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.     

Evaluation of the mutagenic and genotoxic activities of anti-hepatitis B analogs of beta-L-adenosine by the Ames test and the Comet assay

beta-L-2'-deoxyadenosine (beta-L-dA), beta-L-2',3'-dideoxyadenosine (beta-L-ddA) and its two bis (S-acyl-2-thioethyl; SATE) phosphotriester derivatives, beta-L-2',3'-dideoxyadenosine-5'-monophosphate-bis(MeSATE) and beta-L-2',3'-dideoxyadenosine-5'-monophosphate-bis(tButylSATE) have been previously shown to exhibit potent and selective anti-hepatitis B activity in vitro. None of the four compounds was mutagenic up to 100 microg in the Ames test (microtechnique) using Salmonella typhimurium strains TA 97a, TA 98, TA 100 and TA 102, with and without metabolic activation. In addition, the genotoxicity of beta-LdA and the three other compounds was evaluated in human lymphocytes using the Comet assay, at doses up to 5 microg with or without the addition of a microsomal S9 fraction. None of the four compounds induced DNA strand breakage with and without metabolic activation. In summary, the data clearly demonstrate that the purine nucleoside beta-L-dA, beta-L-ddA and the two prodrugs, beta-L-ddAMP-bis(MeSATE) and beta-L-ddAMP-bis(tButylSATE) are not mutagenic in the Ames test and do not induce DNA damage in human lymphocytes, as assessed by the Comet assay.     

Genotoxicity of phthalates

Many of the environmental, occupational and industrial chemicals are able to generate reactive oxygen species (ROS) and cause oxidative stress. ROS may lead to genotoxicity, which is suggested to contribute to the pathophysiology of many human diseases, including inflammatory diseases and cancer. Phthalates are ubiquitous environmental chemicals and are well-known peroxisome proliferators (PPs) and endocrine disruptors. Several in vivo and in vitro studies have been conducted concerning the carcinogenic and mutagenic effects of phthalates. Di(2-ethylhexyl)-phthalate (DEHP) and several other phthalates are shown to be hepatocarcinogenic in rodents. The underlying factor in the hepatocarcinogenesis is suggested to be their ability to generate ROS and cause genotoxicity. Several methods, including chromosomal aberration test, Ames test, micronucleus assay and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutation test and Comet assay, have been used to determine genotoxic properties of phthalates. Comet assay has been an important tool in the measurement of the genotoxic potential of many chemicals, including phthalates. In this review, we will mainly focus on the studies, which were conducted on the DNA damage caused by different phthalate esters and protection studies against the genotoxicity of these chemicals.     

Resolution of contradiction between in silico predictions and Ames test results for four pharmaceutically relevant impurities

The ICH M7 Guideline requires low level control of mutagenic impurities in pharmaceutical products to minimize cancer risk in patients (ICHM7, 2014). Bacterial mutagenicity (Ames) data is generally used to determine mutagenic and possible carcinogenic potential of compounds. Recently, a publication on experiences of using two in silico systems to identify potentially mutagenic impurities highlighted the importance of performing a critical review of published Ames data utilized as part of a mutagenicity assessment of impurities (Greene et al., 2015). Four compounds (2-amino-5-hydroxybenzoic acid, 2-amino-3-chlorobenzoic acid, methyl 2-amino-4-chlorobenzoate and 4-morpholinopyridine) reported mutagenic were identified in a two system in silico assessment and expert review of the structuresas non-mutagenic. Likely reasons for mutagenicity could not be identified and the purity of the compounds tested was proposed. In the current investigation, the purest available sample of the four compounds was tested in an OECD-compliant Ames test. The compounds were all found to be non-mutagenic. Possible reasons for the discrepancy between previously reported and current results are discussed. Additionally, important points to consider when conducting an expert review of available Ames data are provided particularly in cases where reported Ames results are discrepant with a two system in silico assessment.     

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.     

