The Comet Assay with Multiple Mouse Organs: Comparison of Comet Assay Results and Carcinogenicity with 208 Chemicals Selected from the IARC Monographs and U.S. NTP Carcinogenicity Database

ABSTRACT

The comet assay is a microgel electrophoresis technique for detecting DNA damage at the level of the single cell. When this technique is applied to detect genotoxicity in experimental animals, the most important advantage is that DNA lesions can be measured in any organ, regardless of the extent of mitotic activity. The purpose of this article is to summarize the in vivo genotoxicity in eight organs of the mouse of 208 chemicals selected from International Agency for Research on Cancer (IARC) Groups 1, 2A, 2B, 3, and 4, and from the U.S. National Toxicology Program (NTP) Carcinogenicity Database, and to discuss the utility of the comet assay in genetic toxicology.

Alkylating agents, amides, aromatic amines, azo compounds, cyclic nitro compounds, hydrazines, halides having reactive halogens, and polycyclic aromatic hydrocarbons were chemicals showing high positive effects in this assay. The responses detected reflected the ability of this assay to detect the fragmentation of DNA molecules produced by DNA single strand breaks induced chemically and those derived from alkali-labile sites developed from alkylated bases and bulky base adducts. The mouse or rat organs exhibiting increased levels of DNA damage were not necessarily the target organs for carcinogenicity. It was rare, in contrast, for the target organs not to show DNA damage. Therefore, organspecific genotoxicity was necessary but not sufficient for the prediction of organ-specific carcinogenicity. It would be expected that DNA crosslinkers would be difficult to detect by this assay, because of the resulting inhibition of DNA unwinding. The proportion of 10 DNA crosslinkers that was positive, however, was high in the gastrointestinal mucosa, stomach, and colon, but less than 50% in the liver and lung. It was interesting that the genotoxicity of DNA crosslinkers could be detected in the gastrointestinal organs even though the agents were administered intraperitoneally.

Chemical carcinogens can be classified as genotoxic (Ames test-positive) and putative nongenotoxic (Ames test-negative) carcinogens. The Ames test is generally used as a first screening method to assess chemical genotoxicity and has provided extensive information on DNA reactivity. Out of 208 chemicals studied, 117 are Ames test-positive rodent carcinogens, 43 are Ames test-negative rodent carcinogens, and 30 are rodent noncarcinogens (which include both Ames test-positive and negative noncarcinogens). High positive response ratio (110/117) for rodent genotoxic carcinogens and a high negative response ratio (6/30) for rodent noncarcinogens were shown in the comet assay. For Ames test-negative rodent carcinogens, less than 50% were positive in the comet assay, suggesting that the assay, which detects DNA lesions, is not suitable for identifying nongenotoxic carcinogens. In the safety evaluation of chemicals, it is important to demonstrate that Ames test-positive agents are not genotoxic in vivo. This assay had a high positive response ratio for rodent genotoxic carcinogens and a high negative response ratio for rodent genotoxic noncarcinogens, suggesting that the comet assay can be used to evaluate the in vivo genotoxicity of in vitro genotoxic chemicals. For chemicals whose in vivo genotoxicity has been tested in multiple organs by the comet assay, published data are summarized with unpublished data and compared with relevant genotoxicity and carcinogenicity data.

Because it is clear that no single test is capable of detecting all relevant genotoxic agents, the usual approach should be to carry out a battery of in vitro and in vivo tests for genotoxicity. The conventional micronucleus test in the hematopoietic system is a simple method to assess in vivo clastogenicity of chemicals. Its performance is related to whether a chemical reaches the hematopoietic system. Among 208 chemicals studied (including 165 rodent carcinogens), 54 rodents carcinogens do not induce micronuclei in mouse hematopoietic system despite the positive finding with one or two in vitro tests. Forty-nine of 54 rodent carcinogens that do not induce micronuclei were positive in the comet assay, suggesting that the comet assay can be used as a further in vivo test apart from the cytogenetic assays in hematopoietic cells. In this review, we provide one recommendation for the in vivo comet assay protocol based on our own data.     

