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

Nicotine has been tested in the conventional mouse bone marrow assay. Single doses of 1mg/kg bw or 2mg/kg bw were given by oral intubations and bone marrow was sampled at 24h (1mg/kg) or at 6, 12 and 18 h after treatment (2mg/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 1.1mg/kg bw was called positive for the induction of chromosomal aberrations in mouse bone marrow cells at all sampling intervals, even as early as 6h 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 nicotine is not clastogenic at the doses and time intervals tested in the present experiments.     

Micronucleus assays on 5-fluorouracil and 6-mercaptopurine with mouse peripheral blood reticulocytes.

As part of the 5th collaborative study of the Collaborative Study Group for the Micronucleus Test (CSGMT), the sensitivity and advantages of the micronucleus assay using mouse peripheral blood cells were evaluated using 5-fluorouracil (5-FU) and 6-mercaptopurine (6-MP). The peripheral blood cells were collected from a tail vein of CD-1 male mice just before and 24-120 h after intraperitoneal injection. At 24-h intervals. The maximum incidence of micronucleated reticulocytes (MNRETs) at 50 mg/kg 5-FU was observed 96 h after injection; at 100 mg/kg, the peak was delayed to 120 h, and followed severe bone marrow depression. With 6-MP, maximum MNRETs were observed 48 h after treatment at all doses tested. At dose levels higher than 50 mg/kg, severe bone marrow depression was observed after maximum MNRETs. Though the appearance patterns of MNRETs and the bone marrow depression were different between 5-FU and 6-MP, the positive response of both chemical could be detected with this assay system as well as with the micronucleus test using femoral bone marrow cells.     

The micronucleus test of methyl methanesulfonate with mouse peripheral blood reticulocytes using acridine orange-coated slides.

The usefulness of the micronucleus assay using mouse peripheral blood erythrocytes and acridine orange (AO)-coated slides was evaluated with methyl methanesulfonate (MMS). The micronucleus test was carried out at doses ranging from 20 to 80 mg/kg body weight in CD-1 mice by intraperitoneal injection. Peripheral blood cells were examined from 0 to 72 h after treatment at 12- or 24-h intervals. Bone marrow cells from other mice treated with 80 mg/kg MMS were also sampled at the same times. The frequency of micronucleated reticulocytes (MNRETs) increased dose-dependently at every sampling time except 72 h, and the maximum frequency of MNRETs was observed at about 36 h after treatment. Micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow after a dose of 80 mg/kg were significantly induced at 12 h to 36 h, and the maximum frequency of MNPCEs was observed at 24 h after treatment. The induction of MNRETs was delayed by about 12 h compared to that of MNPCEs in bone marrow, and the maximum frequencies of MNRETs were lower than those of MNPCEs, but the induction of MNRETs by MMS was significant and dose-dependent. It is concluded, therefore, that bone marrow cells could be replaced by peripheral blood cells as material for the micronucleus assay using AO-coated slides.     

Orthovanadate increased the frequency of aneuploid mouse sperm without micronucleus induction in mouse bone marrow erythrocytes at the same dose level

