Clastogenicity Assays Research Articles

Genotoxicity study of high aspect ratio silver nanowires

ABSTRACTThe genotoxicity potential of silver nanowires synthesized via the solution-based polyol method has been investigated. They were found to be non-mutagenic in three Salmonella strains and were not genotoxic in a clastogenicity assay in mice. Residual surfactant was found to have an effect on the toxicological properties of the nanowires by increasing the rate of Ag+ release. Residual surfactant can be easily degraded via a UV treatment.

An assessment of the carcinogenic potential of ezetimibe using nonclinical data in a weight-of-evidence approach

Ezetimibe blocks intestinal absorption of sterols via interaction with the Neimann-Pick C1-Like 1 (NPC1L1) transporter and is approved for use in the treatment of primary hyperlipidemia (heterozygous familial and non-familial), homozygous familial hypercholesterolemia, and homozygous sitosterolemia. A recently completed randomized clinical trial [simvastatin and ezetimibe in aortic stenosis (SEAS)] testing the effectiveness of Vytorin™ (a combination of simvastatin and ezetimibe) in patients with aortic stenosis reported an unexpected safety finding: an increase in overall cancer incidence and cancer-associated mortality (all types) in the treated groups relative to the placebo control. A subsequent meta-analysis utilizing a much larger database from two ongoing clinical trials indicated that the observed findings in the SEAS trial were likely due to chance and not a true drug-induced effect. Nonetheless, it has been suggested by various commentators on the SEAS trial that ezetimibe may be carcinogenic. The extensive nonclinical database for ezetimibe was used to test the hypothesis that ezetimibe may be a direct or indirect carcinogen. Using two different in silico approaches, ezetimibe showed no structural alerts for genetic toxicity or carcinogenicity. Ezetimibe was not genotoxic in two reverse mutation assays, one in vitro clastogenicity assay, and two mouse micronucleus assays. No evidence of proliferative lesions was observed in three species in studies of 1–12 months in duration. Ezetimibe was not carcinogenic in standard 2-year bioassays in mice and rats. Additionally, in these 2-year bioassays, no drug-related non-neoplastic lesions were noted. The absence of drug-induced non-neoplastic or proliferative lesions in these studies indicates that ezetimibe treatment was not associated with findings characteristic of carcinogens (i.e., DNA reactivity or cell proliferation) Administration of pharmacologic doses of ezetimibe to mice did not alter hepatic expression patterns of genes associated with apoptosis, cell proliferation, or epithelial–mesenchymal transition. No evidence of drug-induced tumors was observed in mice in which the molecular target of ezetimibe (NPC 1L1) was knocked out over the life span of the animal. In conclusion, the nonclinical data do not support the proposed hypothesis based on the single observation from the SEAS trial and, rather, support the conclusion that ezetimibe does not represent a carcinogenic hazard to humans using this drug in a therapeutic setting.

In vitro Safety Evaluation and Anticlastogenic Effect of BacoMind™ on Human Lymphocytes

BacoMind (BM) is a standardized extract of Bacopa monnieri, which belongs to the family Scrophulariaceae and is a creeping annual plant found throughout the Indian subcontinent. It has been used by Ayurvedic medicinal practitioners in India for almost 3000 years and is classified as a medharasayana, a substance which improves memory and intellect. With the widespread traditional use as well as scientific validation of Bacopa monnieri for nootropic activity, a bioactive-rich unique phytochemical composition-BacoMind was developed from B. monnieri for use as a cognition and memory enhancing agent. The present study aimed to investigate the in vitro toxicity of this formulation of BacoMind on human lymphocytes and to rule out its possible contribution to mutagenicity. In the present investigation the active ingredients present in BM were identified and quantified by high performance liquid chromatography (HPLC) and high performance thin-layer chromatography (HPTLC). Antioxidant and anticlastogenic properties of BM were studied in vitro with and without metabolic activation. Doses of BM were chosen on the basis of mitotic index (MI) and cytokinesis-block proliferation index (CBPI). Clastogenicity assays were performed at 31.2 microg/mL, 62.5 microg/mL, and 125 microg/mL, while the Salmonella reverse mutation assay (Ames test) was performed at doses of 61.72, 185.18, 555.55, 1666.67, and 5000.00 microg/plate. HPLC and HPTLC analysis of BM revealed the presence of bacoside A3, bacopaside I, bacopaside II, jujubogenin isomer of bacopasaponin C, bacosine, luteolin, apigenin, bacosine, and beta-sitosterol D glucoside. BM demonstrated significant antioxidant activity. The number of chromosomal aberrations and the frequency of micronuclei induced by BM were not statistically significant up to a dose of 62.5 microg/mL. A subsequent dose of 125 microg/mL prior to metabolic activation induced mild clastogenicity, but it was found to be biologically insignificant as this effect was not seen post metabolic activation. BM also demonstrated a dose-dependent protection against the clastogens used in this study using the above tests for clastogenicity. Maximum protection was observed in presence of metabolic activation. Moreover, BM demonstrated no mutagenic effect on the tested strains, as observed in the Ames test. BM protected human lymphocytes against various clastogens. BM also exhibited high antioxidant activity which might be responsible for the observed protective effects against the clastogens since the used clastogens are known to induce their clastogenic effects via production of oxidative radicals.

