Assessment of DNA damage in lymphocytes of workers exposed to X-radiation using the micronucleus test and the comet assay.

The mutagenic and carcinogenic effects of genotoxic agents on exposed people have constituted an increasing concern. Therefore, the objective of this work was to assess DNA damage in lymphocytes of workers exposed to X-radiation using the cytokinesis-blocked micronucleus test and the comet assay (single-cell gel electrophoresis), and to compare these two techniques in the monitoring of exposed populations. The cytokinesis-blocked micronucleus test and the comet assay were employed in the monitoring of 22 workers occupationally exposed to X-radiation in a hospital in southern Brazil. The frequency of dicentric bridges was also measured. The results of both assays and the frequency of dicentric bridges revealed a significant increase in genetic effects on the cells of exposed individuals. Age was significantly correlated with micronucleus frequency and damage index in the comet assay. The concomitant analysis of dicentric bridges when determining micronucleus frequency does not require much extra work, and may serve as a reference to the type of mutagenic effect (clastogenic or aneugenic). The combination of the alkaline comet assay with the cytokinesis-blocked micronucleus test appears to be very informative for the monitoring of populations chronically exposed to genotoxic agents.     

Cytotoxic and mutagenic evaluation of extracts from plant species of the Miconia genus and their influence on doxorubicin-induced mutagenicity: An in vitro analysis

The Miconia genus, a plant widely used for medicine, occurs in tropical America and its extracts and isolated compounds have demonstrated antibiotic, antitumoral, analgesic and antimalarial activities. However, no study concerning its genotoxicity has been conducted and it is necessary to determine its potential mutagenic effects to develop products and chemicals from these extracts. This study assessed the cytotoxicity, mutagenicity and the protective effects of methanolic extracts from Miconia species on Chinese hamster lung fibroblast cell cultures (V79). The cytotoxicity was evaluated using a clonogenic assay. Cultures exposed to the extract of Miconia albicans up to a concentration of 30 μg/mL, M. cabucu up to 40 μg/mL, M. albicans up to 40 μg/mL and M. stenostachya up to 60 μg/mL exhibited a cytotoxic effect on the cells. The clonogenic assay used three non-cytotoxic concentrations (5, 10 and 20 μg/mL) to evaluate mutagenicity and antimutagenicity of the extracts. Cultures were treated with these three extract concentrations (mutagenicity test) or the extract associated with doxorubicin (DXR) (antimutagenicity test) in three protocols (pre-, simultaneous and post-treatments). Distilled water and DXR were used as negative and positive controls, respectively. In the micronucleus (MN) test, a significant reduction was observed in MN frequency in cultures treated with DXR and extracts compared to those receiving only DXR; a significant reduction was also observed for the presence of mutagenicity in all treatments. This study confirmed the safe use of Miconia extracts at the concentrations tested and reinforced the therapeutic properties previously described for Miconia species by showing their protective effects on doxorubicin-induced mutagenicity.     

Investigating the safety of plants used in South African traditional medicine: testing for genotoxicity in the micronucleus and alkaline comet assays

Previous studies indicate that traditional botanical remedies can be valuable for treating human disease. The potential risk from long-term use of such remedies has not, however, been fully investigated, especially in terms of their potential carcinogenic activity. In the present study, 51 South African plant species were selected on the basis of their use in traditional medicine and crude extracts were sequentially prepared from different dried plant parts using dichloromethane followed by 90% methanol. These extracts were tested for genotoxic activity in human peripheral blood lymphocytes using the micronucleus test, with further testing of select extracts using the alkaline comet assay. Screening results indicated the induction of significant numbers of micronuclei by many of the plant extracts. Several samples also induced DNA damage in human white blood cells using the alkaline comet assay. Although a number of these plants are recognised as toxic by traditional healers, several plants that are used in common remedies were found to be genotoxic and potentially dangerous. Environ.     

