Optimized conditions for an in vitro micronucleus (MN) test procedure were defined using a chamber slide that enabled the preparation of fine specimens without undergoing complicating procedures using culture dishes. The issues investigated are 1) the effect of slide materials on the adhesion of cells, 2) the number of seeding cells necessary to obtain an adequate number of cells for observation and 3) effects of hypotonic treatment and fixation on the cytoplasmic: nuclear area ratio. In addition, we determined cell viability in each chamber using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The results of the investigation were as follows: 1) cell adhesion was best using plastic slides, 2) the optimum number of cells for seeding was 6.6×103 cells/cm2, 3) the best condition for hypotonic treatment was incubation in 75 mM KCl at 37°C for 5 min, and 4) the best condition for fixation was treatment of cells twice for about 2 min in icecold methanol containing 6% acetic acid. Finally, the result of the MTT assay correlated with the number of viable cells in chamber as determined by the trypan blue dye exclusion assay. An in vitro MN test was conducted under these conditions using the known clastogens, Mitomycin C and dimethylnitrosamine. These clastogens dose-dependently induced a significant increase in the number of micronucleated cells with positive responses at concentrations approximately 10 times lower than those of the chromosomal aberration test. On the other hand, the frequency of micronucleated cells in the solvent control was stable and low (0.4-1.7%). These results indicated that the in vitro MN test has a high level of sensitivity to clastogens. It was concluded that the in vitro MN test using chamber slides is a rapid, simple and sensitive method to detect clastogens.
Arsenic compounds contained in sea foods have raised public health concerns, because their chronic exposure through dietary intake may increase cancer risk. In the present study, we investigated the in vitro genotoxicity of two inorganic arsenics (arsenite; As[III], arsenate; As[V]) and three organic arsenics (monomethylarsonic acid; MMAA, dimethylarsenic acid; DMAA, trimethylarsine oxide; TMAO) using mouse lymphoma Tk assay (MLA). In the standard MLA with 3 h treatment, exposure to As[III] and As[V] significantly induced Tk-mutants. The genotoxicity of As[III] was over 50-times greater than that of As[V]. Among organic arsenics, on the other hand, only DMAA showed weak genotoxicity with 3 h treatment at high doses. In the 24 h treatment MLA, DMAA and TMAO weakly induced Tk-mutants. These results indicate that inorganic arsenics rather than organic arsenics should be considered for genotoxic risk. We discussed the genotoxic risk of arsenic compounds through dietary intake.
The cytotoxic and genotoxic effects of goniothalamin, a plant styryllactone, were evaluated using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and the Alkaline Comet assay respectively in human leukemic cell lines. Following 72 h of treatment, the IC50 values of goniothalamin in human HL-60 promyelocytic leukemia cells and CEM-SS T-lymphoblastic cells were 4.5 μg/mL and 2.4 μg/mL respectively. The genotoxicity of goniothalamin in both HL-60 and CEM-SS cells was detected as early as 2 h following treatment at IC10 and IC25 concentrations. However, pretreatment with the antioxidant N-acetyl-cysteine (NAC) at 1 mM for 30 minutes did not abrogate genotoxicity of this compound. This result suggests that primary induction of DNA damage by goniothalamin may not involve oxidative damage. In conclusion, our results demonstrate genotoxic damage induced by goniothalamin in leukemic cells. Further studies are needed to ascertain the mode of action of goniothalamin in inducing DNA damage.
Local raw foods such as the salted fishes, dried shrimps, anchovies and the shrimp pastes (belacan) have been used in many Malaysian cookings. In this study, the effects of those foods extracts on the DNA of the Chang liver cells were evaluated using the Single Cell Electrophoresis Assay (Comet Alkaline Assay). Percentage of damage to the DNA was calculated using manual scoring based on the severity of the DNA damage (tail moment). “Belacan” at 62.5 μg/mL showed the strongest damage to the DNA (100±2.13%), followed by the salted fish (100±8.6%), dried anchovies (21.67±8.4%) and the dried shrimps (18.5±3.4%). High salt content could be related to the genotoxicity. Further investigations should be carried out to determine their toxicological profiles to evaluate more of their potential hazards to health.
