Frequency Of Micronuclei In Polychromatic Erythrocytes Research Articles

The rat bone marrow micronucleus test: Statistical considerations on historical negative control data

Recent updates of the OECD Guidelines for the Testing of Chemicals (Section 4: Health Effects) on genotoxicity testing emphasize the use of appropriate statistical methods for data analysis and proficiency proof. Updates also concern the mammalian erythrocyte micronucleus test (OECD 474), as the currently most often performed regulatory in vivo test. As the updated guideline gives high importance to adequate statistical assessment of historical negative control data to estimate validity of experiments and judge results, the present study evaluated statistical methodologies for handling of historical negative control data sets, and comes forward with respective proposals and reference data. Therefore, the working group “Statistics” within the German-speaking “Gesellschaft für Umwelt-Mutationsforschung e.V.” (GUM) compiled a data set of 891 negative control rats from valid OECD 474-studies of four laboratories. Based on these data, Analysis-of-Variance (ANOVA) identified “laboratory” and “strain”, but not “gender” as relevant stratification parameters, and argued for approximately normally distributed micronucleus frequencies in polychromatic erythrocytes per animal. This assumption provided the basis for further specifying one-sided parametric tolerance intervals for determination of corresponding upper historical negative control limits. Finally, the stability of such limits was investigated as a function of the number of experiments performed, using a simulation-based statistical strategy.

Evaluation of reproductive/developmental and repeated dose (subchronic) toxicity and cytogenetic effects in rats of a roofing asphalt fume condensate by nose-only inhalation

A Type III Built-up Roofing Asphalt (BURA) fume condensate was evaluated for subchronic systemic toxicity and reproductive/developmental toxicity screening in Wistar rats, by OECD protocol 422 and OECD cytogenetic protocol 474. Animals were exposed by nose-only inhalation to target concentrations of 30, 100 and 300mg/m3 total hydrocarbons (actual concentrations, 30.0, 100.1 and 297.3mg/m3). The study was performed to assess potential hazards from asphalt fumes to which humans could be exposed during application. No adverse effects were seen for spermology, reproductive or developmental parameters or early postnatal development of offspring from day 1 to 4 postpartum. BURA fume condensate did not induce any significant increases in micronucleus frequency in polychromatic erythrocytes of rat bone marrow nor was neurobehavioral toxicity observed at any dose. Systemic effects were slight and seen at doses above those measured at work sites. The systemic NOAEC of 100mg/m3 for males was based on decreased body weight gain, food consumption and increased absolute and relative lung wet weight correlated with slight histological changes in the lung, primarily adaptive in nature at 300mg/m3. The female NOAEC of 30mg/m3 was based on a statistically significant increase in relative wet lung weight at higher doses, correlated with slight histopathologic effects in the lungs at the highest dose. However, no increase in relative lung weight was seen in breeding females at 100mg/m3.

Evaluation of aneugenic effects of bisphenol A in somatic and germ cells of the mouse

Bisphenol A (BPA) is a synthetic monomer widely used to polymerize polycarbonate plastics and resins. It is shown in vitro to interfere with microtubules, producing aberations in mitotic and meiotic spindles. An increase of meiotic abnormalities in untreated female mice from an experimental colony was temporally correlated with the accidental release of BPA from polycarbonate cages and bottles damaged by inadvertent treatment with harsh alkaline detergents [P.A. Hunt, K.E. Koehler, M. Susiarjo, C.A. Hodges, A. Ilagan, R.C. Voigt, S. Thomas, B.F. Thomas, T.J. Hassold, Bisphenol A exposure causes meiotic aneuploidy in the female mouse, Curr. Biol. 13 (2003) 546–553]. In the present study, potential aneugenic effects of BPA on mouse male and female germ cells and bone marrow cells have been evaluated after acute, sub-chronic or chronic in vivo exposure. Female mice were orally treated with a single BPA dose, with 7 daily administrations or exposed for 7 weeks to BPA in drinking water. No significant induction of hyperploidy or polyploidy was observed in oocytes and zygotes at any treatment condition. The only detectable effect was a significant increase of metaphase II oocytes with prematurely separated chromatids after chronic exposure; this effect, however, had no irreversible consequence upon the fidelity of chromosome segregation during the second meiotic division, as demonstrated by the normal chromosome constitution of zygotes under the same exposure condition. With male mice, no delay of meiotic divisions was found after six daily oral doses of BPA with the BrdU assay. Similarly, no induction of hyperploidy and polyploidy was shown in epydidimal sperm hybrized with probes for chromosomes 8, X and Y, 22 days after six daily oral BPA doses. Finally, two daily oral BPA doses did not induce any increase of micronucleus frequencies in polychromatic erythrocytes of mouse bone marrow. In conclusion, our results do not add evidence to the suspected aneugenic activity of BPA and suggest that other factors or co-factors should be considered to explain the unexpected burst of meiotic abnormalities previously attributed to accidental BPA exposure.

