Tripterygium hypoglaucum (level) Hutch induces aneuploidy of chromosome 8 in mouse bone marrow cells and sperm

Aneuploidy of mouse chromosome 8 induced by a Chinese medicinal herb, Tripterygium hypoglaucum (level) Hutch (THH) was investigated by fluorescence in situ hybridization (FISH) in vivo. Male mice were treated with THH (single i.p. injection) at doses of 120, 240 and 480 mg/kg. Colchicine (COL, 1.5 mg/kg i.p.) was used as a positive control. Bone marrow cells and epididymal sperm were collected 24 h and 22 days after treatment, respectively. Chromosome 8 aneuploidies induced by THH in bone marrow cells and sperm were determined by FISH with a biotin-16-dUTP labelled DNA probe corresponding to the centromeric region of chromosome 8. The hybridized probe was detected with avidin-FITC. The frequencies of trisomy 8 in bone marrow cells were 0.16% in the solvent control group, 0.39% in the COL-treated group and 0.33, 0.41 and 0.41% in the THH-treated groups, respectively. The frequencies of disomy 8 sperm were 0.11% in the solvent control group, 0.27% in the COL-treated group and 0.23, 0.27 and 0.27% in the THH-treated groups, respectively. The experiment showed that induced aneuploidy frequencies were higher in bone marrow cells than in sperm with COL and the two higher doses of THH (P < 0.05). All groups were significantly different from the corresponding solvent controls (P < 0.01-0.001), but there was no dose-related increase in either cell type. Considering the present results together with our previous studies, it appears that THH is a potent mammalian aneugen which may pose a genetic risk to human patients.     

Chromosomal composition of micronuclei in mouse NIH 3T3 cells treated with acrylamide, extract of Tripterygium hypoglaucum (level) hutch, mitomycin C and colchicine, detected by multicolor FISH with centromeric and telomeric DNA probes

The chromosomal composition of micronuclei (MN) induced by the model mutagens mitomycin (MMC) and colchicine (COL) as well as by acrylamide (AA) and the traditional Chinese medicine Tripterygium hypoglaucum (level) hutch (THH) in NIH 3T3 cells was analyzed by multicolor fluorescence in situ hybridization (FISH) using DNA probes for the centromere repeated minor satellite DNA and the telomeric hexamer repeat (TTAGGG). The majority of MN (78.6%) from treatment with MMC (0.1 microg/ml) did not show centromeric signals, reflecting the clastogenic action of MMC. Following treatment with COL (0.1 microg/ml), 74.5% of the MN showed centromeric signals and several telomeric signals, indicating that MN induced by this well-known aneugen were mainly composed of whole chromosomes. After treatment with AA (100, 200 and 400 microg/ml) both MN containing whole chromosomes and MN containing acentric fragments were found to increase in a dose-dependent manner, demonstrating that AA is not only a clastogen but also an aneugen. THH induced a high frequency of MN harboring whole chromosomes at all concentrations tested (5, 10 and 20 microl/ml) and produced a dose-dependent increase in fragment-containing MN, indicating that THH has both aneugenic and clastogenic potential.     

Investigation of genotoxic activity of trans-dehydrocrotonin, a clerodane diterpene from Croton cajucara

The genotoxic action of three doses of trans-dehydrocrotonin (t-DCTN), an active ingredient obtained from the bark extracts of an Amazon native plant, Croton cajucara, were examined in Swiss mouse bone marrow cells in vivo, submitted to acute intraperitoneal treatment, by micronucleus (MN) and chromosomal aberration (CA) tests. The statistical tests (Anova and Tukey) made to compare the results obtained in each of the three doses of t-DCTN with the negative-control group showed that the frequencies of MN and mitotic index were equal to the negative-control and that the frequencies of CA were lower than that observed in the negative-control. Therefore, based on our results it can be said that t-DCTN is not genotoxic nor cytotoxic to mouse bone marrow cells, submitted to acute intraperitoneal treatment in vivo. Teratogenesis Carcinog. Mutagen. 19:377-384, 1999.     