In vitro and in silico toxicity evaluation of bioactive 4′-aminochalcone derivatives

The 4′-aminochalcones compounds are open-chain flavonoids structures which have shown a known array of pharmacological activities, such as antibacterial, antifungal, anti-inflammatory and antitumor effects. There is little toxicological information available about these compounds in the literature. Therefore, the investigation of toxic effects of three 4′-aminochalcone derivatives was performed using in silico and in vitro assays. In silico provided results that indicated the occurrence of mutagenic and genotoxic effects. In vitro tests, using Cellular Proliferation and Viability, Micronucleus, and DNA damage by Comet assay, showed that the compounds studied also present mutagenic and genotoxic effects, which confirm the result determined by the in silico analysis. The use of experimental and computational models is complementary to each other and the results determined for 4′-aminochalones suggest that the chalcones should also be carefully considered since they show some risks to cause toxic effects to human cells.     

Chemical composition and genotoxic activity of petroleum derivatives collected in two working environments

Pitch and bitumen, two complex petroleum derivative mixtures, were studied for both their chemical composition and their mutagenic/DNA damaging activity. While bitumen revealed no genotoxic effect and low polycyclic aromatic hydrocarbons (PAHs) concentration, petroleum pitch showed a high concentration of mutagenic/carcinogenic PAHs, and also an elevated mutagenic activity when assayed by the Ames test, in the presence of postmitochondrial rat liver fractions. The in vitro mutagenic activity was detectable as frameshift mutation by assaying the pitch both as an in toto mixture and after HPLC fractionation, the most polar fractions being the most active. In contrast, both derivatives showed no in vivo DNA damage in rat liver, using the DNA alkaline elution technique and the fluorometric assay of DNA unwinding.     

Effects of prochloraz on DNA damage,lipid peroxidation and antioxidant system in vitro

Prochloraz is a broad-spectrum contact imidazol fungicide used against several diseases in wheat, barley and oleaginous plants but also for treatment of flower production. Although prochloraz has endocrine disrupting and hepatocarcinogenic effects, there is lack of data on toxic effects of prochloraz. Therefore, we aimed to investigate the DNA damage effects of prochloraz in NRK-52E cells by using Ames and Comet assay. By using a standard alkaline Comet assay procedure, there was no DNA damage observed after 24?h prochloraz exposure. It also showed that prochloraz caused neither base-pair substitution nor frame shift mutations by using TA98, TA100 strains, respectively, with/without metabolic activation in Ames assay. Both Comet and Ames assays, the exposure concentrations were 12.5, 25, 50 and 100?µM. IC₅₀ value of prochloraz was determined as 110.76?µM in NRK-52E cells by MTT cytotoxicity test. Also, we evaluated possible effects of prochloraz on lipid peroxidation, reduced glutathione (GSH), oxidized glutathione (GSSG) and antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Rd) in NRK-52E cells at 1–50?µM concentrations. Prochloraz induced lipid peroxidation and altered glutathione contents and antioxidant enzyme activities in NRK-52E cells. Our results indicated that prochloraz showed no evidence of mutagenicity and DNA damage; however, some alterations were observed on lipid peroxidation and antioxidant systems in prochloraz treatment.     

Evaluation of genotoxic potential of avarol,avarone, and its methoxy and methylamino derivatives in prokaryotic and eukaryotic test models

In this study, mutagenic and genotoxic potential of anti-tumor compounds avarol, avarone, and its derivatives 3′-methoxyavarone, 4′-(methylamino)avarone and 3′-(methylamino)avarone was evaluated and compared to cytostatics commonly used in chemotherapy (5-fluorouracil, etoposid, and cisplatin). Mutagenic potential of selected hydroquinone and quinones was assessed in prokaryotic model by the SOS/umuC assay in Salmonella typhimurium TA1535/pSK1002. Genotoxic potential was also assessed in eukaryotic models using comet assay in human fetal lung cell line (MRC-5), human adenocarcinoma epithelial cell line (A549), and in human peripheral blood cells (HPBC). The results indicated that avarol and avarone do not exert mutagenic/genotoxic potential. Among the studied avarone derivatives, mutagenic potential was detected by SOS/umuC test for 3′-(methylamino)avarone, but only after metabolic activation. The results of comet assay indicated that 3′-methoxyavarone and 3′-(methylamino)avarone have a significant impact on the level of DNA damage in the MRC-5 cell line. Genotoxic potential was not observed in A549 cells or HPBC probably due to a different uptake rate for the compounds and lower in metabolism rate within these cells.     

