Mouse Bone Marrow Micronucleus Test Research Articles

Protective effects of chlorogenic acid, curcumin and β-carotene against γ-radiation-induced in vivo chromosomal damage

The mouse bone marrow micronucleus test was carried out to evaluate the possible role of the dietary constituents chlorogenic acid (CGA), curcumin (CR) and β-carotene (BC) in modulating the in vivo chromosomal damage induced by γ-radiation. The results obtained suggest that oral administration of CGA (50, 100 and 200 mg/kg b.w.), CR (5, 10 and 20 mg/kg b.w.) and BC (0.5 and 2.5 mg/kg b.w.) to mice can significantly reduce the frequencies of micronucleated polychromatic erythrocytes (Mn PCEs) induced by whole body exposure to γ-radiation (1.15) Gy; 0.05 Gy/s). With CGA and CR, this effect was observed after a single administration either 2 h before or immediately after irradiation. However, with BC a 7-day feeding before irradiation was necessary to obtain a significant reduction in the incidence of Mn PCEs. The protective effects of CGA, CR and BC were observed in bone marrow cells sampled 24, 30 and 48 h after exposure to radiation.

Testing of known carcinogens and noncarcinogens in the Syrian hamster embryo (SHE) micronucleus test in vitro; correlations with in vivo micronucleus formation and cell transformation

Seventy-five chemicals, carcinogens and noncarcinogens, were tested in the SHE (Syrian hamster embryo) micronucleus test in vitro. Substances inducing a reproducible and dose dependent increase in micronucleus frequency were regarded as positive. The acquired data were analyzed for correlations with results obtained from the in vivo mouse bone marrow micronucleus test and from morphological transformation of SHE cells. Out of 48 carcinogens tested 41 (85%) yielded a positive result and out of 17 noncarcinogens all proved negative. For 7 chemicals no carcinogenicity data were available so far; these compounds yielded no response in the mouse bone marrow and in the SHE micronucleus assay. For 3 chemicals only inadequate carcinogenicity data were available. A high degree of concordance with data from the in vivo micronucleus test was found (89%) and the accordance with results from morphological SHE cell transformation was even higher (95%). These findings provide new evidence that the in vitro SHE micronucleus test does in fact represent a short-term test of high predictive value.

Genotoxicity testing of the herbicide Roundup and its active ingredient glyphosate isopropylamine using the mouse bone marrow micronucleus test, Salmonella mutagenicity test, and Allium anaphase-telophase test

The genotoxic potential of the herbicide Roundup and its active agent, glyphosate isopropylamine salt, was studied in three different assays. No clastogenic effects were found in the mouse bone marrow micronuleus test for either of the two agents. In the Salmonella assay only Roundup was tested. It showed a weak mutagenic effect for the concentrations 360 μg/plate in TA98 (without S9) and 720 μg/plate in TA100 (with S9). These concentrations are close to the toxic level. The anaphase-telophase Allium test showed no effect for the glyphosate isopropylamine salt, but a significant increase in chromosome aberrations appeared after treatment with Roundup at concentrations of 1.44 and 2.88 mg/1 when calculated as glyphosate isopropylamine. The most frequent aberrations observed could be characterized as disturbances of the spindle.

Toxicology of Diethylene Glycol Butyl Ether 3. Genotoxicity Evaluation in an In Vitro Gene Mutation Assay and an In Vivo Cytogenetic Test

DGBE was evaluated in a forward gene mutation assay at the HGPRT locus of CHO cells in culture and in an in vivo mouse bone marrow micronucleus test for cytogenetic damage. DGBE did not elicit a positive response in the CHO/HGPRT assay when tested up to a maximum concentration of 5000 μg/ml with and without an external metabolic activation system (S-9). In the micronucleus test employing three post-treatment bone marrow sampling times (24, 48, and 72 hr), DGBE was ineffective in increasing the incidence of micronucleated polychromatic erythrocytes (MN-PCE) when tested in both sexes up to a maximum tolerated dose of 3300 mg/kg body weight. Thus, these data and those of others indicate a general lack of genotoxic potential for DGBE in short-term tests.

Genotoxic activity of 1-chloromethylpyrene in stomach epithelium in vivo: insensitivity of the stomach scintillation UDS assay.