Cytogenetic studies of PCB77 on brown trout (Salmo trutta fario) using the micronucleus test and the alkaline comet assay

Polychlorinated biphenyls (PCBs) are stable pollutants, whichcan be found in almost every compartment of terrestrial andaquatic ecosystems. They are very lipophilic and therefore havethe potency of accumulating in the fat stores of animals. Themechanisms by which PCBs exert their adverse effects are stillunclear. It is known that PCBs induce some important biotransformationenzymes, but their mutagenic properties are still controversial.The DNA breakage and clastogenic potency of a planar PCB77 (3,3', 4, 4'-tetrachlorobiphenyl) was determined in vivo in fish,using the single cell gel electrophoresis or comet assay andthe micronucleus test, on erythrocytes of the brown trout exposedfor 3, 9 and 14 days to initial PCB concentrations of 780 and918 pg/ml, dissolved in the water. Blood was taken by a caudalpuncture and the erythrocytes were either deposited in an agarosegel (0.6%) for the comet assay or smeared directly on slidesfor the micronucleus test. Five fish were studied per treatmentand 50 and 2000 erythrocytes per concentration and per animalwere analysed for the comet assay and the micronucleus testrespectively. Ethyl methanesulphonate (EMS) at a concentrationof 25 mg/I water was used as a positive control. Although EMSinduced a statistically significant increase of single strandbreaks in the comet assay, in neither of the two tests used,were mutagenic effects due to PCB exposure observed. ³To whom correspondence should be addressed     

Evaluation of the genotoxicity of river sediments from industrialized and unaffected areas using a battery of short-term bioassays

The present investigation evaluated the capacity of the Salmonella mutagenicity test, the comet assay, and the micronucleus assay to detect and characterize the genotoxic profile of river sediments. Three stations were selected on an urban river (Bouches du Rhône, France) exposed to various sources of industrial and urban pollution (StA, StB, and StC) and one station on its tributary (StD). One station in a nonurban river was included (REF). The concentrations of 16 polycyclic aromatic hydrocarbons (PAHs) were determined by HPLC, and the genotoxicity of the sediments was monitored by the Salmonella mutagenicity test (TA98 + S9, YG1041 ± S9), the comet assay, and the micronucleus assay on CHO cells. Chemical analysis showed that the total PAH concentrations ranged from 23 μg kg^?1 dw (REF) to 1285 μg kg^?1 dw (StD). All the sediments were mutagenic in the Salmonella mutagenicity test. The mutagenicity was probably induced by the presence of nitroarenes (StA, StB, StC, and StD) and aromatic amines (REF) as deduced from the mutagenicity profiles of strains YG1041 ± S9 and TA98 + S9. The comet assay revealed direct DNA lesions in REF, StA, and StB sediments and metabolization‐dependent DNA damage in StC and StD. The micronucleus assay showed an absence of clastogenicity for StA ± S9 and StC‐S9, and a significant clastogenicity ± S9 for the three other stations. The genotoxicity ranking determined by the comet assay + S9 matched the ranking of total and carcinogenic PAH concentrations, and this assay was found to be the most sensitive. Environ. Mol. Mutagen., 2008. © 2008 Wiley‐Liss, Inc.     

Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero.     

Genotoxicity of azidoalanine in mammalian cells

Sodium azide mutagenesis is mediated through a metabolic intermediate in bacteria and plant species. However, very little is known about the interaction of this intermediate with nucleic acids, its genotoxic potential, or its mechanism of action, especially in mammalian cells. Chinese hamster cells and normal human skin fibroblasts were treated with extracts from Salmonella typhimurium or Hordeum vulgare (barley) containing a crude mutagenic metabolite, as well as with synthetically produced azidoalanine. The cells were evaluated for the induction of sister chromatid exchanges and the ability to perform unscheduled DNA synthesis. With the purified azidoalanine and the azide-treated extracts from Hordeum vulgare, there was a statistically significant increase in the frequency of sister chromatid exchanges observed in both Chinese hamster cells and human fibroblasts. This increase was about twofold, as compared with the control. On the other hand, there was no detectable genotoxic response when cells were exposed to azide-treated extract from Salmonella typhimurium. The results imply that azidoalanine and the crude mutagenic metabolite from Hordeum vulgare are weakly genotoxic in mammalian cells.     