A simplified method for the induction of 8-azaguanine resistance in Salmonella typhimurium

The selection of induced 8-azaguanine (8-AG) resistant mutants, recently developed as a test system in Salmonella typhimurium, has been adapted to the plate incorporation assay technique usually employed in the Ames test. The sensitivity of the procedure has been checked by testing known mutagenic compounds (ethyl methanesulfonate (EMS), N-methyl-N'-mtro-N-nitrosoguanidine (MNNG), 2-nitrofluorene (2-NF), 4-nitroquinoline-1-oxide (4NQO), mitomycin C (MMC) and comparing the increases in mutant numbers with those obtained in the Ames test: the results so far obtained show a lower sensitivity of the 8-AG system, more notable for strains lacking R factor plasmid.Five chemicals (Natulan, Paraquat, Diquat, 4-chloro-benzotrifluoride (4-Cl-BTF), 3-nitro-4-chloro-benzotrifluoride (3-n-4-Cl-BTF), not mutagenic in the Ames test but known or suspected to be mutagenic or carcinogenic in other systems, have been also checked: two of them, the herbicides Paraquat and Diquat, were positive in the 8-AG resistance test.     

Genotoxicity evaluation of locally produced dental porcelain – An in vitro study using the Ames and Comet assays

The aim of this study was to determine the genotoxicity of a locally produced dental porcelain (Universiti Sains Malaysia, Malaysia) using the Ames and Comet assays. In the Ames assay, four genotypic variants of the Salmonella strains (TA98, TA100, TA1537 and TA1535) carrying mutations in several genes were used. The dental porcelain was incubated with these four strains in five different doses both in the presence and absence of metabolic activation (S9) and the result was assessed based on the number of revertant colonies. Concurrently, appropriate positive controls were used so as to validate the test. The average number of revertant colonies per plate treated with locally produced dental porcelain was less than double as compared to that of negative control. In the Comet assay, L929 (CCL-1 ATCC, USA) mouse fibroblast cells were treated with the dental porcelain in three different concentrations along with concurrent negative and positive controls. The tail moment which was used as a measurement of DNA damage was almost equal to that of the negative control, suggesting that the locally produced dental porcelain did not induce any DNA damage. The results indicated that the locally produced dental porcelain is non-genotoxic under the present test conditions.     

Mutagenicity and clastogenicity evaluation of tacrine by Ames and micronucleus assays

Tacrine was evaluated for its mutagenic and clastogenic activities using the Ames bacterial reverse-mutation assay and the rodent bone marrow micronucleus assay. Tacrine was tested for mutagenic potential at six different concentrations, with 1,250 µg/plate as the highest concentration, followed by five lower concentrations with 2-fold spacing. In clastogenic evaluation, tacrine was administered orally to Wistar rats for 2 days at 5, 10, and 20?mg/kg body weights to assess micronucleus induction in bone marrow erythrocytes. In the Ames assay, tacrine showed nonmutagenicity in four tester strains of Salmonella typhimurium viz. TA98, TA100, TA102, and TA1535, but it was found to be mutagenic in the TA1537 tester strain, both in the presence and absence of a metabolic activation system. Tacrine was found to be nonclastogenic on bone marrow cells of rats at all doses tested and was found to be mutagenic in only the TA1537 strain of Salmonella.     