The objective of the current study was to investigate the ability of orthovanadate to induce aneuploidy in mouse sperm and micronuclei in mouse bone marrow cells at the same dose levels. The BrdU-incorporation assay was performed to test if the chemical treatment altered the duration of the meiotic divisions. It was found that orthovanadate (25mg/kg bw) treatment did not cause meiotic delay. To determine the frequencies of hyperhaploid and diploid sperm, male mice were treated by intraperitoneal (i.p.) injection with 5, 15 or 25mg/kg bw orthovanadate and sperm were sampled from the Caudae epididymes 22 days later. Fluorescence in situ hybridization (FISH) was performed with DNA-probes for chromosomes 8, X or Y. Significant increases in the frequencies of total hyperhaploid sperm (p<0.01) were found with 15 and 25mg/kg bw orthovanadate, indicating induced non-disjunction during male meiosis. The dose-response was described best by a linear equation. Orthovanadate did not significantly increase the frequencies of diploid sperm at any of the three doses tested, indicating that no complete meiotic arrest occurred. Orthovanadate was investigated also by the micronucleus test at i.p. doses of 1, 5, 15 or 25mg/kg bw, followed by bone marrow sampling 24h after treatment. None of the orthovanadate doses caused a significant increase in the rates of micronuclei (MN). Since the results show that orthovanadate induced non-disjunction during male meiosis without an accompanying induction of MN in bone marrow erythrocytes under the present experimental conditions and doses, it is concluded that male germ cells (meiosis) are more sensitive to the aneugenic effects of orthovanadate than somatic cells (mitosis). However, induction of micronuclei was reported in the literature with orthovanadate, vanadylsulfate and ammonium metavanadate, which contradicts the notion that vanadium compounds might be unique germ cell aneugens.     

Sex differences in micronucleus induction with hycanthone methanesulfonate in bone marrow cells of mice.

The clastogenic effect of the antischistosomal drug hycanthone methanesulfonate was studied with the micronucleus test in mouse bone marrow cells. Male and female (102/El x C3H/El)F1 mice were treated with single i.p. injections. Bone marrow was sampled 18, 24 and 30 h after treatment with 100 mg/kg. The highest micronucleus yield occurred at 24 h. The dose response for micronucleus induction at 24 h after treatment was non-linear for doses between 5 and 300 mg/kg. The lowest effective dose was 5 mg/kg for females and 10 mg/kg for males. The experiments revealed a significantly higher sensitivity of female mice for the induction of micronuclei in polychromatic erythrocytes by hycanthone methanesulfonate. This result supports the recommendation to use both sexes for quantitative assessment of genotoxicity in the micronucleus test.     

Validation of the mouse peripheral blood micronucleus assay using acridine orange supravital staining with urethane.

The mouse peripheral blood micronucleus assay using acridine orange supravital staining was compared with the standard bone marrow assay using urethane (ethyl carbamate)-treated mice. Urethane was intraperitoneally injected to CD-1 and BDF1 mice at doses ranging from 62 to 1000 and 62 to 250 mg/kg, respectively. Peripheral blood was collected from the tail 0, 24, 48, and 72 h and bone marrow cells were smeared at 24 and 42 h after the treatment. Although the response of micronucleus induction in peripheral reticulocytes was delayed by about 24 h compared to that in bone marrow polychromatic erythrocytes, the maximum frequencies of micronucleated young erythrocytes were comparable. Therefore, the peripheral blood micronucleus assay using the acridine orange supravital staining method may provide a good alternative to the conventional bone marrow assay.     

Validation of the mouse peripheral blood micronucleus assay using acridine orange supravital staining with urethane

The mouse peripheral blood micronucleus assay using acridine orange supravital staining was compared with the standard bone marrow assay using urethane (ethyl carbamate)-treated mice. Urethane was intraperitoneally injected to CD-1 and BDF₁ mice at doses ranging from 62 to 1000 and 62 to 250 mg/kg, respectively. Peripheral blood was collected from the tail 0, 24, 48, and 72 h and bone marrow cells were smeared at 24 and 42 h after the treatment. Although the response of micronucleus induction in peripheral reticulocytes was delayed by about 24 h compared to that in bone marrow polychromatic erythrocytes, the maximum frequencies of micronucleated young erythrocytes were comparable. Therefore, the peripheral blood micronucleus assay using the acridine orange supravital staining method may provide a good alternative to the conventional bone marrow assay.     