Investigation of cytotoxic, apoptosis-inducing, genotoxic and protective effects of the flavonoid rutin in HTC hepatic cells

Rutin is a flavonoid with antioxidant, vasodilatory, anti-inflammatory and immune-stimulating activities. To study the toxicity of rutin and its protective effect, this work investigated the cytotoxic, apoptosis-inducing, genotoxic and protective effects of rutin in HTC cells. In the MTT assay, the highest concentration tested (810 μM) showed cytotoxicity after 72 h of treatment, where cell viability and cell proliferation was diminished. None of the concentrations of rutin tested induced apoptosis after 24 h treatment. The highest concentration of rutin after 24 h treatment induced DNA damage, shown in the comet assay, but did have a genotoxic effect in the micronucleus test. Rutin was tested against the pro-carcinogenic agent benzo(a)pyrene, at concentrations of 90, 270 and 810 μM, and was found to reduce induced DNA damage significantly. This protective effect of rutin against a pro-carcinogen, suggests an important biological activity for this compound, which can contribute to human health through the diet.     

Mutagenicity of chili extract and capsaicin in short-term tests

Vanillin, capsaicin and chili extracts were tested for mutagenicity in Salmonella typhimurium histidine-deficient tester strains TA 98, TA 100, TA 1535, TA 1537, and TA 1538. Vanillin was nonmutagenic, whereas chili extract and capsaicin were mutagenic with metabolic activation. Capsaicin, an active component of chili extract, was the more potent mutagen. The positive samples were also tested in two mammalian test systems: the micronucleus test and the 8-azaguanine-resistant mutagenesis assay that used V79 Chinese hamster cells. It was observed that both were negative for the latter test at the dose level tested, whereas in the micronucleus test, only capsaicin was positive near the LD50 dose. Capsaicin also inhibited DNA synthesis in the testes of Swiss mice injected at two dose levels.     

Protective effects of β-glucan extracted from barley against benzo[a]pyrene-induced DNA damage in hepatic cell HepG2

The aim of the present study was to assess the genotoxic and antigenotoxic effect of β-glucan (BG) extracted from barley. The genotoxicity of BG was tested in the single-cell gel electrophoresis assays (SCGE)/HepG2 test system. Moreover, the protective effects of BG against the genotoxicity of B[a]P were studied to delineate its mechanism of antigenotoxicity using four different treatment protocols – pre-treatment, simultaneous simple, simultaneous with pre-incubation, and post-treatment. The results showed that the compound itself was devoid of mutagenic activity at the three lower concentrations studied (1, 5, and 25 μg/mL); however, genotoxic and cytotoxic effects were seen at 100 and 200 μg/mL, respectively. In combination experiments with B[a]P, pronounced inhibition of DNA migration in the SCGE assay was observed in the two simultaneous treatments, and a smaller reduction was observed in the two other treatments. Thus, the data suggest that BG acts through binding to the genotoxic compound or capturing free radicals produced during its activation. However, the protective effects observed with pre-treatment and post-treatment suggest that the BG may be modulating cell metabolism.     

In vitro evaluation of the genotoxic and cytotoxic effects of artesunate, an antimalarial drug, in human lymphocytes

Artesunate is one of the main antimalarial drugs used in several countries. It is a semisynthetic compound derived from artemisinin, a substance extracted from the Chinese plant, Artemisia annua L. Despite the widespread use of artesunate as an antimalarial drug, there is a lack of data regarding its genotoxic effects in human lymphocytes. Therefore, in this study, we used the comet assay and micronucleus test to evaluate the possible genotoxic effects of artesunate in cultured human lymphocytes. In addition, cell death by necrosis and apoptosis was also assessed. Cells exposed to different concentrations of artesunate showed a significant concentration-dependent increase (P < 0.05) in DNA damage index and micronuclei frequency. A significant increase in the frequency of apoptotic and necrotic cells was also observed. Our results showed that artesunate is a genotoxic and cytotoxic compound in cultured human lymphocytes.     