Toxicogenomics discrimination of potential hepatocarcinogenicity of non‐genotoxic compounds in rat liver

Long‐term carcinogenicity testing of a compound is exceedingly time‐consuming and costly, and requires many test animals, whereas the Ames test, which is based on the assumption that any substance that is mutagenic may also exert carcinogenic potential, is useful as a short‐term screening assay but has major drawbacks. Although, in fact, 90% of compounds that give a positive Ames test cause cancer in laboratory animals, a good proportion of compounds that give a negative Ames test are also carcinogens; that is, there is no good correlation between carcinogenicity and negative Ames test results. As an alternative to these two approaches, we have tried applying toxicogenomics to predict the carcinogenicity of a compound from the gene expression profile induced in vivo. To establish our model, male Sprague–Dawley rats were orally administered test compounds (12 hepatocarcinogens and 26 non‐hepatocarcinogens) for 28 days. Analysis of liver gene expression data by Support Vector Machines (SVM) dividing compounds into ‘for training’ and ‘for test’ (20 cases assigned randomly) allowed a set of marker genes to be tested for prediction of hepatocarcinogenicity. The developed prediction model was then validated with reference to the concordance rate with training data and test data, and a good performance was obtained. We will have new gene expression data and continue the validation of our model. Copyright © 2012 John Wiley & Sons, Ltd.     

Mutagenicity of aromatic amines and amides with chemical models for cytochrome P450 in Ames assay

Chemical models for cytochrome P450, consisting of water-insoluble or water-soluble iron porphyrin plus an oxidant, have been used to detect the mutagenicity of promutagens in genotoxicity assays. The procedure for using chemical models for cytochrome P450 as substitutes for the S9 mix in the Ames assay have been already established. Aromatic amines and amides require metabolic activation by cytochrome P450 when they exert their mutagenicity in Salmonella typhimurium strains. In this study, we optimized the conditions of the assay using a water-soluble chemical model, 5,10,15,20-tetrakis(1-methylpyridinium-4-yl)porphyrinatoiron(III) pentachloride (4-MPy), plus tert-butyl hydroperoxide (t-BuOOH), magnesium monoperoxyphthalate, or iodosylbenzene, by comparing the mutagenicity of 2-aminofluorene (AF) in the Ames test. The model with 4-MPy/t-BuOOH showed the highest AF mutagenic potency. The chemical model activated 2-naphthylamine, 4-aminobiphenyl, and benzidine in S. typhimurium TA98. In aromatic amides, the model with 4-MPy/t-BuOOH weakly activated 2-acetylaminofluorene (AAF). To detect higher mutagenicity of aromatic amides, we used a higher concentration of 4-MPy/t-BuOOH by a factor of 5 over that used for aromatic amines, and then detected the mutagenicity of AAF, 2-acetylaminoanthracene, and 2-acetylamino-9-fluorenone. Furthermore, we concluded that the AAF mutagenicity in the presence of 4-MPy/t-BuOOH is derived from N-hydroxylacetylamino compounds.     

LACK OF DNA BINDING IN THE RAT NASAL MUCOSA AND OTHER TISSUES OF THE NASAL TOXICANTS ROFLUMILAST,A PHOSPHODIESTERASE 4 INHIBITOR,AND A METABOLITE, 4-AMINO-3,5-DICHLOROPYRIDINE,IN CONTRAST TO THE NASAL CARCINOGEN 2,6-DIMETHYLANILINE*