Effect of tetrandrine on micronucleus formation and sister-chromatid exchange in both in vitro and in vivo assays

The genotoxicity of tetrandrine, a drug potentially useful for the treatment of silicosis, was studied using the micronucleus and the sister-chromatid exchange (SCE) assay systems. Cultured Chinese hamster lung (V79) cells were used for the in vitro micronucleus and sister-chromatid exchange studies. Mouse bone marrow was used for the in vivo micronucleus assay and mouse spleen cells for the in vivo/in vitro sister-chromatid exchange analysis. The results show that SCE levels in V79 and in spleen cells were significantly elevated by treatment with tetrandrine at doses above 0.08 mg/ml and 100 mg/kg bw, respectively. Increased tetradrine-induced SCE in vitro was metabolic activation dependent. Tetrandrine failed to induce micronuclei at any of the doses tested. A decrease of replicative index with an increase in the concentration of tetrandrine was found both in vitro and in vivo. These results indicate that tetrandrine is a weak indirect-acting genotoxicant.     

Induction of micronuclei by Zearalenone in Vero monkey kidney cells and in bone marrow cells of mice: protective effect of Vitamin E

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin mainly produced by Fusarium graminaerum, found as a world-wide contaminant mainly of corn and wheat. Previous studies have demonstrated that among several other effects on animals and humans, ZEN also displays hepatotoxicity, immunotoxicity and nephrotoxicity. ZEN is mainly known as a hormonal disrupter due to its estrogenic activities and consequent toxicity for reproduction. Furthermore, mutagenic and genotoxic proprieties of ZEN were disclosed recently, the molecular mechanisms of which are not yet well understood. In the present study, the genotoxic potential of ZEN was evaluated using genotoxicity tests: the 'cytokinesis block micronucleus assay' in Vero monkey kidney cells and the 'in vivo mouse bone marrow micronucleus assay'. In cultured cells treated with 5, 10 and 20 microM ZEN, the frequency of binucleated micronucleated cells (BNMN) was assessed in 1000 binucleated cells and in mice given oral doses of 10, 20 and 40 mg/kg bw, the frequency of polychromatic erythrocytes micronucleated (PCEMN) in bone marrow cells was assessed in 2000 polychromatic erythrocytes (PCE). The potential prevention of ZEN-induced effects by 25 microM Vitamin E (Vit E) was also evaluated.In vivo, doses of 10, 20 and 40 mg/kg bw ZEN representing, respectively 2, 4 and 8% of the LD50 (LD50 of ZEN in mice is 500 mg/kg bw), were administered to animals either with or without pre-treatment with Vit E (216.6 mg/kg bw) in order to evaluate its preventive potential.ZEN was found to induce micronuclei (MN) in a dose-dependent manner in cultured Vero cells as well as in mouse bone marrow cells. The present data emphasise the likely clastogenic pathway among the molecular mechanisms that underlay the ZEN-induced genotoxicity. Vit E was found to prevent partially-from 30 to 50%-these toxic effects, most likely acting either as a structural analogue of ZEN or as an antioxidant.     

Chemoprotective effects of carnosine against genotoxicity induced by cyclophosphamide in mice bone marrow cells

The protective effects of carnosine as a natural dipeptide were investigated in mouse bone marrow cells against genotoxicity induced by cyclophosphamide. Mice were injected with solutions of carnosine at three different doses (10, 50 and 100?mg kg(-1) bw) for five consecutive days. On the fifth day of treatment, mice were injected cyclophosphamide and killed after 24?h. The frequency of micronuclei in polychromatic erythrocytes and the ratio of polychromatic erythrocyte/polychromatic erythrocyte?+?normochromatic erythrocyte [PCE/(PCE?+?NCE)] were evaluated by May-Grunwald/Giemsa staining. Histopathology of bone marrow was examined in mice treated with cyclophosphamide and carnosine. Carnosine significantly reduced micronucleated polychromatic erythrocytes (MnPCEs) induced by cyclophosphamide at all three doses. Carnosine at dose of 100?mg kg(-1) bw reduced MnPCEs 3.76-fold and completely normalized the PCE/(PCE?+?NCE) ratio. Administration of carnosine inhibited bone marrow toxicity induced by cyclophosphamide. It appeared that carnosine with protective activity reduced the oxidative stress and genotoxicity induced by cyclophosphamide in bone marrow cells of mice. Copyright ? 2012 John Wiley & Sons, Ltd.     

