Incidence Of Micronucleated Polychromatic Erythrocytes Research Articles

Genotoxicity Evaluation of Capsaicin-Containing (CP) Pharmacopuncture, in an In Vivo Micronucleus Test.

ObjectivesCapsaicin-containing (CP) pharmacopuncture was developed to treat neuropathic pain. This study was conducted to assess the toxicity of CP extract for pharmacopuncture, using a micronucleus test.MethodsFirst, a dose range finding study was conducted. Then an in vivo micronucleus test was performed to determine the induction of micronuclei in mouse bone marrow cells after intramuscular administration of CP twice with a 24-hour interval to 8-week-old ICR mice. A high dose of 0.2 mL/animal was selected, and this was sequentially diluted by applying a geometric ratio of 2 to produce two lower dose levels (0.1 and 0.05 mL/animal). In addition, negative and positive control groups were set up, and an HPLC analysis was conducted to confirm the capsaicin content of CP.ResultsThe incidence of micro-nucleated polychromatic erythrocytes in polychromatic erythrocytes in the CP-treated group was similar to that in the negative-control group, while that in the positive-control group was significantly greater. In addition, the ratio of polychromatic erythrocytes to total erythrocytes in the CP treatment group and the positive control group was not significantly different from the negative control group. In the HPLC analysis, capsaicin in the CP was identified through a comparison with the retention time of the capsaicin standard of 27 min.ConclusionCP did not show any indication of any potential to induce micronuclei formation in bone marrow cells of ICR mice under the conditions of this study. Further toxicity studies are necessary to ensure the safety of the use of CP in clinical practice.

Evaluation of Genotoxicity of Water and Ethanol Extracts from Rhus verniciflua Stokes (RVS).

Rhus verniciflua Stokes (RVS), one of traditional medicinal plants in Asia, was found to have pharmacological activities such as antioxidative and antiapoptotic effects, raising the possibility for the development of a novel class of anti-cancer drugs. Thus, potential genotoxic effects of RVS in three short-term mutagenicity assays were investigated, which included the Ames assay, in vitro Chromosomal aberration test, and the in vivo Micronucleus assay. In Ames test, the addition of RVS water extracts at doses from 313 up to 5000 mg/plate induced an increase more than 2-fold over vehicle control in the number of revertant colonies in TA98 and TA1537 strains for detecting the frame-shift mutagens. The similar increase in reversion frequency was observed after the addition of RVS ethanol extracts. To assess clastogenic effect, in vitro chromosomal aberration test and in vivo micronucleus assay were performed using Chinese hamster lung cells and male ICR mice, respectively. Both water and ethanol extracts from RVS induced significant increases in the number of metaphases with structural aberrations mostly at concentrations showing the cell survival less than 60% as assessed by in vitro CA test. Also, there was a weak but statistically significant increase in number of micronucleated polychromatic erythrocytes (MNPCEs) in mice treated with water extract at 2000 mg/kg while ethanol extracts of RVS at doses of up to 2000 mg/kg did not induce any statistically significant changes in the incidence of MNPCEs. Therefore, our results lead to conclusion that RVS acts as a genotoxic material based on the available in vitro and in vivo results.

Studies of the Toxicological Potential of Tripeptides (L-Valyl-L-prolyl-L-proline and L-lsoleucyl-L-prolyl-L-proline): VII. Micronucleus Test of Tripeptides-Containing Casein Hydrolysate and Lactobacillus helveticus-fermented Milk Powders in Rats and Mice