Development of a co-culture model of mouse primary hepatocytes and splenocytes to evaluate xenobiotic genotoxicity using the medium-throughput Comet assay

To date, only a limited number of toxicological studies have focused on the establishment and validation of in vitro genotoxicity screening systems using primary hepatocytes, and the results of these studies have been inconsistent. Therefore, the aim of this study was to develop an effective co-culture model of mouse-derived primary hepatocytes and splenocytes for screening chemicals for genotoxicity using the medium-throughput Comet assay. This cocultured model was constructed and verified using known genotoxic and non-genotoxic compounds as positive and negative controls, respectively. Cytotoxicity was measured using Cell Counting Kit-8 and lactate dehydrogenase methods. DNA damage was detected using both alkaline and formamidopyrimidine DNA glycosylase (FPG) Comet assays. Compared with the controls, DNA strand breaks and FPG-sensitive sites showed significant concentration-dependent increases in genotoxic-agent-treated groups. In contrast, DNA damage remained unchanged in non-genotoxic-agent-treated groups. In addition, different types of genotoxic agents resulted in different time-dependent DNA lesions. Our results indicated that the % tail DNA indicating both DNA strand breaks and FPG-sensitive sites might be effective markers for predicting chemical-induced DNA damage and oxidative DNA damage using the cocultured model of hepatocytes and splenocytes. Collectively, these findings provide reliable experimental data for the establishment of in vitro genotoxicity screening methods.     

Genotoxicity and antigenotoxicity of cashew (Anacardium occidentale L.) in V79 cells

The use of plants for the treatment of diseases continues to rise although there are few studies providing proof of these effects. One of these plants is the Anacardium occidentale, popularly known as the cashew. The present study evaluated the possible genotoxic and protective activities of cashew stem bark methanolic extract, in vitro, using methyl methanesulfonate (MMS) as a positive control, to compare possible mechanisms of DNA damage induction in the Comet assay. The antigenotoxicity protocols used were pre, simultaneous and post-treatment in relation to MMS. In genotoxicity and antigenotoxicity assessments, besides MMS, PBS was used as the negative control and three concentrations of the A. occidentale extract (500 μg/mL, 1000 μg/mL and 2000 μg/mL) were used on Chinese hamster lung fibroblasts (V79 cells). The Comet assay revealed that the two lowest concentrations tested presented no genotoxic activity, whereas the highest presented genotoxicity. All of the concentrations showed protective activity in simultaneous and post-treatment in relation to MMS. Further studies are required to identify the substances that comprise the extract and more clearly comprehend the antigenotoxic mechanism detected in this study.     

The evaluation of possible role of endocrine disruptors in central and peripheral precocious puberty

Exposure to environmental chemicals can affect genetic and epigenetic molecular pathways and may cause altered growth and development. Among those exposures, endocrine-disrupting chemicals (EDCs) are of particular concern as humans are abundantly exposed to these chemicals by various means in every period of life. Several well-known environmental chemicals, including phthalates and bisphenol A (BPA), are classified as EDCs. These EDCs are suggested to play roles in early onset of puberty in girls. The aim of this study is to determine plasma phthalate (di(2-ethylhexyl)phthalate [DEHP] and its main metabolite mono(2-ethylhexyl)phthalate [MEHP]) and urinary BPA levels in girls with idiopathic central precocious puberty (CPP) and peripheral precocious puberty (PPP). This study was performed on newly diagnosed idiopathic central precocious puberty (CPP) patients (n?=?42) and peripheral precocious puberty (PPP) (n?=?42) patients, who were admitted to Keçiören Training and Research Hospital, Clinic of Pediatric Endocrinology between August 2012 and –July 2013. Nonobese healthy girls (n?=?50) were used as the control group. Urinary BPA levels were not statistically different in control, PPP and CPP groups (medians 10.91, 10.63 and 10.15?μg/g creatinine, respectively; p?>?0.05). Plasma DEHP levels were significantly higher in PPP group when compared to control. Plasma MEHP levels were not significantly different in control and PPP groups (p?>?0.05). However, in CPP group, both plasma DEHP and MEHP levels were significantly higher than control and PPP groups. This study showed that phthalates might play a role in the occurence of CPP in girls.     

