Region-specific chromatin decondensation and micronucleus formation induced by 5-azacytidine in human TIG-7 cells.

A human diploid lung fibroblast cell strain, TIG-7, has a heteromorphic chromosome 15 with an extra short arm carrying a homogeneously staining region (15p+hsr). We demonstrated previously that the 15p+hsr consists of an inactive and G+C-rich rDNA cluster characterized by fluorescence in situ hybridization (FISH) and various chromosome banding techniques. Thus, it was suggested that the region could contain highly methylated DNA. To observe methylation status on the target region directly under the microscope, we used a demethylating agent, 5-azacytidine (5-azaC), to induce decondensation of the chromatin containing methylated DNA. At 24 h after treatment with 0.5 microM 5-azaC, marked decondensation of the 15p+hsr was observed in almost all of the metaphases. Furthermore, we observed micronuclei, which were equivalent to the rDNA of the 15p+hsr demonstrated by FISH in the same preparation. In contrast, the DNA cross-linking agent mitomycin C (MMC) preferentially induced 15p+hsr-negative micronuclei. These findings indicated that chromatin decondensation and subsequent DNA strand breakage induced by the demethylating effect of 5-azaC led specifically to 15p+hsr-positive micronuclei.

Validation study of the in vitro micronucleus test in a Chinese hamster lung cell line (CHL/IU).

We conducted a collaborative validation study, under the auspices of the Japanese Ministry of Labour, on the in vitro micronucleus test to see if it could be used as an alternative to the in vitro chromosome aberration test for evaluation of chemical safety. We used the Chinese hamster lung cell line (CHL/IU), which is the most widely used system for the latter test in Japan, and evaluated 66 chemicals, including clastogens and polyploidy inducers. The cytochalasin B cytokinesis blocking method, which is commonly used in human lymphocyte culture, was applied to the established cell line, but did not improve the detection of chemically-induced micronuclei in continuously growing cells. The highest micronucleus frequencies were obtained at 48 or 72 h continuous treatments. In short treatments (6 h), a 42 h recovery time yielded the best responses. Concordance between the results of the micronucleus test and the chromosomal aberration test was satisfactorily high (88.7%), and we concluded that the in vitro micronucleus test could be used in place of the chromosomal aberration test as a simple and rapid method for detecting clastogens and aneugens in vitro. We also propose a protocol for the test.

Application of laser scanning cytometry to the analysis of chromosomal aberrations induced by benzo[a]pyrene in CHO-WBLT cells.

BACKGROUND: A recently developed laser scanning cytometry technique was applied to cytometric studies to detect rapidly stable chromosomal aberrations induced by a carcinogen in a Chinese hamster fibroblast cell line, CHO-WBLT. METHODS: Individual chromosomes were collected from metaphase cells by a syringe technique and spread on slides. The DNA content of each chromosome stained with propidium iodide was measured with a laser scanning cytometer (LSC). A characteristic DNA histogram, designated as the "laser scanning karyotype (LSK)," was obtained from about 20,000 chromosomes of CHO-WBLT cells. Each chromosome was confirmed morphologically under the microscope by using a "re-location" system built into the LSC. RESULTS: A total of 21 chromosomes, including marker chromosomes specific to the cell line, were assigned to 10 major peaks in the LSK, which was analogous to the karyotype demonstrated with the classical Q-banding technique. In contrast, clonal sublines isolated after exposure to the carcinogen benzo[a]pyrene showed LSKs different from those found in untreated control cells, and seven of 20 clones were found to be abnormal, with a small number of chromosomal translocations and/or deletions, which were confirmed by Q-banding. CONCLUSIONS: The laser scanning cytometry technique was employed to detect stable chromosomal aberrations in CHO-WBLT cells after treatment with benzo[a]pyrene. The results obtained with this technique were comparable to those obtained by Q-banding; therefore, this method may be useful for rapid primary screening to detect stable, abnormal karyotypes induced by environmental chemicals and/or radiation.

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.

Thiopronin reduces the frequencies of neocarzinostatin-induced chromosomal aberrations and sister chromatid exchanges in Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells were treated in the G1 phase of the cell cycle with different concentrations of neocarzinostatin (NCS) alone or in combination with N-(2-mercaptopropionyl)-glycine (thiopronin; TP). TP reduces the frequencies of NCS-induced chromosomal aberrations (CA) and of sister chromatid exchanges (SCE) significantly when added to the cultures simultaneously (TPsim), 1 min (TP1) or 10 min (TP10) after the addition of NCS. The addition of TP 30 min (TP30) or 60 min (TP60) after NCS reduces the frequencies of SCE, but not of CA. Our results indicate that the induction of CA and SCE by NCS is partially based on different mechanisms.

