In vivo sister chromatid exchange induced by 1,2-dichloroethane on bone marrow cells of mice

In vivo sister chromatid exchange induced by 1,2-dichloroethane (DCE) was studied in bone marrow cells of mice after acute treatment for 24 hr. With the exception of the lowest concentration (0.5 mg/kg), each treated series exhibited a statistically significant increase in SCEs when compared with the control.     

Quinoline-induced chromosome aberrations and sister chromatid exchanges in rat liver

Induction of chromosome aberrations and sister chromatid exchanges (SCEs) was studied in hepatocytes of F344 rats exposed in vivo to the hepatocarcinogen quinoline (Q). Hepatocytes were isolated 4–48 hr after a single dose of 200 mg/kg body weight or 24 hr after 28 repeated doses (once a day) of 25–200 mg/kg body weight/day by gastric intubation, and allowed to proliferate in Williams' medium E supplemented with epidermal growth factor. Cells were fixed after a culture period of 48 hr. A single dose of Q induced chromosome aberrations in up to 22% of metaphase cells, and SCEs with a frequency of up to 1.27 per chromosome 12 hr after the dose, while the control values were 1% and 0.63 per chromosome, respectively. Treatment with 28 repeated doses of Q induced significant chromosome aberrations and SCEs dose-dependently. Cytogenetic damage induced in the liver by repeated doses of Q was greater than induced by a single dose. Furthermore, Q induced replicative DNA synthesis in the liver, but failed to induce micronucleus formation in the bone marrow. The noncarcinogen 8-hydroxyquinoline was also examined and found to be essentially non-genotoxic to rat liver. These results show that Q is a genotoxic carcinogen to rat liver and the present method of in vivo cytogenetic assay should be useful for evaluating the genotoxicity of hepatocarcinogens. Environ. Mol. Mutagen. 30:459–467, 1997 © 1997 Wiley-Liss, Inc.     

Clastogenic effect of fenfluramine in mice bone marrow cells in vivo

Fenfluramine, an amphetamine derivative used in the treatment of obesity, has been evaluated in vivo in the bone marrow cells of Swiss albino mice using two cytogenetic endpoints for assessing its genotoxic and clastogenic potentials. Concentrations of 0.75, 1.5, 3.0, and 5.0 mg/kg b.w. were administered orally for the study of sister chromatid exchange frequencies and chromosome aberrations (CA). SCE frequencies showed a positive dose response; 1.5 mg/kg being the minimum effective concentration. Fen caused a prolongation of cell cycle at all concentrations. Except for the minimum therapeutic dose (0.75 mg), all other doses (1.5, 3.0, and 5.0 mg) showed a significant increase in the percentage of damaged cells over that of the vehicle control. The degree of clastogenicity was directly proportional to the dosage used and inversely related with the duration of treatment. A gradual reduction of the clastogenic potential was observed after 12 and 24 hr of exposure, indicating that the maximum effect occurs at the middle or late synthetic phase of the cell cycle. This study, probably the first detailed screening of the drug for its genotoxicity, shows that Fen is moderately clastogenic and a DNA damaging agent in vivo.     

The kinetics of in vivo sister chromatid exchange induction in mouse bone marrow cells by alkylating agents: cyclophosphamide

Administration of cyclophosphamide (5, 10, 20 and 25 mg/kg body weight) to male CD-1 mice 2 hr after subcutaneous implantation of a 5-bromo-2'-deoxyuridine (BrdUrd) pellet (55 mg) resulted in a dose-dependent increase in sister chromatid exchanges (SCE) in bone marrow cells. Treatment with cyclophosphamide (15 mg/kg body weight) at the time of BrdUrd implantation and 2, 6.5, and 13 hr post-BrdUrd implantation resulted in the induction of approximately 19 SCE/cell indicating that the bone marrow SCE response was independent of the time of administration. Treatment with cyclophosphamide (15 mg/kg body weight) at 26, 19, 13, and 6 hr prior to BrdUrd implantation resulted in baseline SCE (3.3 SCE/cell) at 26 hr with an increasing number of SCE/cell with decreasing time prior to BrdUrd implantation. These results compare favorably with those obtained by Kram et al [1981] with mitomycin C (MMC) using a similar protocol. The time-dependent induction of SCE is qualitatively similar for CP and MMC, both of which are bifunctional alkylating agents metabolically activated by oxidation and reduction, respectively, and suggests that these two compounds may induce SCE by a similar mechanism.     

Developmental toxicity of the topoisomerase inhibitor, etoposide, in rabbits after intravenous administration.