Micronucleus test and comet assay for the evaluation of zebrafish genomic damage induced by erythromycin and lincomycin

An enormous quantity of pharmacologically active principles are currently being introduced into the environment, with consequent escalation of environmental problems, but only a small number of studies are focusing on an assessment of their genotoxic effects. The aim of this article is to assess the genotoxic effects of erythromycin, lincomycin, and of a combination of these two antibiotics on the genome of the zebrafish. The genotoxicity of the two antibiotics was assessed by applying the micronucleus test to erythrocytes and performing a Comet assay on erythrocytes and hepatocytes. The fish were exposed to antibiotics at different concentrations and times of exposure, under standard laboratory conditions. Depending on the different experimental conditions, erythromycin and lincomycin induced a significant increase in DNA migration (tail moment) and a significant increase in micronuleus frequency. We also conducted an analysis on the activation of repair mechanisms when the genotoxic agent was removed. Only a few of the cells displayed a decrease in damage under these test conditions.     

Genotoxicity of marine sediments in the fish hepatoma cell line PLHC-1 as assessed by the Comet assay

The main goal of this study was to test the usefulness of the Comet assay in the PLHC-1 hepatoma fish cell line as a tool for detecting the presence of genotoxic compounds in contaminated marine sediments. The system has been tested using both model chemicals (benzo[a]pyrene (B[a]P) and ethyl methanesulfonate (EMS)) and extracts of sediment samples obtained with solvent dichloromethane/methanol. For all of the analysed sediment extracts as well as for the model chemicals a concentration dependent genotoxic effect was observed. The sediment with the highest observed genotoxic potential was additionally extracted using various solvents in order to test which class of compounds, according to their polarity, is most responsible for the observed genotoxic effect. Non-polar solvents (cyclohexane and dichloromethane) yielded stronger genotoxic effect but the highest level of DNA damage was determined after exposure to sediment extract obtained with the solvent mixture dichloromethane/methanol which extracts a wide range of contaminants. Our results indicate that the PLHC-1 cell line is a suitable in vitro model in sediment genotoxicity assessment and encourage the use of fish cell lines as versatile tools in ecogenotoxicology.     

Evaluation of the protective effect of ascorbic acid on nitrite- and nitrosamine-induced cytotoxicity and genotoxicity in human hepatoma line

Nitrites are ubiquitous environmental contaminants present in drinking water and foods. Nitrosamines can be formed endogenously from nitrate and nitrite and secondary amines or may be present in food, tobacco smoke, and drinking water. The major goal of this work was to evaluate the cytotoxic, reactive oxygen species (ROS)-producing and genotoxic effects of nitrite and nitrosamines and the possible protection by ascorbic acid in HepG2 cells. It was found that nitrite, N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), and N-nitrosomorpholine (NMOR) decreased cell viability, increased intracellular ROS production, and caused genotoxicity. Compared to untreated cells as determined by alkaline Comet assay, nitrite, NDMA, NDEA, and NMOR raised the tail intensity up to 1.18-, 3.79-, 4.24-, and 4.16-fold, respectively. Ascorbic acid (AA, 10 μM) increased cell viability and reduced ROS production significantly (p?<?0.05). Additionally, AA treatment decreased the tail intensity caused by nitrite, NDMA, NDEA, and NMOR to 33.74%, 58.6%, 44.32%, and 43.97%, respectively. It can be concluded that ascorbic acid was able to reduce both tail intensity and tail moment in all of the nitrosamine treatments, particularly in NDMA. AA protected HepG2 cells against genotoxic effects caused by nitrosamines. This protection might be through different mechanisms, some of which are not still understood in depth. The future interest will be to understand which pathways are influenced by antioxidants, particularly by AA, and the outcomes of this prevention in other cell line types.     

Comparative assessment of the SOS Chromotest kit and the Mutatox test with the Salmonella plate incorporation (Ames test) and fluctuation tests for screening genotoxic agents