The possible role of acetyltransferase in the induction of cytogenetic effects by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in cultured Chinese hamster cells.

When metabolically activated, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a heterocyclic amine isolated from cooked food, is clastogenic in cultured Chinese hamster and human cells. Secondary metabolites of PhIP are formed via acetyltransferase (AT) and sulfotransferase (ST) activity; however, which is responsible for its clastogenic effect is unknown. We addressed this question. We used a parental Chinese hamster lung cell line and three sublines transfected with different AT genes to test the clastogenic (i.e., micronucleus-inducing) effects of metabolically activated PhIP and 7,12-dimethylbenz[a]anthracene (DMBA) in the presence and absence of pentachlorophenol (PCP), a ST inhibitor. PhIP was significantly more clastogenic in the three AT-enriched sublines than in the parental line (p < 0.001). DMBA (a ST-activated mutagen), on the other hand, equally induced MNs in all the cell lines. When PCP was added to the test system, the MN-induction ability of DMBA, but not of PhIP, decreased significantly (p < 0.001). These findings strongly suggest that PhIP clastogenicity is due to AT activity and not to ST activity.

Re-evaluation of chromosomal aberration induction on nine mouse lymphoma assay "unique positive' NTP carcinogens.

In a collaborative study organized under the JEMS MMS, nine mouse lymphoma assay (MLA) "unique positive' NTP rodent carcinogens were re-evaluated by an in vitro chromosomal aberration assay using Chinese hamster lung fibroblast cells (CHL/IU). Six of nine chemicals induced chromosomal aberrations; bromodichloromethane, chlorendic acid and isophorone induced structural aberrations, and chlorodibromomethane, pentachloroethane and 1,1,1,2-tetrachloroethane induced numerical aberrations (polyploidy). These six chemicals, therefore, are not uniquely positive in the MLA. The difference between the NTP results and ours might be due to the use of different cell lines and protocols, and in some cases, to different interpretations of polyploidy. The remaining three chemicals, benzyl acetate, cinnamyl anthranilate and trichloroethylene, were negative in this study.

Cytogenetic effects of a food mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-beta]pyridine (PhIP), and its metabolite, 2-hydroxyamino-1-methy-6-phenylimidazo[4,5-beta]pyridine (N-OH-PhIP), on human and Chinese hamster cells in vitro.

2-Amino-1-methyl-6-phenylimidazo[4,5-beta]pyridine (PhIP) induced structural chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) in human lymphocytes and human diploid fibroblasts (TIG-7) at concentrations above 12.5 microgram /ml in the presence of rat S9 mix. PhIP also elevated the frequencies of SCEs in human lymphocytes in the presence of rat S9 at concentrations above 2.0 microgram/ml with dose-dependency. A proximate form of metabolites of PhIP, 2-hydroxy-amino-1-methyl-6-phenylimidazo[4,5-beta]pyridine (N-OH-PhIP), caused CAs in human and Chinese hamster fibroblast cells in the absence of S9 mix at concentrations above 0.75 microgram/ml and 1.25 microgram/ml, respectively, which were 10 times lower than the effective concentration of PhIP. No marked differences were observed in the cytogenetic sensitivity to N-OH-PhIP between human and Chinese hamster cells, except between lymphocytes obtained from different donors.

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ), a carcinogenic pyrolysate, induces chromosomal aberrations in Chinese hamster lung fibroblasts in vitro.

The ability of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) to induce chromosomal aberrations (CAs) in Chinese hamster lung fibroblast (CHL/IU) cells in vitro was examined. On incubation with rat S9 (2.5-10%, v/v) for 3 h, followed by a recovery culture period of 21 h, IQ caused significant induction of CAs at a concentration 20 micrograms/ml, but had less effect at 40 micrograms/ml. With longer recovery culture times such as 27-33 h, however, IQ was much more effective at 40 micrograms/ml. No significant induction was observed with 1 or 6 h treatments followed by 23 or 18 h recovery cultures, respectively. On incubation without S9, only weak CA induction by IQ was observed. These results show that IQ is a clastogen and that its clastogenic effect varied with the experimental conditions, such as the time of exposure and the time of recovery culture. The cell cycle perturbation effect is suggested to be one of the critical factors for the detection of the clastogenic potential of IQ.