Comet Assay Protocol Research Articles

Determination of the Effect of Thymoquinone on DNA Damage in Kidney Cells Treated to High Glucose Depending on Time and Dosage by Comet Assay

The purpose of this study was to assess the anti-genotoxic potential of thymoquinone (TQ) against DNA damage in NRK-52E cells treated with high glucose using the comet assay technique single cell gel electrophoresis method. Cells were propagated by regular passages in in vitro conditions. TQ proliferative concentration (10μM) and IC25 (3rd-hour: 550 mM, 12th-hour: 240 mM, 24th-hour: 200 mM) and IC50 (3rd-hour: 760 mM, 12th-hour: 400 mM, 24th-hour: 280 mM) values for each hour of high glucose and were determined separately with MTT method. At these concentrations, the cells were divided into control(C), Thymoquinone (TQ), high glucose(G) and high glucose plus thymoquinone (GT) groups; It was incubated with the indicated substances for 3, 12, 24 hours. DNA damage was evaluated by applying the comet assay protocol and the results were calculated as DNA damage index (DDI). While DDI levels were observed to be significantly higher (p<0.05) in all groups administered high glucose compared to the control, a significant decrease was determined in all groups in which TQ was added along with high glucose. It was determined that high concentrations of glucose had genotoxic effects on kidney cells, and TQ administration together with high glucose, depending on concentration and time, had a significant effect on reducing DNA damage. However, it was concluded that the application of only thymoquinone significantly increased the DDI value compared to the control, and this was a data worth investigating in future studies. Additionally, TQ inhibited DNA damage. These results demonstrated the importance of TQ against nephrotic syndrome with its high antioxidant properties.

Evaluation of imidacloprid (Confidor OD®) genotoxicity in Chrysoperla externa eggs (Neuroptera: Chrysopidae) through comet assay

The comet assay allows the analysis of DNA damage caused by different genotoxins. This assay has recently gained interest because of its ease of studying the interactions of xenobiotics with different organisms.Chrysoperla externa (Hagen, 1861) is a species of great economic relevance because it is a predator of major agricultural pests during its larval stage. Neonicotinoids are the most important chemical class of insecticides introduced into markets. A previous imidacloprid toxicity assessment on C. externa showed that this neonicotinoid insecticide reduced the egg viability. The objective of this study was to analyze the genotoxicity of Confidor OD® (imidacloprid 20% a.i., LS, Bayer CropScience) on the biological control agent C. externa at DNA level using the comet assay as an ecotoxicological biomarker. A comet assay protocol has been developed for this species at first time. For the bioassays, the commercial product formulated Confidor OD® was used at two concentrations: 100 and 180 mg/l of the active ingredient. Selected eggs were dipped in a Confidor OD® solution for 15 s. Descriptors evaluated in the comet assay were damage index, % DNA damage, and tail length. The damage index did not show any significant differences between the different concentrations evaluated, but differences were observed for tail length, because at higher concentrations of Confidor OD®, there were greater DNA breaks. The DNA of the cells from treated eggs analyzed at 48 h and 96 h of development showed the same % DNA damage; that is, they had no recovery capacity. Application of Confidor OD® to C. externa eggs produced irreparable breaks at the DNA level. The technique adjusted for C. externa can be used in other beneficial insects to study pesticide genotoxicity using a comet assay.

Visualizing DNA single- and double-strand breaks in the Flash comet assay by DNA polymerase-assisted end-labelling.

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.

Four Decades of the Comet Assay: pH Optimum of Lysis Buffer Still Needs to be Elucidated.