Genotoxicity of TiO(2) anatase nanoparticles in B6C3F1 male mice evaluated using Pig-a and flow cytometric micronucleus assays

In vivo micronucleus and Pig-a (phosphatidylinositol glycan, class A gene) mutation assays were conducted to evaluate the genotoxicity of 10 nm titanium dioxide anatase nanoparticles (TiO(2)-NPs) in mice. Groups of five 6-7-week-old male B6C3F1 mice were treated intravenously for three consecutive days with 0.5, 5.0, and 50 mg/kg TiO(2)-NPs for the two assays; mouse blood was sampled one day before the treatment and on Day 4, and Weeks 1, 2, 4, and 6 after the beginning of the treatment; Pig-a mutant frequencies were determined at Day -1 and Weeks 1, 2, 4 and 6, while percent micronucleated-reticulocyte (%MN-RET) frequencies were measured on Day 4 only. Additional animals were treated intravenously with three daily doses of 50 mg.kg TiO(2)-NPs for the measurement of titanium levels in bone marrow after 4, 24, and 48 h of the last treatment. The measurement indicated that the accumulation of the nanoparticles reached the peak in the tissue 4 h after the administration and the levels were maintained for a few days. No increase in either Pig-a mutant frequency of the frequency of %MN-RETs was detected, although the %RETs was reduced in the treated animals on Day 4 in a dose-dependent manner indicating cytotoxicity of TiO(2)-NPs in the bone marrow. These results suggest that although TiO(2)-NPs can reach the mouse bone marrow and are capable of inducing cytotoxicity, the nanoparticles are not genotoxic when assessed with in vivo micronucleus and Pig-a gene mutation tests.     

Cytogenetic effects of ribavirin on mouse bone marrow

The micronucleus test and mitotic chromosome analysis were used to study the in vivo mutagenic activity of ribavirin on bone marrow cells of Swiss albino mice. To determine the incidence of micronuclei, mice were injected i.p. twice, at an interval of 24 h. with the drug at doses of 20, 100 and 200 mg/kg. Animals were killed 6 h after the second dose and bone marrow was examined for the presence of micronuclei in developing erythrocytes. Ribavirin significantly (P less than 0.05) induced micronuclei in polychromatic erythrocytes at all doses. A study was conducted to investigate the cytogenetic effect of the drug on mitotic chromosomes. Ribavirin at 200 mg/kg/day was administered to mice for 3 and 5 days. Repeated treatment with the high dose of ribavirin produced a highly significant (P less than 0.02) increase in abnormal metaphase spreads. The results indicate that ribavirin is mutagenic to bone marrow cells of mice as evaluated by the micronucleus test and by chromosome analysis.     

The genotoxic and cytotoxic effects of nicotine in the mouse bone marrow

The objective of the present study was to investigate the potential of nicotine to induce micronucleated polychromatic erythrocytes (MNPCE) in bone marrow of male and female mice. Cyclophosphamide at 40mg/kg was used as positive control clastogen. Single doses of 4, 8 or 16mg/kg nicotine were given via oral intubation and bone marrow was sampled at 18, 24, 30, 36 and 48h after treatment. Cyclophosphamide yielded the expected positive results. Despite the evident signs of acute toxicity shown by the animals, mainly at the 8 and 16mg/kg doses of nicotine, and the reduction in the % PCE, the results show that the MNPCE frequency in male and female mice was not affected by treatment with any of the selected doses of nicotine, in either of the sampling times 18 or 24h. However, at 30 and 36h after treatment, the MNPCE showed significant increases in both genders after doses of 8 and 16mg/kg. A sex-dependent response was recorded, with males having more MNPCE than females after treatment with 8 or 16mg/kg nicotine and sampling at 30h. However, at 36h more MNPCE were induced in females than in males, suggesting different degrees of dose interaction in the sexes under the conditions of the assay. The response was directly correlated with bone-marrow toxicity, as greater bone-marrow suppression was noted in females than in males when 36h samples were examined. By 48h recovery was observed even though the cytotoxicity was high. These findings suggest that nicotine at high doses and after prolonged time intervals is genotoxic and cytotoxic for mouse bone marrow.     