In the rat micronucleus test, it was reported that phenacetin induced micronuclei in peripheral blood, but not in bone marrow. In this study, we used the new methylene blue/acridine orange (N/AO) staining method and the acridine orange (AO) staining method to examine whether phenacetin induces micronuclei in rat bone marrow. Utilizing the method of N/AO staining, reticulocytes were classified into Type I to IV, and then micronucleated reticulocytes (MNRETs) of each type were counted. Micronucleated polychromatic erythrocytes (MNPCEs) were detected in AO-stained specimens. After a single oral administration of phenacetin, MNRETs were mainly increased in Type II and Type III reticulocytes at 24 and 48 hours, respectively, but were not increased at 72 h after administration at a dose of 2000 mg/kg. In addition, the frequency of MNRETs in reticulocytes of Type I and Type II was increased at doses of 500, 1000 and 2000 mg/kg, and the MNRETs frequency at 2000 mg/kg was 3-fold higher than that of MNPCEs at the same dose. After two or four repeated doses of phenacetin, MNRETs were mainly increased in both Type II and Type III reticulocytes at 24 h after final administration. Similar magnitude increases in the frequencies of MNRETs in Type I and Type II reticulocytes, and in Type III reticulocytes and MNPCEs were found for both two and four repeated doses. These results indicate that phenacetin can induce micronuclei in rat bone marrow and that the N/AO staining method in combination with micronucleus scoring in the restricted type(s) of reticulocytes is useful in the rat micronucleus test.
The suppressive effect of (-)-epigallocatechin gallate (EGCG), the major polyphenolic constituent present in green tea, on 7,12-dimethylbenz[a]anthracene (DMBA)-induced chromosome aberrations (CA) in rat bone marrow cells was studied. Rats given EGCG before the DMBA injection displayed a considerably suppressed frequency of DMBA-induced CA in their bone marrow cells. The suppressive effect of EGCG (60 mg/kg body weight) given 24 h before was observed 24, 30, 48 and 72 h after the DMBA injection, but not at the early period (6, 12 and 18 h) after the DMBA treatment. On the other hand, EGCG (60 mg/kg body weight) given 0.5 h before DMBA suppressed DMBA-induced CA at all periods after the DMBA injection. The suppression of EGCG given 24 h or 0.5 h before was observed for all doses of DMBA (25, 50, 75 and 100 mg/kg) investigated. EGCG given at 60 mg/kg body weight 0.5 h before the DMBA injection showed greater suppressive effect than the same dose given 24 h before. The suppressive effect of EGCG given 0.5 h before was dosedependent in the range of 20-60 mg/kg body weight. Methyl methanesulfonate (MMS: direct-acting carcinogen)-induced CA were not suppressed by EGCG. The administration of dehydroepiandrosterone (DHEA), a typical substrate for hydroxysteroid sulfotransferases, 0.5 h before DMBA injection also significantly suppressed DMBA-induced CA but DHEA given 24 h before did not. These results suggest that EGCG has two different suppression mechanisms for DMBA-induced CA depending on the administration time. The suppression of DMBA-induced CA by EGCG given 24 h or 0.5 h before may result from the modification of microsomal enzyme system or the inhibition of sulfotransferase activity by EGCG, respectively.
Symposium: Spontaneous mutagenesis: its mechanisms and implications in carcinogenesis
Cancers are diseases due to somatic mutations of two types; activation of oncogenes and inactivation of tumor suppressor genes. In childhood, retinoblastoma arises due to two mutations of the Rb genes, one is hereditary or somatic and the other is somatic. However, most cancers arise at older ages as a result of a succession of several mutations. For example, incidence of colorectal cancer increases with about fifth power of age. This relationship means that colorectal cancer arises as a result of successive five mutations in oncogenes and tumor suppressor genes. Incidences of colon cancers are very high in rich countries and low in poor countries including Japan at the years before 1970, showing a strong linear correlation between daily meat consumptions and colon cancer rates. This correlation seems to indicate that daily consumption of high amounts of meat stimulates clonal expansion of precancerous cells, resulting in formation of malignant colon cancers. This may be the case of a Darwinian selection of fitter cells. In an epidemiologic study carried out in 1984 on Japanese Zen priests, who rarely took meat in daily diet, they showed significantly lower mortality from all causes and cancers of respiratory tracks than men in the general Japanese public. Incidence of cancer of intestine except rectum for the Zen monks was lower, though not statistically significant, than that of general Japanese men. These results indicate that cancer incidences may be prevented considerably by change in our life style.