The proper course and reproducibility of diagnostic techniques depend on narrowly defined reaction conditions, including the reaction pH. Nevertheless, numerous assays are affected by an inaccurately defined reaction pH. Buffers are sometimes suggested for use outside their useful pH ranges, which complicates the reproducibility of results because the buffering capacity is insufficient to retain the disclosed pH. Here, we focus on the comet assay lysis buffer. Comet assay is broadly used for quantifying DNA breaks in eukaryotic cells. The most widespread comet assay protocols employ lysis of the cells before electrophoresis in a buffer containing Triton X-100, a high concentration of NaCl, sodium sarcosinate, EDTA, and Tris, with some modifications. However, nearly all researchers report that they use Tris buffer at pH 10, and some report the pH of the Tris additive alone. Alternatively, others report the pH of the final lysis buffer. However, the lysis solution used in the comet assay is buffered at a pH outside the useful range of Tris. Tris-based buffers have a useful pH range of 7.0 - 9.0. The buffer composed of 10 mM Tris has pKa 8.10 at 25°C and 8.69 at 4°C. The cell lysis conditions used in nearly all modifications of comet assay protocols remain imprecise and uncritically employed. Despite the pH of the lysis buffer likely has negligible effect on the detection of DNA breaks, precise lysis conditions are highly important for the use of comet assay in the detection of base modifications, which are often unstable and sensitive to pH.

Reconstructed human intestinal comet assay, a possible alternative in vitro model for genotoxicity assessment.

The aim of the present study was to evaluate the compatibility of reconstructed 3D human small intestinal microtissues to perform the in vitro comet assay. The comet assay is a common follow-up genotoxicity test to confirm or supplement other genotoxicity data. Technically, it can be performed utilizing a range of in vitro and in vivo assay systems. Here, we have developed a new reconstructed human intestinal comet (RICom) assay protocol for the assessment of orally ingested materials. The human intestine is a major site of food digestion and adsorption, first-pass metabolism as well as an early site of toxicant first contact and thus is a key site for evaluation. Reconstructed intestinal tissues were dosed with eight test chemicals: ethyl methanesulfonate (EMS), ethyl nitrosourea (ENU), phenformin hydrochloride (Phen HCl), benzo[a]pyrene (BaP), 1,2-dimethylhydrazine hydrochloride (DMH), potassium bromate (KBr), glycidamide (GA), and etoposide (Etop) over a span of 48 h. The RICom assay correctly identified the genotoxicity of EMS, ENU, KBr, and GA. Phen HCl, a known non-genotoxin, did not induce DNA damage in the 3D reconstructed intestinal tissues whilst showing high cytotoxicity as assessed by the assay. The 3D reconstructed intestinal tissues possess sufficient metabolic competency for the successful detection of genotoxicity elicited by BaP, without the use of an exogenous metabolic system. In contrast, DMH, a chemical that requires liver metabolism to exert genotoxicity, did not induce detectable DNA damage in the 3D reconstructed intestinal tissue system. The genotoxicity of Etop, which is dependent on cellular proliferation, was also undetectable. These results suggest the RICom assay protocol is a promising tool for further investigation and safety assessment of novel ingested materials. We recommend that further work will broaden the scope of the 3D reconstructed intestinal tissue comet assay and facilitate broader analyses of genotoxic compounds having more varied modes of actions.

Measuring DNA modifications with the comet assay: a compendium of protocols.

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.

Calibration of the comet assay using ionising radiation

Several trials have attempted to identify sources of inter-laboratory variability in comet assay results, aiming at achieving more equal responses. Ionising radiation induces a defined level of DNA single-strand breaks (per dose/base pairs) and is used as a reference when comparing comet results but relies on accurately determined radiation doses. In this ring test we studied the significance of dose calibrations and comet assay protocol differences, with the object of identifying causes of variability and how to deal with them. Eight participating laboratories, using either x-ray or gamma radiation units, measured dose rates using alanine pellet dosimeters that were subsequently sent to a specialised laboratory for analysis. We found substantial deviations between calibrated and nominal (uncalibrated) dose rates, with up to 46% difference comparing highest and lowest values. Three additional dosimetry systems were employed in some laboratories: thermoluminescence detectors and two aqueous chemical dosimeters. Fricke’s and Benzoic Acid dosimetry solutions gave reliable quantitative dose estimations using local equipment. Mononuclear cells from fresh human blood or mammalian cell lines were irradiated locally with calibrated (alanine) radiation doses and analysed for DNA damage using a standardised comet assay protocol and a lab-specific protocol. The dose response of eight laboratories, calculated against calibrated radiation doses, was linear with slope variance CV= 29% with the lab-specific protocol, reduced to CV= 16% with the standard protocol. Variation between laboratories indicate post-irradiation repair differences. Intra-laboratory variation was very low judging from the dose response of 8 donors (CV=4%). Electrophoresis conditions were different in the lab-specific protocols explaining some dose response variations which were reduced by systematic corrections for electrophoresis conditions. The study shows that comet assay data obtained in different laboratories can be compared quantitatively using calibrated radiation doses and that systematic corrections for electrophoresis conditions are useful.