The developmental effect of the topoisomerase inhibitor, etoposide, was investigated in pregnant rabbits given intravenous doses during early organogenesis. Does received 0, 0.25, 0.5, 1, or 2 mg/kg/day on days 7 through 9 of gestation. Fetal parameters were evaluated on day 28 of gestation. Live fetuses were examined for gross, visceral, and skeletal malformations and variations. In addition, telencephalon in embryos 8 h following the final treatment was examined histologically. No change in general condition was observed in any does, but a significant decrease in body weight gain during the pregnancy and enlargement of the liver resulting from marked fatty change were observed in does treated with etoposide at 2 mg/kg/day. Etoposide had neither lethal nor growth retarded effects on embryos/fetuses. However, axial skeletal malformation and extra ribs had a low incidence but were significant in the group treated with etoposide at 2 mg/kg/day, whereas no significant increases in external malformations in term fetuses nor in pyknotic cells in the ventricular zone of telencephalon in embryos were noticed in any etoposide-treated groups. It was concluded that anatomical defects (skeletal malformation or variation) in rabbits were induced by intravenous etoposide treatment during early organogenesis and that they occurred in the presence of maternal toxicity.     

The genotoxic effect of potassium metabisulfite using chromosome aberration, sister chromatid exchange, micronucleus tests in human lymphocytes and chromosome aberration test in bone marrow cells of rats

Potassium metabisulfite (PMB) is used as an antimicrobial substance in many kinds of foods. In the present study, the effects of PMB on chromosome aberrations (CAs), sister chromatid exchanges (SCEs), and micronucleus (MN) formation in human lymphocytes and as well as its effect on CAs in bone marrow cells of rats were investigated. The human lymphocytes were treated with 25, 50, 100, and 200 microg/ml of PMB for 24 and 48 hr. PMB was also intraperitoneally (ip) injected to the rats as a single dose of 150, 300, and 600 mg/kg body weight (b.w.) for 12 and 24 hr before sacrifice. PMB induced abnormalities such as structural and numerical (total) CAs, SCEs, and MN formations in a dose dependent manner in the lymphocytes of the 24- and 48-hr treatment periods. In addition, PMB showed a cytotoxic effect by decreasing the replication index (RI), mitotic index (MI) and nuclear division index (NDI) in a dose dependent manner in human lymphocytes. The compound induced CA as well and decreased the MI in bone marrow cells of rats. It might be concluded that PMB had a high genotoxic and cytotoxic risk.     

Sister chromatid exchanges induced by tertiary butyl hydroquinone in bone marrow cells of mice

Tertiary butyl hydroquinone (TBHQ)--a phenolic antioxidant, was evaluated by assessing the induction of sister chromatid exchanges (SCEs) in bone marrow metaphase cells of mice. Six concentrations, 0.5, 2, 20, 50, 100, and 200 mg/kg, of TBHQ were injected intraperitoneally. A positive dose-response effect in the SCE frequency was observed using the Cochran-Armitage trend test. Two mg/kg of TBHQ was found to be the minimum effective dose. Study of the cell-cycle kinetics showed a delay in cell cycle induced by the higher concentrations of TBHQ. Thus, TBHQ was found to be a DNA damage-inducing agent and also a cytotoxic chemical in vivo in mice.     

Cytogenetic studies of ethyl acrylate using C57BL/6 mice

The clastogenicity of ethyl acrylate (EA) was examined in vivo by injecting i.p. five male C57BL/6 mice per dose group with either 125, 250, 500 or 1000 mg/kg EA dissolved in saline. Controls received solvent only. Acrylamide (100 mg/kg), because of its similarity in structure and mode of action to EA, was used as a positive control. Twenty-four hours after injection, the animals were anesthetized and the spleens aseptically removed. Splenocytes were isolated on density gradients and cultured with concanavalin A to stimulate cell division. In half the cultures bromodeoxyuridine was added at 21 h for analysis of chromosome aberrations (CAs) in first division cells and sister chromatid exchange (SCE) in second division cells. In the remaining cultures cytochalasin B was added to produce binucleated cells for scoring of micronuclei (MN). There was no significant increase in SCE or CAs at any of the doses of EA examined. At the highest dose examined (1000 mg/kg), EA did cause a small but significant increase in binucleated cell MN. Acrylamide caused an increase in MN and SCEs in splenocytes. Because others have found EA to be clastogenic in vitro, isolated splenocytes were exposed to a wide range of concentrations of EA during the G0 stage of the cell cycle or 23 h after mitogen stimulation during the late G1 or early S phase of the cell cycle. Although EA was toxic for both exposure regimens, significant increases in chromatid-type aberrations were found only when the target cells were treated 23 h after mitogenic stimulation. No statistically significant increase in SCE frequency was found after either treatment regimen. These data suggest that EA is only clastogenic at near toxic concentrations during a specific stage of the cell cycle.     

