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Spontaneously occurring cytogenetic aberrations were compared in 48- and 72-h human lymphocyte cultures from a group of 20 normal male volunteers. Mitotic indices for both cultures times were also evaluated. BUdR (5-bromo, 2-deoxyuridine) labelled metaphases from three men were utilized to determine the cell cycle kinetics in the medium used. There was no statistically significant difference between 48- and 72-h cultures among the majority of aberration categories considered. Possibly, the lymphocyte populations at the two different culture times maintained a consistent yield level. Mitotic indices in 72-h cultures were invariably higher than those in 48-h cultures. An overwhelming number of first division metaphases were observed at 48 h of incubation; however, approximately 22% of the lymphocytes did not enter into mitosis until 72 h in culture. A number of difficulties with regard to utilizing BUdR-labelled first division metaphases for cytogenetic analyses are discussed.Key words: BUdR, lymphocytes, mitosis, culture.

Probucol (4,4′-(isopropylidenedithio)bis(2,6-di- t -butylphenol)), a cholesterollowering drug, was evaluated for cytogenetic toxicity in the bone-marrow cells of Sprague-Dawley rats. Male and female rats were fed diets containing 0, 200, 400, or 800 mg/kg body weight/day for 8 consecutive days. Animals treated with trimethylphosphate served as positive controls. Femoral bone-marrow specimens were aspirated from all animals for cytogenetic evaluation. Analysis of the data generated by this study indicated that the incidence of cytogenetic aberrations in the bone marrow of rats was not affected by administration of probucol within the present treatment regimen.

This report presents the cytogenetic findings in bone marrow cells of rats exposed to styrene vapor. Male and female Sprague-Dawley rats were exposed to 0, 600 and 1000 ppm of styrene vapor by inhalation 6 hr per day, 5 days a week, for a period of one yr. Blind scoring of metaphase spreads prepared from bone marrow cells collected at the end of the last exposure revealed that neither the 600 ppm nor the 1000 ppm exposures to styrene vapor produced an incidence of chromosomal anomalies higher than those occurring spontaneously. It is interpreted that styrene is non-clastogenic within the present exposure regimen.

Peripheral blood samples from Sprague-Dawley rats gave successful lymphocyte growth in GIBCO: 1A, RPMI 1640, and Eagle's minimum essential medium (MEM) culture media. Various growth conditions, cytokinetics, and sister chromatic exchange (SCE) induction were studied using reconstituted GIBCO 1A only. Neither methoxyflurane anesthesia of the rats before sampling nor washing of the cells with phosphate buffered saline affected the mitotic index. Cultures treated with [3H]thymidine showed the lymphocytes entering into DNA synthesis after approximately 24 h. The time at which BUdR (5-bromo-2' deoxyuridine) was added, i.e. 0 vs. 24 h incubation, had minimal effect on the mitotic index of cultures harvested at 48 h. However, when harvest was extended to 72 h, mitotic activity was greater in the cultures treated with BUdR at 24 h. No significant differences in mitotic index and the number of average lymphocyte division were detected in cultures exposed to 0.3 to 0.5 microgram/ml BUdR at 24 h and harvested at 72 h. Although SCE frequencies increased in the presence of BUdR, the baseline level of SCEs was estimated to be 5 to 6/cell. Average generation time of the lymphocytes dividing between 48 and 72 h was 16.5 h. Because of its simplicity of culture and the reproducible nature of its in vitro growth kinetics, the Sprague-Dawley rat lymphocyte is a suitable model for cytogenetic investigations.

High doses of a test chemical sometimes affect the food consumption of the treated animals even to the point of starvation. The effect of such a non-specific toxicological stress on the bone marrow micronucleus test was investigated in male and female Sprague-Dawley rats. A 42-h starvation had a noticeable effect on the ratio of polychromatic to normochromatic erythrocytes (PCE and NCE); the values in the starved animals were approx. 77% of the negative controls. An effect of starvation on the frequency of micronucleated erythrocytes (MN-E) was not discernable from the results of the present experiment.

In vitro cytogenetic assays are widely conducted to assess the mutagenic potential of chemicals. Chinese hamster ovary (CHO) cells or human lymphocytes are often used for these assays; however, these cell types have certain limitations. In order to evaluate an alternate cell system, cultured rat lymphocytes were treated for 4 h at 48 h of incubation with a variety of direct- and indirect-acting clastogens in the presence or absence of an exogenous mammalian activation system. Cytogenetic effects of in vitro physiological alterations such as medium hypertonicity or pH changes were also evaluated. The background aberration rate of rat lymphocytes is approximately 2%, and they respond positively to both direct- and indirect-acting clastogens. In contrast to CHO cells, however, neither the hyperosmolality nor pH changes in the treatment media have significant effect on background aberrations. Unlike samples of human blood, rat blood can be collected under well-controlled environmental conditions. Because of the easy access to rat blood samples, the simplicity of culture, the reproducible nature of its in vitro growth, the positive response to known clastogens and negative response to media pH changes or hyperosmolalities, the rat lymphocyte in vitro chromosomal assay presented is an optimal system to assess the mutagenic potential of chemicals.