Lack of clastogenic/genotoxic effects of Baccharis dracunculifolia extract on Swiss mouse peripheral blood cells

Baccharis dracunculifolia De Candole (DC) (Asteraceae) is indigenous throughout southeastern Latin America and is used by local people in traditional medicine. This plant is known to be the source of resin for the highly valued Brazilian green propolis. As no information is available on the safety of high doses of B. dracunculifolia extract, we evaluated the mutagenic potential of high doses of this plant extract in vivo on peripheral blood cells of Swiss mice using the comet assay and the micronucleus test. The extract was administered by gavage at doses of 1000, 1500 and 2000 mg/kg body weight. Peripheral blood cell samples were collected 4 and 24 h after treatment for the comet assay (genotoxicity assay), and at 48 and 72 h for the micronucleus test (clastogenicity assay). The B. dracunculifolia extract was devoid of clastogenic/genotoxic activity at all doses.

Genetic Toxicity Assessment: Employing the Best Science for Human Safety Evaluation Part III: The Comet Assay as an Alternative to In Vitro Clastogenicity Tests for Early Drug Candidate Selection

Early screening of drug candidates for genotoxicity typically includes an analysis for mutagenicity in bacteria and for clastogenicity in cultured mammalian cells. In addition, in recent years, an early assessment of photogenotoxicity potential has become increasingly important. Also, for screening purposes, expert computer systems can be used to identify structural alerts. In cases where structural alerts are identified, mutagenicity testing limited to bacteria can be conducted. The sequence of computer-aided analysis and limited testing using bacteria allows for screening a comparatively large number of drug candidates. In contrast, considerably more resources, in terms of supplies, technical time, and the amount of a test substance needed, are required when screening for clastogenic activity in mammalian cells. In addition, the relatively large percentage of false positive results for rodent carcinogenicity associated with clastogenicity assays is of considerable concern. As a consequence, mammalian cell-based alternatives to clastogenicity assays are needed for early screening of mammalian genotoxicity. The comet assay is a relatively fast, simple, and sensitive technique for the analysis of DNA damage in mammalian cells. This assay seems especially useful for screening purposes because false positives associated with excessive toxicity appear to occur less frequently, only relatively small amounts of a test compound are needed, and certain steps of the test procedure can be automated. Therefore, the in vitro comet assay is proposed as an alternative to cytogenetic assays in early genotoxicity/photogenotoxicity screening of drug candidates.

The effect of PHA stimulation on lymphocyte sub-populations in whole-blood cultures

Although lymphocytes in phytohaemagglutinin (PHA)-stimulated whole blood cultures are routinely used to assess genotoxin-induced chromosome damage, very little information is available on the effect of PHA on the various cell populations present, and there appear to be no data for the protocols used in routine genotoxicity assays. In this study, we used flow cytometric analysis to examine the size/complexity of the white blood cell (WBC) population and the expression of key antigenic markers by the lymphocytes over a 96-h period following PHA stimulation. The changes in the WBC population are complex, and would seem to represent different populations dying out, remaining static or starting to divide. The initial decrease seen in overall cell numbers probably reflects death of the neutrophil and monocyte populations. The subsequent increase in cell numbers appears to be due to division of the lymphocytes and, by 96 h post-stimulation, they comprise about half the total cell number and, as expected, > 90% are activated T-cells; it seems reasonable to assume that these represent the target cells in genotoxicity assays. Although we do not suggest that these findings should alter the routine conduct of clastogenicity assays using PHA-stimulated whole-blood cultures, they indicate that such tests are empirical and that closer investigation will only confirm their relatively imprecise nature.