Induction of sister chromatid exchanges by cypermethrin and carbosulfan in bone marrow cells of mice in vivo

The public health effects of pesticides cannot be denied. However, the undesired effects of chemical pesticides have been recognized as a serious public health concern during the past decades. The present study describes the genotoxic effects of two pesticides, namely cypermethrin and carbosulfan, in a murine test system in vivo. The test parameter used was analysis of sister chromatid exchanges (SCE) in bone marrow cells. Both cypermethrin (5, 10 and 20 mg/kg) and carbosulfan (1.25, 2.5 and 5 mg/kg) induced significant increases in the frequency of SCEs (P < 0.001). However, no significant dose-response correlation could be found for either of the pesticides. Carbosulfan induced a cell cycle delay, as evidenced by an increase in average generation time accompanied by accumulation of cells in the first division cycle, but cypermethrin did not induce any such response. The present study indicates that carbosulfan has a higher potential to cause genetic alterations than cypermethrin in mice and may also pose a mutagenic risk to human beings.     

Modification of clastogenicity of three known clastogens by garlic extract in mice in vivo

The anticlastogenic activity of crude extract of garlic (Allium sativum L.) was studied in bone marrow cells of mice. Male laboratory-bred Swiss albino mice were given one of three concentrations of the freshly prepared extract (100 mg, 50 mg, and 25 mg/kg body weight) as a dietary supplement by gavage for 6 consecutive days. On the seventh day the mice were administered a single acute dose of two known clastogens, mitomycin C(1.5 mg/kg) and cyclophosphamide (25 mg/kg) or sodium arsenite (2.5 mg/kg), simultaneously with garlic extract. After 24 hr, chromosome preparations were made from the bone marrow cells. The endpoint studied were chromosomal aberrations and damaged cells. Garlic extract alone induced a low level of chromosomal damage. The clastogenicity of all three mutagens were reduced significantly in the animals which had been given garlic extract as dietary supplement. The extent of reduction was different for the three clastogens and may be attributed to the interaction with the different components of the extract. © 1993 Wiley-Liss, Inc.     

Evidence for an indirect effect of radiation on mammalian chromosomes. III. UV- and x-ray-induced sister chromatid exchanges in heterokaryons

The hypothesis that chromosomes may be damaged indirectly by radiation was examined by assaying sister chromatid exchange, (SCE) frequency in heterokaryons between irradiated and unirradiated mouse and Chinese hamster cells. One cell line was UV or x irradiated, then fused to unirradiated BrdU-labeled cells of the other line; SCEs in the unirradiated set were scored in heterokaryons. A dose-dependent increase was consistently observed; the magnitude of which suggested that 25% of UV-induced and up to 60% of x-ray-induced SCEs are indirectly induced. Medium transfer experiments, cell mixing, and fusion with irradiated chick erythrocyte ghosts suggested that unirradiated chromosomes in heterokaryons are damaged by a stable, nondiffusible cytoplasmic component contributed by the irradiated cell.     

Evidence that SCEs induced by mutagens do not occur at the same locus in successive cell cycles: Lack of cancellation in three-way stained CHO chromosomes

An approach based on the synchronization of CHO cells after a first cell cycle incorporating a relatively low amount of bromodeoxyuridine (BrdUrd) into DNA, followed by mutagenic treatment and subsequent culture for second and third generations of BrdUrd incorporation for the scoring of sister chromatid exchanges (SCEs) per cell cycle in three-way differentially (TWD) stained chromosomes, has been used to investigate the possible concellation of SCEs. Cancellation is expected to occur if two mutagen-induced SCEs occur at exactly the same site in subsequent rounds of replication. Lesions in DNA seem to persist and are able to induce SCE throughout two cell cycles after treatment with the three mutagens tested—mitomycin C (MMC), ethyl methanesulfonate (EMS) and ultraviolet (UV) light—though this latter agent was shown as only moderately persistent. Our results seem to indicate that SCEs induced by these mutagens do not take place at the same locus in successive cell generations, as assessed by a lack of SCE cancellation. © 1994 Wiley-Liss, Inc.     