Extruded micronuclei induced by colchicine or acrylamide contain mostly lagging chromosomes identified in paintbrush smears by minor and major mouse DNA probes

In the mouse bone marrow micronucleus assay, it was studiedwhether micronuclei (MN) could be expelled from polychromaticerythrocytes (PCE) in a similar way to the main nucleus. Toavoid the disrupting centrifugation step of the conventionalbone marrow preparation procedure, the paintbrush techniquewas used in the present experiments. With May–Grünwald–Giemsastaining of paintbrush slides, 5% of the colchicine (COL)-inducedMN were found attached to the outside membranes of PCE and wereregarded as extruded. Of the acrylamide (AA)-induced MN, 22%were extruded. After fluorescence in situ hybridization (FISH)of a total of 300 MN per chemical treatment with the mouse minorand major satellite DNA probes, 9.7% MN were extruded in theCOL group and 8.3% MN were extruded in the AA group. FISH showedthat 76% of the retained COL-induced MN were signal-positive,indicating that they contained entire chromosomes. With A A,29% minor-positive and 28.3% major-positive retained MN werefound, confirming its known clastogenicity. However, the observedfrequency of signal-positive MN (1.7 MNPCE_pos/ 1000 PCE) inthe AA group was about three times higher than in the control(0.5 MNPCE_pos/1000 PCE) which indicates that AA has aneugenicpotential. FISH analysis of the extruded MN showed 72–100%major as well as minor signals. It is concluded that expelledMN contain mostly entire chromosomes. ³To whom correspondence should be addressed     

DNA content determination of micronucleated polychromatic erythrocytes induced by clastogens and spindle poisons in mouse bone marrow and peripheral blood

The frequencies and DNA distributions of micronuclei (MN) inpolychromatic erthrocytes (PCEs) from bone marrow (BM) and peripheralblood (PB) of mice after four different treatments were determinedby flow cytometry. PCEs were differentiated from normochromaticerythrocytes (NCEs) using the fluorescent RNA stain Thiazoleorange, while MN in erythrocytes were detected with the DNAstain Hoechst 33342. The treatments were X-irradiation (1 Gy)cyclophosphamide (CPA; 30 mg/kg) vincristine sulphate (VCR;0.08 mg/kg) and cholchicine (COL; 1 mg/kg). All treatments showedincreased frequencies of micronucleated PCEs at 30 h after treatmenthi BM and at 50 h in PB. The clastogens(X-irradiation and CPA)and the spindle poisons (VCR and COL) could be grouped accordingto the fluorescent characteristics of the induced MN as wellas the relative frequency of small (0.5—2% of the diploidDNA content) and large (2—10%) MN. In PB the relativefrequency of large MN was lower than in BM indicating that theywere partly eliminated before entrance into the peripheral circulation. ³To whom correspondence should be addressed     

Flow cytometric detection of micronuclei induced by chemicals in poly- and normochromatic erythrocytes of mouse peripheral blood

In order to standardize automated scoring for the in vivo micronucleus(MN) test a flow cytometric method which recognized micronucleatedpolychromatic erythrocytes (MPCE) and micronucleated normochromaticerythrocytes (MNCE) in mouse peripheral blood developed by Grawéet al. (1992) has been modified and applied. Blood samples werepurified with 35% percoll solution and stained with the RNA-specificdye thiazole orange (TO) and with the DNA-specific dye Hoechst33342 (HO) for dual laser flow cytometry. The TO fluorescentsignals permitted the discrimination between polychromatic andnormochromatic erythrocytes (PCE and NCE). Cytotoxic effectscould be assessed by the reduction of PCE counts. The blue fluorescentsignals of HO permitted the scoring of MN. The MPCE and MNCEwere flow sorted for microscopic analysis and showed that 95%of the sorted cells actually contained MN. Three model chemicals,the clastogen mitomycin C, the aneugen colchicine and the industrialchemical acrylamide were tested at 24 h intervals after singleintraperitoneal injection up to 72 h after treatment of male(l02/ElC3H/EI)F₁ mice. All three chemicals showed a dose-relatedmaximum of the MPCE frequencies at 48 h while the MNCE frequenciesstayed within the control range up to 72 h. The data obtainedwith the flow cytometric method were in good agreement withpublished results. The flow cytometric technique presented hereis a fast, accurate and automated method for quantifying MPCEand MNCE in peripheral blood as an indicator of cytogeneticdamage induced hi the bone marrow and scored in peripheral bloodsamples. With minor modifications the technique will also beapplicable to bone marrow samples.     