The influence of vinblastine treatment on the formation of radiation-induced micronuclei in mouse bone marrow.

The combined effects of vinblastine sulphate (VBL) and gamma radiation treatments on the induction of micronuclei in polychromatic erythrocytes (MPCE) and normochromatic erythrocytes (MNCE) and changes in polychromatic/normochromatic erythrocyte ratio (P/N ratio) in mouse bone marrow, were studied. Compared to double distilled water (DDW) injected controls, 0.05 mg/kg b.wt. of vinblastine alone significantly increased the frequency of MPCE and MNCE. When VBL was administered before exposure to various doses of gamma radiation the frequency of micronuclei (MPCE and MNCE) increased with the increasing radiation dose up to a dose of 4 Gy, but this increase levelled off at 3 to 4 Gy irradiation. The frequency of MPCE was found to be significantly lower at 3 and 4 Gy radiation when compared to the DDW+irradiated group. A significant dose dependent decline in the P/N ratio was observed in the DDW+irradiated group. VBL treatment before irradiation resulted in a more significant decline in the P/N ratio than that of DDW+irradiated group. The formation of micronuclei (MPCE and MNCE) increased from 8 h post-exposure and continued to increase up to 28 h. There was a sharp decline in the frequency of micronuclei (MPCE and MNCE) at 32 h post-irradiation, in both DDW + 1 Gy irradiated and VBL + 1 Gy irradiated groups; then it declined gradually up to 72 h post-exposure without restoration to normal level. The P/N ratio declined with time in all three groups (VBL alone, DDW+irradiation, and VBL+irradiation) without restoration to normal level up to 72 h post-treatment, except in the DDW+irradiated group, where it was normal.

Nicotine is not clastogenic at doses of 1 or 2 mg/kg body weight given orally to male mice

Nicotne has been tested in the conventional mouse bone marrow assay. Single doses of 1 mg/kg bw or 2 mg/kg bw were given by oral intubations and bone marrow was sampled at 24 h (1 mg/kg) or at 6, 12 and 18 h after treatment (2 mg/kg). Nicotine treatment did not increase the micronucleus frequencies in polychromatic erythrocytes while the positive control compound mitomycin C yielded the expected result. These data contradict the only published in vivo study of nicotine in which 1.1 mg/kg bw was called positive for the induction of chromosomal aberrations in mouse bone marrow cells at all sampling intervals, even as early as 6 h after treatment. It is discussed that aberration scoring is a matter of subjectivity and depends on strict discrimination criteria between gaps and true DNA discontinuities, i.e. breaks. International collaboration has shown that micronucleus scoring is less subjective, hence more reliable. Therefore it is concluded that nocotine is not clastogenic at the doses and time intervals tested in the present experiments.

Induction of micronuclei by alkaloids extracted from Senecio brasiliensis and stored for 23 years

In the present study, we report the results of an investigation on pyrrolizidine alkaloids extracted from Senecio brasiliensis (Sprengel) Less., which were stored for more than 23 years under variable conditions of temperature and humidity and exposed to light. Both the crude alkaloid (integerrimine+retrorsine+impurities) and pure integerrimine conserved the ability to induce acute toxicity in mice, leading to the death of the animals in less than 24 h. The alkaloids also conserved the potential to induce significant increases in micronucleus frequencies in polychromatic erythrocytes of mouse bone marrow compared to the negative control. The administration of alkaloids to lymphocyte cultures blocked with cytochalasin-B showed no significant increase in micronucleus frequency in binucleated cells, probably due to the lack of a metabolic activation mechanism. However, an antimitotic effect was observed.