The objective of these in vivo experiments was to assess the mutagenic potential of tripeptides as reflected by the ability of the test compound to induce the formation of micronuclei in mouse polychromatic erythrocytes. The test agents used in these experiments were (1) powdered Aspergillus oryzae protease casein hydrolysate (CH) and (2) powdered Lactobacillus helveticus-fermented milk (FM). Both test agents contain two tripeptides, L-valyl-L-prolyl-L-proline (VPP) and L-isoleucyl-L-prolyl-L-proline (IPP). Male Sprague-Dawley rats (five per group) were administered doses of 0, 500, 1000, or 2000 mg (0, 3, 6, or 12 mg VPP plus IPP)/kg body weight (BW)/day CH by oral gavage for 2 days. Male CD-1 mice (six per group) received a single oral gavage dose of 0, 500, 1000, or 2000 mg (0, 0.8, 1.6 or 3.3 mg VPP plus IPP)/kg BW of FM. Positive-control agents were cyclophosphamide (10 mg/kg, intraperitoneal [i.p.]) in rats and mitocycin C (2 mg/kg, i.p.) in mice. Twenty-four hours after the second dose of CH, or the dose of cyclophosphamide to rats, or FM or mitocycin C to mice, bone marrow cells were fixed and examined for the presence of polychromatic erythrocytes (PCEs) and the presence or absence of micronucleated PCEs (MNPCEs). Administration of CH to rats and FM to mice produced neither changes in body weights nor signs of systemic toxicity. Similarly, neither CH nor FM caused statistically significant variations in the incidences of either PCEs or MNPCEs. Both positive-control agents caused unequivocal increases in the incidence of MNPCEs and cyclophosphamide significantly reduced the percent of rat erythrocytes appearing as PCEs. The results of these micronucleus assays conducted with either powdered CH or FM in rats and mice, respectively, show that neither form of the tripeptides possesses the potential to induce micronuclei formation in these rodent species.

Eugenol as An In Vivo Radioprotective Agent

In the present work, an attempt has been made to evaluate the possible in vivo radioprotection by eugenol. Swiss albino mice were administered different doses of eugenol (75,150 and 300 mg/kg) before exposure to 1.5 Gy of gamma radiation. The micronucleus test was carried out to determine the genetic damage in bone marrow. Our results demonstrated significant reduction in the frequencies of micronucleated polychromatic erythrocytes (MnPCEs) with all three eugenol doses. Eugenol (150 mg/kg) was also tested against different doses of radiation (0.5, 1, 1.5, and 2 Gy) and was found to afford significant radioprotection. Reduction in the incidence of MnPCEs could be noticed up to 72 h postirradiation (1.5 Gy). Moreover, the level of peroxidative damage and the specific activities of lactate dehydrogenase (LDH) and methylglyoxalase I (Gly I) were observed in the liver of mice treated with eugenol for seven days in comparison to untreated mice. The results revealed that eugenol exerted significant protection against oxidative stress. This possibility was further supported by the enhanced response of Gly I and the lowered activity of LDH. The present findings suggested that eugenol has a radioprotective potential.

Ascorbic acid reduces the frequency of iron induced micronuclei in bone marrow cells of mice

Iron is a potent oxidant that can lead to the formation of genotoxic lipid peroxides. Ascorbic acid, which enhances dietary iron absorption, has been suggested to enhance the oxidant effects of iron and to directly lead to the formation of lipid peroxides. The combined effects of dietary iron and ascorbic acid on genotoxicity were investigated by measuring the frequency of micronuclei in the bone marrow cells of C3H/He mice. In addition, liver iron concentration was measured in all treated groups. Three weeks old mice were fed diets for 3 weeks containing iron at 100 or 300 mg/kg diet in the form of FeSO 4 that were supplemented either with or without ascorbic acid (15 g/kg diet). The results of the bone marrow micronucleus test revealed that the high iron diet resulted in an increased frequency of micronucleated polychromatic erythrocytes (MnPCEs) as compared to low iron. Ascorbic acid supplementation in the low iron diet did not show any effect on incidence of MnPCEs and protected against the increased frequency of MnPCEs induced by the high iron diet. However, liver iron concentration was significantly increased only in the high iron treated and ascorbic acid supplemented group as compared to all other groups. These results demonstrate that ascorbic acid protects against the clastogenic effects of iron.

In vivo micronucleus assays on 2,4-dichlorophenoxyacetic acid and its derivatives

The potential for 2,4-D and seven of its salts and esters to induce cytogenetic abnormalities in mammalian cells in vivo was investigated in the mouse bone marrow micronucleus test. All the test materials were administered to male and female mice by oral gavage and the frequencies of micronucleated polychromatic erythrocytes (MN-PCE) in the bone marrow were determined at intervals of 24, 48 and 72 h following dosing. There were no significant increases in the incidence of MN-PCE in the treated mice at any of the bone marrow sampling times. These results are consistent with the reported lack of in vitro genetic toxicity for these materials in various in vitro genotoxicity assays as well as the absence of carcinogenic potential for 2,4-D in both mice and rats.