1-Methyl-2-pyrrolidinone (NMP) does not induce structural and numerical chromosomal aberrations in vivo

1-Methyl-2-pyrrolidinone induces aneuploidy in yeast, but only under special treatment conditions. Other genotoxic effects have not been found in vitro, and in vivo no data are available in the literature. Therefore, NMP was investigated in the mouse micronucleus test and the Chinese hamster bone marrow test for structural and numerical chromosomal aberrations. These tests can detect both types of alterations as demonstrated by appropriate positive control substances (cyclophosphamide, vincristine sulfate and benomyl). NMP at single oral doses up to 3800 mg/kg body weight (∼ 80% of the LD₅₀) did not lead to an increase either in micronucleated erythrocytes or in structural or numerical chromosomal aberrations when bone marrow was sampled 16, 24 and 48 h after treatment in the micronucleus test or after 24 and 48 h for karyotype analysis.     

Comparison of single/multiple-dose protocols using triethylenemelamine and procarbazine hydrochloride for the mouse bone marrow micronucleus test

Treatment of mice with a single dose of either 4.8 mg/kg of triethylenemelamine (TEM) or 348 mg/kg of procarbazine hydrochloride (PC) induced higher frequencies of micronucleated polychromatic erythrocytes (MPE) after 48 h than after 24 h. The same observation was made when animals were treated with 1.6 or 8 mg/kg of TEM or 116 or 580 mg/kg of PC for 2 consecutive days (double-dose protocol). Surprisingly, the third dose of either 1.6 or 8 mg/kg of TEM caused lower MPE frequencies at the 72-h than at the 48-h sampling time. The observation that lower MPE frequencies after 72 h were also accompanied by reduced bone marrow toxicity might have reflected a drug-related adaptive reaction of the animals, for example the induction of detoxifying enzymes. Mean MPE frequencies as well as bone marrow toxicity were also slightly decreased after the third dose of either 116 or 580 mg/kg of PC, but statistical analysis showed no differences between the 48-h and the 72-h sampling times as regards the MPE frequencies and bone marrow toxicity. In addition to the high mean MPE frequency observed after 2 doses of 116 mg/kg of PC at the 48-h sampling time, a late increase in micronucleus induction was also seen after triple dosing at the 96-h sampling time. The present experiments with TEM and PC showed similar sensitivity for the multiple-dose assays when compared with the single-dose micronucleus test. In the case of the triple-dose assay, bone marrow toxicity proved to be a critical factor for appropriate dose selection. The computerized image analysis system was a convenient and time-saving tool for the automatic scoring of large quantities of cells for micronuclei as well as for the evaluation of bone marrow depression from the entire cell population analyzed for micronuclei.     

Inhibitory effects of coffee on the genotoxicity of carcinogens in mice

The mouse bone marrow micronucleus test was carried out to evaluate the possible inhibitory effects of 3 doses (125, 250 and 500 mg/kg) of standard instant coffee on the in vivo genotoxicity of 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (BP), aflatoxin B1 (AFB1) and urethane (UR). Coffee was orally administered twice, 2 and 20 h before the carcinogens were injected intraperitoneally. From the results obtained, it was evident that the administration of 250 and 500 mg coffee/kg body weight could significantly inhibit the in vivo genotoxicity of these carcinogens. A linear dose response was observed for the inhibitory effect of coffee. Furthermore, inhibition of genotoxicity by coffee was observed in bone marrow cells which were sampled at 6-h intervals (48, 54, 60, 66 and 72 h) from the time of peak induction of micronuclei by DMBA.     

