Micronucleus Test In Human Lymphocytes Research Articles

In vitro genotoxicological evaluation of protein-rich powder derived from Xanthobacter sp. SoF1.

One way of limiting the environmental impact of food production and improving food security is to replace part of the animal- or plant-based protein in the human diet with protein sourced from microorganisms. The recently discovered bacterium Xanthobacter sp. SoF1 (VTT-E-193585) grows autotrophically using carbon dioxide gas as the only carbon source, yielding protein-rich biomass that can be processed further into a powder and incorporated into various food products. Since the safety of this microbial protein powder for human consumption had not been previously assessed, its genotoxic potential was evaluated employing three internationally recognized and standardized studies: a bacterial reverse mutation test, an in vitro chromosomal aberration assay in human lymphocytes, and an in vitro micronucleus test in human lymphocytes. No biologically relevant evidence of genotoxicity or mutagenicity was found.

X-ray Structures and Feasibility Assessment of CLK2 Inhibitors for Phelan-McDermid Syndrome.

CLK2 inhibition has been proposed as a potential mechanism to improve autism and neuronal functions in Phelan-McDermid syndrome (PMDS). Herein, the discovery of a very potent indazole CLK inhibitor series and the CLK2 X-ray structure of the most potent analogue are reported. This new indazole series was identified through a biochemical CLK2 Caliper assay screen with 30k compounds selected by an in silico approach. Novel high-resolution X-ray structures of all CLKs, including the first CLK4 X-ray structure, bound to known CLK2 inhibitor tool compounds (e.g., TG003, CX-4945), are also shown and yield insight into inhibitor selectivity in the CLK family. The efficacy of the new CLK2 inhibitors from the indazole series was demonstrated in the mouse brain slice assay, and potential safety concerns were investigated. Genotoxicity findings in the human lymphocyte micronucleus test (MNT) assay are shown by using two structurally different CLK inhibitors to reveal a major concern for pan-CLK inhibition in PMDS.

Assessment of in vivo and in vitro genotoxicity of glibenclamide in eukaryotic cells.

Glibenclamide is an oral hypoglycemic drug commonly prescribed for the treatment of type 2 diabetes mellitus, whose anti-tumor activity has been recently described in several human cancer cells. The mutagenic potential of such an antidiabetic drug and its recombinogenic activity in eukaryotic cells were evaluated, the latter for the first time. The mutagenic potential of glibenclamide in therapeutically plasma (0.6 μM) and higher concentrations (10 μM, 100 μM, 240 μM and 480 μM) was assessed by the in vitro mammalian cell micronucleus test in human lymphocytes. Since the loss of heterozygosity arising from allelic recombination is an important biologically significant consequence of oxidative damage, the glibenclamide recombinogenic activity at 1 μM, 10 μM and 100 μM concentrations was evaluated by the in vivo homozygotization assay. Glibenclamide failed to alter the frequency of micronuclei between 0.6 μM and 480 μM concentrations and the cytokinesis block proliferation index between 0.6 μM and 240 μM concentrations. On the other hand, glibenclamide changed the cell-proliferation kinetics when used at 480 μM. In the homozygotization assay, the homozygotization indices for the analyzed markers were lower than 2.0 and demonstrated the lack of recombinogenic activity of glibenclamide. Data in the current study demonstrate that glibenclamide, in current experimental conditions, is devoid of significant genotoxic effects. This fact encourages further investigations on the use of this antidiabetic agent as a chemotherapeutic drug.

Toxicological and Genotoxicity Assessment of a Dihydroquercetin-Rich Dahurian Larch Tree (Larix gmelinii Rupr) Extract (Lavitol).

Safety assessment is reported of an orally ingested dihydroquercetin-rich extract (Lavitol) derived from the Dahurian larch tree, used as a food additive and as a dietary supplement ingredient. Dihydroquercetin, a potent antioxidant, is also known as taxifolin. The results of genotoxicity and toxicological tests (Comet assay, micronucleus test in human lymphocytes, chromosomal aberration test, subacute 7-day oral toxicity study, subchronic 90-day toxicology study with histopathologies, and, prenatal and postnatal developmental toxicity studies) on the extract provide further support for the safety of its consumption as a food supplement and food additive.