Selection and Evaluation of an Appropriate Model for Screening Adenosine Analogs for Chromosome Aberrations

As part of a preclinical safety assessment profile, the adenosine analog SC-46267, a candidate antihypertensive drug, was tested for potential genotoxicity in a series of in vitro and in vivo assays. Negative results were obtained in the Salmonella/microsome and CHO/HGPRT point mutation assays and in an acute mouse micronucleus test. However, in the presence of exogenous metabolism, positive results in the mouse lymphoma L5178Y assay and an in vitro chromosome aberration assay with purified rat lymphocytes indicated that the analog was a potential clastogen. Both adenosine and the analog were subsequently tested in rat lymphocytes using whole-blood versus purified lymphocyte cultures. High concentrations of adenosine and the adenosine analog induced chromosome aberrations in the purified lymphocytes. However, neither compound induced chromosome damage when cultured in the presence of whole blood, regardless of the presence or absence of the metabolic activation system. These results were similar to those reported in the literature that suggested that high concentrations of adenosine and other naturally occurring nucleotides can produce nonphysiological perturbations of nucleotide pools that are clastogenic in purified cultures of lymphocytes but not in whole-blood cultures. Our studies appear to support the hypothesis that adenosine deaminase, present at normal physiological levels in whole blood, is capable of detoxifying excessive levels of adenosine. Both compounds were also negative in a 10-day repeated dose mouse micronucleus test, which further ensures that the positive in vitro findings are not biologically significant. An in vitro clastogenicity assay using whole blood appears to be more appropriate than use of purified lymphocytes for screening adenosine analogs. The results from this series of experiments demonstrate the importance of considering culture conditions when designing in vitro test protocols. Furthermore, this study exemplifies the need to explore the mechanisms of activity as well as in vivo relevance of in vitro results.

Genetic toxicology of tricyclic carboxamides, a new class of DNA binding antitumour agent

( N-[2-(Dimethylamino)ethyl]acridine-4-carboxamide (acridine carboxamide; NSC 601316) is an acridine-derived experimental antitumour agent with curative properties against Lewis lung carcinoma in mice. Although it intercalates into DNA and also appears to interact with topoisomerase II, its DNA binding properties appear distinct from other acridine derivatives such as the clinical antitumour drug, amsacrine. The mutagenic properties of acridine carboxamide, together with three related compounds containing either 9-aminoacridine or phenazine chromophores, were studied at the 6-thioguanine and ouabain loci in cultured V79 Chinese hamster fibroblasts. Each compound, when tested at concentrations causing up to 90% kill, had weak but significant activity at the 6-thioguanine but not at the ouabain locus. All drugs were potent inducers of micronuclei, indicating high clastogenic activity. There was a highly significant relationship between mutation frequency (as resistance to 6-thioguanine) and either cytotoxicity (measured as d 37 in a clastogenicity assay) or clastogenicity. A broader range of compounds was also tested for microbial mutagenicity. In Salmonella typhimurium strains, none were mutagenic in TA98, TA100 or TA102 but several were mutagenic in TA1537, a frameshift tester strain. Some drugs also caused ‘petite’ mutagenesis in Saccharomyces cerevisiae. In general, compounds with the phenazine chromophore, which has no positive charge, were the most mutagenic in these systems. However, activity was not related to mammalian mutagenicity or antitumour effect. The results suggest that in mammalian cells, the cytotoxicity, clastogenicity and mutagenic activity of these drugs are mediated by similar mechanisms to those for amsacrine analogues, probably involving the enzyme DNA topoisomerase II.

Fish cell line (ULF-23HU) derived from the fin of the central mudminnow (Umbra limi): Suitable characteristics for clastogenicity assay