Technical Report on the request for technical assistance in relation to the safety of hydroxyanthracene derivatives

In accordance with Article 31 of Regulation (EC) No 178/2002, the European Commission asked EFSA to provide technical assistance in the context of its 'Opinion on the safety of hydroxyanthracene derivatives for use in food' (EFSA ANS Panel, 2018). In this regard, EFSA was asked to assess whether the data from two new scientific publications presented by the Italian Society of Toxicology were sufficient to revise the conclusions of EFSA on the safety of hydroxyanthracene derivatives for use in food. The scientific publications under assessment were two in vivo comet assays in mice conducted on Aloe-emodin and dried whole Aloe ferox juice, respectively. The results of the study with aloe-emodin were considered inconclusive for the induction of DNA strand breaks with the standard comet assay protocol. Results obtained with the modified comet assay protocol with a lesion-specific enzyme (hOGG1) suggest that aloe-emodin can induce oxidative DNA damage in colon cells in vivo. The lack of DNA damage observed in the study on Aloe ferox juice was considered of low relevance with respect to the genotoxicity of hydroxyanthracene derivatives, because of the low concentrations of hydroxyanthracene derivatives and the potential counteracting effect of other components present in the juice (e.g. antioxidants). Based on the available evidence, EFSA concluded that the new information presented does not justify a revision of the conclusions of the EFSA ANS Panel Opinion on hydroxyanthracene derivatives.

Assessment of phytotoxic, genotoxic and enzyme inhibition potential of.

To evaluate Sterculia diversifolia stem bark and leaves for phytotoxic, genotoxic and enzymes inhibition potential. Phytotoxic activity of both stem bark and leaves were screened using Lemna minor. The genotoxic activity of Sterculia diversifolia stem bark and leaves extracts were tested using comet assay protocol while enzyme inhibition activity of crude extract and various fractions of both stem bark and leaves were evaluated using acetyl cholinesterase, lipoxygenase, β-glu-curonidase, urease, xanthine oxidase and carbonic anhydrase. Phytotoxic activity showed significant results in dose dependant manner in both stem bark (ethyl acetate and n-butanol) and leaves (ethyl acetate, n-butanol and n-hexane) fractions. In genotoxic activity, dichloromethane fraction showed significant activity followed by ethyl acetate fraction. Acetyl cholinesterease inhibitory activity showed significant results in both stem bark and leaves fractions, while significant lipoxygenase inhibition was shown by ethyl acetate, dichloromethane, crude extract and n-hexane fractions of both stem bark and leaves. β-glucuronidase, urease and carbonic anhydrase inhibitory activity showed highly significant results in ethyl acetate fraction of both stem bark and leaves, while xanthine oxidase inhibition was shown by dichloromethane fraction of stem bark and leaves extracts. This study emphasizes the important phytotoxic, genotoxic and enzyme inhibition effects of Sterculia diversifolia stem bark and leaves. Hence, it is clear that Sterculia diversifolia stem bark and leaves possess phytotoxic, genotoxic and enzyme inhibitory agents.

Cell culture and variations in the comet assay do not affect the genomic integrity of adipose-derived stem cells

The application of quality control tests, such as the comet assay, are essential when adipose-derived stem cells are cultured for therapeutic purposes. However, the steps involved in the development of this assay should be investigated, in order to reduce their influence on genomic damage in cells. The study aimed to evaluate if the cell culture process causes DNA damage, and if variations in the lysis time and pH of the electrophoresis buffer interfere in the genotoxicity results. Four different comet assay protocols were evaluated, and the effects of lysis time and pH conditions of the electrophoresis buffer solution were stated as follows: 2 hours and pH 12; 24 hours and pH 12; 2 hours and pH ≥ 13 and 24 hours and pH ≥ 13. The tail moment was analyzed, and results indicated that at the time cells were detached from the flasks, there was little damage to the DNA in the adipose-derived stem cells, which was confirmed by evaluation of the expression of mRNA genes involved in damage and repair processes of genetic material. Also, the tail moment values ​​did not show significant differences among the four evaluated protocols (p < 0.05), with no indication of damage when compared to the positive control (p < 0.05). Thus, any of the tested protocols can be applied in genotoxicity tests with adipose-derived stem cells, without causing damage to them.