Genuine needs for rapid, simple, and cost-efficient biotesting procedures to screen an ever-increasing number of chemicals and environmental samples are making the search for such assays a constant endeavor. With respect to genotoxicity screening, we compared, in this study, the performance of two novel assays (Vibrio fischeri M169 Mutatox? assay and the Escherichia coli PQ37 SOS Chromotest kit assay) with two well-established Ames testing procedures (plate incorporation and fluctuation assays). Testing material included 14 chemicals (10 potentially directly acting and 4 indirectly acting compounds) reflecting different chemical classes (2 inorganics, 2 pesticides, 2 halogenated hydrocarbons, 2 alkylating agents, 2 aromatic amines, 1 chlorophenol, and 3 polycyclic aromatic hydrocarbons). Comparative assessment criteria included (1) interprocedural agreement in detecting presence or absence of genotoxicity, (2) accuracy in being able to recognize animal (non)carcinogens, and (3) sensitivity (detection of lowest actively genotoxic concentration). In terms of qualitative responses, both the SOS Chromotest (86% agreement) and Mutatox assays (93% agreement) were good predictors of the Ames testing mutagenicity. For their capability to correctly discriminate between (non)carcinogens, accuracy was 82% (9 of 11 chemicals) for Mutatox, 73% (8 of 11 chemicals) for Ames testing, and 64% (7 of 11 chemicals) for the SOS Chromotest. In general, the Salmonella-based assays proved more sensitive (6 times out of 9 chemicals) than the Mutatox (3 times out of 9 chemicals) and the SOS Chromotest (never more sensitive). Overall, this study demonstrates reliable performances by both the SOS Chromotest and Mutatox for chemical genotoxicity screening when results are referenced to the well-validated Ames assay. Although additional comparative data with other chemicals will be required, it appears likely that these more practical and cost-efficient procedures can be presently useful to screen genotoxic activity of various xenobiotics and environmental samples. © 1994 by John Wiley & Sons, Inc..     

Assessment of the in vitro and in vivo genotoxicity of extracts and indole monoterpene alkaloid from the roots of Galianthe thalictroides (Rubiaceae)

Roots of Galianthe thalictroides K. Schum. (Rubiaceae) are used in folk medicine in the State of Mato Grosso do Sul, Brazil, for treating and preventing cancer. To gain information about the genotoxicity of extracts (aqueous and EtOH), the CHCl₃ phase resulting from partition of the EtOH extract and the indole monoterpene alkaloid 1 obtained from this plant. The genotoxicity of 1 and extracts was evaluated in vivo through the Drosophila melanogaster wing Somatic Mutation and Recombination Test – SMART, while in vitro cytotoxic (MTT) and Comet assays were performed only with alkaloid 1. The results obtained with the SMART test indicated that the aqueous extract had no genotoxic activity. The EtOH extract was not genotoxic to ST descendants but genotoxic to HB ones. The CHCl₃ phase was genotoxic and cytotoxic. Alkaloid 1 showed significant mutational events with SMART, in the cytotoxicity assay (MTT), it showed a high cytotoxicity for human hepatoma cells (HepG2), whereas for the Comet assay, not showing genotoxic activity. The ethanol extract was shown to be genotoxic to HB descendants in the SMART assay, while the results obtained in this test for the monoterpene indole alkaloid 1 isolated from this extract.     

Effects of single-wall carbon nanotubes in human cells of the oral cavity: Geno-cytotoxic risk

Single-wall carbon nanotubes (SWCNTs) are one of the most extensively produced carbon materials and the environmental, public and professional exposure is therefore dramatically increasing. Consequently the studies on bio-effects and safety of SWCNTs are highly needed. The goal of this study was investigate the effects in vitro of SWCNTs in cells of the oral cavity, never employed in this research field. We exposed human gingival fibroblasts to 50, 75, 100, 125, 150 μg/ml SWCNTs for 24 h and we investigated genotoxicity (Comet assay and micronucleus test), cytotoxicity, oxidative stress, as reactive oxygen species (ROS) generation, and stress response, as Heat shock protein 70 (Hsp70) expression. SWCNTs produced genotoxic effects at all doses, even if detected with different sensitiveness by the two tests, and at the two highest doses induced a strong decrease of the cell proliferation and cell survival, causing apoptosis too. Furthermore, we proved the ability of these nanomaterials to induce oxidative stress and Hsp70 expression. Finally, by inhibition of Hsp70 expression, we demonstrated that this heat shock protein conferred protection against SWCNT geno-cytotoxicity.     

