Teratogenesis, carcinogenesis, and mutagenesis最新文献

A cytogenetic study was carried out with 5-azacytidine (5-azaC) and etoposide (VP-16) in CHO-K1 and XRS-5 (mutant cells deficient for double-strand break rejoining) cell lines to verify the interaction effects of the drugs in terms of induction of chromosomal aberrations. 5-azaC is incorporated into DNA causing DNA hypomethylation, and VP-16 (inhibitor of topoisomerase II enzyme) is a potent clastogenic agent. Cells in exponential growth were treated with 5-azaC for 1 h, following incubation for 7 h, and posttreatment with VP16 for the last 3 h. In K1 cells, the combined treatments induced a significant reduction in the aberrations induced in the X and "A" (autosome) chromosomes, which are the main target for 5-azaC. However, in XRS-5 cells, the drug combination caused a significant increase in the aberrations induced in those chromosomes, but with a concomitant reduction in the randomly induced-aberrations. In addition, each cell line presented characteristic cell cycle kinetics; while the combined treatment induced an S-arrest in K1 cells, alterations in cell cycle progression were not found for XRS-5, although each drug alone caused a G2-arrest. The different cell responses presented by the cell lines may be explained on the basis of the evidence that alterations in chromatin structure caused by 5-aza-C probably occur to a different extent in K1 and XRS-5 cells, since the mutant cells present a typical hyper-condensed chromosome structure (especially the X- and "A" chromosomes), but, alternatively, 5-aza-C could induce reactivation of DNA repair genes in XRS-5 cells.

The spontaneous mutation rate in the male germ-line increases with age. The reason for this is unknown, but presumably involves an age-related degeneration in the efficacy of cellular processes. To investigate the possibility that rates of apoptosis and genetic damage (represented by aneuploidy) might vary with age in mice, the testes and sperm of 2- and 12-month-old male MF-1 mice were examined by a modified TUNEL technique and 3-colour sperm-FISH assay, respectively. Sperm were labeled with probes to chromosomes 8, X and Y and 20,000 sperm scored from each of 5 animals per group. A significant increase in gonosomal disomy was found in the aged mice, especially X-X-8. This suggests that advanced paternal age is associated primarily with meiosis II rather than meiosis I disjunction errors. Neither diploidy nor autosomal disomy was affected in the older group. The rate of germ cell apoptosis (apoptotic cells per seminiferous tubule cross-section per animal per group) was higher in the old mice than controls, but not significantly. Considerable inter-animal variability was observed in the older group. The finding of an increase in levels of sperm aneuploidy is novel for 1-year-old mice and confirms the genotoxic effect of ageing in mice. Since apoptosis is assumed to eliminate cells with unrepaired damage, it may be that the apoptotic response in older mice is compromised, resulting in the higher levels of aneuploidy in sperm. However, given the inter-animal variability in testicular germ cell apoptosis, this awaits confirmation.

The year 2001 witnessed the sequencing of 90% of the euchromatic region in the human genome but the ultimate goal to delineate the positions of all genes is yet to be achieved. Fluorescence In Situ Hybridization (FISH) is one of the methods for localizing genes on chromosomes. In the present study, diagnostic utility of single-, dual-, and multicolor FISH was evaluated for prenatal diagnosis, cancer genetics, and screening of various congenital anomalies (sex chromosomal and autosomal). Centromeric probes for chromosomes X and Y were used for screening minor aneuploid cell lines (XXY, XO, and XXX) in the cases of primary amenorrhea and suspected Klinefelter syndrome. The cases with ambiguous genitalia were analyzed using a probe specific for the sex-determining region (SRY). Suspected cases of Down syndrome were subjected to FISH using probe specific for chromosome 21. FISH was also used to study gene alterations in retinoblastoma and myeloid leukemias. Prenatal diagnosis was done to screen for aneuploidies of chromosomes 13, 18, 21, X, and Y using FISH on uncultured cells from amniotic fluid and chorionic villi sampling. The screening for common aneuploidies was extended to abortuses from spontaneous abortions. Using FISH, low-level mosaicism could be identified in some cases of primary amenorrhea and suspected Klinefelter syndrome. Submicroscopic gene rearrangements could be detected using FISH in cases of ambiguous genitalia and cancers. Further interphase FISH could provide results within 24 hours. To conclude, FISH adds to the diagnostic utility of routine cytogenetics and its use on interphase nuclei overcomes the difficulty of conventional cytogenetics, thereby reducing the time between sampling and diagnosis to 24 hr.

