Comparative in vivo and in vitro sister chromatid exchange studies in Chinese hamster bone marrow and spleen cells

The sister chromatid exchange (SCE) assay in bone marrow and spleen cells of Chinese hamsters was used to evaluate the differences between in vivo and in vivo/in vitro (exposure of animals to chemical followed by culturing of cells) conditions. Cyclophosphamide, a mutagenic carcinogen, caused dose-related SCEs both in vivo and in vivo/in vitro. In the in vivo group, both bone marrow and spleen cells showed approximately a five-fold increase in SCEs over controls following 40 mg cyclophosphamide/kg treatment. The same dose, under in vivo/in vitro conditions, caused about three- and six-fold increases in SCEs over controls in bone marrow and spleen cells, respectively. While the extent of cyclophosphamide-induced SCEs (after subtraction of baseline level) in bone marrow is approximately the same under both conditions, the response was significantly higher in spleen cells in vivo/in vitro than in vivo. Under in vitro conditions, treatment of bone marrow and spleen primary cell cultures with a direct acting mutagen, trinitrofluorenone, caused significant dose-related increases in SCEs in both cell types in an equivalent manner. The replicative indices under these experimental conditions remained almost the same. Thus, this study indicates the potential usefulness of Chinese hamster bone marrow and spleen cells for in vivo and in vitro comparative studies with the same tissue to better assess the genotoxic hazard of chemicals.     

Neocarzinostatin induces chromosomal aberrations and sister chromatid exchanges in Chinese hamster ovary cells

Chinese hamster ovary (CHO) cells were treated with neocarzinostatin(NCS) and analyzed for chromosomal aberrations and sister chromatidexchanges (SCE). After treatment the cells were recovered for9, 20, 26 or 30 h. NCS induces chromosomal aberrations and SCE.SCE were much more frequent in cells with chromosome type aberrationsat 20 h recovery time than in those with chromatid type aberrationsat 9 h recovery time. In second post-treatment cells at 26 or30 h recovery time NCS induced chromosomal aberrations but onlyfew SCE.     

Preparation of Chinese hamster bone marrow and spleen primary cell cultures for sister chromatid exchange and chromosomal aberration studies

Summary Procedures for preparing and culturing Chinese hamster bone marrow and spleen cells for cytogenetic studies are described. Animals are killed by cervical dislocation, then the bone marrow is flushed from femora and tibia with Ham's F12 medium into centrifuge tubes. Bone marrow cells are collected by low-speed centrifugation (285 ×g). Approximately one million cells are cultured in a 30-ml Falcon flask with 5 ml complete medium containing 3.95 ml Ham's F12 medium, 1.0 ml fetal bovine serum (FBS), and 0.05 ml penicillin-streptomycin (5000 U/ml and 5000 µg/ml stock). Spleens are obtained aseptically, transferred to centrifuge tubes containing 2 ml of RPMI 1640 and smashed with a sterile spatula. The debris is removed, cells are collected by low-speed centrifugation (285 ×g), and washed three times with phosphate buffered saline supplemented with 2% heat inactivated FBS. Approximately one million cells are suspended in a 30-ml Falcon flask with 5 ml complete medium containing 3.70 ml RPMI 1640. 1.0 ml heat inactivated FBS, 0.05 ml penicillin-streptomycin, 0.05 ml of 200 mM l-glutamine solution, 1 × 10^–5 M 2-mercaptoethanol, and 0.20 ml lipopolysaccharide. For sister chromatid exchange analysis, 20 µM of 5-bromo-2 -deoxyuridine is also added to the culture medium for 24 to 28 h for bone marrow cells and 36 to 40 h for spleen cells. These culture systems can also be used for chromosomal aberration studies.     

Induction of sister chromatid exchanges by ergot compounds in Chinese hamster ovary cells in vitro

Five ergot alkaloids, d-lysergic acid derivatives, were tested for the induction of sister chromatid exchange (SCE) frequencies in cultured Chinese hamster ovary cells. The concentrations of the test substances ranged between 10(-5) and 10(-8) M. Ergotamine, ergonovine, and methylergonovine induced a significantly high number of SCEs at all the concentrations used, while with ergocristine this occurred only at the highest concentration. alpha-Ergocryptine failed to induce SCEs at any concentration. It is therefore surmised that ergotamine, ergonovine, and methylergonovine are effective inducers, ergocristine is a weak inducer, and alpha-ergocryptine is a noninducer in Chinese hamster ovary cells in vitro.     

