Miriam F. Bryant

Induction of micronuclei by X-radiation in human, mouse and rat peripheral blood lymphocytes.

We compared the radiosensitivity of human, rat and mouse peripheral blood lymphocytes (PBLs) by analyzing micronuclei (MN) in cytochalasin B-induced binucleated (BN) cells. For each species and dose 4-ml aliquots of whole blood were X-irradiated to obtain doses of 38, 75, 150 or 300 cGy. Controls were sham-irradiated. After exposure to X-rays, mononuclear leukocytes were isolated using density gradients and cultured in RPMI 1640 medium containing phytohemagglutinin to stimulate mitogenesis. At 21 h cytochalasin B was added to produce BN PBLs, and all cultures were harvested at 52 h post-initiation using a cytocentrifuge. Significant dose-dependent increases in the percentage of micronucleated cells and the number of MN per BN cell were observed in all three species. The linear-quadratic regression curves for the total percentage of micronucleated cells for the three species were similar; however, the curve for the mouse PBLs had a larger quadratic component than either of the curves for the rat or human PBLs. Although the correlation between the percentage of cells with MN and those with chromosome aberrations was high (r2 greater than 0.95), the mouse and rat PBLs were over twice as efficient as human PBLs in forming MN from presumed acentric fragments. These data indicate that the induction of MN in BN cells following ionizing radiation is similar in human, rat and mouse PBLs, but care must be taken in using the MN results to predict frequencies of cells with chromosomal aberrations.

Cytogenetic studies of mice exposed to styrene by inhalation

The data for the in vivo genotoxicity of styrene (STY) are equivocal. To evaluate the clastogenicity and sister-chromatid exchange (SCE)-inducing potential of STY in vivo under carefully controlled conditions, B6C3F1 female mice were exposed by inhalation for 6 h/day for 14 consecutive days to either 0, 125, 250 or 500 ppm STY. One day after the final exposure, peripheral blood, spleen, and lungs were removed and cells were cultured for the analysis of micronucleus (MN) induction using the cytochalasin B-block method, chromosome breakage, and SCE induction. Peripheral blood smears were also made for scoring MN in erythrocytes. There was a significant concentration-related elevation of SCE frequency in lymphocytes from the spleen and the peripheral blood as well as in cells from the lung. However, no statistically significant concentration-related increases were found in the frequency of chromosome aberrations in the cultured splenocytes or lung cells, and no significant increases in MN frequencies were observed in binucleated splenocytes or normochromatic erythrocytes in peripheral blood smears.

Inhalation studies of the genotoxicity of trichloroethylene to rodents

Trichloroethylene (TCE) (CAS No. 79-01-6) is an industrial solvent used in degreasing, dry cleaning, and numerous other medical and industrial processes. Controlled inhalation studies were performed using male C57BL/6 mice and CD rats to determine if TCE can induce cytogenetic damage in vivo. Animals were exposed in groups of five to target concentrations of either 0, 5, 500, or 5000 ppm TCE for 6 h. Tissue samples were taken between 18 and 19 h post exposure. Peripheral blood lymphocytes (PBLs) in rats and splenocytes in mice were cultured and analyzed for the induction of sister-chromatid exchanges, chromosome aberrations, and micronuclei (MN) in cytochalasin B-blocked binucleated cells. Bone marrow polychromatic erythrocytes (PCEs) were analyzed for MN. The only positive response observed was for MN in rat bone marrow PCEs. TCE caused a statistically significant increase in MN at all concentrations, inducing an approximate fourfold increase over control levels at 5000 ppm. TCE was also cytotoxic in rats, causing a significant concentration-related decrease in the ratio of PCEs/normochromatic erythrocytes. This study indicates that there may be species-specific cytogenetic effects attributed to TCE inhalation exposure. In follow-up studies, CD rats were exposed for 6 h/day over 4 consecutive days to either 0, 5, 50 or 500 ppm TCE. No statistically significant concentration-related increases in cytogenetic damage were observed. While the MN frequencies in the 4-day study were comparable to those at the equivalent concentrations in the 1-day study, they were not significantly elevated due to an unusually high MN frequency in the controls. A subsequent replication of the 1-day 5000 ppm TCE exposure with rats again showed a highly significant increase in MN frequencies compared to concurrent controls.

A comparison of sister-chromatid exchange in mouse peripheral blood lymphocytes exposed in vitro and in vivo to phosphoramide mustard and 4-hydroxycyclophosphamide

Cyclophosphamide (CP) and two of its known metabolites, 4-hydroxycyclophosphamide (4-OHCP) and phosphoramide mustard (PAM), were analyzed for their ability to induce sister-chromatid exchanges (SCEs) in mouse peripheral blood lymphocytes (PBLs) in vitro and in vivo. At equimolar concentrations, CP is a more potent SCE inducer in vivo than PAM and PAM and 4-OHCP induce equal numbers of SCEs in a dose-dependent manner. The present study also shows that these metabolites of CP are more potent SCE inducers than CP itself in vitro. This relationship might be explained by the differences in pharmacokinetics of these compounds.

Comparison of sister-chromatid exchange frequencies in mouse T- and B-lymphocytes after exposure to 4-hydroxycyclophosphamide or phosphoramide mustard.

The present study was designed to investigate the genotoxicity of 4-hydroxycyclophosphamide (4-OHCP) and phosphoramide mustard (PAM), both reactive metabolites of cyclophosphamide (CP), for possible differences in SCE-inducing activity in mouse T- and B-lymphocytes. Mouse peripheral blood lymphocytes were isolated and stimulated to divide with either phytohemagglutinin (T-cell mitogen) or lipopolysaccharide (a polyclonal B-cell activator). Significant concentration-dependent increases in SCE frequencies were observed for both 4-OHCP and PAM with both mitogens, with 4-OHCP being almost twice as potent as PAM. There was no difference in SCE response between T- and B-lymphocytes after exposure to either PAM or 4-OHCP. These data do not support the idea that the difference in SCE response in T- and B-lymphocytes by CP in vivo is due to differential responses to either of the proposed putative metabolites of CP.