Evaluation of cytotoxicity and genotoxicity induced by oleic acid-coated iron oxide nanoparticles in human astrocytes

Iron oxide nanoparticles (ION) are gaining importance as diagnostic and therapeutic tool of central nervous system diseases. Although oleic acid-coated ION (O-ION) have been described as stable and biocompatible, their potential neurotoxicity was scarcely evaluated in human nervous cells so far. The primary aim of this work was to assess the molecular and cellular effects of O-ION on human astrocytes (A172 cells) under different experimental conditions. An extensive set of cyto- and genotoxicity tests was carried out, including lactate dehydrogenase release assay, cell cycle alterations, and cell death production, as well as comet assay, γH2AX assay, and micronucleus (MN) test, considering also iron ion release capacity and alterations in DNA repair ability. Results showed a moderate cytotoxicity related to cell cycle arrest and cell death promotion, regardless of serum presence. O-ION induced genotoxic effects, namely primary DNA damage, as detected by the comet assay and H2AX phosphorylation, but A172 cells were able to repair this particular damage because no chromosome alterations were found (confirmed by MN test results). Accordingly, no effects on the DNA repair ability were observed. The presence of serum proteins did not influence O-ION toxicity. Iron ions released from the O-ION surface seemed not to be responsible for the cytotoxic and genotoxic effects observed. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.     

Evaluation of the capacity of the SCGE assay to assess the genotoxicity of biomaterials

The comet test or SCGE assay, which is already widely used in other areas, has never been used to evaluate the mutagenic potential of medical biomaterials in the final form. The purpose of our study was thus to assess the comet test as a means of assessing the genotoxic potential of finished medical biomaterials. We used silicone elastomers with increasing concentrations of 4-nitroquinoline oxide, a genotoxic agent. Hydrogen peroxide was used as the positive control, and tissue culture polystyrene as the negative control. In our study, the comet test did not detect a significant difference in genotoxicity between the pure elastomer and the same elastomer containing 0.01 mg/ml 4-nitroquinoline oxide, but did detect a significant difference between two elastomers containing 0.01 and 0.3 mg/ml of 4-nitroquinoline oxide, respectively. Since, the surface properties of the samples were identical, only the chemical composition may have caused significant differences in mutagenicity. Whatever the cause of the genotoxicity detected by the SCGE assay, testing finished biomaterials using the comet assay makes it possible to evaluate interactions between biomaterials and living tissues that are much closer to actual application conditions.     

Development of test systems for the detection of compounds that prevent the genotoxic effects of heterocyclic aromatic amines: preliminary results with constituents of cruciferous vegetables and other dietary constituents.

Over the past decades, strong efforts have been made to identify dietary constituents that protect against the genotoxic effects of heterocyclic aromatic amines (HAAs). However, most of the methods that have been used, in particular in vitro assays that require the addition of exogenous enzyme homogenates, have only a limited predictive value because important protective mechanisms are not adequately represented and may give misleading results. Therefore, we attempted to develop improved test systems, namely assays, with human hepatoma cells and single-cell gel electrophoresis (SCGE) tests with rats. Genotoxicity tests with human derived Hep G2 cells reflect the genotoxic effects of HAAs better than other in vitro systems. They also enable the detection of protective effects since the human derived hepatoma cells possess phase I and phase II enzymes that are involved in the activation/ detoxification of the amines. The most appropriate endpoint for experiments with Hep G2 cells appears to be micronucleus induction, but protocols for other endpoints are available as well. The second promising model is the SCGE ("comet") assay with rats that was used successfully to measure protective effects of constituents of cruciferous vegetables against 2-amino-3-methylimidazo[4,5-flquinoline (IQ) in the liver and in the colon mucosa. The present study describes the experimental design of the new approaches, as well as results obtained with various dietary constituents.     