An acknowledged weakness of current testing programmes for genotoxic hazard has been the potential insensitivity of the established mouse bone marrow micronucleus test and rat liver unscheduled DNA synthesis (UDS) assays to direct-acting or short-lived mutagens, which may be consumed at the site of initial contact. In such cases, in vivo test systems sampling tissues such as the skin or the stomach would provide valuable data. To test these principles a stomach UDS assay was evaluated using the potent locally active mutagen 1-chloromethylpyrene (1-CMP). Contrary to expectations, no UDS response was observed 16 h following 1-CMP dosage by oral gavage. To confirm the integrity of the 1-CMP used for the stomach UDS assay, a sample of the stored chemical was re-evaluated in vitro and shown to be still strongly positive in the Ames assay and to have alkylating activity at least 15 min after incubation at stomach acid pH. No UDS response was observed when test dose levels were reduced or when earlier sampling times were used. Other genotoxic endpoints were examined in stomach. 32P-Postlabelling analysis revealed high levels of adduct formation in gastric DNA. An assay utilizing electrophoresis of DNA (the comet assay) showed the occurrence of DNA damage following dosing with 1-CMP in vivo. These positive results confirmed that 1-CMP should be regarded as a potential in vivo genotoxin. The failure to detect a UDS response to 1-CMP in stomach was investigated; a strong UDS response was observed in an in vitro hepatocyte UDS assay of 1-CMP indicating that the rat was capable of repairing 1-CMP-derived DNA adducts. Pretreatment of rats with hydroxyurea depressed the level of incorporation of thymidine into DNA both in negative and positive [methyl-N-nitrosoguanidine (MNNG)] controls. The results of these studies indicated that the protease digestion method employed did not selectively or efficiently sample those cells with any UDS response to 1-CMP or MNNG, and the activity seen for the latter was most likely due to the presence of S phase cells within the digests. As a result of the finding that UDS responses were not demonstrated for the potent direct-acting mutagens 1-CMP and MNNG, the protease digestion/scintillation method for stomach UDS does not appear to have general value in a screening programme for locally active genotoxic agents.

Evaluation of a three‐exposure mouse bone marrow micronucleus protocol: Results with 49 chemicals

Forty-nine chemicals were tested in a mouse bone marrow micronucleus test that employed three daily exposures by intraperitoneal injection. Bone marrow samples were obtained 24 hr following the final exposure. Twenty-five rodent carcinogens and 24 noncarcinogens were selected randomly from the 44 carcinogens and 29 noncarcinogens used by Tennant et al. (Science 236:933-941, 1987) to evaluate the performance of four in vitro genetic toxicity tests. As in that study of in vitro tests, the micronucleus tests were conducted with coded chemicals and test results (positive or negative) were determined prior to decoding. This study was conducted as part of an effort to assess the ability of the micronucleus test to discriminate between rodent carcinogens and noncarcinogens and to determine its potential role, in combination with other short-term tests, in identifying genotoxic chemicals that present a carcinogenic hazard. Nine chemicals were judged to be positive in the micronucleus test. This relatively low number of positive results, along with published and unpublished results from rodent micronucleus and chromosome aberration assays on several of these 49 chemicals, contributed to the conclusion that a single micronucleus test protocol is not adequate to detect all chemicals capable of inducing chromosomal damage in the bone marrow. However, a combination of two relatively simple assays such as the Salmonella and micronucleus tests can provide important information on the genetic toxicity of test chemicals and may provide guidance on the need for and the nature and extent of future toxicity studies.

Evaluation of correlation between human carcinogenicity and the mouse bone marrow micronucleus test with acrylonitrile (ACN)

Evaluation of correlation between human carcinogenicity and the mouse bone marrow micronucleus test with acrylonitrile (ACN)

Detection of micronuclei after exposure to mitomycin C, cyclophosphamide and diethylnitrosamine by the in vivo micronucleus test in mouse splenocytes

A micronucleus detection test using mouse splenocytes has been adapted from a method previously carried out using human lymphocytes. An ex vivo protocol was chosen: male C57B16 mice were treated with various compounds. Splenocytes were then isolated and placed in culture for 48 h and stimulated with concanavalin A and conditioned medium. The cytokinesis-block method reported by Fenech and Morley was used to detect and score micronuclei in the proliferating lymphocytes (3 μg/ml of cytochalasin B for 16 h). Three mutagenic clastogens, mitomycin C (MMC), a direct alkylating agent (0.4, 0.8 and 1.6 mg/kg), cyclophosphamide (CP), an indirect alkylating agent (25, 50 and 100 mg/kg) and diethylnitrosamine (DEN), an indirect alkylating agent with labile metabolites (25, 50 and 100 mg/kg), were tested at four sampling times (2, 4, 8 and 15 days). All three compounds were detected from 48 h after treatment. This method was indeed able to detect clastogenic compounds normally detected by the mouse bone marrow micronucleus test (MMC, CP) as well as a compound with labile metabolites which is not usually detected by this test (DEN). Maximum micronucleus induction was observed after 4 days for MMC, 2 days for CP and 15 days for DEN. This method thus appears to offer a potentially useful toxicological test for assessing in vivo clastogenicity.

