Can the comet assay be used reliably to detect nanoparticle‐induced genotoxicity?

The comet assay is a sensitive method to detect DNA strand breaks as well as oxidatively damaged DNA at the level of single cells. Today the assay is commonly used in nano‐genotoxicology. In this review we critically discuss possible interactions between nanoparticles (NPs) and the comet assay. Concerns for such interactions have arisen from the occasional observation of NPs in the “comet head”, which implies that NPs may be present while the assay is being performed. This could give rise to false positive or false negative results, depending on the type of comet assay endpoint and NP. For most NPs, an interaction that substantially impacts the comet assay results is unlikely. For photocatalytically active NPs such as TiO₂, on the other hand, exposure to light containing UV can lead to increased DNA damage. Samples should therefore not be exposed to such light. By comparing studies in which both the comet assay and the micronucleus assay have been used, a good consistency between the assays was found in general (69%); consistency was even higher when excluding studies on TiO₂ NPs (81%). The strong consistency between the comet and micronucleus assays for a range of different NPs—even though the two tests measure different endpoints—implies that both can be trusted in assessing the genotoxicity of NPs, and that both could be useful in a standard battery of test methods. Environ. Mol. Mutagen. 56:82–96, 2015. © 2014 Wiley Periodicals, Inc.     

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     

Vehicle and positive control values from the in vivo rodent comet assay and biomonitoring studies using human lymphocytes: Historical database and influence of technical aspects

There is increased interest in the in vivo comet assay in rodents as a follow‐up approach for determining the biological relevance of chemicals that are genotoxic in in vitro assays. This is partly because, unlike other assays, DNA damage can be assessed in this assay in virtually any tissue. Since background levels of DNA damage can vary with the species, tissue, and cell processing method, a robust historical control database covering multiple tissues is essential. We describe extensive vehicle and positive control data for multiple tissues from rats and mice. In addition, we report historical data from control and genotoxin‐treated human blood. Technical issues impacting comet results are described, including the method of cell preparation and freezing. Cell preparation by scraping (stomach and other GI tract organs) resulted in higher % tail DNA than mincing (liver, spleen, kidney etc) or direct collection (blood or bone marrow). Treatment with the positive control genotoxicant, ethyl methanesulfonate (EMS) in rats and methyl methanesulfonate in mice, resulted in statistically significant increases in % tail DNA. Background DNA damage was not markedly increased when cell suspensions were stored frozen prior to preparing slides, and the outcome of the assay was unchanged (EMS was always positive). In conclusion, historical data from our laboratory for the in vivo comet assay for multiple tissues from rats and mice, as well as human blood show very good reproducibility. These data and recommendations provided are aimed at contributing to the design and proper interpretation of results from comet assays. Environ. Mol. Mutagen. 55:633–642, 2014. © 2014 Wiley Periodicals, Inc.     

Persistence of DNA damage in the freshwater mussel Unio pictorum upon exposure to ethyl methanesulphonate and hydrogen peroxide

An important endpoint in assessing pollution-related toxicity is genotoxicity. To obtain insight into the time-course of oxidative- and alkylation-induced DNA damage in the freshwater mussel, Unio pictorum, mussels were exposed for 24 hr to concentration gradients of pro-oxidant hydrogen peroxide (H(2)O(2)) and a mono-functional alkylating agent, ethyl methanesulfonate (EMS). DNA damage was assessed in haemocytes immediately upon exposure and over the recovery period of up to 72 days by means of comet and micronucleus assays. Following exposure to H(2)O(2), DNA damage as detected by the comet assay returned to control values after one day, except for the mussels exposed to the highest dose when damage was detectable for the next 3 days. In contrast, alkylation-induced DNA damage was detectable even after 72 days of recovery in de-chlorinated water, with a dose-response relationship observable throughout the whole recovery period. Micronucleus frequency was the highest on Day 3 after exposure to EMS; it decreased considerably by Day 7 and returned almost to the control levels 19 days after exposure, while no significant induction of micronuclei was observed in mussels exposed to H(2)O(2). Although the comet assay is considered a biomarker of recent genotoxic exposure, detecting DNA damage of shorter longevity than with the micronucleus assay, results presented here show that in the case of alkylation damage the comet assay reveals genotoxic exposure of U. pictorum in a dose-dependent manner even after 2 months.     