Bone marrow micronucleus assay in Brown-Norway rats exposed to diphenyl-methane-4,4'-diisocyanate.

Four groups of young adult male Brown-Norway rats (strain: BN/RijHsd) were either exposed whole-body (WB) to filtered air (negative control) or to respirable aerosols of monomeric diphenylmethane-4,4'-diisocyanate (MDI) at actual breathing zone concentrations of 9.2 +/- 1.5 and 118 +/- 8.6 mg/m3. One additional group was exposed to 11,0 +/- 14.4 mg/m3 MDI using a nose-only (NO) mode. Exposure was 1 h/day, one exposure per week on 3 consecutive weeks. MDI aerosols were generated using either a condensation (WB) or a dispersion-condensation (NO) principle with resultant MMADs of 2.4-3.1 microm and 1.2 microm (GSD approximately l.5), respectively. Humidity ranged from approximately 40% (WB) to approximately 5% (NO). Positive controls received cyclophosphamide and colcemid. Micronuclei in polychromatic erythrocytes (MN-PCE) were counted in bone marrow smears prepared after the final exposure on post-exposure days 1, 2 and 7 and stained with acridine orange or Wright-Giemsa. Both the WB-exposure regimen and the 7-day sampling time point were based upon a previous study in which a significant increase in MN-PCE was reported to occur. Rats exposed to 118 (WB) and 110 mg/m3 MDI (NO) exhibited signs of respiratory distress, including hypothermia, and increased lung weights when compared to WB-exposed rats. The intensity of changes appeared to be slightly more pronounced in NO-exposed rats. At no time point did this study provide any evidence of an MDI-induced effect on the frequency of MN-PCE. No differences in outcome existed following staining with acridine orange or Wright-Giemsa. There was an absence of any effect on the frequency of mast cells and their frequency was low enough not to interfere with the outcome of study. Positive control groups exhibited significant increases in MN-PCE. In summary, monomeric MDI aerosol did not induce cytogenetic damage in Brown-Norway rats when investigated according to current testing guidelines.

Inhibition of cytochrome P450 2E1 decreases, but does not eliminate, genotoxicity mediated by 1,3-butadiene.

1,3-Butadiene (BD), a rodent carcinogen, is metabolized to mutagenic and DNA-reactive epoxides. In vitro data suggest that this oxidation is mediated by cytochrome P450 2E1 (CYP2E1). In this study, we tested the hypothesis that oxidation of BD by CYP2E1 is required for genotoxicity to occur. Inhalation exposures were conducted with B6C3F1 mice using a closed-chamber technique, and the maximum rate of butadiene oxidation was estimated. The total amount of butadiene metabolized was then correlated with the frequency of micronuclei (MN). Three treatment groups were used: (1) mice with no pretreatment; (2) mice pretreated with 1,2-trans-dichloroethylene (DCE), a specific CYP2E1 inhibitor; and (3) mice pretreated with 1-aminobenzotriazole (ABT), an irreversible inhibitor of cytochromes P450. Mice in all 3 groups were exposed to an initial BD concentration of 1100 ppm, and the decline in concentration of BD in the inhalation chamber with time, due to uptake and metabolism of BD, was monitored using gas chromatography. A physiologically based pharmacokinetic model was used to analyze the gas uptake data, estimate V(max) for BD oxidation, and compute the total amount of BD metabolized. Model simulations of the gas uptake data predicted the maximum rate of BD oxidation would be reduced by 60% and 100% for the DCE- and ABT-pretreated groups, respectively. Bone marrow was harvested 24 h after the onset of the inhalation exposure and analyzed for frequency of micronuclei in polychromatic erythrocytes (MN-PCE). The frequency of MN-PCE per 1000 PCE in mice exposed to BD was 28.2 +/- 3.1, 19.8 +/- 2.5, and 12.3 +/- 1.9, for the mice with no pretreatment, DCE-pretreated mice and ABT-pretreated mice, respectively. Although inhibition of CYP2E1 decreased BD-mediated genotoxicity, it did not completely eliminate genotoxic effects. These data suggest that other P450 isoforms may contribute significantly to the metabolic activation of BD and resultant genotoxicity.