The comet assay with multiple mouse organs: comparison of comet assay results and carcinogenicity with 208 chemicals selected from the IARC monographs and U.S. NTP Carcinogenicity Database

The comet assay is a microgel electrophoresis technique for detecting DNA damage at the level of the single cell. When this technique is applied to detect genotoxicity in experimental animals, the most important advantage is that DNA lesions can be measured in any organ, regardless of the extent of mitotic activity. The purpose of this article is to summarize the in vivo genotoxicity in eight organs of the mouse of 208 chemicals selected from International Agency for Research on Cancer (IARC) Groups 1, 2A, 2B, 3, and 4, and from the U.S. National Toxicology Program (NTP) Carcinogenicity Database, and to discuss the utility of the comet assay in genetic toxicology. Alkylating agents, amides, aromatic amines, azo compounds, cyclic nitro compounds, hydrazines, halides having reactive halogens, and polycyclic aromatic hydrocarbons were chemicals showing high positive effects in this assay. The responses detected reflected the ability of this assay to detect the fragmentation of DNA molecules produced by DNA single strand breaks induced chemically and those derived from alkali-labile sites developed from alkylated bases and bulky base adducts. The mouse or rat organs exhibiting increased levels of DNA damage were not necessarily the target organs for carcinogenicity. It was rare, in contrast, for the target organs not to show DNA damage. Therefore, organ-specific genotoxicity was necessary but not sufficient for the prediction of organ-specific carcinogenicity. It would be expected that DNA crosslinkers would be difficult to detect by this assay, because of the resulting inhibition of DNA unwinding. The proportion of 10 DNA crosslinkers that was positive, however, was high in the gastrointestinal mucosa, stomach, and colon, but less than 50% in the liver and lung. It was interesting that the genotoxicity of DNA crosslinkers could be detected in the gastrointestinal organs even though the agents were administered intraperitoneally. Chemical carcinogens can be classified as genotoxic (Ames test-positive) and putative nongenotoxic (Ames test-negative) carcinogens. The Ames test is generally used as a first screening method to assess chemical genotoxicity and has provided extensive information on DNA reactivity. Out of 208 chemicals studied, 117 are Ames test-positive rodent carcinogens, 43 are Ames test-negative rodent carcinogens, and 30 are rodent noncarcinogens (which include both Ames test-positive and negative noncarcinogens). High positive response ratio (110/117) for rodent genotoxic carcinogens and a high negative response ratio (6/30) for rodent noncarcinogens were shown in the comet assay. For Ames test-negative rodent carcinogens, less than 50% were positive in the comet assay, suggesting that the assay, which detects DNA lesions, is not suitable for identifying nongenotoxic carcinogens. In the safety evaluation of chemicals, it is important to demonstrate that Ames test-positive agents are not genotoxic in vivo. This assay had a high positive response ratio for rodent genotoxic carcinogens and a high negative response ratio for rodent genotoxic noncarcinogens, suggesting that the comet assay can be used to evaluate the in vivo genotoxicity of in vitro genotoxic chemicals. For chemicals whose in vivo genotoxicity has been tested in multiple organs by the comet assay, published data are summarized with unpublished data and compared with relevant genotoxicity and carcinogenicity data. Because it is clear that no single test is capable of detecting all relevant genotoxic agents, the usual approach should be to carry out a battery of in vitro and in vivo tests for genotoxicity. The conventional micronucleus test in the hematopoietic system is a simple method to assess in vivo clastogenicity of chemicals. Its performance is related to whether a chemical reaches the hematopoietic system. Among 208 chemicals studied (including 165 rodent carcinogens), 54 rodents carcinogens do not induce micronuclei in mouse hematopoietic system despite the positive finding with one or two in vitro tests. Forty-nine of 54 rodent carcinogens that do not induce micronuclei were positive in the comet assay, suggesting that the comet assay can be used as a further in vivo test apart from the cytogenetic assays in hematopoietic cells. In this review, we provide one recommendation for the in vivo comet assay protocol based on our own data.     

Mechanisms of genotoxicity. A review of in vitro and in vivo studies with engineered nanoparticles

Engineered nanoparticles (NPs) are widely used in different technologies but their unique properties might also cause adverse health effects. In reviewing recent in vitro and in vivo genotoxicity studies we discuss potential mechanisms of genotoxicity induced by NPs. Various factors that may influence genotoxic response, including physico-chemical properties and experimental conditions, are highlighted. From 4346 articles on NP toxicity, 112 describe genotoxicity studies (94 in vitro, 22 in vivo). The most used assays are the comet assay (58 in vitro, 9 in vivo), the micronucleus assay (31 in vitro, 14 in vivo), the chromosome aberrations test (10 in vitro, 1 in vivo) and the bacterial reverse mutation assay (13 studies). We describe advantages and potential problems with different methods and suggest the need for appropriate methodologies to be used for investigation of genotoxic effects of NPs, in vitro and in vivo.     