Caffeic Acid Genotoxicity: Correlation of the Pig-a Assay with Regulatory Genetic Toxicology In Vivo Endpoints

Caffeic acid is found in variety of fruits and vegetables. It is considered as possible human carcinogen (Group 2B). It is negative in Ames and mouse micronucleus (MN), but positive in mouse lymphoma and chromosomal aberration assays. The objective of this study was to evaluate the in vivo genotoxicity of caffeic acid using three different endpoints: in vivo MN, Pig-a, and comet assay. Two sets of six rats per group were administered vehicle (0.5% hydroxypropyl methylcellulose), 500, 1,000, or 2,000 mg/kg/day of caffeic acid for three consecutive days via oral gavage. One set of animals was used for the Pig-a and MN assay and the other set was used for the comet assay. N-Ethyl N-Nitrosourea was used as positive control for the Pig-a and MN assay, and ethyl methanesulfonate for the comet assay. From one set of animals, peripheral blood was collected on Days −1, 14, and 30 for the Pig-a assay and on Day 4 for the MN assay. The other set of animals was euthanized 3 hr after the last dose; liver and blood were collected for the comet assay. A statistically significant increase in the MN frequency was observed at 2,000 mg/kg/day. No increase in the red blood cells (RBC^CD59-) or reticulocytes (RET^CD59-) Pig-a mutant frequencies was observed on Days 14 or 30. No increase in DNA strand breaks was observed in the peripheral blood or liver in the comet assay. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, 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.     

PIG-A gene mutation as a genotoxicity biomarker in human population studies: An investigation in lead-exposed workers

The rodent Pig-a gene mutation assay has demonstrated remarkable sensitivity in identifying in vivo mutagens, while much less is known about the value of the human PIG-A assay for risk assessment. To obtain more evidence of its potential as a predictive biomarker for carcinogen exposure, we investigated PIG-A mutant frequencies (MFs), along with performing the Comet assay and micronucleus (MN) test, in 267 workers occupationally exposed to lead. Multivariate Poisson regression showed that total red blood cell PIG-A MFs were significantly higher in lead-exposed workers (10.90 ± 10.7 × 10⁻⁶) than in a general population that we studied previously (5.25 ± 3.6 × 10⁻⁶) (p < .0001). In contrast, there was no increase in lymphocyte MN frequency or in DNA damage as measured by percentage comet tail intensity in whole blood cells. Current year worker blood lead levels (BLL), an exposure biomarker, were elevated (232.6 ± 104.6 μg/L, median: 225.4 μg/L); a cumulative blood lead index (CBLI) also was calculated based on a combination of current and historical worker BLL data. Chi-square testing indicated that PIG-A MFs were significantly related to CBLI (p = .0249), but independent of current year BLL (p = .4276). However, % comet tail intensity and MN frequencies were better associated with current year BLL than CBLI. This study indicates that the PIG-A assay could serve as biomarker to detect the genotoxic effects of lead exposure and demonstrates that a battery of genotoxicity biomarkers having mechanistic complementarity may be useful for comprehensively monitoring human carcinogenic risk.     

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.     

Testing of acetaminophen in support of the international multilaboratory in vivo rat Pig-a assay validation trial

Acetaminophen, a nonmutagenic compound as previously concluded from bacteria, in vitro mammalian cell, and in vivo transgenic rat assays, presented a good profile as a nonmutagenic reference compound for use in the international multilaboratory Pig-a assay validation. Acetaminophen was administered at 250, 500, 1,000, and 2,000 mg·kg⁻¹·day⁻¹ to male Sprague Dawley rats once daily in 3 studies (3 days, 2 weeks, and 1 month with a 1-month recovery group). The 3-Day and 1-Month Studies included assessments of the micronucleus endpoint in peripheral blood erythrocytes and the comet endpoint in liver cells and peripheral blood cells in addition to the Pig-a assay; appropriate positive controls were included for each assay. Within these studies, potential toxicity of acetaminophen was evaluated and confirmed by inclusion of liver damage biomarkers and histopathology. Blood was sampled pre-treatment and at multiple time points up to Day 57. Pig-a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as CD59-negative RBC and CD59-negative RET frequencies, respectively. No increases in DNA damage as indicated through Pig-a, micronucleus, or comet endpoints were seen in treated rats. All positive controls responded as appropriate. Data from this series of studies demonstrate that acetaminophen is not mutagenic in the rat Pig-a model. These data are consistent with multiple studies in other nonclinical models, which have shown that acetaminophen is not mutagenic. At 1,000 mg·kg⁻¹·day⁻¹, Cmax values of acetaminophen on Day 28 were 153,600 ng/ml and 131,500 ng/ml after single and repeat dosing, respectively, which were multiples over that of clinical therapeutic exposures (2.6–6.1 fold for single doses of 4,000 mg and 1,000 mg, respectively, and 11.5 fold for multiple dose of 4,000 mg) (FDA 2002). Data generated were of high quality and valid for contribution to the international multilaboratory validation of the in vivo Rat Pig-a Mutation Assay.     