In vivo persistence of sister chromatid exchanges (SCE) induced by gamma rays in mouse bone marrow cells

The sister chromatid exchange (SCE) frequencies induced in bone marrow cells by in vivo irradiation with gamma rays before or after bromodeoxyuridine (BrdUrd) incorporation were compared. The frequency of SCE at different postirradiation times was also measured in bone marrow cells in vivo, irradiated before BrdUrd incorporation. Increased sensitivity to SCE induction by radiation was found in cells after BrdUrd incorporation for one cycle when compared with cells irradiated before BrdUrd incorporation. The increased SCE frequency persisted for at least 72 hr after the initial irradiation, implying that the gamma ray-induced lesion(s) capable of eliciting an SCE are persistent and cannot be easily repaired.     

Induction of sister chromatid exchanges and chromosome damage by gossypol in bone marrow cells of mice

The chromosome-damaging potential of gossypol was evaluated by scoring sister chromatid exchanges (SCEs), determining the percentage of pulverized metaphases and the mitotic index in bone marrow cells of mice. Bone marrow cells were collected approximately 21 hours after the intraperitoneal (0,20,40,80, or 160 micrograms/g) and oral (0,40,80, or 160 micrograms/g) administration of gossypol acetic acid. Irrespective of the dosing schedule (single or multiple doses), the vehicle used (physiological saline, corn oil, or 10% aqueous ethanol), and the route of administration, the mean SCE count per cell was significantly higher (P less than 0.05) in gossypol-treated groups than their control counterparts. At 80 and 160 micrograms/g dose levels, the occurrence of metaphase chromosome pulverization was significantly greater, while mitotic index values were markedly lower than those of the corresponding control values. The results suggest that gossypol is a potentially mutagenic and clastogenic agent in murine bone marrow cells.     

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.     

Diethylstilbestrol-diphosphate induces chromosomal aberrations but not sister chromatid exchanges in murine bone marrow cells in vivo

Diethylstilbestrol diphosphate (DES-dp) clastogenesis was examined in the bone marrow of C57B1/6 male and female mice. Significant and sex-related dose effects were observed for the induction of chromatid-type chromosomal aberrations and for the inhibition of cellular proliferation. Females were more sensitive to the effects of DES-dp than males when assessed for either induced chromosomal aberrations or proliferative inhibition. Contrary to other published results, we did not observe either an increase in sister chromatid exchanges or an increased incidence of aneuploidy. Ovariectomy reduced the ability of DES-dp to inhibit cellular proliferation and decreased the high degree of variability between animals at high doses of DES-dp. The results of our studies show that DES is a clastogenic agent in vivo which may relate to its carcinogenicity.     

The longevity of chemically induced sister chromatid exchanges in chinese hamster ovary cells

The persistence of the lesions leading to sister chromatid exchanges (SCEs) following acute exposure of Chinese hamster ovary (CHO) cells to direct-acting chemical mutagens was measured. The results revealed that these lesions (and consequently SCEs) are rapidly eliminated from cells. SCE levels fell to near control values by the third or fourth day (six and eight cell cycles, respectively) following exposure of CHO cells to quinacrine mustard (QM) and mitomycin C (MMC). In contrast, cells exposed to methyl methanesulfonate (MMS) showed a small but significant increase in SCE level over control up to and including the fifth day following exposure (approximately ten cell cycles), suggesting that the behavior of these lesions in cells is influenced, at least in part, by the mechanism by which a specific agent interacts with DNA. The possibility that the decline in SCE level was due to the loss of cell populations with high numbers of exchanges was eliminated by the assessment of cloning efficiencies of treated and control cultures.     

A comparison of induced sister chromatid exchange levels in Chinese hamster ovary cells and cultured human lymphocytes

Human lymphocytes and Chinese hamster ovary cells were exposed to DNA damaging agents for two cell cycles, and the induced sister chromatid exchange (SCE) rate was compared. CHO cells showed a significant increase in SCE following treatment with bleomycin and vincristine whereas human lymphocytes did not. Both CHO cells and lymphocytes showed an increase in SCE with 5-bromodeoxyuridine (BrdUrd) and tritiated uridine (3H-Urd) but the increase was greater in CHO cells. SCE levels were similar after exposure to proflavine and colcemid did not increase the exchange frequency in either cell type. Apparently CHO cells are more sensitive than human lymphocytes to the actions of bleomycin, vincristine, BrdUrd, and 3H-Urd. SCE analysis in response to mutagens and carcinogens should therefore be based on more than one cell type.     