Automatic analysis of the micronucleus test in primary human lymphocytes using image analysis

The in vitro micronucleus test (MNT) is a well-established test for early screening of new chemical entities in industrial toxicology. For assessing the clastogenic or aneugenic potential of a test compound, micronucleus induction in cells has been shown repeatedly to be a sensitive and a specific parameter. Various automated systems to replace the tedious and time-consuming visual slide analysis procedure as well as flow cytometric approaches have been discussed. The ROBIAS (Robotic Image Analysis System) for both automatic cytotoxicity assessment and micronucleus detection in human lymphocytes was developed at Novartis where the assay has been used to validate positive results obtained in the MNT in TK6 cells, which serves as the primary screening system for genotoxicity profiling in early drug development. In addition, the in vitro MNT has become an accepted alternative to support clinical studies and will be used for regulatory purposes as well. The comparison of visual with automatic analysis results showed a high degree of concordance for 25 independent experiments conducted for the profiling of 12 compounds. For concentration series of cyclophosphamide and carbendazim, a very good correlation between automatic and visual analysis by two examiners could be established, both for the relative division index used as cytotoxicity parameter, as well as for micronuclei scoring in mono- and binucleated cells. Generally, false-positive micronucleus decisions could be controlled by fast and simple relocation of the automatically detected patterns. The possibility to analyse 24 slides within 65h by automatic analysis over the weekend and the high reproducibility of the results make automatic image processing a powerful tool for the micronucleus analysis in primary human lymphocytes. The automated slide analysis for the MNT in human lymphocytes complements the portfolio of image analysis applications on ROBIAS which is supporting various assays at Novartis.

Genotoxicity profile of fexinidazole--a drug candidate in clinical development for human African trypanomiasis (sleeping sickness)

The parasitic disease human African trypanomiasis (HAT), also known as sleeping sickness, is a highly neglected fatal condition endemic in sub-Saharan Africa, which is poorly treated with medicines that are toxic, no longer effective or very difficult to administer. New, safe, effective and easy-to-use treatments are urgently needed. Many nitroimidazoles possess antibacterial and antiprotozoal activity and examples such as tinidazole are used to treat trichomoniasis and guardiasis, but concerns about toxicity including genotoxicity limit their usefulness. Fexinidazole, a 2-substituted 5-nitroimidazole rediscovered by the Drugs for Neglected Diseases initiative (DNDi) after extensive compound mining of public and pharmaceutical company databases, has the potential to become a short-course, safe and effective oral treatment, curing both acute and chronic HAT. This paper describes the genotoxicity profile of fexinidazole and its two active metabolites, the sulfoxide and sulfone derivatives. All the three compounds are mutagenic in the Salmonella/Ames test; however, mutagenicity is either attenuated or lost in Ames Salmonella strains that lack one or more nitroreductase(s). It is known that these enzymes can nitroreduce compounds with low redox potentials, whereas their mammalian cell counterparts cannot, under normal conditions. Fexinidazole and its metabolites have low redox potentials and all mammalian cell assays to detect genetic toxicity, conducted for this study either in vitro (micronucleus test in human lymphocytes) or in vivo (ex vivo unscheduled DNA synthesis in rats; bone marrow micronucleus test in mice), were negative. Thus, fexinidazole does not pose a genotoxic hazard to patients and represents a promising drug candidate for HAT. Fexinidazole is expected to enter Phase II clinical trials in 2012.

The Genotoxic Effect of the New Acaricide Etoxazole

Etoxazole is a member of the diphenyl oxazoline class of insecticide was newly developed for use on pome fruits, cotton and strawberries as a acaricide. In the present study, genotoxic effects of acaricide etoxazole (ETX) (miticide/ovicide) were investigated using chromosome aberration (CA) test, sister chromatid exchange (SCE) test and micronucleus test in human lymphocytes. ETX induced the CAs at all concentrations (5, 10 and 20 microg/ml) for 24 h and also induced the CA at the highest concentration (20 microg/ml) for 48 h only. The inducing the CAs for 48 hours treatment period was dose-dependent. Besides, it induced the SCE at all concentrations and treatment periods in a dose-dependent manner as well. Although, ETX decreased the mitotic index (MI) at all concentration and treatment periods dose-dependently, while it did not decrease the replication index (RI) when compared to the negative and solvent controls. In addition, ETX induced the micronucleus at all concentrations except 5 microg/ml for 48 h. This inducing was in a dose-dependent manner as well. In conclusion, it can be concluded that ETX has a potential genotoxic effects in cultured human peripheral lymphocytes.

