G. Krishna

In vivo cytogenetic studies on mice exposed to ethylene dibromide

The pesticide, ethylene dibromide (EDB), was evaluated with in vivo cytogenetic assays to determine its genotoxicity. CD1 male mice were exposed to EDB through intraperitoneal injections. Bone marrow cells isolated from femora were analyzed for sister-chromatid exchange (SCE), chromosome aberration and micronucleus formation. The results showed that only certain concentrations of EDB tested caused a slight but significant increase in SCEs and chromosome aberrations. However, these increases were not dose-related. No increase in the polychromatic erythrocytes with micronuclei was observed following EDB exposure. Also, EDB did not cause cell-cycle delay in comparison with controls. Thus, it appears that EDB is not an effective genotoxic agent in vivo in mice.

Comparative in vivo and in vitro genotoxicity studies of airborne particle extract in mice

The genotoxicity of an acetone extract of locally collected airborne particles was evaluated both in vitro and in vivo using the sister-chromatid exchange (SCE) assay in mice. At the highest concentration (5.36 mg/5 ml culture), the extract caused approximately a 3-fold increase in SCEs over controls in mouse bone marrow and spleen primary cells in vitro. However, the same airborne particle extract did not induce a significant increase in the SCE level over controls in vivo in mouse bone marrow and spleen cells when administered intraperitoneally or through oral gavage. This indicates that bone marrow and spleen primary cell cultures can be used in in vitro genotoxicity studies of complex mixtures, and that the genotoxicity of airborne particles detected in the in vitro system cannot always be detected in vivo with the same cell types. In addition, the same acetone extract of airborne particles caused dose-related his+ revertants in the strain TA98 of Salmonella typhimurium, both with and without S9 activation. The significant finding of this study is that the in vitro genotoxicity results of airborne particle extract may not be very meaningful in an in vivo situation.

In vivo and in vivo/in vitro kinetics of cyclophosphamide-induced sister-chromatid exchanges in mouse bone marrow and spleen cells

In several acute and chronic exposures to various chemicals in vivo and in vitro, the average sister-chromatid exchange (SCE) frequencies in human, mouse, rat, and rabbit lymphocytes generally decrease with time following treatment. The rate of this decline varies, but little data have been published pertaining to the comparative kinetics of SCEs both in vivo and in vivo/in vitro (exposure of animals to the test compound and culturing of cells) simultaneously in the same tissues. In this study, a single dose of cyclophosphamide (40 mg/kg) was injected for varying periods (6-48 h) and its effects, as assessed by the induction of SCEs, were analyzed under both in vivo and in vivo/in vitro conditions in mouse bone marrow and spleen cells. In vivo, the cyclophosphamide-induced SCEs increased with increasing time up to 12 h, stayed at approximately the same level until 24 h, and then decreased with increase in post-exposure time. However, the SCE levels remained significantly higher than controls at 48 h post-exposure time in both bone marrow and spleen cells. Under in vivo/in vitro conditions, the SCEs in bone marrow decreased with increase in post-exposure time until reaching control values by 48 h post exposure. However, in spleen cells, the decrease in SCE level was gradual, and by 48 h post-exposure time, the cells still had approximately 6 times higher SCEs than the control values. These results suggest that there are pharmacokinetic differences for cyclophosphamide in mouse bone marrow and spleen. Also, there is a differential SCE response to cyclophosphamide under in vivo and in vivo/in vitro conditions.

Murine bone marrow culture system for cytogenetic analysis

A mouse bone marrow culture system for examining genotoxicity of agents by first exposing animals in vivo then growing cells in vitro is presented. This assay can also be used for in vitro and/or for the in vivo and in vitro comparative cytogenetic studies. The protocol involves culturing of approximately 1,000,000 nucleated cells obtained from mice tibia and femora in 5 ml of Hams F-12 medium containing 20% fetal bovine serum, 10% whole uterus extract from pregnant mice and 1% penicillin-streptomycin. The use of flasks and mouse uterus extract for culturing are important steps for higher mitotic yield. The addition of 20 microM BrdU for 24 h helps in the differentiation of sister chromatids for sister-chromatid exchange (SCE) analysis. Cyclophosphamide, given to mice through intraperitoneal injection, induced significant dose-related SCEs in culture. Trinitrofluorenone, a direct-acting mutagen, caused dose-related SCEs in in vitro bone marrow cell culture.

Preparation of mouse bone marrow primary cultures for sister chromatid exchange and chromosomal aberration studies

A procedure for preparing and culturing mouse bone marrow cells for cytogenetic studies is described. Animals are killed by cervical dislocation, the bone marrow is flushed from femora and tibia with Hams F12 medium into centrifuge tubes. Bone marrow cells are collected by low-speed centrifugation (285 ×g). One million cells are suspended in a 30-ml Falcon flask with complete medium containing 3.45 ml Hams F12 medium, 1 ml fetal bovine serum, 0.05 ml penicillin-streptomycin, and 0.5 ml whole uterus extract from pregnant mice. For sister chromatid exchange analysis, 20 µM of 5-bromo-2′ -deoxyuridine is also added to the culture medium for 24 to 28 h. The cell cycle is approximately 12 to 14 h, in culture. This culture system can also be used for chromosomal aberration studies.

In vivo induction of sister chromatid exchanges in mice by nitrosated coal dust extract.

The genotoxicity of coal dust extract nitrosated with sodium nitrite (NaNO2) was investigated in mice with the in vivo sister chromatid exchange (SCE) assay system. The SCEs in bone marrow cells of mice were examined following single and double oral dosings of coal dust extract, NaNO2, and nitrosated coal dust extract. Coal dust extract and NaNO2 separately did not cause significant increases of SCEs either in single or in double dosings. Nitrosated coal dust extract in single doses did not increase SCEs but in two doses significant increases in SCEs were observed (P less than 0.02). The mutagenicity of the same extracts was tested in Salmonella typhimurium with the Ames tester strain TA98. Coal dust extract was either non- or weakly mutagenic and NaNO2 was nonmutagenic. The nitrosated coal dust extract caused pronouced increases in his+ revertants both with and without rat liver S9 activation. These findings provide additional evidence that nitrosation of ingested coal dust which may occur in the stomach environment could be one of the factors leading to the higher incidence of gastric cancer in coal miners.

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

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 Hams 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 Hams 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 mMl-glutamine solution, 1 × 10−5M 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.

Sister-chromatid exchange studies on direct- and indirect-acting clastogens in mouse primary cell cultures

An in vitro sister-chromatid exchange (SCE) assay using mouse primary bone marrow and spleen cells was conducted with both direct- and indirect-acting genotoxic agents. 2,4,7-Trinitrofluorenone, a direct-acting genotoxic agent, induced a significant dose-related increase in SCEs. In both bone marrow and spleen cells, 2.0 micrograms/ml caused an approx. 3-fold increase in SCE level over control values. Cyclophosphamide, an indirect-acting genotoxicant which requires metabolic activation for its clastogenicity, induced a significant increase in SCEs in the presence of S9 from liver of rats pretreated with Aroclor-1254. A dose of 2 micrograms/ml resulted in a 2-fold increase in bone marrow and a greater than 5-fold increase in spleen cells. Benzo[a]pyrene, another indirect-acting genotoxicant, also induced significant dose-related SCE responses in both cell types. It seems that primary bone marrow and spleen cell culture systems can detect both direct- and indirect-acting genotoxicants and may be useful for routine and/or comparative cytogenetic studies.