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.     

A critical Assessment of the Genotoxicity Profile of the Fungicide Tricyclazole

Tricyclazole (8-methyl-[1,2,4]triazolo[3,4-b][1,3]benzothiazole) is a fungicide used globally on rice for treatment of the seasonal rice blast disease. Human exposure to this fungicide can occur via dietary and nondietary routes. In a battery of in vitro assays, tricyclazole did not induce gene mutations in bacteria (Ames test) or at the Hprt locus of CHO cells. It was also negative for the induction of micronuclei in human lymphocyte cultures and unscheduled DNA synthesis (UDS) in primary rat hepatocyte. Paradoxically, tricyclazole induced a mutagenic response at the Tk locus of the mouse lymphoma L5178Ycells (MLA), which occurred equally among small/large colony phenotypes. Selection of preexisting mutants leading to a false-positive response in the MLA was ruled out in follow-up experiments. In vivo, tricyclazole was negative in the rat liver UDS assay, mouse bone micronucleus test and a transgenic (MutaMouse) gene mutation assay in glandular stomach, liver, and kidney. Other supporting evidence for the lack of genotoxicity for tricyclazole comes from an in vivo study for sister chromatid exchanges in Chinese hamsters, and a dominant lethal test in the male germ cells of mice. The combined evidence from the genotoxicity studies together with the evidence from toxicokinetic, carcinogenicity, developmental, and reproductive toxicity studies confirm that mutagenicity does not occur in relevant in vivo systems. Data were also compared to potential animal and human exposure, mechanistic data on biological targets and data on analogues, confirming adequacy of the available data for hazard identification and risk assessment. Environ. Mol. Mutagen. 61:300–315, 2020. © 2019 Wiley Periodicals, Inc.     

Proposal of an in vivo comet assay using haemocytes of Drosophila melanogaster

This study presents the first application of an in vivo alkaline comet assay using haemocytes of Drosophila melanogaster larvae. These cells, which play a role similar to that of mammalian blood, can be easily obtained and represent an overall exposure of the treated larvae. To validate the assay, we evaluated the response of these cells to three well‐known mutagenic agents: ethyl methanesulfonate (EMS), potassium dichromate (PD), and gamma radiation (γ‐irradiation). Third‐instar Drosophila larvae were exposed to different concentrations of EMS (1, 2, and 4 mM) and PD (0.5, 1, and 2.5 mM) and to different doses of γ‐irradiation (2, 4, and 8 Gγ). Subsequently, haemolymph was extracted from the larvae, and haemocytes were isolated by centrifugation and used in the comet assay. Haemocytes exhibited a significant dose‐related increase in DNA damage, indicating that these cells are clearly sensitive to the treatments. These results suggest that the proposed in vivo comet test, using larvae haemocytes of D. melanogaster, may be a useful in vivo assay for genotoxicity assessment. Environ. Mol. Mutagen., 2011. © 2010 Wiley‐Liss, Inc.     