Genotoxicity of Aspartame

In the present study, the genotoxic effects of the low‐calorie sweetener aspartame (ASP), which is a dipeptide derivative, was investigated using chromosome aberration (CA) test, sister chromatid exchange (SCE) test, micronucleus test in human lymphocytes and also Ames/Salmonella/ microsome test. ASP induced CAs at all concentrations (500, 1000 and 2000 µg/ml) and treatment periods (24 and 48 h) dose‐dependently, while it did not induce SCEs. On the other hand, ASP decreased the replication index (RI) only at the highest concentration for 48 h treatment period. However, ASP decreased the mitotic index (MI) at all concentrations and treatment periods dose‐dependently. In addition, ASP induced micronuclei at the highest concentrations only. This induction was also dose‐dependent for 48 hours treatment period. ASP was not mutagenic for Salmonella typhimurium TA98 and TA100 strains in the absence and presence of S9 mix.

Comparative studies on genotoxicity and antigenotoxicity of natural and synthetic β-carotene stereoisomers

To evaluate the practical value of natural β-carotene (NβC) and to elucidate the apparent discrepancy between epidemiological observations and intervention trials on the role of β-carotene (βC) in tumor prevention, the genotoxicity and the antigenotoxicity of NβC and synthetic βC crystal (SβCC) stereoisomers were studied comparatively using chromosome aberration analysis and the micronucleus test in human lymphocytes in vitro. NβC was extracted from the halotolerant algae Dunaliella salina. The NβC crystal (NβCC) preparation is about 70% all- trans (TβC) and 8% 9- cis (CβC). The NβC oil (NβCO) preparation is about 40% all- trans and 38% 9- cis. SβCC is more than 97% all- trans, and the 9- cis can not be detected. The mixture of βC (βCM) preparation is 74% SβCC and 26% NβC. Our results show no genotoxicity of 1–30 μg/ml NβCC, but this concentration of NβCC inhibited significantly γ-ray-induced micronucleus formation in human lymphocytes in vitro. One to thirty μg/ml NβCO was most effective against both γ-ray-induced and spontaneous micronucleus formation. However, no influence of NβCO on spontaneous chromosome aberrations in human lymphocytes in vitro was observed. NβCO suppressed significantly mitomycin C (MMC)-induced chromosome aberrations. One to thirty μg/ml SβCC induced a dose-dependent increase in micronucleus frequency, and also inhibited γ-ray-induced micronucleus formation. No effect of βCM on spontaneous chromosome aberrations was found. One to thirty μg/ml βCM is more effective against MMC-induced chromosome aberrations than NβCO. These results suggest that CβC might play a critical role in the genotoxicity and antigenotoxicity of SβCC and NβC. The genotoxic activity of SβCC might be involved in carcinogenesis. NβC or βCM could be of practical value in tumor prevention and supplementary treatment.

Using the micronucleus assay to detect genotoxic effects of metal ions.

The lymphocyte micronucleus assay was used to measure the average frequency of micronuclei in a population and thus assess genotoxic effects. Data from 174 persons give an average value of 16.4 +/- 7.3, and a slight age-dependence was observed. To detect combined environmental mutagen injuries the micronucleus assay was used to study the effects of metal compounds. Cadmium ions increased the micronucleus frequency linearly after incubation with whole blood in vitro with 10(6)-10(-3) M concentrations for 30 min. Similarly, a linear increase in micronucleus frequency was detected with 10(-3)-10(-1) M mercury ions. Concerning the biological effect of selenium, it was found that neither sodium selenite nor selenium dioxide induced increases at concentrations of 10(-7)-10(-6) M; 10(-5) M caused a slight increase; 10(-4) M, however, destroyed the cells. These results suggest that the human lymphocyte micronucleus test can be used to assess genotoxic injuries due to environmental effects in human lymphocytes.

Analysis of eight known or suspected aneugens by the in vitro human lymphocyte micronucleus test.

In the light of the CEC aneuploidy programme eight known or suspected aneugens were evaluated with the human lymphocyte 'in vitro' micronucleus test using the cytochalasin-B technique. Only colchicine, chloral hydrate and hydroquinone induced dose-dependent increases in micronucleus frequencies. The other five chemicals (cadmium chloride, econazole, pyrimethamine, thiabendazole and thimerosal) were all negative, both with and without S9, and treated in G1 or G2 phase. These data are in good agreement with results from different 'in vivo' studies. However, discrepancies were found between the results from this study of human lymphocytes and those of other cultured cell types where econazole, cadmium chloride, pyrimethamine and thiabendazole induce a significant increase of micronuclei.

A modified method and staining technique for the in vitro micronucleus test in human lymphocytes using cytochalasin B

A modified method and staining technique for the in vitro micronucleus test in human lymphocytes using cytochalasin B