Sampling times in micronucleus testing

A series of micronucleus inducers were evaluated in the mouse bone marrow micronucleus test to determine if a 72-h sampling time enhances the sensitivity for detecting genotoxic agents. Male and female Swiss albino mice were dosed once with 7,12-dimethylbenz[ a]anthrace, 6-mercaptopurine, benzo[ a]pyrene, benzene, cyclophosphamide, 2-acetylaminofluorene, tubulazole, or mitomycin C. According to the EEC and OECD guidelines, the mice were killed at 24, 48 and 72 h after dosing. All test compounds induced an increase in the number of micronucleated polychromatic erythrocytes at 24 and/or 48 h. From the results obtained, it was evident that the 72-h sampling time does not enhance the sensitivity of the micronucleus test. The present data show that for screening purposes two sampling times at 24 and 48 h are sufficient to detect clastogens as well as aneugens. Although quantitative differences were found in sensitivity to micronucleus inducers between male and female mice, no qualitative differences were observed between the two sexes.

Prior bleeding enhances the sensitivity of the in vivo micronucleus test

It has been reported that the sensitivity of the in vivo mouse bone marrow micronucleus test can be increased by inducing erythropoiesis with exogenous erythropoietin prior to treatment (Suzuki et al., 1989). In these studies we demonstrate that removing approximately 0.5 ml of blood from an adult male BDF1 mouse, another method for increasing the rate of erythropoiesis, synergistically increased the frequency of bone marrow micronucleated polychromatic erythrocytes induced by mitomycin C, with maximal enhancement occurring when the mutagen was given 24 h after bleeding. This enhancement response was also demonstrated for benzo[a] pyrene and dimethylnitrosamine but not for 2-acetylaminofluorene. These results indicate that bleeding mice prior to chemical treatment is a simple method for increasing the sensitivity of the micronucleus assay.

Influence of vegetable oil vehicles on bone-marrow proliferation in the mouse micronucleus test

The detection of genotoxins in the mouse bone-marrow micronucleus (MN) test is sensitive to factors which may inhibit bone-marrow proliferation. We have shown that three commercially available cooking oils (olive, peanut and sunflower seed oils), commonly used as vehicles in toxicological tests, were able to induce a cytotoxic effect in mouse bone marrow. The effects observed were reversible and the magnitude of the responses varied with the oil administered. The results suggest a need to examine vehicle effects when conducting the MN test.

Methyl carbamate: Negative results in mouse bone-marrow micronucleus test

Methyl carbamate: Negative results in mouse bone-marrow micronucleus test

Simulation study of the effects of multiple treatments in the mouse bone marrow micronucleus test

The effect of multiple treatment with chemicals in the micronucleus test was evaluated by simulation involving an estimation of the additive accumulation of micronucleated polychromatic erythrocytes (MNPCEs) on the basis of time-response data available on single treatments with mitomycin C, 1-β- D-arabinofuranosylcytosine, 6-mercaptopurine, and methotrexate. The frequency of MNPCEs calculated for different multiple treatment regimens by the model could predict the effects observed in real experiments. On the other hand, the effect of multiple treatments on bone marrow depression, expressed as a decrease in the frequency of polychromatic erythrocytes, was exponential according to both the simulation and actual data. These results suggest that although increasing the number of treatments may additively enhance the MNPCE response obtained with some agents it may, in the case of bone marrow-toxic chemicals and doses, make micronucleus analysis more time-comsuming and even impossible due to the exponential decrease of analyzable cells, especially in the case of manual scoring.

Genotoxic evaluation of letimide with the mouse bone marrow micronucleus test

Letimide, 3[2-(diethylamino)ethyl]-2 H-1,3-benzoxazine-2,4(3 H)-dione, a new analgesic, is a cyclic derivative of a salicylamide with a higher pharmacological potency than acetylsalicylic acid. In this study we evaluated its clastogenic activity using the micronucleus test in vivo. Our results did not show any clastogenic effect produced by letimide as compared with control mice and animals treated with cyclophosphamide.

Multiple dosing effects of benzo[ a]pyrene in the mouse bone marrow micronucleus test

Multiple dosing effects of benzo[ a]pyrene in the mouse bone marrow micronucleus test

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 B 1 (AFB 1) 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.