The phosphodiesterase 4 inhibitor Roflumilast (B9302-107) (RF) and its metabolite 4-amino-3,5-dichloropyridine (ADCP) produced nasal toxicity in preclinical safety studies with rats. The purpose of this study was to assess the possible formation of DNA adducts, by RF and ADCP, in the nasal mucosa, liver and testes of male rats using the ³²P-postlabeling assay. For comparison, rats were exposed to the DNA-reactive carcinogens 2,6-dimethylaniline (DMA), also known as 2,6-xylidine, a nasal carcinogen, and the aromatic amine carcinogens 4,4′-methylene-bis(2-chloroaniline) (MOCA), which yields monocyclic DNA adducts, and 2-acetylaminofluorene (2-AAF). In the case of RF, possible sources of DNA adducts include the parent molecule and its ADCP moiety by enzymatic N-hydroxylation and sulfation, reactions typical of carcinogenic aromatic amines. 4-Acetoxylamino-3,5-dichloropyridine (N-acetoxy-ADCP), a chemically activated derivative of ADCP, was prepared and used to modify DNA which was then used to establish the chromatographic conditions with which to reliably detect whether or not such adducts were formed metabolically from RF and ADCP. Similarly, a standard N-hydroxy-DMA was prepared, but the corresponding N-acetoxy derivative was unstable and decomposed during synthesis. Both N-hydroxy-DMA and N-acetoxy-ADCP were mutagenic in the Salmonella typhimurium Ames assay using strain TA100 without an exogenous bioactivation system, with the former being more potent. N-hydroxy-ADCP was essentially inactive in this assay. For the ³²P-postlabeling assay, male Wistar rats were exposed to the test substances and carrier control compounds by intragastric instillation at the selected dose levels for 7 days. Subsequently, the nasal mucosa, liver, and testes of the rats exposed to the test or control compounds were extirpated, the DNA extracted and the samples postlabeled. The patterns of adducts formed with the test compounds were compared to those formed in N-acetoxy-ADCP- and N-hydroxy-DMA-adducted DNA, which were assayed by both nuclease P₁ and butanol enhancement methods. Based upon the similarity of results from the two enhancement methods, only the former was used for the in vivo studies. No evidence was obtained for the formation of DNA adducts from RF or its metabolites, specifically ADCP, under the conditions of these assays despite the ability to detect adducts from DNA modified chemically with N-acetoxy-ADCP and DNA adducts from the other compounds in their target organs. In the absence of a pattern of compound-related spots, we conclude that RF does not form DNA adducts having the potential to initiate neoplasia in these three tissues.     

DNA fragmentation induced by all‐trans retinoic acid and its steroidal analogue EA‐4 in C2C12 mouse and HL‐60 human leukemic cells in vitro

We have recently shown that retinoic acid induces micronucleation mainly via chromosome breakage (Alakhras et al. Cancer Lett 2011; 306: 15–26). To further study retinoic acid clastogenicity and evaluate DNA damaging potential we investigated the ability of (a) all‐trans retinoic acid and its steroidal analogue EA‐4 to induce DNA fragmentation by using Comet assay under alkaline unwinding and neutral condition electrophoresis, and (b) the retinoids under study to induce small (0–1 kb) DNA fragments. Two cell lines, C2C12 mouse cells and HL‐60 human leukemic cells were used in this study. We found that all‐trans retinoic acid and its steroidal analogue EA‐4 (a) provoke DNA migration due to DNA fragmentation as it is shown by the increased values of Comet parameters, and (b) induce significantly small‐size fragmented genomic DNA as indicated by the quantification of necrotic/apoptotic small DNA segments in both cell systems. A different response between the two cell lines was observed in relation to retinoid ability to increase the percentage of DNA in the tail as well as break DNA in to small fragments. Our findings confirm the ability of retinoic acid to provoke micronucleation by disrupting DNA into fragments, among which small pieces of double‐stranded DNA up to 1 kb are identified. Copyright © 2013 John Wiley & Sons, Ltd.     

