M. Ishidate

The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides

Ultra-vital staining with acridine orange (AO) is introduced into the micronucleus assay with mouse peripheral blood cells. Peripheral blood was stained vitally by dropping whole blood on an AO-coated slide and covering the sample with a coverslip. With this method, reticulocytes are identified easily by their red fluorescing reticulum structure. The distinction between young and mature erythrocytes was clearer and less subjective than the distinction between polychromatic and normochromatic erythrocytes by Giemsa staining or by conventional AO fluorescent staining. Although the induction of micronucleated peripheral reticulocytes (MNRETs) was delayed by about 12 h compared to that of micronucleated polychromatic erythrocytes (MNPCEs) in the bone marrow, the frequencies of MNRETs and MNPCEs were almost identical at each optimal sampling time. It is concluded that bone marrow cells can be replaced by peripheral blood as material for the micronucleus assay.

Kinetics of micronucleus formation in relation to chromosomal aberrations in mouse bone marrow

A simulation analysis of the kinetics of micronucleus formation in polychromatic erythrocytes in mouse bone marrow was performed after a single administration of 3 chemicals--mitomycin C (MMC), 6-mercaptopurine (6-MP) and 1-beta-D-arabinofuranosylcytosine (Ara-C)--with different modes of action. The time-response patterns in the incidence of chromosomal aberrations and micronuclei after treatment with each chemical were compared and subjected to the simulation study with 3 parameters. Two of them, the time between the final mitotic metaphase of the erythroid series and nucleus expulsion (T1), and the duration of the polychromatic erythrocyte (PCE) stage in the bone marrow (T2), were almost identical for the 3 chemicals. However, the coefficients of formation rate of micronucleated cells resulting from cells with chromosomal aberration(s) (k) differed: Ara-C differed from the other two. These results indicate that chromosomal aberrations, especially chromatid breaks and probably gaps, induced by this chemical, effectively contribute to micronucleus formation. The DNA content of micronuclei was also compared to the length of acentric fragments induced by Ara-C and it was found that their distributions were comparable. These findings strongly suggest that chromosomal aberrations induced by chemicals are essential events for the induction of micronuclei in the PCE of bone marrow.

Colchicine-like effect of diethylstilbestrol (DES) on mammalian cells in vitro ☆

Diethylstilbestrol (DES), a synthetic estrogen, showed colchicine-like effects in vitro on cells of the cell lines such as Chinese hamster fibroblast of thymus origin (CHT), rat liver (DL), rat erythroblastic leukemia (EDEN-1/TC) and HeLa-S3. Metaphase arrest was induced 3 h after treatment with 15 microgram/ml of DES and polyploid or polynucleated cells were prominently observed more than 24 h after treatment. The arrest, however, was reversible when the agent was removed from the medium. Tetraploid karotypes induced by DES in CHT cells consisted of all double sets of diploid chromosome constitution except one chromosome marker. By clonal selection, several hypotetraploid sublines were successfully isolated from a CHT cell population after the treatment with 15 microgram/ml of DES for 48 h. Some comparative studies of cytological effects of DES with those induced by colcemid indicated that the DES effect was also a mitotic inhibition similar to colchicine.

Clastogenicity in vitro of the Na, K, Ca and Mg salts of saccharin; and of magnesium chloride; consideration of significance

The sodium, potassium, calcium and magnesium salts of saccharin, and magnesium chloride, have been shown to be clastogenic to Chinese hamster lung (CHL) fibroblasts in vitro, but only at elevated dose levels (8-16 mg/ml). Saccharin acid was inactive to the limits of its solubility (4 mg/ml). When the data are expressed in terms of ionic concentration, each salt showed a similar clastogenic potency. This suggests that ionic effects induced by these salts in the assay medium may be the critical determinant of the clastogenic effects seen, rather than that the saccharin moiety presents a genotoxic insult to the chromosomes of the cells. The metal-chelating agents EDTA and EGTA were non-clastogenic, but the disodium salt of EDTA showed weak activity prior to toxicity at 0.5 mg/ml. The absence of a clastogenic response for the salts of saccharin at dose levels lower than 4 mg/ml is discussed within the context of the threshold-dependent tumour-promoting activity of high dose levels of sodium saccharin to the bladder of male rats. The doubtful value of conducting in vitro clastogenicity studies at dose levels greater than 10(-2) M is discussed.

Chromosome aberration assays in genetic toxicology testing in vitro

The chromosome aberration test using cultured mammalian cells is one of the sensitive methods to predict environmental mutagens and/or carcinogens, and is a complementary test to the Salmonella/microsome assay (Ames test). From our recent survey of 951 chemicals which have been tested for their clastogenicity in cultured mammalian cells such as Chinese hamster fibroblasts or human lymphocytes, it was noted that 47% of them are consistently positive either with or without metabolic activation. When the test was performed using the cell line CHL/IU, 39.2% (292/745) were found to be positive. However, 8% (36/447) of such clastogens were positive only at an extremely high concentration of more than 10 mM. About 11% (48/447) of clastogens such as diethylstilbestrol (DES) and methyl AalphaC (Glob-P-1) induced mainly polyploid cells. Most chemicals induced chromatid-type aberrations, some induce only break-type aberrations at relatively high dose levels, but others induce more exchange-type aberrations at relatively low dose levels. Clastogenic activities were compared among different clastogens, using the D20 value, which is the minimum dose (mg/ml) at which aberrations were found in 20% of metaphases. In addition, the translocation (TR) value was calculated from the incidence of cells with exchange-type aberrations. It was suggested that possible carcinogens are included in the group of compounds with relatively low D20 values, but with high TR values. Karyological analysis was performed, using a FISH painting probe prepared from No. 1 chromosome of CHO cells, on the clonal subline isolated after treatment with benzo(a)pyrene. However, no specific changes common to the agent were detected. Laser scanning cytometry (LSC) was also applied to screen for abnormal karyotypes. A translocation between particular chromosomes was reflected by the deletion of a DNA peak.