Effects of aniline hydrochloride in the mouse bone marrow micronucleus test after oral administration

Mice are non-responsive to aniline-induced carcinogenicity. However, the present paper shows that aniline hydrochloride induces micronuclei in the bone marrow of male CRH mice 24 h after a single oral dose of 1000 mg/kg (in terms of free base) though not at the lower doses of 400 and 500 mg/kg. No micronucleus inductions was found 48 h after oral dosing. A positive response was also seen 24 h after a single i.p. dose of 380 mg/kg aniline. The high oral dose of aniline needed for micronucleus induction, the presence of micronuclei with abnormal morphology and the large inter-animal variability found within this group may possibly indicate a more complex mechanism of micronucleus induction than simple clastogenicity.

The differential clastogenicity of Solvent Yellow 14 and FD & C Yellow No. 6 in vivo in the rodent micronucleus test (observations on species and tissue specificity)

Solvent Yellow 14 is carcinogenic in rats, inducing neoplastic nodules of the liver, but is non-carcinogenic in mice. The present paper shows that Solvent Yellow 14 induces micronuclei in the bone marrow of rats after a single oral dose of 250 mg/kg and above. In mice, however, there was no increased incidence of micronuclei after single oral doses of up to 2000 mg/kg Solvent Yellow 14, thus reflecting the species specific carcinogenic effect of the compound. The structurally related azo dye FD & C Yellow No. 6 is noncarcinogenic to rats and mice and gave a negative result in both rat and mouse bone marrow micronucleus tests after a single oral dose of up to 2000 mg/kg. The rat bone marrow micronucleus test is therefore capable of discrimination between the carcinogenic and the non-carcinogenic azo dye. A negative result was obtained for Solvent Yellow 14 in an in vivo liver unscheduled DNA synthesis assay after oral doses up to 1000 mg/kg. This result demonstrates the inability of the two in vivo assays used to predict target organ specificity seen in the cancer bioassay.

Induction of micronuclei in mouse bone marrow cells: An evaluation of nucleoside analogues used in the treatment of AIDS

Various nucleoside analogues are being used or are being considered for use as therapeutic drugs to inhibit replication of the HTLV-III/LAV virus in infected human cells. Here, the ability of seven nucleoside analogues, a combination of two analogues, and two other therapeutic compounds to induce genotoxic and cytotoxic damage in vivo was evaluated in the mouse bone marrow micronucleus test. Using a 3-consecutive-day oral treatment protocol, almost all of the test chemicals induced a significant increase in the frequency of micronucleated polychromatic erythrocytes (MN-PCE) in male B6C3F1 mice, ranked in decreasing potency as 6-thioguanine greater than Cytarabine HCl greater than 3'-azido-3'-deoxythymidine (AZT)/2',3'-dideoxycytidine combination = AZT greater than Ribavirin = 2',3'-didehydro-3'-deoxythymidine greater than 2',3'-dideoxyadenosine = 2',3'-dideoxycytidine. The frequency of MN-PCE was not increased significantly by treatment with 2',3-dideoxyinosine (DDI) or pentamidine isethionate (PI). The differential ability of AZT and DDI to induce MN in mouse bone marrow was verified from peripheral blood smears prepared from subchronic (90 day) oral studies. The lack of genotoxic activity by DDI was route-specific since, when tested by intraperitoneal injection, a small but significant increase in MN-PCE was observed. A number of these chemicals induced a significant depression in erythropoiesis. However, there was not a significant correlation between the increase in MN-PCE and the depression in the percentage of PCE. This lack of a correlation suggests that factors other than DNA damage may contribute to the inhibition in the rate of erythropoiesis. The presence of increased levels of micronuclei in bone marrow PCE following treatment with various nucleoside analogues suggests that intrinsic genotoxic activity in mammalian cells should be one factor considered during drug selection for AIDS therapy.

Cytogenetic effect of the thiocarbamate herbicides butylate, molinate and vernolate in the mouse bone marrow micronucleus test

Three thiocarbamate herbicides, butylate ( S-ethyl-diisobutylthiocarbamate), vernolate ( S-propyl dipropylthiocarbamate) and molinate ( S-ethyl- N, N-hexamethylenethiocarbamate) were assayed for cytogenetic effect in the mouse bone marrow micronucleus test. Butylate was inactive in bone marrow, vernolate caused a marginal increase in the incidence of micronucleated polychromatic erythrocytes only at a high toxic dose level. Molinate, the N, N-hexamethylene derivative was, however, strongly active in the bone marrow, causing a high frequency of micronucleated erythrocytes, even at subtoxic concentrations.