The aim of this work was to test the cytotoxicity of newly synthesized cis-type complexes of platinum(II) and palladium(II) dichloride with methyl 3,4-diamine-2,3,4,6-tetradeoxy-alpha-L-lyxohexopyranoside, [M(C(7)H(16)N(2)O(2))Cl(2)].H(2)O, against two mouse lymphoma cell lines (L5178Y) differing in their double strand breaks and nucleotide excision repair ability. cis- Diaminedichloroplatinum (CDDP) was used as a reference compound. The toxicity of Pt(C(7)H(16)N(2)O(2))Cl(2) appeared to be similar for both cell lines: IC(50) is 8 microM for L5178Y-R cells and 12 microM for L5178Y-S cells, respectively. In contrast, the palladium complex was found to be more toxic for the LY-R cells than for the LY-S cells. The cytotoxicity of both compounds was compared with their ability to induce DNA crosslinks, as measured by the modified comet assay. CDDP caused retardation of the DNA migration induced by 2 Gy of the X-irradiation in a dose-dependent manner. The ability of Pd(C(7)H(16)N(2)O(2))Cl(2) to retard X-ray induced DNA migration was more pronounced than its platinum analogue and CDDP (see Fig. 6). However, this was not reflected in the toxicity of the compound. Such results indicate that these two compounds may cause a different type of DNA damage and/or that the DNA damage caused by the palladium(II) compound was dealt with in a different manner from that induced by the platinum(II) complex.

Mutant spectra analysis was conducted with spontaneous hisG46 revertants of Salmonella typhimurium strain YG1029 and revertants induced by the plant- and mammalian S9-activation of benzidine and 4-aminobiphenyl (4-ABP). Under preincubation conditions, YG1029 cells were exposed to benizidine or 4-ABP with mammalian S9 activation or to a high molecular weight fraction that contained the plant-activated products. The induced revertants were isolated at mutagen concentrations that caused an increased mutant frequency of approximately 4- to 10-fold above background. Genomic DNA from each revertant was isolated and the hisG region was amplified using polymerase chain reaction (PCR). Using a series of specific probes and a modified version of the ECL3's-oligolabelling and detection system, each of the six possible base-pair substitution mutations at hisG46 that leads to a reversion event was determined. Of the YG1029 spontaneous revertants, transition mutations were 31.8% and transversion mutations were 68.2%. The YG1029 spontaneous mutant spectrum differed significantly from the spontaneous spectrum of TA1535 but did not significantly differ from the spontaneous TA100 mutant spectrum. The differences of the spontaneous mutant spectra among these highly related strains illustrate that the introduction of the plasmid pKM101 into S. typhimurium increased the frequency of transversions (CCC-->ACC; CCC-->CAC) and reduced site 2 (CCC-->CTC) transitions. With plant-activated benzidine, 21.1% of recovered revertants resulted from transitions and 78.9% from transversions while S9 activated-benzidine induced revertants were recovered as 14.2% from transition and 85.8% from transversion mutations. Plant-activated 4-ABP recovered 20.0% transitions and 80.0% transversions. S9-activated 4-ABP-induced 21.4% transitions and 78.6% transversions. Chi-square analysis of mutant spectra indicated that the DNA lesions that resulted in reversion at the hisG46 allele induced by plant-activated benzidine or 4-ABP were different from those generated after mammalian S9 activation of these promutagens. The plant-activated benzidine and 4-ABP induced statistically identical mutant spectra. Also, the mammalian-activated benzidine and 4-ABP induced statistically similar mutant spectra. These data show that the plant-activated and mammalian-activated aromatic amine products inflicted different types or distributions of DNA lesions that were reflected in the resulting induced mutant spectra.

Many natural agents including fruits and vegetables are known to provide protection from different degenerative diseases including cancer, by preventing damage to the cellular components. The effect of two important dietary agents, alpha tocopherol, and the flavonoid quercetin, along with two commonly consumed vegetables, bitter gourd and tomato, were investigated on spontaneous and dimethylbenz(a)anthracene (DMBA)-induced DNA damage in murine lymphocytes in vitro. DNA damage was determined by single cell gel electrophoresis (comet assay). The rationale for such an approach for this study is that DNA damage can lead to genetic disorders that occur at different stages of carcinogenesis and protection from such damages may in the long run help to prevent development of cancer. Both alpha tocopherol and quercetin as single agents were found to be potent inhibitors of DNA damage (spontaneous and carcinogen induced) in a dose-dependent manner. Fresh juices of bitter gourd and tomato could also protect from DMBA-induced DNA damage but not as effectively as the single agents. The anticarcinogenic role of nutrients as well as non-nutrient dietary components need to be explored more extensively. The Comet assay is a simple, fast, and reliable method to determine the protective effect against DNA damage, one of the prerequisites for carcinogenesis.