Induction of sister chromatid exchanges in murine colonic tissue

As cancers of the large bowel arise primarily in the epithelial cells of the colon, measurement of genetic damage induced in those cells may aid in elucidating contributing factors in the etiology of the disease. Accordingly, procedures for the in vivo measurement of sister chromatid exchanges (SCE) have been adapted for use with colonic cells. Agar-coated 5-bromo-2'-deoxyuridine (BrdUrd) tablets were implanted (s.c.) in mice and colonic tissue prepared for measurement of SCE 48 hr later. When mice were treated by intraperitoneal injection with 20 mg/kg 1,2-dimethylhydrazine (DMH), a colon carcinogen, a significant increase over control SCE frequencies was found when treatment with carcinogen occurred in the interval from 14 hr before to 36 hr following BrdUrd administration. Treatment with DMH 2 hr following implantation of BrdUrd produced a dose-dependent increase in SCE frequencies, with the highest dose (20 mg/kg) resulting in frequencies approximately twice those of controls. Dimethylhydrazine was not found to be effective in inducing SCE in bone marrow cells of mice. As SCE can be induced by carcinogens and mutagens, measurement of SCE in colonic epithelium may be useful in identifying potential colon carcinogens.     

Induction and reduction of sister chromatid exchange by CCNU in human lymphocytes in vitro

Sister chromatid exchange (SCE) was studied in human lymphocytes treated with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in vitro. A dose-dependent increase of SCE was observed in cells exposed to 10(-5) - 10(-4) M CCNU. The maximal increase was 25-35 SCEs/cell over the control level, which is similar to the increase found in patients treated with CCNU in vivo. In the presence of rat liver microsomes (S-9 fraction) the frequency of CCNU-induced SCE was slightly higher than in parallel cultures without S-9, suggesting that microsomal metabolism may enhance the rate of decomposition of CCNU into reactive products. The CCNU-induced increase of SCE was greater in cells treated for longer time periods (up to 70 hr) than in cells subjected to a 1-hr treatment. This effect was most pronounced at higher concentrations of the drug (5 X 10(-5) M). The frequency of CCNU-induced SCE was also found to be dependent on the time of treatment in the cell cycle. A treatment for 1 hr during early G1-phase (about 20 hr before the first S-phase) gave rise to a higher increase of SCE than a 1 hr treatment immediately before or during the first or second S-phase. Thus, the CCNU-induced DNA damage leading to SCE seems to persist and may even increase during the prereplicative phase of the cell cycle. After replication in BrdUrd-free medium, the frequency of CCNU-induced SCEs decreased to the control level. The present results, taken together with other studies of strand break and cross-link formation by CCNU in mammalian cells in vitro, suggest that the major SCE-inducing damage by CCNU is DNA interstrand cross-links. These lesions then appear to be slowly removed, if at all, during the prereplicative phase of the cell cycle, and to disappear during or after replication in BrdUrd-free medium in vitro.     

Different behavior of SCE-eliciting lesions induced by low and high doses of busulfan

Our previous studies suggested a dose-dependent transition in the types of DNA lesions induced by busulfan, as measured using the comet assay and by micronuclei analyses. The aim of the present study was to investigate the dose-dependent induction of different sister-chromatid exchange-eliciting lesions; lesions were distinguished by their efficiency in producing sister-chromatid exchange (SCE), and by their reparability during G1. Synchronously dividing murine salivary gland cells were assayed in vivo. Groups of mice were intraperitoneally injected with either 30 or 80 micromol busulfan/kg body weight solution at early or late G1. The rate of SCE/micromol busulfan/kg body weight obtained by exposure at late G1 with the high dose was twice that of the low dose. SCE induction during early G1 was higher than at late G1 with both doses; only the low-dose response was statistically significant. The frequency distribution of SCEs per cell demonstrated that cells exposed at the late G1 phase showed typical profiles that closely fit a Gaussian curve. However, an irregular profile was obtained for cells treated during early G1, which showed some cells with high-SCE frequency. Cells treated in early G1 have more time to repair lesions before DNA synthesis; therefore, the results suggest that instead of repair, secondary SCE-eliciting lesions during G1 were produced, especially at the lower dose. The results obtained in this study indicate that there are dose-dependent differences in the types of SCE-eliciting lesions induced by busulfan.     

Sister chromatid exchange in vivo in mice: I. The influence of increasing doses of bromodeoxyuridine

Bromodeoxyuridine (BrdUrd) pellets weighing either 20, 25, 30, 35, 40, 45, or 53 mg were implanted subcutaneously in strain DBA/2J male mice. Femoral bone marrow cells were analyzed for sister chromatid exchange (SCE) and chromosome aberration frequencies, mitotic indices, and cellular replication kinetics. Small but statistically significant BrdUrd dose-dependent increases of SCEs were observed, whereas chromosome aberrations were induced at low frequencies and occurred independently of BrdUrd dose. The mitotic index remained relatively constant for all doses. A slight inhibition of cellular replication, as manifested by a reduction in third division cells at the 40- and 45-mg doses, was observed. The use of an intense fluorescent light source in the fluorescence-plus-Giemsa technique provided consistently good sister chromatid differentiation at pellet doses substantially lower than those previously used by other investigators.     