Assessment of DNA damage and oxidative stress among traffic conductors and coal miners.

Objective:To assess the DNA damage and oxidative stress among traffic conductors and coal miners.Methods:An analytical cross-sectional survey was conducted in Karak, Pakistan from March to October 2019. A total of 240 individuals participated in the study with an age range between 17 to 55 years. Among the total sample, 60 participants had exposure to traffic pollution while 60 were mine workers. Two control groups, consisting of 60 individuals each, were also recruited for comparison with the two exposure groups. Comet assay protocols were performed for assessing DNA damage and oxidative stress (length of DNA tail, levels of Superoxide Dismutase (SOD), Malondialdehyde (MDA) and Glutathione (GSH)). Data was analyzed using T-test on statistix 9.0 software.Results:The DNA tail length in traffic conductors ranged from 26.83-30.55µm (Mean=28.69 µm while their control group had DNA tail length of 7.98-9.26µm (Mean= 8.62). There was significant difference (P <0.001) between exposure and control group. The DNA length recorded in coal mine workers and their control group was ranged from 29.06-31.26µm (Mean=30.16µm) and 9.42-10.22µm (Mean=9.82), respectively. There was significant difference (P <0.001) between the two groups. As compared to control groups, both exposure groups have high levels of Superoxide Dismutase and Malondialdehyde and low levels of Glutathione. The finding was statistically significant (P <0.001).Conclusion:Increased inhalational exposure to air pollutants via working in traffic or coal mines can impose higher oxidative stress and DNA damage among workers as compared to the general population.

USING SPERM DOUBLE-STRANDED DNA TO PREDICT EMBRYO PLOIDY

To demonstrate how the presence of double-stranded DNA breaks (dsDNA) in spermatozoa can be used to predict embryo ploidy. Over the last 12 months, a prospective pilot study was carried out on semen samples to assess dsDNA levels. These samples were also concurrently assessed to determine total DNA damage. A correlation between the two assays was established. Couples were divided into two groups according to the adopted dsDNA threshold, and clinical outcomes were recorded and compared. Consenting men had their ejaculates screened for dsDNA rates utilizing an in-house protocol of the neutral Comet assay, assessing at least 200 spermatozoa. Concurrently, samples were assessed by terminal deoxynucleotidyl dUTP transferase nick-end labeling (TUNEL) with a commercial kit, screening at least 500 spermatozoa. ICSI was performed in the standard fashion. Resulting blastocysts were biopsied on day 5 or 6 post-insemination for preimplantation genetic testing for aneuploidy (PGT-A) using next-generation sequencing. The initial pilot study reported an average total DNA fragmentation by TUNEL of 11.3±6% and a dsDNA average of 2.2±3% by neutral Comet assay. The results of TUNEL and dsDNA presented with a linear correlation (R2=0.96; P<0.05), and dsDNA values were extrapolated. After establishing a threshold of 3%, 231 couples treated in 381 ICSI/PGT-A cycles were compared according to above or below threshold. A total of 173 couples (maternal age, 37.2±4 years; paternal age, 38.9±5 years) with normal dsDNA levels underwent 296 ICSI cycles, yielding a fertilization rate of 76.9%; 1,562 embryos were screened by PGT-A. This resulted in 574 normal (36.7%), 827 abnormal (52.9%), and 161 mosaic embryos (10.3%). There have been 171 frozen embryo transfers (FET) of euploid blastocysts thus far, with a 51.6% implantation rate and a 55.6% clinical pregnancy rate (CPR). A total of 48 couples with abnormal dsDNA levels underwent 85 ICSI cycles. They had a comparable maternal age of 38.0±5 years, but a much older male partner (43.2±8 years; P<0.0001). They achieved a fertilization rate of 72.7% (P<0.01), with 375 embryos biopsied for PGT-A. A total of 115 were euploid (30.7%; P<0.05), 222 were aneuploid (59.2%; P<0.05), and 38 were mosaic (10.1%). Interestingly, FET cycles from these cases trended toward lower implantation (43.9%) and CPRs (47.4%). This study confirmed the relevance of dsDNA on structural chromosomal abnormalities and therefore the proportion of euploid embryos. Examining dsDNA of the male gamete can provide important information on the prospects of successful embryo implantation.