8-oxo-7,8-dihydroguanine (8-oxoG) is a potent mutagenic lesion that forms at elevated levels in cellular DNA and is repaired with low efficiency in human cells. Unlike its human counterpart, the Drosophila S3 ribosomal/repair protein is endowed with a vigorous 8 oxoG repair activity that is associated to beta,delta-elimination AP lyase activity. We have recently observed that pure GST-tagged Drosophila S3 protein can significantly accelerate the in vitro repair of 8 oxoG performed by human and mouse cell extracts [Cappelli et al., unpublished data]. In this work, we have transfected Chinese hamster cells with mammalian expression vectors containing the Drosophila S3 cDNA. The cells synthesized both S3 mRNA and protein but no improved repair of 8 oxoguanine was observed. Factors important for the proper expression of Drosophila genes in mammalian cells are discussed.

India is one of the 12 mega diversity countries in the world so it has a vital stake in conservation and sustainable utilization of its biodiversity resources. Plant secondary metabolites have been of interest to man for a long time due to their pharmacological relevance. With this in view, the bark powder of Acacia auriculiformis, A. nilotica, Juglans regia, and the fruit powder of Terminalia bellerica, T. chebula, Emblica officinalis, and a combination drug "Triphala," which are known to be rich in polyphenols, were tested for their antimutagenic activities. Antimutagenic activities of the extracts were estimated by employing the plate incorporation Ames Salmonella histidine reversion assay by using the frame shift mutagen tester strain TA98 and base pair substitution strain TA100 against direct acting mutagens (NPD, sodium azide), and the S9-dependent mutagen 2-aminofluorene(2AF). Acetone extracts of all the plants exhibited significant antimutagenic activities among the other extracts tested, but an acetone extract of Acacia nilotica showed a marked anti-mutagent effect. Furthermore, it was more effective against indirect acting mutagen, 2AF, in both TA98 and TA100 tester strains of Salmonella typhimurium than against the direct acting mutagens. The results indicate that an acetone extract of bark and fruit of the medicinal plants under study harbors constituents with promising antimutagenic/anticarcinogenic potential that could be investigated further.

The aim of this study was to investigate the chemoprotective effects of mustard sprouts on benzo(a)pyrene [B(a)P]-induced DNA damage in the single cell gel electrophoresis (SCGE)/Hep G2 assay. This model combines the advantages of the SCGE assay with that of human-derived cells that possess inducible phase I and phase II enzymes. Treatment of the cells with small amounts of mustard juice (0.1-1.25 microl/ml) and B(a)P reduced the genotoxic effect of the carcinogen in a dose-dependent manner. Contrary to the results with the juice, unexpected synergistic effects were observed with allyl isothiocyanate (AITC, 0.3 microM), a breakdown product of sinigrin, which is contained in black mustard and many other cruciferous vegetables. Although these concentrations of AITC did not cause DNA damage per se, pronounced dose-dependent DNA damage was seen with higher concentrations of AITC (>or= 25 microM). In parallel with the comet assays, also enzyme measurements were carried out which showed that exposure of the cells to mustard juice (2.0 microl/ml) causes a moderate induction of ethoxyresorufin-O-deethylase, and more pronounced (approximately 2-fold) increase of the activity of glutathione-S-transferase. In conclusion, our findings indicate that i) mustard juice is highly protective against B(a)P-induced DNA damage in human derived cells and ii) that induction of detoxifying enzymes may account for its chemoprotective properties. iii) Furthermore, our findings show that the effects of crude juice can not be explained by its allyl isothiocyanate contents.

Metanil yellow (MY) and malachite green (MG) are textile dyes, which, despite a ban, are used as food-coloring agents. MY and MG have promoter effects on the development of hepatic preneoplastic lesions induced by N-nitrosodiethylamine (DEN). Tumor-promoting agents are not mutagenic but may alter the expression of genes whose products are associated with hyper-proliferation, tissue remodeling, and inflammation. Cell cycle controls normally function to ensure the integrity of the genome and arrest of cells at G1/S or G2/M checkpoints until all the prerequisite events are completed. In order to understand the mechanism(s) of tumor promotion by MY and MG, we have studied the levels of PCNA, a marker of cell proliferation and cell cycle regulatory proteins, cyclin D1, and its associated kinase, cdk4, cyclin B1, and associated kinase, cdc2. Immunohistochemical staining showed an elevated level of PCNA in animals administered MY and MG subsequent to DEN treatment. Western and Northern blot hybridization showed an increased expression of both cyclin D1 and its associated kinase cdk4, and cyclin B1 and its associated kinase cdc2, in livers of rats administered MY and MG after administration of DEN as compared to untreated or DEN controls. The increased level of mRNA was due to the increased rate of transcription of these genes as studied by run-on transcription assay. These data obtained by the in vivo model of liver tumor development provide strong evidence for a link between dysregulation of the two critical checkpoints of the cell cycle as one of the possible mechanism(s) during tumor promotion by malachite green and metanil yellow.