Activity of the human carcinogens benzidine and 2-naphthylamine in triple- and single-dose mouse bone marrow micronucleus assays: results for a combined test protocol

The activities of the human bladder carcinogens benzidine and 2-naphthylamine in the mouse bone marrow micronucleus assays using a limited test protocol (oral dosing to male mice, sampling 24 h later) have recently been established. As a contribution to the International Collaborative Study on the evaluation of the sensitivity of the triple-dose micronucleus test protocol it was decided to re-evaluate benzidine and 2-naphthylamine using a combined triple- and single-dose test protocol. Benzidine gave a clear positive response in male mice 24 h after 3 daily doses of 150 and 300 mg/kg. A single dose of 900 mg/kg of benzidine gave a weaker response 24 h after dosing. In the case of 2-naphthylamine a stronger positive response was observed 24 h after a single dose of 600 mg/kg as compared to 3 daily doses of 200 or 400 mg/kg. There was no significant difference in the increased positive response observed for a single dose of 30 mg/kg of cyclophosphamide compared with 3 successive daily doses of 10 mg/kg. Based on the present data the combined triple/single-dose micronucleus test protocol is strongly supported.     

Genotoxic effect of Anovlar 21, an oral contraceptive, on mouse bone marrow

Anovlar 21, a combination drug containing the oestrogen ethinyloestradiol and the progestin norethisterone acetate, was studied for its in vivo genotoxic effect on the bone marrow cells of Swiss albino mice. The chromosomal aberration assay and the micronucleus test were employed for the study. 0.08, 0.4, 0.8, 1.6, 3.2, 4.8, 6.4 and 8.0 mg/kg/day of the drug was orally administered for 15 consecutive days to mice. Bone marrow preparations were made 24 h after the final feeding. The lowest dose, 0.08 mg/kg, represents the human therapeutic range. Marrow preparations of mice fed 0.8 mg/kg/day for 15 days were made at 6, 12, 24, 48 and 96 h, and 1, 2 and 3 weeks and a time-yield analysis was carried out. Statistically significant increases in chromosomal aberrations were observed in animal groups fed doses of greater than or equal to 0.4 mg/kg/day. In the time-response study, the maximum frequency of aberrations was noted at 24 h, thereafter decreasing gradually with increasing time. But the drug did not induce a significant increase in the number of micronuclei in bone marrow erythrocytes at any of the doses or time intervals studied.     

Micronucleus test with ethyl methanesulfonate in mouse peripheral blood reticulocytes stained supravitally using acridine orange-coated slides.

A new method for the micronucleus test using peripheral blood reticulocytes stained supravitally using acridine orange-coated slides was evaluated in male CD-1 mice treated with ethyl methanesulfonate (EMS) at doses of 100, 200, 300, and 400 mg/kg. Peripheral blood samples were taken 0, 24, 48, 72, and 96 h after treatment from each mouse without killing. The frequencies of micronucleated reticulocytes increased dose-dependently with the peak at 48 h after treatment. These results indicate that, at least for EMS, the new method used here can be an alternative to the conventional method using bone marrow polychromatic erythrocytes.     

Micronucleus test with ethyl methanesulfonate in mouse peripheral blood reticulocytes stained supravitally using acridine orange-coated slides

A new method for the micronucleus test using peripheral blood reticulocytes stained supravitally using acridine orange-coated slides was evaluated in male CD-1 mice treated with ethyl methanesulfonate (EMS) at doses of 100, 200, 300, and 400 mg/kg. Peripheral blood samples were taken 0, 24, 48, 72, and 96 h after treatment from each mouse without killing. The frequencies of micronucleated reticulocytes increased dose-dependently with the peak at 48 h after treatment. These results indicate that, at least for EMS, the new method used here can be an alternative to the conventional method using bone marrow polychromatic erythrocytes.