Genotoxicity evaluation of titanium dioxide nanoparticles using the Ames test and Comet assay

Titanium dioxide nanoparticles (TiO₂‐NPs) are being used increasingly for various industrial and consumer products, including cosmetics and sunscreens because of their photoactive properties. Therefore, the toxicity of TiO₂‐NPs needs to be thoroughly understood. In the present study, the genotoxicity of 10nm uncoated sphere TiO₂‐NPs with an anatase crystalline structure, which has been well characterized in a previous study, was assessed using the Salmonella reverse mutation assay (Ames test) and the single‐cell gel electrophoresis (Comet) assay. For the Ames test, Salmonella strains TA102, TA100, TA1537, TA98 and TA1535 were preincubated with eight different concentrations of the TiO₂‐NPs for 4 h at 37 °C, ranging from 0 to 4915.2 µg per plate. No mutation induction was found. Analyses with transmission electron microscopy (TEM) and energy‐dispersive X‐ray spectroscopy (EDS) showed that the TiO₂‐NPs were not able to enter the bacterial cell. For the Comet assay, TK6 cells were treated with 0–200 µg ml^–1 TiO₂‐NPs for 24 h at 37 °C to detect DNA damage. Although the TK6 cells did take up TiO₂‐NPs, no significant induction of DNA breakage or oxidative DNA damage was observed in the treated cells using the standard alkaline Comet assay and the endonuclease III (EndoIII) and human 8‐hydroxyguanine DNA‐glycosylase (hOGG1)‐modified Comet assay, respectively. These results suggest that TiO₂‐NPs are not genotoxic under the conditions of the Ames test and Comet assay. Published 2012. This article is a US Government work and is in the public domain in the USA.     

Experimental evidence of false-positive Comet test results due to TiO2 particle – assay interactions

Abstract

We have studied the genotoxicity of TiO₂ particles with a Comet assay on a unicellular organism, Tetrahymena thermophila. Exposure to bulk- or nano-TiO₂ of free cells, cells embedded in gel or nuclei embedded in gel, all resulted in a positive Comet assay result but this outcome could not be confirmed by cytotoxicity measures such as lipid peroxidation, elevated reactive oxygen species or cell membrane composition. Published reports state that in the absence of cytotoxicity, nano- and bulk-TiO₂ genotoxicity do not occur directly, and a possible explanation of our Comet assay results is that they are false positives resulting from post festum exposure interactions between particles and DNA. We suggest that before Comet assay is used for nanoparticle genotoxicity testing, evidence for the possibility of post festum exposure interactions should be considered. The acellular Comet test described in this report can be used for this purpose.     

Aspects of nitrogen dioxide toxicity in environmental urban concentrations in human nasal epithelium

Cytotoxicity and genotoxicity of nitrogen dioxide (NO₂) as part of urban exhaust pollution are widely discussed as potential hazards to human health. This study focuses on toxic effects of NO₂ in realistic environmental concentrations with respect to the current limit values in a human target tissue of volatile xenobiotics, the epithelium of the upper aerodigestive tract.Nasal epithelial cells of 10 patients were cultured as an air-liquid interface and exposed to 0.01 ppm NO₂, 0.1 ppm NO₂, 1 ppm NO₂, 10 ppm NO₂ and synthetic air for half an hour. After exposure, genotoxicity was evaluated by the alkaline single-cell microgel electophoresis (Comet) assay and by induction of micronuclei in the micronucleus test. Depression of proliferation and cytotoxic effects were determined using the micronucleus assay and trypan blue exclusion assay, respectively.The experiments revealed genotoxic effects by DNA fragmentation starting at 0.01 ppm NO₂ in the Comet assay, but no micronucleus inductions, no changes in proliferation, no signs of necrosis or apoptosis in the micronucleus assay, nor did the trypan blue exclusion assay show any changes in viability. The present data reveal a possible genotoxicity of NO₂ in urban concentrations in a screening test. However, permanent DNA damage as indicated by the induction of micronuclei was not observed. Further research should elucidate the effects of prolonged exposure.     