Classification of micronuclei in murine erythrocytes: immunofluorescent staining using CREST antibodies compared to in situ hybridization with biotinylated gamma satellite DNA.

Micronuclei (MN) in erythrocytes of mouse bone marrow cells were induced in vivo by the spindle poisons colchicine (COL) and vinblastine (VBL), by hydroquinone (HQ) and by the alkylating agent mitomycin C (MMC). Two different methods were applied to detect whole chromosomes with centromeric proteins or chromatin in MN to discriminate between spindle damaging or clastogenic activity of these chemicals. One method determined the fraction of MN with centromeric chromatin by immunofluorescent staining using antikinetochore antibodies (CREST staining). The other method applied non-radioactive in situ hybridization with a novel DNA probe. The fractions of MN that showed positive signals by either technique thus indicating with a high probability the presence of whole chromosomes instead of acentric fragments, were in good agreement for COL, VBL and HQ. After application of MMC, however, 4.5% of the MN were CREST-positive, while 29% gave a positive hybridization signal. The results suggest, that kinetochores may have lost certain centromeric antigens due to treatment with MMC so that MN containing whole chromosomes appear CREST-negative. The presented in situ hybridization scheme using satellite DNA is a more direct detection and is advantageous to the CREST staining technique in that it is unaffected by damage of kinetochore or centromeric function.     

Effects of vinblastine and colchicine on male rat meiosis in vivo: Disturbances in spindle dynamics causing micronuclei and metaphase arrest

We have studied the effects of vinblastine sulfate (VBL) and colchicine (COL) on male rat in vivo and in vitro meiosis. A novel methodology based on isolating a segment of seminiferous tubules containing meiotically dividing spermatocytes was applied. During meiotic divisions at stage XIV of rat spermatogenesis, both chemicals induced only low frequencies of micronu-clei (MN), 0.8–3.2 MN/1,000 spermatids. Fluorescence in situ hybridization experiments in mice with the mouse centromere-specific y-sat-ellite DNA probe showed that 50.7% of VBL-induced MN and 56.6% of COL-induced MN were centromere positive, indicating that the MN induced by both chemicals contained detached chromosomes. The inhibition of cell proliferation was determined by counting the number of cells arrested at metaphase during the first meiotic (MI) or the second meiotic (MII) division. VBL was found to be a potent inducer of cell death while COL was not. The direct effects of VBL and COL on the meiotic spindles were evaluated using immunohistochemistry with anti-a-tubulin and confocal microscopy. In the control animals a significant difference was observed between the mean length of metaphase spindles of MI and MII Both were dramatically decreased 6 hr after treatment with 2.0 mg/kg of VBL and 0.8 mg/kg of COL, respectively. At 18 hr after COL injection the spindles had about the same length as in the controls. However, the VBL-induced shortening was even more evident at 18 hr for both Ml and Mll. The possible reasons for observed differences between the two chemicals and between meiosis and mitosis are discussed. © 1995 Wiley-Liss, Inc.     

Mutagenicity of an organophosphate insecticide acephate--an in vivo study in chicks

The chromosome aberration assay (CA) in bone marrow cells andthe micronucleus test (MNT) in both bone marrow and peripheralblood erythrocytes have been carried out for the evaluationof the clastogenic potential of acephate (asataf) in a chickin vivo test system. Technical grade acephate was administeredto evaluate dose-responses (25, 50 and 100 mg/kg), route-responses(i.p. and p.o.) and time-responses (6, 24 and 48 h). A comparisonof CA frequencies after acute and chronic dosing was also performed.Only 50 mg/kg of acephate induced significant bone marrow chromosomeaberrations after 24 h exposure while all three doses inducedsignificant increases in micronuclei in both bone marrow andperipheral blood erythrocytes in i.p. route only. The presenteddata confirm our earlier reports that the neonatal chicken testsystem is a convenient model and can be used as an alternativeto mammalian systems for screening some environmental contaminantsfor genotoxicity. ¹To whom correspondence should be addressed     

Evaluation of the mutagenicity of the anti-inflammatory drug salicylazosulfapyridine (SASP)

