Morphological changes in hepatic Ito cells after parenteral treatment with carmellose sodium in rats.

Carmellose sodium (1.5%), dissolved in physiological saline, was given for 4 days via oral, subcutaneous or intraperitoneal routes. In the rats treated with carmellose sodium by the parenteral route, hepatic sinusoidal component cells and free macrophages were swollen. The degree of the swelling was more severe in the rats treated by the intraperitoneal route than by the subcutaneous route. The swollen sinusoidal component cells which engulfed the carmellose sodium-related substance were classified as Kupffer's cells or Ito cells by immunohistochemical reaction. Ito cells were isolated from a 5-week-old rat and were cultured in Dulbecco's modified Eagle's medium (DMEM). Some of the Ito cells which were cultured in DMEM containing 0.3% carmellose sodium showed phagocytic properties; vesicles in their cytoplasm and other Ito cells which were cultured in DMEM containing India ink contained some carbon particles.

Micronucleus test with vincristine sulfate and colchicine in peripheral blood reticulocytes of mice using acridine orange supravital staining.

The induction of micronuclei in mouse peripheral blood reticulocytes (RETs) was studied with the spindle poisons vincristine sulfate (VINC) and colchicine (COL) using acridine orange (AO) supravital staining. Each chemical was studied independently in two laboratories using the same protocol. Blood samples were prepared at 0, 24, 48, and 72 h after a single intraperitoneal treatment with VINC (0.0625, 0.125, and 0.25 mg/kg) or COL (0.25, 0.5, 1.0, and 2.0 mg/kg). Both VINC and COL induced micronucleated RETs (MNRETs) significantly and dose-dependently with a peak at 48 h after treatment. Maximum frequencies of micronucleated polychromatic erythrocytes (MNPCEs) were observed 24 h after treatment with VINC; thus, the transition time from MNPCEs to MNRETs was about 24 h. Both spindle poisons gave comparable results in the paired laboratories, indicating that the present AO supravital staining method is highly reproducible.

Enhancement of cytogenetic and cytotoxic effects on multidrug-resistant (MDR) cells by a calcium antagonist (verapamil).

The cytogenetic effects of a calcium antagonist, verapamil, on anticancer antibiotic-induced chromosomal damage and cytotoxicity were studied in multidrug-resistant (MDR) Chinese hamster ovary (CHO) cells in vitro. Nine colchicine-resistant (CHr) sublines were obtained by stepwise culturing with increasing concentrations of colchicine. Compared with the parent CHO cells, CHr sublines exhibited an approximately 2.6- to 120-fold higher resistance to colchicine. CHr sublines were cross-resistant to mitomycin C (MMC), actinomycin D (ACD), daunomycin (DM), bleomycin (BLM) and adriamycin (ADM). These anticancer antibiotics are known to induce chromosomal aberrations in various cell types. However, one MDR subline, CHr-500, showed resistance to induction of chromosomal aberrations by MMC. In CHr-500 cells, verapamil at a non-toxic concentration of 10 micrograms/ml enhanced the MMC-induced chromosomal damage and cytotoxicity to the levels seen in the sensitive parent cells. The increase in chromosomal damage in the presence of verapamil was correlated with the increase in cytotoxicity.

Micronucleus test with vincristine administered by intraperitoneal injection and oral gavage.

The effects of vincristine sulfate (VINC) on micronucleus induction were studied in 2 strains of mice (MS/Ae: CD-1) following intraperitoneal (i.p.) or oral administration (p.o.) of the chemical. On the basis of a small-scale acute toxicity study and a pilot micronucleus experiment, the full-scale micronucleus test was performed with a sampling time of 24 h at doses of 0.063, 0.125, 0.25 and 0.5 mg/kg (i.p.) and 1.25, 2.5, 5.0 and 10 mg/kg (p.o.). The maximum frequency of micronucleated polychromatic erythrocytes was 7.15% in MS/Ae mice and 4.98% in CD-1 mice at 5.0 mg/kg p.o. in both cases. The maximum frequencies by the i.p. route (9.93% in MS/Ae mice; 11.68% in CD-1 mice) occurred at 0.25 mg/kg and 0.125 mg/kg, respectively. Although the doses showing a positive response were different between the 2 routes, VINC induced micronuclei very efficiently at all doses tested by both administration routes in both strains.

Spontaneous expulsion of micronuclei by enucleation in the micronucleus assay.

The effect of enucleation on the frequency of micronuclei induced by mitomycin C (MMC) and vincristine (VCR) was examined in mouse L-929 cells enucleated with cytochalasin B (Cyt-B). Approximately 30% of the L-929 cells became enucleated cells during the 8-h incubation in medium containing 8 micrograms/ml of Cyt-B. Using this enucleation technique, we estimated the reduction rate of 2 mutagen-induced micronuclei by enucleation. Treatment with MMC caused a dose-dependent induction of micronuclei in L-929 cells, with the reduction rate being 38.6% at the lowest dosage (0.0125 microgram/ml), which induced mostly mono-micronuclei in L-929 cells, and 6.8% at the highest dosage (0.1 microgram/ml), which induced many multi-micronuclei. Furthermore, VCR also induced micronuclei in a dose-dependent way in L-929 cells, and the same tendency for micronucleus reduction as with MMC was observed. The reduction rate of micronucleated cells by enucleation was estimated to be about 31-39% when the micronucleated cells contain mono-micronuclei. Therefore, the rate of reduction is affected by the number of micronuclei per cell, and the reduction depends on the increase in the number of micronuclei per cell.

In vitro micronucleus method with erythropoietin-differentiated erythrocytes.

A new in vitro micronucleus method has been developed for the detection of chromosomal damage in mouse bone-marrow erythrocytes which are differentiated with erythropoietin (EPO). After the hemolysis of bone-marrow erythrocytes, newly-formed polychromatic erythrocytes (PCEs) were observed in cultures incubated in the presence of EPO. Using the erythroid differentiation technique, increased frequencies of micronuclei were detected in PCEs after treatment with mitomycin C. In addition, the in vitro micronucleus method was found to be more sensitive than the in vitro chromosomal aberration test on Chinese hamster cells. The results show that the in vitro micronucleus method using EPO-differentiated erythrocytes is a highly sensitive and useful system for the detection of mutagens.