochloraz is a known endocrine disruptor causing developmental toxicity with multiple mechanisms of action. However, data are scarce concerning other toxic effects. Since oxidative stress response, with formation of reactive oxygen species (ROS), is a common mechanism for different toxic endpoints, e.g. genotoxicity, carcinogenicity and teratogenicity, the aim of this study was to investigate if prochloraz can induce oxidative stress and/or DNA damage in human cells. A cell culture based in vitro model was used to study oxidative stress response by prochloraz, as measured by the activity of the nuclear factor erythroid 2-related factor 2 (Nrf2), a key molecule in oxidative defense mechanisms. It was observed that prochloraz induced oxidative stress in cultured human adrenocortical H295R and hepatoma HepG2 cells at non-toxic concentrations. Further, we used Comet assay to investigate the DNA damaging potential of prochloraz, and found that non-toxic concentrations of prochloraz induced DNA damage in HepG2 cells. These are novel findings, contradicting previous studies in the field of prochloraz and genotoxicity. This study reports a new mechanism by which prochloraz may exert toxicity. Our findings suggest that prochloraz might have genotoxic properties.

A bioactivity-guided screening of Sri Lankan plants in the search for novel antibacterial and anticancer agents

Evidence of nuclear uptake and increased DNA damage in human lung epithelial cells after low dose exposure to reactive titanium dioxide nanoparticles

Ethnopharmacological relevance: Leptopyrum fumarioides has been used in the traditional medicine of Mongolia for the treatment of various diseases, including drug intoxications. However, since there is only sparse information about its chemistry, active components, and pharmacological and toxicological effects, the major aim of the present study employing mouse lymphoma cells was to evaluate the genotoxic and antigenotoxic/antioxidative effects of extracts and components isolated from this plant.Material and methods: A crude methanol extract was separated into three different sub-extracts: dichloromethane, n-butanol, and water. The major constituent of the n-butanol extract, i.e., the flavone luteolin-7-O-glucoside and a mixture of the most abundant compounds in the dichloromethane sub-extract were then isolated. DNA damage was evaluated using the comet assay; the antioxidant activity was evaluated using the DPPH radical scavenging assay.Results: The crude methanol extract, the dichloromethane sub-extract and the mixture of compounds isolated from the latter fraction, increased the level of DNA damage after three hours of exposure. In contrast, no increase in DNA damage was observed in the cells that had been exposed to the n-butanol and water sub-extracts, or to the pure flavone. When non-DNA damaging concentrations of extracts and compounds were tested together with the DNA damaging agent catechol, all sub-extracts were found to reduce the catechol-induced DNA damage (the flavone was then found to be the most effective protective agent). The n-butanol sub-extract and the flavone were also found to have the most prominent antioxidative effects.Conclusion: Based on the results from the present study, components in Leptopyrum fumarioides were found to protect the DNA damage induced by catechol, probably by acting as potent antioxidants. (C) 2014 Elsevier Ireland Ltd. All rights reserved.

The mutagenicity of 4-nitro-o-phenylenediamine (4-NOPD) and o-phenylenediamine (OPD) was compared using the Mouse Lymphoma Assay (MLA) with or without metabolic activation (S9). As expected, OPD was found to be a more potent mutagen than 4-NOPD. To evaluate possible mechanisms behind their mutagenic effects, the following end points were also monitored in cells that had been exposed to similar concentrations of the compounds as in the MLA: general DNA damage (using a standard protocol for the Comet assay); oxidative DNA damage (using a modified procedure for the Comet assay in combination with the enzyme hOGG1); reactive oxygen species (ROS; using the CM-H2DCFDA assay); and the balance of the nucleotide pool (measured after conversion to the corresponding nucleosides dC, dT, dG and dA using high-performance liquid chromatography). Both compounds increased the level of general DNA damage. Again, OPD was found to be more potent than 4-NOPD (which only increased the level of general DNA damage in the presence of S9). Although less obvious for OPD, both compounds increased the level of oxidative DNA damage. However, an increase in intracellular ROS was only observed in cells exposed to 4-NOPD, both with and without S9 (which in itself induced oxidative stress). Both compounds decreased the concentrations of dA, dT and dC. A striking effect of OPD was the sharp reduction of dA observed already at very low concentration, both with and without S9 (which in itself affected the precursor pool). Taken together, our results indicate that indirect effects on DNA, possibly related to an unbalanced nucleotide pool, mediate the mutagenicity and DNA-damaging effects of 4-NOPD and OPD to a large extent. Although induction of intracellular oxidative stress seems to be a possible mechanism behind the genotoxicity of 4-NOPD, this pathway seems to be of less importance for the more potent mutagen OPD.

Diet and oral supplements comprise two distinct sources of antioxidants known to prevent oxidative stress. Beneficial effects from antioxidants have been seen for patients at risk for type 2 diabetes.The aim of this thesis was to evaluate the positive effects of antioxidants against oxidative stress and DNA damage in type 2 diabetes subjects. We also used antioxidants as tools to determine the mechanisms behind genotoxicity induced by mutagenic pro-oxidative agents in mouse lymphoma cells. Several techniques were used to measure oxidative stress and DNA damage, but the main technique used was alkaline comet assay.The results showed that the fruit and vegetable intake was inversely related to oxidative stress in type 2 diabetes subjects. However, oral supplementary intake of 20 antioxidants did not decrease oxidative stress biomarkers.In studies on mouse lymphoma cells, using the alkaline comet assay, DNA damage was induced by catechol and o-phenylenediamine (OPD), while 4-nitro-o-phenylenediamine (4-NOPD) induced only oxidative damage, showing different mechanisms of action behind the mutagenicity of the compounds. Also, oxidative stress was induced by catechol and 4-NOPD, whereas imbalances in the nucleotide pool were seen after exposure to OPD or 4-NOPD. Addition of antioxidants together with these pro-oxidants showed that β-carotene was able to reduce DNA damage at low concentrations of catechol, but increased DNA damage at high concentration. In comparison, addition of α-tocopherol slightly decreased catechol-induced DNA damage at all concentrations of catechol. However, no effect of α-tocopherol was seen on OPD-or 4-NOPD-induced DNA damage.In conclusion, antioxidants from fruits and vegetables, but not from oral supplements, reduced oxidative stress in type 2 diabetes patients, suggesting fruits and vegetables being a healthier source for antioxidant-intake, as compared to oral supplements. Different mechanisms of action for mutagenic pro-oxidants were shown in mouse lymphoma cells, introducing the nucleotide pool as an interesting target for oxidative stress. Reduction of catechol-induced DNA damage by β-carotene or α-tocopherol was shown, with a pro-oxidative action of β-carotene at high concentration of catechol, Interestingly, α-tocopherol was not able to decrease OPD- or 4-NOPD-induced DNA damage, supporting different mechanisms of action behind the genotoxicity from the three pro-oxidants.