Environmental nitration processes enhance the mutagenic potency of aromatic compounds

This work is an attempt to establish if aromatic nitration processes are always associated with an increase of genotoxicity. We determined the mutagenic and genotoxic effects of Benzene (B), Nitrobenzene (NB), Phenol (P), 2‐Nitrophenol (2‐NP), 2,4‐Dinitrophenol (2,4‐DNP), Pyrene (Py), 1‐Nitropyrene (1‐NPy), 1,3‐Dinitropyrene (1,3‐DNPy), 1,6‐Dinitropyrene (1,6‐DNPy), and 1,8‐Dinitropyrene (1,8‐DNPy). The mutagenic activities were evaluated with umuC test in presence and in absence of metabolic activation with S9 mix. Then, we used both cytokinesis‐blocked micronucleus (CBMN) assay, in combination with fluorescent in situ hybridization (FISH) of human pan‐centromeric DNA probes on human lymphocytes in order to evaluate the genotoxic effects. Analysis of all results shows that nitro polycyclic aromatic hydrocarbons (PAHs) are definitely environmental genotoxic/mutagenic hazards and confirms that environmental aromatic nitration reactions lead to an increase in genotoxicity and mutagenicity properties. Particularly 1‐NPy and 1,8‐DNPy can be considered as human potential carcinogens. They seem to be significant markers of the genotoxicity, mutagenicity, and potential carcinogenicity of complex PAHs mixtures present in traffic emission and industrial environment. In prevention of environmental carcinogenic risk 1‐NPy and 1,8‐DNPy must therefore be systematically analyzed in environmental complex mixtures in association with combined umuC test, CBMN assay, and FISH on cultured human lymphocytes. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

On the mechanism of genotoxicity of ethephon on embryonic fibroblast cells

Ethephon is one of the most widely used plant growth regulator in agriculture that its application has been increased in recent years. Many reports have raised concern over the safety of this organophosphorus compound. The aim of the current study was to assess the potential genotoxic effect of ethephon on murine embryonic fibroblast (MEF) cell line, using two genotoxicity endpoints: γH2AX expression and comet assay. γH2AX served as an early and sensitive biomarker of genotoxic damage. Oxidative stress biomarkers, including reactive oxygen species (ROS), lipid peroxidation (LPO) and total antioxidant capacity were also examined. The results showed a significant increase in cell proliferation, 24?h post-treatment with 10, 40,160?μg/ml ethephon, while at the higher concentrations cytotoxic effect was observed. The γH2AX expression and γH2AX foci count per cell were significantly increased at non-cytotoxic concentrations of ethephon, accompanied with increased DNA damage as illustrated by comet assay. LPO and ROS levels were elevated only at 160?μg/ml and higher doses. The results interestingly showed that low non-cytotoxic doses of ethephon promoted DNA damage inducing cell proliferation, raising the possibility of ethephon mutagenicity. The genotoxic effect of ethephon at low doses might not relate to oxidative damage and that increased in the level of ROS and LPO generation at higher doses could account for the cytotoxic effect of ethephon. Taken together, our study provides strong in vitro evidence on potential genotoxicity of ethephon at low doses. More precise studies are needed to clarify the mutagenic effect of chronic exposure to ethephon.     

Tinospora cordifolia, a safety evaluation

Tinospora cordifolia is one of the indispensable medicinal plants used in veterinary folk medicine/Ayurvedic system of medicine for the treatment of diverse diseases and recommended for improving the immune system by means of body resistance. In the current study, we evaluated the genotoxic risk of the aqueous extract of T. cordifolia (TC) in a battery of four different genotoxicity tests viz., Ames, in vitro chromosome aberration (CA), rodent bone marrow micronucleus (MN), and Comet assay. Experimental results confirmed that in Ames test up to 5000 μg/plate of TC did not exhibit any mutagenic effect in Salmonella typhimurium mutant strains (TA97a, TA98, TA100, TA102, and TA1535). In CA assay, TC was not clastogenic to human peripheral blood lymphocytes up to a concentration of 3000 μg/ml. In MN and Comet assays, TC was pre-treated for 7 days at three dose levels (150, 200 and 250 mg/kg body weight) orally to male Balb/c mice. The results showed that TC treatment did not display clastogenicity and DNA damaging effect in bone marrow erythrocytes and peripheral blood lymphocytes respectively.     