Comet assay protocol for Bombus atratus fat body and pericardial cells (Hymenoptera, bombini) at a safe concentration of mercury

The decline of the Bombus population is closely related to the presence of environmental pollutants. Among these pollutants, trace metals represent a major concern, which includes mercury, a known genotoxic substance. The induction of genotoxicity may be demonstrated by the comet assay (a.k.a. single-cell gel electrophoresis), a simple and sensitive method for DNA damage estimating. The current work provided, for the first time, a protocol of comet assay for Bombus atratus using mercury as a standard chemical at safe concentrations according to the Environment National Council of Brazil, and the World Health Organization. Bees were collected and divided into three groups (n = 11 each), in which the exposed groups received a 0.2 ppb or a 1 ppb of mercury solution, and the control group received water. The bioassay was performed for 48 h at controlled temperature and humidity conditions, according to the OECD guideline toxicological test method for B. terrestris. The samples were stained with different dyes to observe the efficacy of each one. Variations of parameters in methodology, such as concentration and time of exposure to lysis solution as well as the electrophoretic process, allowed the observation of comets at different levels. DAPI and acridine orange presented an unstable fluorescence, and silver nitrate dye was more effective. Therefore, the comet assay was shown to be an effective method to evaluate genotoxic effects in bees. The obtained results may be helpful for the establishment of a suitable protocol for future genotoxicity assessment in neotropical bees using different doses of xenobiotics.

Technical Updates to the Comet Assay In Vivo for Assessing DNA Damage in Zebrafish Embryos from Fresh and Frozen Cell Suspensions.

The Single-Cell Gel Electrophoresis, simply known as the Comet assay, is a sensitive and quick technique used to quantitate DNA damage, widely used to assess the effects of genotoxicants and mutagens in animal cells. Still, performing the assay on peripheral or cultured cells is far more expeditious and cost effective than solid tissue, especially from small biological model like the zebrafish embryo. The current work describes and validates a highly cost-effective protocol of the updated Comet assay designed for zebrafish embryos. Compared with the few previous applications of the Comet assay on this biological model, the present method successfully simplifies the process of cell harvesting and resuspending, producing a much higher yield of viable nucleoids with reduced basal DNA damage, even from a small number of embryos, and compatible with scoring with safe fluorescent dyes. Additionally, the protocol can be just as easily performed on freshly harvested cells of cells frozen in dimethyl sulfoxide (DMSO)-containing physiological buffer, without a significant increase of DNA damage, which is another highly relevant update, especially for researchers handling high numbers of samples.

Evaluation of the Major Steps in the Conventional Protocol for the Alkaline Comet Assay.

Single cell gel electrophoresis, also known as the comet assay, has become a widespread DNA damage assessment tool due to its sensitivity, adaptability, low cost, ease of use, and reliability. Despite these benefits, this assay has shortcomings, such as long assay running time, the manipulation of multiple slides, individually, through numerous process steps, the challenge of working in a darkened environment, and reportedly considerable inter- and intra-laboratory variation. All researchers typically perform the comet assay based upon a common core approach; however, it appears that some steps in this core have little proven basis, and may exist, partly, out of convenience, or dogma. The aim of this study was to critically re-evaluate key steps in the comet assay, using our laboratory’s protocol as a model, firstly to understand the scientific basis for why certain steps in the protocol are performed in a particular manner, and secondly to simplify the assay, and decrease the cost and run time. Here, the shelf life of the lysis and neutralization buffers, the effect of temperature and incubation period during the lysis step, the necessity for drying the slides between the electrophoresis and staining step, and the need to perform the sample workup and electrophoresis steps under subdued light were all evaluated.