2-Hydroxypyridine N-Oxide is not genotoxic in vivo

2-Hydroxypyridine N-oxide (HOPO) is an important coupling reagent used in pharmaceutical synthesis. Our laboratory previously reported HOPO as equivocal in the Ames assay following extensive testing of multiple lots of material. Given the lack of reproducibility between lots of material and the weak increase in revertants observed, it was concluded that it would be highly unlikely that HOPO would pose a mutagenic risk in vivo. The purpose of the current investigation was to assess experimentally in rats the mutagenic (Pig-a mutation induction) and more broadly genotoxic (micronucleus and comet induction) potential of HOPO. Rats were administered HOPO (0, 50, 150, 300, and 500 mg/kg/day) by oral gavage for 28 days. At the end of study, the following parameters were assessed: frequency of Pig-a mutant red blood cells and reticulocytes, frequency of peripheral blood micronuclei, and the incidence of comet formation in liver. Toxicokinetic data collected on study Days 1 and 28 demonstrated systemic exposure to HOPO. Although there were no overt clinical signs, animals treated with HOPO showed a dose-related decrease in body weight gain. There were no increases observed in any of the genotoxicity endpoints assessed. The results from this study further support the conclusion that in the context of pharmaceutical synthesis, HOPO should not be considered a mutagenic impurity but rather controlled as a normal process-related impurity. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.     

Evaluation of a suitable DNA damage biomarker for human biomonitoring of exposed workers

The objective of this study was to identify a sensitive and noninvasive biomarker of early genotoxic effects, for health risk assessment of workers exposed to mixtures of low doses of xenobiotics. We studied 30 workers exposed to antineoplastic drugs, 57 workers exposed to different mixtures of polycyclic aromatic hydrocarbons (PAHs) (41 airport workers and 16 paving workers) and 76 controls. Comet and micronucleus (MN) tests were performed on lymphocytes and exfoliated buccal cells. The MN assay on lymphocytes did not show significant differences between exposed and controls, while the MN assay on exfoliated buccal cells showed higher values in workers exposed to antineoplastics as compared with controls (0.85 vs. 0.48, P = 0.042). The comet assay on lymphocytes showed a higher comet percentage value (18.11 vs. 11.24 in controls, P = 0.001) and mean tail moment (TM) value (21.84 vs. 16.72 in controls, P = 0.003) in individuals exposed to PAHs as compared with controls; no significant differences were found in exposed to antineoplastics. The comet assay on exfoliated buccal cells did not show significant differences between exposed and control groups for comet percentages, whereas the TM value was higher in workers exposed to PAHs (55.1 vs. 32.31 for controls, P < 0.001). These results show that exfoliated buccal cells, obtained by a noninvasive procedure, represent robust target cells to assess the occupational exposure to inhalable mixture of chemicals at low doses. The comet assay seems to be suitable to promptly evaluate the genotoxic effects of PAHs mixtures that also contain volatile substances. The MN test is suitable to evaluate the effects of antineoplastics. Environ. Mol. Mutagen. 2009. © 2009 Wiley‐Liss, Inc.     

Gamma-H2AX immunofluorescence for the detection of tissue-specific genotoxicity in vivo

The phosphorylation of histone H2AX in Serine 139 (gamma-H2AX) marks regions of DNA double strand breaks and contributes to the recruitment of DNA repair factors to the site of DNA damage. Gamma-H2AX is used widely as DNA damage marker in vitro, but its use for genotoxicity assessment in vivo has not been extensively investigated. Here, we developed an image analysis system for the precise quantification of the gamma-H2AX signal, which we used to monitor DNA damage in animals treated with known genotoxicants (EMS, ENU and doxorubicin). To compare this new assay to a validated standard procedure for DNA damage quantification, tissues from the same animals were also analyzed in the comet assay. An increase in the levels of gamma-H2AX was observed in most of the tissues from animals treated with doxorubicin and ENU. Interestingly, the lesions induced by doxorubicin were not easily detected by the standard comet assay, while they were clearly identified by gamma-H2AX staining. Conversely, EMS appeared strongly positive in the comet assay but only mildly in the gamma-H2AX immunofluorescence. These observations suggest that the two methods could complement each other for DNA damage analysis, where gamma-H2AX staining allows the detection of tissue-specific effects in situ. Moreover, since gamma-H2AX staining can be performed on formalin-fixed and paraffin-embedded tissue sections generated during repeated-dose toxicity studies, it does not require any further treatments or extra procedures during dissection, thus optimizing the use of resources and animals. Environ. Mol. Mutagen. 60:4–16, 2019. © 2018 Wiley Periodicals, Inc.     