Salicylazosulfapyridine, commonly known as sulfasalazine or SASP, is an anti-inflammatory drug that is widely used in the treatment of diseases such as ulcerative colitis and Crohn's disease. Increases in sister chromatid exchanges (SCE) and micronuclei (MN) frequencies have been reported in lymphocytes of patients maintained on SASP therapy for up to 21 months. We have tested SASP for its ability to induce chromosome aberrations (ABS) and SCE in cultured Chinese hamster ovary (CHO) cells, ABS in mouse bone marrow cells, and MN in erythrocytes from both bone marrow and peripheral blood of mice. In vitro assays for ABS and SCE were negative. In vivo, SASP administered by single gavage at doses up to 1000 mg/kg did not increase ABS in bone marrow cells of male B6C3F1 mice; however, increases in MN were observed in the peripheral blood erythrocytes of male and female B6C3F1 mice administered 675, 1350 or 2700 mg/kg SASP by gavage for 90 days. Weak but significant dose-related increases in MN were also observed in the bone marrow cells of male B6C3F1 mice administered 500, 1000 and 2000 mg/kg SASP for 3 days. These positive findings in mice support the role of SASP in the induction of MN and SCE in humans, and suggest the need for further evaluation of possible adverse human health effects associated with SASP therapy.     

Comparative mutagenic and genotoxic effects of three antimalarial drugs, chloroquine, primaquine and amodiaquine

Comparative mutagenic and genotoxic effects of three antimalarialdrugs, chloroquine, primaquine and amodiaquine, were assessedin the Ames mutagenicity assay (in strains TA97a, TA100, TA102and TA104) and in vivo sister chromatid exchange (SCE) and chromosomeaberration (CA) assays in bone marrow cells of mice. These arethe most commonly used antimalarial drugs available at presentthroughout the world. The results of the bacterial mutagenicityassays showed a very weak mutagenic effect of all three drugsin Salmonella strains TA97a and TA100 both with and withoutS9 mix and in TA104 only with S9 mix. The results of the invivo SCE and CA assays indicate that these three drugs are genotoxicin bone marrow cells of mice. ³To whom correspodence should be addressed. Tel: +91 33 473 3491; Fax: +91 33 473 5197; Email: iichbio{at}giascl01.vsnl.net.in     

Detection of aneugenic and clastogenic potential of X-rays, directly and indirectly acting chemicals in human hepatoma (Hep G2) and peripheral blood lymphocytes, using the micronucleus assay and fluorescent in situ hybridization with a DNA centromeric probe

In human hepatoma (Hep G2) cells and peripheral blood lymphocytes(HPBL) the cytokinesis-blocked micronuclei (MN) and fluorescentin situ hybridization (FISH) assays were applied to study aneugenicand clastogenic potentials of X-rays, directly and indirectlyacting chemicals. Induction of MN was studied in vitro followingtreatment with X-rays, directly acting chemicals, such as methylmethanesulphonate(MMS), colchicine (COL), vincristine sulphate (VCS) and vinblastinesulphate (VBS), and indirectly acting agents, such as cyclophosphamide(CP), hexamethylphosphoramide (HMPA), 2-acetylaminofluorene(2-AAF) and 4-acetylaminofluorene (4-AAF). Depending on thepresence of the fluorescent signal in the MN following FISHwith a human DNA centromeric probe, MN in the binucleated HepG2 cells and lymphocytes were scored as centromere-positiveor centromere-negative, representing an aneugenic and clastogenicevent respectively. In the controls     

Induction of micronuclei by lambda-cyhalothrin in Wistar rat bone marrow and gut epithelial cells

We have investigated the genotoxicity of lambda-cyhalothrin (LCT) (CAS registry No. 91465-08-06), a pyrethroid insecticide, in bone marrow cells and in colonic crypt epithelial cells of groups of four rats per dose treated in vivo by gavage at doses of 0.8, 3.06 and 6.12 mg/kg body weight (body wt). We measured genotoxicity using the micronucleus (MN) assay, scoring 2000 polychromatic erythrocytes (PCEs) per animal for bone marrow and 1000 colonic crypt epithelial cells per animal for the colon. We assessed cytotoxicity in bone marrow by calculating the ratio of PCEs to normochromatic erythrocytes, and in the colonic crypt epithelium by observing the frequency of binucleate cells and the mitotic index in 1000 cells. Apoptosis in colonic crypt epithelial cells was measured by observing the frequency of karyorrhexis and karyolysis in 1000 cells. We found that LCT induced a statistically significant dose-related increase in MN formation in the bone marrow and the colonic crypt. The colonic epithelium was more sensitive to the clastogenic effects of LCT than the bone marrow as judged by the significantly higher frequencies of MN in the colon than in the bone marrow at doses of 3.06 and 6.12 mg/kg body wt.     