In vivo protection by cimetidine against fast neutron-induced micronuclei in mouse bone marrow cells

We have previously shown that cimetidine is capable of reducing the clastogenic effect of γ-rays. In this research the radioprotective property of this drug was examined against low doses of fast neutrons using the miconucleus assay. Swiss albino male mice (12 weeks old) were irradiated by fast neutrons emitted from a 241Am- 9Be source. The absorbed doses were 1.5, 2.25, 3.375 and 5.06 cGy at a dose rate of 0.718 cGy/h. Two hours prior to neutron irradiation mice were treated by cimetidine at a concentration of 15 mg/kg body weight injected i.p. Mice were sacrificed by cervical dislocation at different post-irradiation times (24, 48 and 72 h). The results obtained show that the frequency of neutron-induced micronuclei in polychromatic erythrocytes (PCEs) is significantly higher than those of control groups ( P<0.05) at the neutron doses used in these experiments. Moreover, cimetidine effectively reduced (1.5–2-fold) the frequency of micronuclei in PCE ( P<0.05). These results show that cimetidine can protect bone marrow cells against clastogenic effects of low dose fast neutrons and hence high linear energy transfer (LET) radiation. The mechanism by which cimetidine reduces the clastogenic effects of fast neutrons is not fully understood. It might act through a free radical scavenging mechanism associated with the amplification of the glutathione system.

Studies on the genotoxicity of molybdenum salts in human cells in vitro and in mice in vivo.

Molybdenum is an essential element in plants and animals as a cofactor for enzymes. Molybdenum trioxide is used in metallurgical processes, in cosmetics as a pigment, and in contact lens solution, yet limited information is available on molybdenum genotoxicity. In the present study the micronucleus (MN) assay in human lymphocytes and mouse bone marrow and the dominant lethal assay in mice were used to assess the genotoxic effects of molybdenum salts in vitro and in vivo. Two salts of molybdenum were tested in whole blood cultures. Ammonium molybdate was more potent than sodium molybdate in causing a dose-dependent decrease in viability and replicative index and an increase in MN formation in binucleated lymphocytes (P < 0.001). A dose-response in both kinetochore-positive MN (caused by chromosome lagging) and kinetochore-negative MN (associated with chromosome breakage) was observed. Based on the results of a toxicity study of sodium molybdate, two doses, 200 and 400 mg/kg, were assessed in the bone marrow MN assay in mice (two i.p. injections 24 and 48 hr prior to euthanasia). A modest but statistically significant increase in MN frequency in polychromatic erythrocytes was observed (P < 0.05). The same treatment protocol was used to analyze dominant lethality. A dose-dependent increase in postimplantation loss represented mostly by early resorptions was observed the first week after treatment (P = 0.003). These preliminary data suggest that sodium molybdate induces dominant lethality at the postmeiotic stage of spermatogenesis. Overall, molybdenum salts produced moderately positive results both in vitro in human cells and in vivo in mice.

Effect of repeated benzene inhalation exposures on benzene metabolism, binding to hemoglobin, and induction of micronuclei

Metabolism of benzene is thought to be necessary to produce the toxic effects, including carcinogenicity, associated with benzene exposure. To extrapolate from the results of rodent studies to potential health risks in man, one must know how benzene metabolism is affected by species, dose, dose rate, and repeated versus single exposures. The purpose of our studies was to determine the effect of repeated inhalation exposures on the metabolism of [ 14C]benzene by rodents. Benzene metabolism was assessed by characterizing and quantitating urinary metabolites, and by quantitating 14C bound to hemoglobin and micronuclei induction. F344 N rats and B6C3F 1 mice were exposed, nose-only, to 600 ppm benzene or to air (control) for 6 hr/day, 5 days/week for 3 weeks. On the last day, both benzene-pretreated and control animals were exposed to 600 ppm, 14C-labeled benzene for 6 hr. Individual benzene metabolites in urine collected for 24 hr after the exposure were analyzed. There was a significant decrease in the respiratory rate of mice (but not rats) pretreated with benzene which resulted in lower levels of urinary [ 14C]benzene metabolites. The analyses indicated that the only effects of benzene pretreatment on the metabolite profile in rat or mouse urine were a slight shift from glucuronidation to sulfation in mice and a shift from sulfation to glucuronidation in rats. Benzene pretreatment also had no effect, in either species, on formation of [ 14C]benzene-derived hemoglobin adducts. Mice and rats had similar levels of hemoglobin adduct binding, despite the higher metabolism of benzene by mice. This indicates that hemoglobin adduct formation occurs with higher efficiency in rats. After 1 week of exposure to 600 ppm benzene, the frequency of micronucleated, polychromatic erythrocytes (PCEs) in mice was significantly increased. Exposure to the same level of benzene for an additional 2 weeks did not further increase the frequency of micronuclei in PCEs. These results indicate that repeated exposures to benzene, such as might be encountered by humans as a result of occupational or environmental exposures, are not likely to change or increase benzene metabolism.

