Teratogenesis, carcinogenesis, and mutagenesis最新文献

Inadequate doses or prolonged chemotherapy can be cytotoxic or genotoxic to cancer patients, increasing the risk for the development of a second cancer, particularly acute leukemia. The association between therapeutic and genotoxic properties of oncocalyxone A (Onco A), make cytotoxic tests (mitotic index and chromosomal aberrations) fundamental in the accompaniment of the effects of this active compound. Therefore, the aim of the present study was to determine the genotoxic action of Onco A in vitro, during different phases of the cell cycle, utilizing primary cultures of lymphocytes of healthy individuals. The results showed that Onco A is cytotoxic during the cell cycle phases G1, G1/S, and S, however, not in G2. Onco A did not demonstrate a genotoxic effect in any of the cell cycle phases at the concentration studied. It is concluded that during the period of exposure, this active substance inhibits DNA synthesis and consequently cell division. Therefore, the absence of such genotoxicity for Onco A in the tests performed in this study provides important information in regard to the therapeutic use of this agent. Further studies are necessary to better understand the molecular mechanism of action of Onco A.

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 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.

Thalassaemia is a heterogeneous group of inherited anaemias, characterised by a reduction or total absence of one or more of the globin chains of haemoglobin. Individuals with thalassaemia major require regular blood transfusions in order to maintain their haemoglobin concentration at an appropriate level. An essential treatment in parallel with transfusions is iron chelation therapy to remove excess iron deposited in tissues from the transfused blood. The high iron levels in these patients make free oxygen radicals accessible, for example, through Fenton-type chemistry, and generate superoxide and hydroxyl radicals. Increased oxygen radical capacity is known to be associated with cancer and ageing. In a previous study, it has been shown that peripheral blood lymphocytes isolated from a sickle/beta thal double heterozygote-sickle phenotype thalassaemia patient, who was not undergoing chelation therapy, showed increased sensitivity to the effects of oxygen radicals and iron salts by comparison with lymphocytes from normal controls. Furthermore, in a later study, this patient also showed increased sensitivity to the dietary food mutagen 3-amino-1-methyl-5H-pyridol(4,3-b)indole (Trp-P-2) when compared to the control. The present study, therefore, investigated whether the above observation could be duplicated using different food mutagens in different thalassaemia genotypes. The effect of the food mutagens 2-amino-2-methylimidazolo(4,5-f)quinolone (IQ) and 2-amino-1-methyl-6-phenylimidazol(4,5-b)pyridine (PhIP) on lymphocytes of three different thalassaemia patients, a beta-thalassaemia major, a beta-thalassaemia/Hb E, and an alpha-thalassaemia trait with a 3.7-kb deletion, who were not undergoing chelation therapy were investigated using the Comet assay. All three thalassaemia genotypes showed increased sensitivity to both IQ and PhIP in comparison to the control, although with PhIP at the highest two concentrations (50 and 75 microM) the differences monitored with the alpha-thalassaemia trait were found not to be statistically significant (P > 0.05).

In order to determine differences in repair after treatment with DNA damaging agents, normal and cancer cells were selected for analysis of single strand breaks and DNA crosslinks using the Comet assay. Normal human lymphocytes, human colorectal adenocarcinoma SW620 cells, lung carcinoma A549, and H460 cell lines were exposed to an ethylating agent (ethylmethane sulfonate [EMS]), and a cross-linking agent (mitomycin C [MMC]). Differences in repair profiles of DNA damage demonstrated using the comet assay were observed in human lymphocytes and tumour cell lines with both mutagens. Results were also indicative that MMC repair is concentration-dependent. It was also apparent that normal cells repair DNA damage more readily than tumour cells. Repair also varied between different cell lines. To investigate the mechanistic differences of these two chemicals, flow cytometry studies were undertaken in tumour cells, namely cell cycle analysis and frequency of micronuclei induction (FMN). A G2M phase block was clearly evident following treatment with EMS at all concentrations tested. With MMC, an initial arrest of cells in G2M was accompanied by a build-up in S-phase over longer exposure periods. Also, at the highest mutagen doses there were different patterns of micronuclei induction. Thus, using the mutagens with different mechanisms of action highlighted the differences in repair patterns between normal and tumour cells.

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.

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.