Identification of kinetochore-containing (CREST+) micronuclei in mouse bone marrow erythrocytes.

A methodology for the characterization of kinetochore-containing (CREST+) micronuclei (MN), based on the use of antikinetochore antibodies (derived from CREST patients) and indirect immunofluorescence, was applied to mouse bone marrow erythrocytes. The proposed protocol allows us to obtain fluorescent signals of good quality and highly reproducible data. The clastogenic agent mitomycin C (MMC; 1 mg/kg body wt) and the two aneugenic compounds chloral hydrate (CH; 200 mg/kg body wt) and colchicine (COL; 1 mg/kg body wt) were used to verify the sensitivity of this approach to chemicals with different mechanisms of action. These compounds were tested at a 20 h time interval from treatment and all of them were able to significantly increase (P less than 0.001) the frequency of MN in polychromatic erythrocytes. Of the MN observed in preparations from control animals, 45% were CREST+ and this percentage increased significantly (P less than 0.001) after treatment with CH or COL. On the contrary, only 22% CREST+ MN were found after treatment with MMC (statistical comparison with the control value: P less than 0.001). The CREST characterization of MN induced in vivo in mouse bone marrow allows us to infer the origin of MN formation, thus contributing to the identification of aneugenic agents.     

Micronucleus induction in mouse and rat fetuses treated transplacentally during histogenesis with mitomycin C and 7,12-dimethylbenz(a)anthracene

During histogenesis, mouse and rat fetuses were treated transplacentally with mitomycin C (MMC) and 7,12-dimethylbenz(a)anthracene (DMBA). The micronucleus (MN)-inducing effects of MMC were analysed in mouse fetal blood and liver; the effects of DMBA were analysed in mouse fetal and rat fetal blood and in maternal bone marrow of both species. Both test substances were clearly clastogenic during the period of development in which the embryos were analysed--i.e., MMC from gestational day 14 until day 18 in mice and DMBA on days 14 and 17 in mice and days 16 and 19 in rats. In mouse fetal liver and blood the MMC-induced MN frequencies did not vary significantly during the whole period. MMC was more effective in fetal blood than in fetal liver. DMBA-induced MN frequencies in maternal bone marrow were slightly higher in rats than in mice. Compared to maternal bone marrow, fetal MN frequencies were about four to five times higher in rats but less than two times higher in mice. Thus, rat fetuses were far more susceptible to the clastogenic action of DMBA than mouse fetuses. These results are discussed with respect to fetal development and maternal/fetal metabolism.     

Clastogenic effects of known and suspect spindle poisons studied by chromosome analysis in mouse bone marrow cells

The present study was performed within the project 'Genomic Mutations' (sponsored by the Commission of the European Communities) in order to gather all possible experimental information on 10 chemicals selected on the basis of their possible capacity to induce aneuploidy. An analysis of chromosomal aberrations was carried out in bone marrow cells of mice with the first five chemicals: colchicine (COL), econazole (EZ), chloralhydrate (CH), hydroquinone (HQ) and diazepam (DIAZ). The experiments were performed in parallel to micronucleus tests with the objective to distinguish a positive micronucleus response due to chromosomal breakage from that obtained by lagging chromosomes. The results of the micronucleus tests will be reported elsewhere. COL, CH, EZ and DIAZ showed no clastogenic effects in mouse bone marrow cells after single intraperitoneal injection. Polyploid cells were significantly more frequent after COL treatment. HQ showed a dose-dependent induction of chromosomal aberrations at 6 and 24 h after treatment. After 24 h, cells with multiple aberrations up to complete chromosome fragmentation were frequently observed. They indicate that a small fraction of the cell population, probably related to a specific stage of the cell cycle, was particularly sensitive to HQ. A sex difference in clastogenic response to HQ was not observed. It is concluded that of the five chemicals tested only HQ was clastogenic in mouse bone marrow cells under the present experimental conditions.     