Enhancement of benzene clastogenicity by praziquantel in mice

Praziquantel (PQ) is a commonly used drug to treat patients with schistosomiasis. Previous studies using cells in vitro have shown that PQ can enhance the mutagenic activities of known mutagens. We have conducted a cytogenetic - urine metabolite study to determine the in vivo clastogenic and co-clastogenic potential of PQ with a ubiquitous environmental contaminant, benzene (BZ). 16 groups of adult male ICR mice (5 animals per group) were used. They were negative control, solvent controls (cremophore E1 3%, olive oil and combined), positive control (BZ 440 mg/kg b.w.) and 11 exposed groups. To test for clastogenicity of PQ, mice were treated orally with 100, 400, 800 and 1200 mg/kg b.w. PQ and sacrificed 30 h later for determination of micronuclei (MN) frequency in bone-marrow polychromatic erythrocytes (PCE). None of these PQ doses induced an increase of MN frequency. On the other hand, BZ induced, as expected, a high frequency of MN (46.4 ± 6.34/1000 PCE). The enhancement effect of PQ was tested in 7 groups of mice using 3 different protocols. Mice were treated with 440 mg/kg b.w. BZ and 1 h later with 0, 100, 200, 400, 800 and 1200 mg/kg b.w. PZ. In another group, 800 mg/kg PQ was administered at 3 h after BZ exposure. In the last group, PQ (800 mg/kg) was administered at 1 h prior to BZ exposure. Results from the first combined exposure group showed a significant PQ dose-dependent increase in the frequency of MN in PCE ( p < 0.05). The increase with the two high doses of praziquantel is significantly higher ( p < 0.05) than the MN frequencies in the benzene control and the expected value based on the additive effects of the two agents. Studies with other combined treatment groups showed that the induction of MN was highest when PQ was administered at 1 h before BZ exposure. Moreover, the presence of BZ metabolites (muconic acid, phenol, catechol and hydroquinone) in urine was studied in 6 of the combined treatment groups. This metabolite study revealed that PQ enhanced the metabolism of BZ towards the pathway to form muconaldehyde which is converted to muconic acid in urine. In conclusion, our study showed that PQ is not a clastogen but can enhance the clastogenic activity of BZ in vivo by shifting the metabolic pathways of BZ towards formation of muconaldehyde which may be responsible for the enhancement effect.

Cytogenetic effects of pesticides: III. Induction of micronuclei in mouse bone marrow by the insecticides cypermethrin and rotenone

The production of micronuclei in mouse bone marrow by the pyrethroid insecticide, cypermethrin and the botanical insecticide, rotenone was examined. Three routes of administration were used for the insecticides: intraperitoneal, oral and dermal. The different routes of treatment with cypermethrin and rotenone caused toxicity of marrow as indicated by a significant increase in the percentage of polychromatic erythrocytes (PEs) over that of the control. Cypermethrin showed mutagenic potential as evidenced by a positive response in the micronucleus assay. Oral administration of the insecticide at a dietary level of 900 ppm for 7 and 14 consecutive days as well as double and multiple (total 4) dermal treatments (360 mg/kg body wt.) induced a statistically significant increase in the frequency of PEs with micronuclei. The conducted intraperitoneal (i.p.) treatments with cypermethrin: single injection at 60 and 180 mg/kg body wt., double and multiple injections (total 3) at 60 mg/kg body wt. did not affect the percentage of PEs with micronuclei. The different treatments with rotenone: single, double and multiple (i.p.) injections (total 3) at 2 and 3 mg/kg body wt., oral administration for 14 consecutive days at dietary level of 225 ppm and multiple dermal treatments (total 4) with 135 mg/kg body wt. showed no effect on the frequency of micronuclei in PEs.