Optimization of the alkaline comet assay for easy repair capacity quantification of oxidative DNA damage in PBMC from human volunteers using aphidicolin block

Assessment of DNA repair capacity (DRC) upon ex vivo challenge of peripheral blood mononuclear cells (PBMC) with oxidative damage inducing agents, as evaluated by the comet assay, is widely used as biomarker to assess the antioxidant status in human studies. Here, the alkaline comet assay was now optimized for easy and time saving detection of repair capacity upon oxidative stress-induced DNA damage using the DNA polymerase inhibitor aphidicolin (APC) to block repair of hydrogen peroxide (H2O2) induced DNA damage. Addition of a DMSO-containing DNA damage stop solution was found suitable to replace washing steps for H2O2 removal before APC block. Cell treatment with APC at 6 μM did not impact baseline DNA damage but could reliably block DNA repair after H2O2 challenge in both fresh and cryopreserved samples thus omitting the use of a starting time point control. Under the conditions used, frozen cells, with or without an additional 4 h rest, showed the same repair capacity as their fresh counterpart. The intra assay coefficient of variation (CV) was 3.3%. To provide proof of principle, the modified assay was applied to cryopreserved PBMC from 19 participants of a short-term Brassica diet intervention study investigating potential health promoting effects of the food intervention. Then, a 33% increase in DRC (p ≤ 0.01) could be shown in samples after intervention (mean ± SD: 5.82 ± 1) as compared to baseline (mean ± SD: 4.38 ± 1.21). Individual samples from baseline and intervention showed an inter-individual CV of 27.65% (baseline) and 17.26% (intervention). Taken together this modified comet assay protocol allows the facilitated detection of DNA repair in fresh or cryopreserved human PBMC samples with a good sensitivity and reliability and could be useful in human studies addressing the antioxidant status and repair capacity of PBMC.

Acute toxic effects of ruthenium (II)/amino acid/diphosphine complexes on Swiss mice and zebrafish embryos

Anticancer potential of ruthenium complexes has been widely investigated, but safety evaluation studies are still scarce. Despite of ruthenium-based anticancer agents are known to cause fewer side effects compared to other metal-based drugs, these compounds are not fully free of toxicity, causing mainly nephrotoxicity. Based on the promising results from antitumor activity of the complexes [Ru(L-Met)(bipy)(dppb)]PF6 (RuMet) and [Ru(L-Trp)(bipy)(dppb)]PF6 (RuTrp), for the first time we investigated the toxicity profile of these complexes in rodent and zebrafish models. The acute oral toxicity was evaluated in Swiss mice. The mutagenic and genotoxic potential was determined by a combination of Micronucleus (MN) and Comet assay protocols, after exposure of Swiss mice to RuMet and RuTrp in therapeutic doses. Zebrafish embryos were exposed to these complexes, and their development observed up to 96 h post-fertilization. RuMet and RuTrp complexes showed low acute oral toxicity. Recorded behavioral changes were not recorded, nor were macroscopic morphological changes or structural modifications in the liver and kidneys. These complexes did not cause genetic toxicity, presenting a lack of micronuclei formation and low DNA damage induction in the cells from Swiss mice. In contradiction, cisplatin treatment exhibited high mutagenicity and genotoxicity. RuMet and RuTrp showed low toxicity in the embryo development of zebrafish. The RuMet and RuTrp complexes demonstrated low toxicity in the two study models, an interesting property in preclinical studies for novel anticancer agents.

The next three decades of the comet assay: a report of the 11th International Comet Assay Workshop.

The International Comet Assay Workshops are a series of scientific conferences dealing with practical and theoretical aspects of the Comet Assay (single-cell gel electrophoresis)-a simple method for detecting DNA strand breaks. The first paper describing such an assay was published over 30 years ago in 1984 by Swedish researchers O. Ostling and K. J. Johanson. Appropriately, the theme for the 2015 meeting was looking to the future: 'The Next 3 Decades of the Comet Assay'. The programme included 25 oral and 43 poster presentations depicting the latest advances in technical developments as well as applications of the comet assay in genotoxicity testing (in vitro and in vivo) and biomonitoring of both humans and the environment. Open discussion sessions based on questions from the participants allowed exchange of practical details on current comet assay protocols. This report summarises technical issues of high importance which were discussed during the sessions. We provide information on ways to improve the assay performance, by testing for cytotoxicity, by using reference samples to reduce or allow for inter-experimental variation, and by standardising quantification of the damage, including replicates and scoring enough comets to ensure statistical validity. After 30 years of experimentation with the comet assay, we are in a position to control the important experimental parameters and make the comet assay a truly reliable method with a wealth of possible applications.