Comparing the genotoxicity of a potentially carcinogenic and a noncarcinogenic PAH,singly, and in binary combination,on peripheral blood cells of the European sea bass

Research on the toxicological mechanisms of polycyclic aromatic hydrocarbons (PAHs) deemed carcinogenic and noncarcinogenic has mostly been developed for individual compounds even though, in the environment, PAHs invariably occur in mixtures. The present work aimed at understanding the interaction effects of two model PAHs, the potentially carcinogenic benzo[b]fluoranthene (B[b]F) and the noncarcinogenic phenanthrene (Phe) to a marine fish (the sea bass Dicentrarchus labrax). The study endeavoured an ecologically‐relevant scenario with respect to concentrations and contaminant matrix, sediments, which are the main reservoirs of these substances in the environment, due to their hydrophobic nature. For the purpose, 28‐day laboratorial bioassays with spiked sediments (with individual and combined PAHs at equitoxic concentrations) were conducted. Genotoxicity was determined in peripheral blood through the “Comet” assay and by scoring erythrocytic nuclear abnormalities (ENA). The results showed that exposure to either PAHs induced similar levels of DNA strand breaks, although without a clear dose‐ or time‐response, likely due to the low concentrations of exposure and potential shits in PAH bioavailability during the assays. However, clastogenic/aneugenic lesions were only observed in fish exposed to B[b]F‐spiked sediments. Conversely, the combination assays revealed a supra‐additive effect especially at chromosome level, linked to concentrations of PAHs in water. A decrease in DNA‐strand breakage was observed over time during all assays, revealing some ability of fish to cope with this DNA lesion. Overall, the findings show that low‐moderate concentrations of sediment‐bound mixed PAHs may significantly increase the hazard of mutagenesis even when the individual concentrations indicate low risk, especially considering that chromosome‐level damage is unlikely to be repaired, leading to the fixation of DNA lesions upon prolonged exposures. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1307–1318, 2016.     

Detection of DNA adducts derived from the reactive metabolite of furan, cis-2-butene-1,4-dial

Furan is a toxic and carcinogenic compound used in industry and commonly found in the environment. The mechanism of furan's carcinogenesis is not well-understood and may involve both genotoxic and nongenotoxic pathways. Furan undergoes oxidation by cytochrome P450 to cis-2-butene-1,4-dial, which is thought to mediate furan's toxic effects. Consistently, cis-2-butene-1,4-dial readily reacts with glutathione, amino acids, and nucleosides. To determine the importance of DNA alkylation in furan-induced carcinogenesis, we developed an assay for the detection of cis-2-butene-1,4-dial-derived DNA adducts. DNA samples were treated with O-benzyl-hydroxylamine, which reacts with the aldehyde functionality of the DNA adducts. Enzyme hydrolysates of these samples were then analyzed by capillary electrospray tandem mass spectrometry with selected reaction monitoring. The dCyd and dAdo adducts were detected in digests of DNA treated with nanomolar concentrations of cis-2-butene-1,4-dial. In addition, these adducts were present in DNA isolated from Ames assay strain TA104 treated with mutagenic concentrations of cis-2-butene-1,4-dial. These data support the hypothesis that cis-butene-1,4-dial is a genotoxic metabolite of furan. This method will allow us to explore the role of these adducts in furan-induced carcinogenesis.     

Comparative mutagenicity of apicidin and apicidin derivatives (SD-0203 and SD-2007), histone deacetylase inhibitors

The fungal metabolite apicidin [cyclo(N-O-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8-oxodecanoyl)] is known to inhibit histone deacetylase (HDAC). In this study, the genotoxicity of apicidin and its derivatives were tested using three tests: a bacterial reverse mutation assay (Ames test), an in vitro chromosome aberration (CA) test, and an in vivo micronucleus (MN) test. Apicidin was negative in the Ames test in the presence and absence of the microsomal metabolizing enzyme system. Apicidin induced a significant increase in the total chromosome aberrations in Chinese hamster ovary (CHO) cells. In the MN test, apicidin induced mutagenic activity at the highest dose (1000 microM/kg). The apicidin derivatives SD-0203 and SD-2007 did not induce mutagenic activity in the Ames test and no significant mutagenic potency was observed in the CA test. However, these compounds significantly and dose-dependently increased the number of micronucleated polychromatic erythrocytes (MNPCEs) as well as the PCE/(PCE + NCE) ratio in the MN test. These results suggest that apicidin and its derivatives preferentially induce CA and MN but are not effective in the Ames test.