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.     

Sister chromatid exchange and chromosome aberrations in mice after in vivo exposure of green S--a food colorant.

Sister chromatic exchanges (SCE) and chromosome aberrations (CA) in mice after in vivo exposure of Green S were carried out following single acute treatment. Except for the lowest dose (25 mg/kg body weight) a significant increase in the SCEs were observed in all the other doses (50, 100, and 200 mg/kg) tested. In CA study two higher doses (200 and 400 mg/kg) showed a significant increase in CA when compared with control. The minimum effective dose which induced SCE and CA was 50 and 200 mg/kg of body weight, respectively. The trend tests for the evidence of dose response effects were also significant for both SCE and CA. No significant differences were observed in cell replication kinetic (RI) analysis. A significant increase in the mitotic index (MI) was also observed in the highest dose (400 mg/kg) tested when compared with control. Thus the present study indicates that Green S can induce both SCE and CA in vivo in bone marrow cells of mice.     

Investigations into the concept of a threshold for topoisomerase inhibitor-induced clastogenicity

Although the application of the concept of a threshold to risk assessment is widespread, there remains little experimental evidence for the existence of thresholds for genotoxic compounds, other than aneugens. The clastogenicity of topoisomerase inhibitors is believed to result from the transient stabilization of the topoisomerase enzyme with DNA during the catalytic cycle. This leads to the formation of a stabilized cleavage complex, which, in turn, may result in the formation of a DNA strand break. This indirect mechanism of clastogenicity is the basis for the concept of threshold for this class of drug. Using micronucleus induction in L5178Y mouse lymphoma cells as a genotoxic end-point, a three pronged approach was used to examine whether the concept of a threshold for clastogenicity could be demonstrated for topoisomerase type II inhibitors in vitro. This involved (i) the study of mechanism (TARDIS assay), (ii) hypothesis testing versus estimation (i.e. scoring up to 10,000 cells/treatment at concentrations immediately above and below the NOEL for micronucleus induction) and (iii) statistical modelling of the concentration-response curves for micronucleus induction. Several topoisomerase type II inhibitors were investigated with varying clastogenic potencies (etoposide = doxorubicin < genistein < ciprofloxacin). Pragmatic thresholds for clastogenicity in L5178Y cells were defined at 0.00236 microg/ml for etoposide, 0.00151 microg/ml for doxorubicin, 1 microg/ml for genistein and 50 microg/ml for ciprofloxacin. In addition, it was demonstrated that etoposide-induced clastogenicity was concentration and time dependent. These results, along with mechanistic data showing that all of the compounds induced concentration-dependent increases in the formation of topoisomerase II stabilized cleavage complexes, provide a weight of evidence to support a threshold concept for clastogenicity with topoisomerase II poisons.     

Transplacental genotoxicity of triethylenemelamine, benzene, and vinblastine in mice.

Transplacental cytogenetic effects of triethylenemelamine (TEM), benzene, and vinblastine on maternal mice and their fetuses have been investigated using micronucleus and sister chromatid exchange (SCE) as genetic endpoints. CD-1 mice were treated on day 14 and 15 of gestation with TEM (0.125, 0.25, and 0.5 mg/kg), benzene (439,878, and 1,318 mg/kg), and vinblastine (0.5, 1, and 2 mg/kg) by intraperitoneal injection at 24 hr intervals, and sacrificed 40 hr after the first injection. Erythrocytic precursor cells in maternal bone marrow and fetal livers (2-4) from each pregnant mouse were used for the micronucleus and/or the SCE analyses. Significant dose-related increases in both micronuclei and SCE were found in maternal bone marrow and fetal liver following TEM treatment. Benzene at the highest dose (1,318 mg/kg) also caused a significant increase in micronuclei and SCE in both maternal bone marrow and fetal liver cells. The embryonic genotoxic effect of TEM was much higher than that of benzene for both genetic endpoints, and the frequency of micronuclei induced by benzene was higher in fetal liver than in maternal bone marrow cells. Vinblastine, a spindle poison, induced micronuclei but not SCE. Micronuclei induction by vinblastine was 7 fold greater in maternal bone marrow than in fetal liver cells. All three chemicals were cytotoxic in maternal bone marrow cells, but not in fetal liver cells except for TEM, which showed a weak cytotoxicity in fetal liver cells in the micronucleus assay. These results indicate that TEM, benzene, and vinblastine are transplacental genotoxicants in mice.