Antimicrobial Activities and Toxicity of Crude Extract of the Psophocarpus Tetragonolobus Pods

The extract of the Psophocarpus tetragonolobus pods has been tested for antimicrobial activity in a disk diffusion assay on eight human pathogenic bacteria and two human pathogenic yeasts. The extracts of P. tetragonolobus possessed antimicrobial activity against all tested strains. The ethanolic extract of P. tetragonolobus pods was further tested for in vivo brine shrimp lethality test and in vitro sheep erythrocyte cytotoxic assay. The brine shrimp lethality test exhibited no significant toxicity (LC₅₀=1.88 mg/ml) against Artemia salina, whereas sheep erythrocyte test showed significant toxicity. The reason for haemolysis of erythrocyte was discussed. The P. tetragonolobus extract with high LC₅₀ value signified that this plant is not toxic to human. This result also suggested that the ethanolic extract of P. tetragonolobus pods is potential source for novel antimicrobial compounds.     

In vitro and in vivo assessments of the genotoxic potential of 3-chloroallyl alcohol

3-Chloroallyl alcohol (3-CAA) can be found in the environment following the application of plant protection products. 3-CAA is formed in groundwater following the injection of 1,3-dichloropropene, a fumigant used to control nematodes. 3-CAA is also formed, in leafy crops, as a glycoside conjugate following application of the herbicide, clethodim. Human exposure may occur from groundwater used as drinking water or through dietary consumption. To characterize 3-CAA's potential to cause genotoxicity in mammals, in vitro and in vivo studies were conducted. 3-CAA was negative in an Ames test and positive in a mouse lymphoma forward mutation assay. 3-CAA was negative in an acute in vivo CD-1 mouse bone marrow micronucleus assay when administered up to a dose level of 125 mg/kg/day for two consecutive days. In a combined gene mutation assay and erythrocyte micronucleus assay, using transgenic Big Blue® Fischer 344 rats, 3-CAA was administered via drinking water at targeted dose levels of 0, 10, 30, and 100 mg/kg/day for 29 days. Peripheral blood samples, collected at the end of treatment, were analyzed for micronucleus induction in reticulocytes using flow cytometry. Liver and bone marrow samples, collected 2 days after the termination of the treatment, were analyzed for the induction of mutations at the cII locus. 3-CAA did not induce an increase in mutant frequency or micronuclei under the experimental conditions. In conclusion, the mutagenic response observed in the in vitro mouse lymphoma assay is not confirmed in the whole animal. 3-CAA is not considered to pose a mutagenic risk.     

In vivo rodent erythrocyte micronucleus assay. II. Some aspects of protocol design including repeated treatments, integration with toxicity testing, and automated scoring

An expert working group on the in vivo micronucleus assay, formed as part of the International Workshop on Genotoxicity Test Procedures (IWGTP), discussed protocols for the conduct of established and proposed micronucleus assays at a meeting held March 25-26, 1999 in Washington, DC, in conjunction with the annual meeting of the Environmental Mutagen Society. The working group reached consensus on a number issues, including: (1) protocols using repeated dosing in mice and rats; (2) integration of the (rodent erythrocyte) micronucleus assay into general toxicology studies; (3) the possible omission of concurrently-treated positive control animals from the assay; (4) automation of micronucleus scoring by flow cytometry or image analysis; (5) criteria for regulatory acceptance; (6) detection of aneuploidy induction in the micronucleus assay; and (7) micronucleus assays in tissues (germ cells, other organs, neonatal tissue) other than bone marrow. This report summarizes the discussions and recommendations of this working group. In the classic rodent erythrocyte assay, treatment schedules using repeated dosing of mice or rats, and integration of assays using such schedules into short-term toxicology studies, were considered acceptable as long as certain study criteria were met. When the micronucleus assay is integrated into ongoing toxicology studies, relatively short-term repeated-dose studies should be used preferentially because there is not yet sufficient data to demonstrate that conservative dose selection in longer term studies (longer than 1 month) does not reduce the sensitivity of the assay. Additional validation data are needed to resolve this point. In studies with mice, either bone marrow or blood was considered acceptable as the tissue for assessing micronucleus induction, provided that the absence of spleen function has been verified in the animal strains used. In studies with rats, the principal endpoint should be the frequency of micronucleated immature erythrocytes in bone marrow, although scoring of peripheral blood samples gives important supplementary data about the time course of micronucleus induction. When dose concentration and stability are verified appropriately, concurrent treatment with a positive control agent is not necessary. Control of staining and scoring procedures can be obtained by including appropriate reference samples that have been obtained from a separate experiment. For studies in rats or mice, treatment/sampling regimens should include treatment at intervals of no more than 24 hr (unless the test article has a half-life of more than 24 hr) with sampling of bone marrow or blood, respectively, within 24 or 40 hr after the last treatment. The use of a DNA specific stain is recommended for the identification of micronuclei, especially for studies in the rat. In the case of a negative assay result with a non-toxic test article, it is desirable that systemic exposure to the test article is demonstrated. The group concluded that successful application of automated scoring by both flow cytometry and image analysis had been achieved, and defined criteria that should be met if automated scoring is employed. It was not felt appropriate to attempt to define specific recommended protocols for automated scoring at the present time. Other issues reviewed and discussed by the working group included micronucleus assays that have been developed in a number of tissues other than bone marrow. The group felt that these assays were useful research tools that could also be used to elucidate mechanisms in certain regulatory situations, but that these assays had not yet been standardized and validated for routine regulatory application.     