Rationale of genotoxicity testing of nanomaterials: Regulatory requirements and appropriateness of available OECD test guidelines

Abstract

The development of an environmental health and safety risk management system for nanoscale particle-types requires a base set of hazard data. Accurate determination of health and environmental risks of nanomaterials is a function of the integration of hazard and exposure datasets. Recently, a nanoparticle risk assessment strategy was promulgated and the components are described in a document entitled “Nanorisk framework” (www.nanoriskframework.com). A major component of the hazard evaluation includes a proposed minimum base set of toxicity studies. Included in the suggested studies were substantial particle characterization, a variety of acute hazard and environmental tests, concomitant with screening-type genotoxicity studies. The implementation of well-accepted genotoxicity assays for testing nanomaterials remains a controversial issue. This is because many of these genotoxicity tests were designed for screening general macroparticle chemicals and might not be suitable for the screening of nanomaterials (even of the same compositional material). Furthermore, no nanoparticle-type positive controls have been established or universally accepted for these tests. Although it is the comparative results of the test material vs. the negative or vehicle control that forms the basis for interpreting the results and potency of test materials in genetic toxicology assays, the lack of a nanoparticle-type positive control questions the suitability of the tests to identify nanomaterials with genotoxic properties. It is also not possible to establish historical positive control ranges that would confirm the sensitivity of the tests. Although several genetic toxicology tests have been validated for chemicals according to the Organisation for Economic Co-operation and Development (OECD) test guidelines, the relevance of these assays for nanoparticulate materials remains to be determined. In an attempt to remedy this issue, the OECD has established current projects designed to evaluate the relevance and reproducibility of safety hazard tests for representative nanomaterials, including genotoxicity assays (i.e., Steering Group 3 – Safety Testing of Representative Nanomaterials). In this article, we discuss our past approaches and experience in conducting genotoxicity assays (1) for a newly developed ultrafine TiO₂ particle-type; and (2) in a recent inhalation study, evaluating micronucleus formation in rat erythrocytes following exposures to engineered amorphous nanosilica particles. It seems clear that the development of standardized approaches will be necessary in order to determine whether exposures to specific nanoparticle-types are associated with genotoxic events. The appropriateness of available genotoxicity test systems for nanomaterials requires confirmation and standardization. Accordingly, it seems reasonable to conclude that any specific regulatory testing requirements for nanoparticles would be premature at this time.     

Flow cytometric evaluation of the contribution of ionic silver to genotoxic potential of nanosilver in human liver HepG2 and colon Caco2 cells

Exposure to nanosilver found in food‐ and cosmetics‐related consumer products is of public concern because of the lack of information about its safety. In this study, two widely used in vitro cell culture models, human liver HepG2 and colon Caco2 cells, and the flow cytometric micronucleus (FCMN) assay were evaluated as tools for rapid predictive screening of the potential genotoxicity of nanosilver. Recently, we reported the genotoxicity of 20 nm nanosilver using these systems. In the current study presented here, we tested the hypothesis that the nanoparticle size and cell types were critical determinants of its genotoxicity. To test this hypothesis, we used the FCMN assay to evaluate the genotoxic potential of 50 nm nanosilver of the same shape, composition, surface charge and obtained from the same commercial source using the same experimental conditions and in vitro models (HepG2 and Caco2) as previously tested for the 20 nm silver. Results of our study show that up to the concentrations tested in these cultured cell test systems, the smaller (20 nm) nanoparticle is genotoxic to both the cell types by inducing micronucleus (MN). However, the larger (50 nm) nanosilver induces MN only in HepG2 cells, but not in Caco2 cells. Also in this study, we evaluated the contribution of ionic silver to the genotoxic potential of nanosilver using silver acetate as the representative ionic silver. The MN frequencies in HepG2 and Caco2 cells exposed to the ionic silver in the concentration range tested are not statistically significant from the control values except at the top concentrations for both the cell types. Therefore, our results indicate that the ionic silver may not contribute to the MN‐forming ability of nanosilver in HepG2 and Caco2 cells. Also our results suggest that the HepG2 and Caco2 cell cultures and the FCMN assay are useful tools for rapid predictive screening of a genotoxic potential of food‐ and cosmetics‐related chemicals including nanosilver. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.