Evaluation of genetic damage in operating room personnel exposed to anaesthetic gases

Information on potential genetic damage in humans after exposure to waste anaesthetic gases in Indian hospitals is scarce. To evaluate the possible genotoxic effects of waste anaesthetic gases, the chromosomal aberrations analysis and comet assay were studied in peripheral blood lymphocytes in 45 operating room personnel currently employed at a hospital in South India. In addition, the micronucleus test on buccal epithelial cells was also carried out in the same subjects. The exposed group was compared with a group of 45 non-exposed group, matched by age, sex, alcohol consumption and smoking habits. The results showed a statistically significant increase in DNA damage by the comet assay in the exposed group. Chromosome aberrations and micronucleus frequencies also increased significantly in the study subjects in comparison to the controls. Analysis of variance showed that smoking had a significant effect on DNA mean tail length, whereas alcohol consumption, duration of exposure to anaesthetic agents, age and gender had no significant effect. All the confounding factors had significant effect by the micronucleus test. However, smoking, alcohol consumption, age, gender and years of exposure showed no significant effect by the chromosome aberrations test. The results of our study suggest that exposure to waste anaesthetic gases has the potential to cause changes in the human genome.     

Genotoxic effects of three selected black toner powders and their dimethyl sulfoxide extracts in cultured human epithelial A549 lung cells in vitro

Until now, the adverse effects of toner powders on humans have been considered to be minimal. However, several recent reports have suggested possible significant adverse health effects from toner dust inhalation. The aim of this study was to evaluate the genotoxic potential of black toner powders in vitro. For the study of DNA damage, A549 cells were exposed to toner‐powder suspensions and to their DMSO extracts, and then subjected to the comet assay and to the in‐vitro cytokinesis block micronucleus test (CB‐MNvit). Cytotoxic effects of the toner samples were assessed by the erythrosin B assay. Furthermore, size, shape, and composition of the toner powders were investigated. None of the three toner powders or their DMSO extracts reduced cell viability; however, they did induce DNA damage and formed micronuclei at concentrations from 80 to 400 μg cm^?2, although to a varying extent. All toner powders contain considerable amounts of the pigments carbon black and magnetite (Fe₃O₄) as well as small amounts of polycyclic aromatic hydrocarbons (PAHs). The overall results of our in‐vitro study suggest that the investigated toner‐powder samples are not cytotoxic but genotoxic. From the results of the physical and chemical characterization, we conclude that metals and metalloids as components of magnetite, or PAHs as components of the carbon‐bearing material, are responsible for the genotoxic effects. Further research is necessary to determine the relevance of these in‐vitro observations for private and occupational toner powder exposure. Environ. Mol. Mutagen., 2011. © 2010 Wiley‐Liss, Inc.     

Studies of anticancer and antipyretic activity of Bidens pilosa whole plant

Screening of different extracts and fractions from the plant Bidens pilosa Linn. var. (Asteraceae) has been conducted using the in-vitro comet assay for anticancer and the antipyretic action, which was done with in-vivo models. The extract from whole plant was extracted with n-hexane, chloroform and methanol extract (E1 - E3). The extracts were fractioned by column chromatography method and fractioned with ethyl acetate, acetone and water (F1 - F3). All the extracts and fractions were tested for anticancer and antipyretic activity. Among extracts E1 shows remarkable anticancer activity and E3 bears maximum antipyretic activity. In the antipyretic activity, paracetamol was used as the standard test drug. The most promising material (LC50 < 1500 microg/ml) was F1 ethyl acetate fractions of methanolic extract and methanolic crude extract of whole plants. However, little correlation was observed in the degree of antipyretic activity between the test drug and standard drug. In conclusion, the extract obtained from the whole plant of Bidens pilosa showed a significant cytotoxic effect to methanolic extract against Hela cells by in vitro method and showed a comparable antipyretic activity effect to paracetamol in rabbit pyrogen test.     