The evaluation of a multiple dosing protocol for the mouse bone-marrow micronucleus assay using benzidine and 2,6-xylidine

Male ICR mice were treated with 1, 2 or 3 daily doses of either benzidine or 2,6-xylidine. Groups of 5 animals were sacrificed 24 h after the last dose and the bone marrow examined for micronuclei. Benzidine was given at dose levels of 40 and 200 mg/kg and 2,6-xylidine was given at dose levels of 75 and 375 mg/kg. These doses represent 10 and 50% of the respective median lethal doses. Benzidine produced a significant ( p < 0.001) dose related increase in the incidence of micronucleated polychromatic erythrocytes (MPE), while 2,6-xylidine had no effect on the frequency of micronucleated cells. Statistical analyses of the data indicated that the incidence of MPE was independent of the number of doses administered prior to bone marrow harvest.

Relative distribution of mature erythrocytes, polychromatic erythrocytes (PE) and micronucleated PE on mouse bone-marrow smears: control observations

Maps are presented showing the distribution of normal erythrocytes (NE), polychromatic erythrocytes (PE) and micronucleated PE (MPE) on 3 mouse bone-marrow smears. Adjacent areas of each slide were assessed along their whole length, and it was found that both the incidence of MPE and the ratio of PE: NE varied between different regions of the slide. These two variables were essentially independent of each other. The maps give the impression of clusters of islands of MPE on the slide, but these become less significant when the number of erythrocytes in those areas is taken into account. The present data confirm our previous conclusion that the resolving power of the bone-marrow micronucleus assay is related to the number of PE assessed for MPE per slide. Our data also illustrate that in instances where small departures from control values are observed, clarification/confirmation of the result can be obtained by extending the assessment of slides for MPE. It is proposed that the present recognition of the inherent sensitivity contained within this assay necessitates careful examination of the criteria for a positive response. The selection of PE suitable for assessment is discussed, and our recent 24-h oral corn oil control data are analysed.

Enhancement of the sensitivity of the micronucleus test by calcium antagonist

In vitro and in vivo short term mutagenicity tests are widely used for screening environmental and industrial chemicals for carcinogenic potential. It is necessary to evaluate chemical carcinogens with the in vivo mutagenicity test system because of the difficulty of extrapolating the results of in vitro mutagenicity tests to human beings. But the in vivo mutagenicity test system has a low sensitivity for screening carcinogenic potential compared with the in vitro test.The in vivo micronucleus test developed by Schmid is commonly used because of its simplicity, reproducibility, and economic return. We have tried to modify this test to elevate its sensitivity.Verapamil is a calcium antagonist administered to patients with heart disease. It is known that verapamil has the ability to increase the permeability of the cell membrane. It is important that mutagens reach the DNA strands through the cell membrane in order to induce mutagenic changes of cells.The aim of this study is to investigate the effects of a calcium antagonist on the sensitivity of in vivo and in vitro micronucleus tests using vincristine as a mutagen.In the in vivo micronucleus test, both single and consecutive administration of verapamil showed a dose-response relationship between the dose of verapamil or vincristine and the incidence of micronucleated polychromatic erythrocytes (MPCE). However, there were no significant differences in MPCE incidence between single and consecutive administration of verapamil.On the other hand, in in vitro micronucleus test, the longer the incubation time of vincristine and verapamil with cultured bone marrow cells, the higher the MPCE induction rates. The dose-response relationship between the incidence of MPCE and the concentration of verapamil or vincristine was also observed in the in vitro micronucleus test.This present study suggests that the sensitivity of the micronucleus test can be increased by verapamil, a calcium antagonist.

Inactivity of methylene chloride in the mouse bone marrow micronucleus assay.

Methylene chloride (MC) has been evaluated for its ability to induce micronucleated polychromatic erythrocytes (MPEs) in the bone marrow of treated mice. Groups of five male and five female C57BL/6J/Alpk mice were exposed, by gavage, to doses of 4000, 2500 and 1250 mg/kg MC in corn oil, the highest dose-level being selected to be the maximum tolerated dose. Bone marrow samples were taken 24, 36, 48 and 72 h after dosing. No significant increases in the incidence of MPEs over controls were observed for any of the test groups, and it is concluded that MC is not clastogenic in this assay.