Relationship between carcinogenicity in rodents and the induction of sister chromatid exchanges and chromosomal aberrations in Chinese hamster ovary cells

Two independent analyses were carried out to compare the induction of sister chromatid exchanges and of chromosomal aberrations as predictors of carcinogenicity. Using both a classical and a Bayesian approach, as well as by analysis of the structural fragments generated by CASE, an artificial intelligence system, it is included that individually neither of these tests is a satisfactory predictor of carcinogenicity. However, because the analysis revealed that each of the cytogenetic assays responds to a different set of structural features associated with carcinogenicity, it can be concluded that the assays can be included in a battery of tests to improve predictivity.     

High frequency of in vivo sister chromatid exchanges in the bone marrow cells of mice bearing mammary adenocarcinoma dbrB

Sister chromatid exchanges (SCEs) in vivo were evaluated in the bone marrow cells of mice bearing mammary adenocarcinoma dbrB of 0.5 and 1.0 cm³, respectively. It has been observed that the SCE frequency is significantly higher (p < 0.001) in the bone marrow cells of tumor-bearing mice as compared to the bone marrow cells of non-tumor-bearing mice. SCE frequency in the bone marrow cells of mice with 1.0-cm³ tumors did not increase appreciably with tumor size as compared to that in the bone marrow cells of mice with o.5-cm³ tumors. This indicates the presence of mammary carcinoma-induced somatic stress as noted in the genetic material of the bone marrow cells of the host.     

Induction of chromosome aberrations and specific locus mutation but not sister chromatid exchanges in Chinese hamster ovary cells by neocarzinostatin

The induction of chromosome aberrations, sister chromatid exchanges (SCE), cytotoxicity, and 6-thioguanine-resistant mutation by neocarzinostatin (NCS) in Chinese hamster ovary cells was analyzed. It was observed that within the same concentration range of 0.01-0.1 mu/ml NCS, the drug induced a significant increase in all analyzed end-points except in SCE frequencies. There was no increase in SCE frequencies even when the cells were treated at the G1/S border in the first cell cycle and when aberrations were observed in the same cell showing a second cycle differential staining pattern. Our study indicates that the cellular damage induced by NCS leads to expression in chromosome aberrations, cytotoxicity, and mutagenicity but not in sister chromatid exchanges.     

Significant differences in the structural basis of the induction of sister chromatid exchanges and chromosomal aberrations in Chinese hamster ovary cells

The structural basis of the induction of sister chromatid exchanges (SCE) and chromosomal aberrations (Cvt) in Chinese hamster ovary cells was investigated by the CASE (Computer Automated Structure Evaluation) method, an artificial-intelligence-based system. Using the relevant National Toxicology Program data bases CASE identified a set of structural determinants responsible for the induction of SCE and another one for Cvt. A comparison between the structural determinants associated with SCE and Cvt revealed an overlap of only 22.6%, while the overlap between SCE and the determinants of mutagenicity in Salmonella is 54.5%. This indicates a) that the structural bases of the two phenomena differ and b) that it is likely that SCE, but not Cvt, involves a significant electrophilic/DNA-damaging component.     

Increased sister chromatid exchange frequency in bone marrow cells of myelodysplastic syndromes

The frequency and distribution of sister chromatid exchange (SCE) was determined in bone marrow and peripheral lymphocytes of patients with preleukemic myelodysplastic syndromes. Patients with refractory anemia (RA), RA with excess of blasts (REAB), RA with ring sideroblasts, and RA in transformation presented a 2-3-fold increase in SCE frequency in the bone marrow cells. In contrast, lymphocytes from these patients showed only a marginal increase in SCE. Analysis of interchromosomal distribution of SCE indicated a preferential involvement of chromosomes in group C in patients with RA with excess of blasts. Furthermore, the SCE in patients was not found to be influenced by the karyotype status.     

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.     

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.