Antiproliferative potential of aqueous leaf extract of Mucuna pruriens on DMBA-induced breast cancer in female albino rats

BackgroundBreast cancer is the most common cancer and a major cause of death in women, makes up about one-tenth of all new cancer diagnoses worldwide. Breast cancer and other cancer forms are heterogeneous diseases with varied morphological appearances, molecular features, behavior, and response to therapies (using surgery, chemotherapy, radiation therapy and targeted therapy). Despite the achievement of objective responses, the available treatments are associated with significant limitations in safety and efficacy. Several plant products (barks, leaves, flowers, roots, fruits, seeds) have been characterized as effective in cancer chemoprevention with minimal or no side effects. AimsThis study is aimed at evaluating the antiproliferative potential of aqueous leaf extract of Mucuna pruriens on 7,12-dimethylbenzeneanthracene (DMBA)-induced-breast cancer in female albino rats. Materials and methodsM. pruriens, thirty (30) female albino rats and 7,12-dimethylbenzeneanthracene were used for this study. Hot extraction protocol was employed in the preparation of aqueous extract of M. pruriens leaves. Qualitative and quantitative phytochemical screening of aqueous leaf extract of M. pruriens and weight determination was employed using standard protocol. Comet assay protocol was employed to determine level of deoxyribonucleic acid (DNA) fragmentation while hematoxylin and eosin were used for histological assay. ResultsPhytochemical screening revealed the presence of alkaloids, reducing sugar, anthraquinones, flavonoids, saponins, tannins, cardiac glycosides, phenols and steroids. Flavonoid was quantitatively determined to be present in the highest amount. Histological assay revealed the presence of atrophy of seromucous glands with surrounding stromal fibrosis and hyperplasia of serous and mucinous. There was a significant weight difference between the control and treatment groups. Determination of length and weight of isolated tumor from rat induced with DMBA only was 2.4cm and 2.8g respectively. DNA smears obtained from single cell gel electrophoresis (SCGE) suggested possible DMBA-induced damage which was significantly prevented owing to the effect of the leaf extract of M. pruriens. ConclusionThis study has shown that the leaf extract of M. pruriens could be used as a prophylactic measure against DMBA-induced cell proliferation in the breast tissues of female albino rats.

Applicability of the comet assay in evaluation of DNA damage in healthcare providers' working with antineoplastic drugs: a systematic review and meta-analysis.

Unintended occupational exposure to antineoplastic drugs (ANDs) may occur in medical personnel. Some ANDs are known human carcinogens and exposure can be monitored by genotoxic biomarkers. To evaluate the obstacles to obtaining conclusive results from a comet assay test to determine DNA damage among AND exposed healthcare workers. We systematically reviewed studies that used alkaline comet assay to determine the magnitude and significance of DNA damage among health care workers with potential AND exposure. Fifteen studies were eligible for review and 14 studies were used in the meta-analysis. Under random effect assumption, the estimated standardized mean difference (SMD) in the DNA damage of health care workers was 1.93 (95% CI: 1.15-2.71, p<0.0001). The resulting SMD was reduced to 1.756 (95% CI: 0.992-2.52, p<0.0001) when the analysis only included nurses. In subgroup analyses based on gender and smoking, heterogeneity was observed. Only for studies reporting comet moment, I2 test results, as a measure of heterogeneity, dropped to zero. Heterogeneity analysis showed that date of study publication was a possible source of heterogeneity (B=-0.14; p<0.0001). A mixture of personal parameters, comet assay methodological variables, and exposure characteristics may be responsible for heterogenic data from comet assay studies and interfere with obtaining conclusive results. Lack of quantitative environmental exposure measures and variation in comet assay protocols across studies are important obstacles in generalization of results.