Copper oxide nanoparticles and copper sulphate act as antigenotoxic agents in drosophila melanogaster

The biological reactivity of metal and metal oxide nanomaterials is attributed to their redox properties, which would explain their pro‐ or anti‐cancer properties depending on exposure circumstances. In this sense, copper oxide nanoparticles (CuONP) have been proposed as a potential anti‐tumoral agent. The aim of this study was to assess if CuONP can exert antigenotoxic effects using Drosophila melanogaster as an in vivo model. Genotoxicity was induced by two well‐known genotoxic compounds, namely potassium dichromate (PD) and ethyl methanesulfonate (EMS). The wing‐spot assay and the comet assay were used as biomarkers of genotoxic effects. In addition, changes in the expression of Ogg1 and Sod genes were determined. The effects of CuONP cotreatment were compared with those induced by copper sulfate (CS), an agent releasing copper ions. Using the wing‐spot assay, CuONP and CS were not able to reduce the genotoxic effects of EMS exposure, but had the ability to decrease the effects induced by PD, reducing the frequency of mutant twin‐spots that arise from mitotic recombination. In addition, CuONP and CS were able to reduce the DNA damage induced by PD as determined by the comet assay. In general, similar qualitative antigenotoxic effects were obtained with both copper compounds. The antigenotoxic effects of environmentally relevant and non‐toxic doses of CuONP and CS may be explained by their ability to partially restore the expression levels of the repair gene Ogg1 and the antioxidant gene Cu,ZnSod, both of which are inhibited by PD treatment. Environ. Mol. Mutagen. 58:46–55, 2017. © 2016 Wiley Periodicals, Inc.     

Suitability of Long-Term Frozen Rat Blood Samples for the Interrogation of Pig-a Gene Mutation by Flow Cytometry

The rodent blood Pig-a assay has been undergoing international validation for use as an in vivo hematopoietic cell gene mutation assay, and given the promising results an Organization for Economic Co-operation and Development (OECD) Test Guideline is currently under development. Enthusiasm for the assay stems in part from its alignment with 3Rs principles permitting combination with other genotoxicity endpoint(s) and integration into repeat-dose toxicology studies. One logistical requirement and experimental design limitation has been that blood samples required antibody labeling and flow cytometric analysis within one week of collection. In the current report, we describe the performance of freeze–thaw reagents that enable storage and subsequent labeling and analysis of rat blood samples for at least seven months. Data generated from three laboratories are presented that demonstrate rat erythrocyte recoveries in the range of 80–90%. Despite some loss of erythrocytes, Pearson coefficients and Bland–Altman analyses based on fresh blood vs. frozen/thawed matched pairs indicate that mutant cell and reticulocyte frequencies are not significantly affected, as the measurements are highly correlated and exhibit low bias. Collectively, these data support the effectiveness and suitability of a freeze–thaw procedure that endows the assay with several new advantageous characteristics that include: flexibility in scheduling personnel/instrumentation; reliability when shipping samples from in-life facilities to analytical sites; 3Rs-friendly, as blood from positive control animals can be stored frozen to serve as analytical controls; and ability to defer a decision to generate Pig-a data until more toxicological information becomes available on a test substance. Environ. Mol. Mutagen. 60:47–55, 2019. © 2018 Wiley Periodicals, Inc.