A cell line (ULF-23HU) from the fin of the central mudminnow (Umbra limi) was characterized and tested for its suitability to assess cytogenetic damages induced by chemicals in fish. Cells of this line exhibit a fibroblastlike appearance and grew optimal at 25°C in, TC-199 medium containing 10% fetal bovine serum, but slower growth continued down to 4°C, where they could be stored for prolonged periods. Seeding efficiency of ULF-23HU cells on the plastic substratum was approximately 85% in the above culture medium at 25°C. They had a 32-h cell cycle time taken up by a 20-h S period as determined by the autoradiographic analysis of the fraction of labeled mitosis. Cultures showed relatively high mitotic index (0.84 to 2.35%) during exponential growth phase lasting about 7 d. Karyological analysis of the cells at the different subculture passages revealed constant chromosome modal number of 23 consisting of metacentric or submetacentric chromosomes, which were primarily similar to those of in vivo cells, with one additional chromosome. The spontaneous sister chromatid exchange rate was 5.3 per metaphse. When ULF-23HU cells were exposed toN-nitroso-N-methylurea, a clastogen in the mammalian cells, dose-dependent increases both in sister chromatid exchanges and chromosome aberrations were clearly detected. These results on the growth kinetics and cytogenetic characteristics offered the high possibility of the use of this cell line as a suitable in vitro model for clastogenicity studies in fish.

Chloracetamide- N-metholol: an example of an in vitro and in vivo clastogen which is non-mutagenic to Salmonella

The industrial biocide chloracetamide- N-metholol (CAM) has been shown to be non-mutagenic to 6 strains of Salmonella using both the plate-incorporation and a pre-incubation test protocol. Its biocidal activity is unlikely to have influenced these results since Kathon 886, a more potent biocide, was concomitantly detected as mutagenic to strain TA100. In contrast, CAM was weakly clastogenic to human lymphocytes cultured in vitro and elicited a positive response in the mouse bone marrow micronucleus test when assayed using the intraperitoneal, but not the oral route of administration. A positive response was concomitantly observed for the rodent carcinogen and formaldehyde-releasing agent hexamethylphosphoramide (HMPA) in these 2 clastogenicity assays. Data are presented showing the slow hydrolysis of CAM to formaldehyde in vitro, and both [ carbonyl- 14C]CAM and [ metholol- 14C]CAM have been shown to interact covalently with calf-thymus DNA in vitro. It is concluded that CAM may be a direct-acting carcinogen to rodents, but that both the qualitative and quantitative outcome of its bioassay for carcinogenicity will be influenced critically by the bioassay protocol adopted; in particular, by the route of administration selected. These findings emphasize the need to complement the Salmonella gene-mutation assay with an in vitro assay for the induction of chromosomal aberrations if in vivo genotoxins are to be detected efficiently in vitro.

Activity in clastogenicity and SCE assays, and aberration kinetics in chinese hamster V79-E cells

The nitrosoureas 1-methyl-1-nitroso-3-phenylurea, 1-ethyl-1-nitroso-3phenylurea, 1-methyl-1-nitroso-3-( p-fluorophenyl)urea, 1-methyl-1-nitroso-3( p-chlorophenyl)urea, as well as 1-methyl-1-nitroso-3-( p-bromophenyl)urea, as well as their non-nitrosated parent compounds, were checked for induction of chromosomal aberrations and sister-chromatid exchanges in V79-E cells without metabolic activation in vitro. For comparison, methylnitrosourea, ethylnitrosourea and nitrosocarbaryl were included in this study. Whereas the non-nitrosated agents were inactive, the nitroso derivatives were potent clastogens and inducers of SCEs. Clastogenicity parallels SCE induction, but the latter assay is about 10 times more sensitive (based on concentration of substance) than the clastogenicity assay. The dependence of aberration frequency on sampling time, which was studied for 5 nitroso compounds, revealed striking differences. As demonstrated by differential chromatid staining, the lag phase until maximal aberration rates may cover more than 2 cell cycles. Preventive oncological aspects of these nitrosamides and the mechanism of aberration kinetics are discussed.

Activity of citrinin metabolized by rat and human microsome fractions in clastogenicity and SCE assays on Chinese hamster V79-E cells

The mycotoxin citrinin is a potent inducer of chromosomal aberrations in the clastogenicity assay on V79-E cells when metabolized by rat and human liver microsomes. Rat and human liver microsomes, standardized on protein content, activate citrinin at equal levels. 5 × 10−4 M citrinin induces complex translocations in a high frequency as well as defects of chromosomal coiling. Higher concentrations are cytotoxic, lower ones are almost inactive. After metabolization of mycotoxin by rat-kidney microsomes or an S9 mix fraction containing rat liver and kidney microsomes, toxic effects predominate and chromosomal aberrations are diminished. Clastogenic citrinin concentrations do not induce an increase of SCE frequency. Although the mode of action of this mycotoxin on chromosomal structure remains obscure, possible explanations are discussed.