Application of antikinetochore antibody staining (CREST staining) to micronuclei in erythrocytes induced in vivo

Micronuclei (MN) can be formed by acentric chromosome fragments or whole lagging chromosomes. In order to discriminate MN produced by chromosome breakage from those arising from spindle malfunction a staining method using immunofluorescent kinetochore antibodies has been applied successfully in vitro. In the present study MN in polychromatic erythrocytes of mouse bone marrow cells induced in vivo by the spindle poison colchicine (COL) and the clastogen mitomycin C (MMC) were analysed after CREST staining. The staining method was modified by using pretreatment with detergents in order to allow a good penetration of the antibodies into the MN. About 66% of the MN induced by COL in contrast to only 4.5% of the MMC induced MN were CREST-positive. The preliminary results show that the CREST staining is also in MN induced in vivo capable of detecting the origin of MN and to discriminate between the spindle damaging or clastogenic activity of environmental agents. The method described will give supplementary information about MN, it cannot substitute for the common micronucleus test.     

Induction effects of sulfur dioxide inhalation on chromosomal aberrations in mouse bone marrow cells

To investigate the induction of chromosome aberrations (CA) in mouse bone marrow cells by sulfur dioxide (SO(2)) inhalation, mice were treated by SO(2) exposure for 4 h/day for 7 days at various concentrations of SO(2), then mitotic indices and CA in mouse bone marrow cells were analyzed. The present results show that SO(2) might increase the frequencies of CA and aberrant cells in mouse bone marrow in a dose-dependent manner. The frequencies (%) of aberrant cells in mouse bone marrow induced by SO(2) at concentrations of 0, 7, 14, 28 and 56 mg/m(3) were 1.81, 3.00, 3.58, 4.26, 4.86, respectively. At low concentrations SO(2) induced only chromatid-type CA, while at high concentrations SO(2) induced both chromatid-type and chromosome-type CA. SO(2) inhalation decreased the mitotic indices of bone marrow cells. The results imply that SO(2) inhalation may inhibit mitoses and increase CA frequencies of bone marrow cells and that it is a clastogenetic and genotoxic agent. Long exposure to SO(2) pollution at low concentrations in the environment may be a potential risk for induction of cytogenetic damage in vivo in humans.     

Cytogenetic analyses of mice exposed to dichloromethane

Chromosome damage was studied in female B6C3F1 mice exposed to dichloromethane (DCM) by subcutaneous or inhalation treatments. No increase in the frequency of either sister chromatid exchanges (SCEs) or chromosome aberrations (CAs) in bone marrow cells was observed after a single subcutaneous injection of 2,500 or 5,000 mg/kg DCM. Inhalation exposure to DCM for 10 days at concentrations of 4,000 or 8,000 ppm resulted in significant increases in frequencies of SCEs in lung cells and peripheral blood lymphocytes, CAs in lung and bone marrow cells, and micronuclei (MN) in peripheral blood erythrocytes. Lung cell CAs and blood erythrocyte MN reached frequencies of approximately two times control levels. Following a 3-month inhalation exposure to 2,000 ppm DCM, mice showed small but significant increases in lung cell SCEs and peripheral blood erythrocyte MN. These findings suggest that genotoxicity may play a role in the carcinogenicity of DCM in the lungs of B6C3F1 female mice.     

Evaluation of the genotoxic effects of pyrimethamine, an antimalarial drug, in the in vivo mouse

Pyrimethamine is an antimalarial drug and a known teratogenic agent. With this drug, positive and negative results have been reported by various investigators in in vivo and in vitro genotoxicity/mutagenicity assays. In this investigation the genotoxic effects of pyrimethamine (PY) were tested in mice in vivo systems, using the bone marrow micronucleus test (MNT) and the transplacental MN test (TMNT). PY at the highest dose (40 mg/kg) induced statistically significant MN in bone marrow cells at 24 and 48 h. In the transplacental MN test, PY did not induce significant MN in fetal liver or in maternal bone marrow. Teratogenesis Carcinog. Mutagen. 20:65-71, 2000.