Differences in liver homogenates from Donryu, Fischer, Sprague-Dawley and Wistar strains of rat in the drug-metabolizing enzyme assay and the salmonella/hepatic S9 activation test

Comparison studies for detecting differences between liver microsome and S9 preparations from 4 strains (Donryu, Fischer, Sprague-Dawley, Wistar) of young male rats were carried out with pretreatment of the animals by inducers such as PCBs and PB plus 5,6-BF. Each microsome fraction was assayed for the enzymic activity of metabolism of model substrates such as aniline, benzophetamine, BP, DMN and 7-ethoxycoumarin. The hepatic S9 sample was also compared, as regards its metabolizing ability to activate 9 pre-mutagens (2AA, AAF, o-AAT, BP, DAB, DMBA, DMN, m-PDA, quinoline) to directly acting mutagens in the Salmonella/hepatic S9 activation test by using TA98, TA100 and TA1537 strains with or without cytochrome P450 inhibitors (SKF-525A, metyrapone, 7,8-benzo-flavone). In the enzymic assay with PCBs-induced microsomes, BP hydroxylation a strain-specific difference: the microsomes from Fischer and Wistar rats were more effective for metabolizing BP than those from the other strains of rat. The effect of induction by BP plus 5,6-BF for Fischer rats showed relatively higher enzymic activity in the same induction group. Other microsomes prepared from rats with and without induction by PB plus, 5,6-BF did not show a clear-cut strain dependency in the enzymic activities assayed. In the mutation experiments with hepatic S9 samples, the examination of DAB and quinoline revealed a marked strain difference when S9 samples prepared from PCBs-pretreated and PB-plus-5,6-BF-induced rats were used: the S9 sample from Fischer rats was available for activating the two pre-mutagens to directly acting mutagens. No marked difference in the metabolic activation of the remaining 7-pre-mutagens was observed on other S9 preparations. In examinations of mutagenicity activities with the use of three inhibitors, the two S9 preparations made with the two induction methods showed inhibition profiles closely similar to each other. However, there were minor differences in the profiles by these inhibitors. From these findings it was concluded that Fischer rat-liver S9 is useful for detecting mutagens in the metabolic activation test, when induction by PB plus 5,6-BF was used in the Ames Salmonella test.

Species difference in the metabolic activation of phenacetin by rat and hamster liver microsomes

Phenacetin is mutagenic in Salmonella typhimurium TA 100 when liver 9,000 X g supernatant fractions from PCB-treated hamsters instead of rats are used. A mechanism of the species difference in phenacetin mutagenicity was investigated. By high-performance liquid chromatography analysis, it was found that phenacetin is activated to direct-acting mutagens through N-hydroxylation and deacetylation by hamster liver microsomes. Although no significant species difference was observed in N-hydroxylation, rates of deacetylation were 9 to 150 times higher in hamsters than in rats. The results indicate that the marked species difference in phenacetin mutagenicity is due to the difference in deacetylation activity between rat and hamster liver microsomes.

Induction of chromosomal aberrations in active oxygen-generating systems. I. Effects of paraquat in Chinese hamster cells in culture.

A possible role for the superoxide anion radical (O2-) in the clastogenicity of paraquat (PQ) was investigated in cultured Chinese hamster cells. When cells were treated with 0.8 mg/ml of PQ for 3 h followed by 21 h of recovery time, structural chromosome aberrations were induced in about 50% of the metaphases examined. Almost all aberrations were of the chromatid-type and involved exclusively gaps and breaks. The induction of chromosomal aberrations by PQ was enhanced by a 1-h pretreatment with diethyldithiocarbamate, an inhibitor of superoxide dismutase. Diethyl maleate, a glutathione scavenger, also enhanced the induction of chromosomal aberrations, but 3-aminotriazole, an inhibitor of catalase, showed no such effects. Enhanced induction of chromosomal aberrations was also observed when PQ-treated cells were cultured at a high oxygen concentration (80%). The present results suggest that the production of chromosomal aberrations by PQ may be directly or indirectly related to the generation of O2-, but not to the formation of hydrogen peroxide by the dismutation reaction of O2- or of other active oxygen species including the hydroxyl radical and singlet oxygen.

Multiple-dosing effects of benzo[a]pyrene in the mouse bone marrow micronucleus test

Multiple-dosing effects of benzo[a]pyrene (B[a]P) in the micronucleus test were studied using CD-1 male mice. Mice were treated orally once, twice or 3 times with 250, 500, 1000 or 2000 mg/kg, at 24-h intervals. Bone marrow cells were sampled 24 h after the last administration. The present study indicated that the incidence of polychromatic erythrocytes with micronuclei significantly increased more in the group of animals that received B[a]P twice than in those receiving it one or 3 times. The dose of 500 mg/kg B[a]P yielded the greatest response of any dose regimen.