Characterization of the genotoxic potential of formaldehyde in V79 cells

Formaldehyde (FA) is known to be genotoxic and mutagenic in proliferating mammalian cells in vitro. The present study was performed to further characterize its genotoxic potential in the V79 Chinese hamster cell line. The induction of DNA strand breaks and DNA-protein cross-links (DPXs) was measured by the comet assay in relationship to the induction of sister chromatid exchanges (SCEs) and micronuclei (MN). Induction of DNA strand breaks was found neither with the standard protocol of the alkaline comet assay nor with modifications using extended electrophoresis times or proteinase K. The concentration-effect relationship for the genotoxic effects was characterized by fitting different curves to the data. A two-phase regression model fitted best in comparison with a linear or a quadratic model and indicated practical thresholds for the induction of SCE and MN. For the induction of DPX as measured by the comet assay, neither a linear concentration-response relationship nor any of the tested models fitted well to the data. Three repeated treatments with genotoxic concentrations of FA with a 3-h interval led to enhanced levels of DPX and MN while the same treatments with a 24-h interval did not enhance FA genotoxicity but suggested adaptive protection against the DNA-damaging action of FA.     

Studies on the genotoxic effects of crude liver oils from 3 species of Mediterranean sharks by means of in vitro micronucleus test using human lymphocytes

Lymphoid system tumours have been identified in two subjects who used to handle for several years mediterranean shark liver oil and squalen extracted from this oil. Moreover, scientific data, reported in 1959 by Kr?ning, show the induction of lymphoid tumours in C57 B1 mice after exposure of their skin to squalen. These observations rose the question of a possible mutagenic power of shark liver oil. In order to determine the genotoxicity of these oils, in vitro assays have been performed on crude hepatic oil of three species of mediterranean sharks: two benthic sharks, Centrophorus granulosus and Galeus melastomus, and one pelagic specie, Prionace glauca. Genotoxicity of oils have been assayed using a micronucleus test which can detected simultaneously clastogen and aneugen effects. The incubation of human cells with the hepatic crude oils of Centrophorus granulosus increases the rate of the binucleated micronucleated cell in a dose dependent manner. The mean micronucleated cell rate was 9.0%. +/- 1.1 in controls and increased up to 27,1%. +/- 4,0 for the highest concentrations of oil extracts. Similar results have been obtained with crude hepatic oils of Galeus melastomus and Prionace glauca. The results of this experimental study show that the crude liver oils of three species of sharks are genotoxic and confirm a high carcinogenic risk.     

A comparison of genotoxicity of automotive exhaust particles from laboratory and environmental sources

This research (1) ranked the genotoxicity of methylene chloride extracts of laboratory and environmentally collected particles and (2) evaluated the role of collection location and sample composition on genotoxic potency. Samples of exhaust from a spark-ignition automobile, light-duty diesel automobile, and a heavy-duty diesel engine operated in a laboratory on a dynamometer were studied, as well as samples taken in a highway tunnel and outside the same tunnel. The tunnel samples were collected 30 m inside or 56 m outside the exit portal at times when between 70%-95% of the traffic consisted of diesel trucks. In the Ames Salmonella mutagenicity assay, each extract produced a dose-dependent increase in mutagenicity in strain TA-98 without addition of liver S-9 fraction. Extracts from two tunnel samples collected 1 yr apart, and extracts of particles collected outside the tunnel had similar mutagenic activity. The order of mutagenic activity per microgram of extract in TA-98 without S-9 from the lowest to the highest was environmental sample less than or equal to tunnel less than heavy-duty diesel less than light-duty diesel less than spark ignition. Addition of S-9 or testing in Salmonella strains resistant to the mutagenicity of nitroaromatic compounds (TA-98 NR and TA-98 1,8-DNP6) decreased the mutagenic response. With cell killing, sister chromatid exchanges, and mutations as endpoints in Chinese hamster ovary cells (CHO), the order of potency was tunnel less than light-duty less than spark-ignition samples. All three extracts induced a similar amount of mitotic delay per microgram with or without S-9. Enhanced chromosome aberration frequency was detected only in cells exposed to extracts from spark-ignition exhaust. The data indicated that genotoxic activity was detected in each particle extract, that the potency ranking was similar using different genetic endpoints, and that the magnitude of the genotoxic potency was similar.