Mutagenicity of 5-formyluracil in mammalian cells.

5-Formyluracil, a major oxidized form of thymine, was incorporated into a predetermined site of one of the leading and lagging template strands of a double-stranded vector, and the DNA replication efficiency and the mutation frequency of 5-formyluracil in simian COS-7 cells were investigated. 5-Formyluracil did not block DNA replication and was weakly mutagenic in simian cells. 5-Formyluracil primarily elicited base substitutions at the modified positions.     

Mutagenicity and genotoxicity of isatin in mammalian cells in vivo

Isatin (1H-indole-2,3-dione) is a synthetically versatile substrate used for the synthesis of heterocyclic compounds and as a raw material for drug synthesis. Isatin and its derivatives demonstrate anticonvulsant, antibacterial, antifungal, antiviral, and anticancer properties. We evaluated the genotoxic and mutagenic effects of acute (24h) and repeated (14d) exposure to isatin in vivo, using the comet assay and the micronucleus test. Three doses (50, 100, and 150mg/kgb.w.) were administered to mice via gavage. Doses were selected according to the LD(50) of isatin, estimated in a preliminary test to be 1g/kgb.w. To evaluate the results, parametric (ANOVA/Tukey) and non-parametric (Kruskal-Wallis/Dunn's post hoc test) tests were used, according to the nature of the data distribution. At all doses (50, 100 and 150mg/kgb.w.), after acute treatment with isatin, alterations in DNA migration (comet assay) were not observed and mutagenic effects were not seen (micronucleus test on peripheral blood cells). After repeated doses, only the highest dose of isatin (150mg/kgb.w.) induced alterations in the DNA that gave rise to micronuclei in the bone marrow and peripheral blood cells of the mice. Our results show that the mutagenic and genotoxic effects of isatin depend on dose and on period of exposure.     

Mutagenicity and DNA repair of glycidamide-induced adducts in mammalian cells

Glycidamide (GA)-induced mutagenesis in mammalian cells is not very well understood. Here, we investigated mutagenicity and DNA repair of GA-induced adducts utilizing Chinese hamster cell lines deficient in base excision repair (BER), nucleotide excision repair (NER) or homologous recombination (HR) in comparison to parent wild-type cells. We used the DRAG assay in order to map pathways involved in the repair of GA-induced DNA lesions. This assay utilizes the principle that a DNA repair deficient cell line is expected to be affected in growth and/or survival more than a repair proficient cell. A significant induction of mutations by GA was detected in the hprt locus of wild-type cells but not in BER deficient cells. Cells deficient in HR or BER were three or five times, respectively, more sensitive to GA in terms of growth inhibition than were wild-type cells. The results obtained on the rate of incisions in BER and NER suggest that lesions induced by GA are repaired by short patch BER rather than long patch BER or NER. Furthermore, a large proportion of the GA-induced lesions gave rise to strand breaks that are repaired by a mechanism not involving PARP. It is suggested that these strand breaks, which might be the results from alkylation of the backbone phosphate, are misrepaired by HR during replication thereby leading to a clastogenic rather than a mutagenic pathway. The type of lesion responsible for the mutagenic effect of GA cannot be concluded from the results presented in this study.     

Mutagenicity and DNA repair of glycidamide-induced adducts in mammalian cells

Glycidamide (GA)-induced mutagenesis in mammalian cells is not very well understood. Here, we investigated mutagenicity and DNA repair of GA-induced adducts utilizing Chinese hamster cell lines deficient in base excision repair (BER), nucleotide excision repair (NER) or homologous recombination (HR) in comparison to parent wild-type cells. We used the DRAG assay in order to map pathways involved in the repair of GA-induced DNA lesions. This assay utilizes the principle that a DNA repair deficient cell line is expected to be affected in growth and/or survival more than a repair proficient cell.A significant induction of mutations by GA was detected in the hprt locus of wild-type cells but not in BER deficient cells. Cells deficient in HR or BER were three or five times, respectively, more sensitive to GA in terms of growth inhibition than were wild-type cells. The results obtained on the rate of incisions in BER and NER suggest that lesions induced by GA are repaired by short patch BER rather than long patch BER or NER. Furthermore, a large proportion of the GA-induced lesions gave rise to strand breaks that are repaired by a mechanism not involving PARP. It is suggested that these strand breaks, which might be the results from alkylation of the backbone phosphate, are misrepaired by HR during replication thereby leading to a clastogenic rather than a mutagenic pathway. The type of lesion responsible for the mutagenic effect of GA cannot be concluded from the results presented in this study.     

Mutagenicity of methylisocyanate and its reaction products to cultured mammalian cells

Methylisocyanate (MIC) induced mutagenic responses in the absence of exogenous activation in the mouse lymphoma cell forward mutation assay at concentrations as low as 8-24 microM. MIC produced predominantly small mutant colonies, suggesting the possibility of clastogenic activity. The intermediate hydrolysis product, methylamine, was also mutagenic without exogenous activation but required several hundred-fold higher concentrations (ca. 3 mM). N,N'-Dimethylurea, the final product in the reaction of methylisocyanate and water, was totally refractory in either the presence or absence of S9 for concentrations up to 57 mM (5 mg/ml). The ethyl ester of N-methylcarbamic acid was also tested since it was the only available analogue to the highly reactive N-methylcarbamic acid intermediate. This compound was mutagenic only in the presence of S9 at doses exceeding 5-40 microM, which suggested the possibility that the free acid, produced by enzymatic hydrolysis, is also mutagenic. The mutagenic activity of the ester resulted solely in the production of small mutant colonies.     

Mutagenicity of actinomycin D in mammalian cells due to clastogenic effects

Actinomycin D was clastogenic and mutagenic in L5178Y/TK +/- -3.7.2C mouse lymphoma cells. The majority of the mutants were small colonies, indicating that actinomycin D acts primarily by a clastogenic mechanism.     

Mutagenicity of a unique apurinic/apyrimidinic site in mammalian cells.

Abasic sites are common DNA lesions produced either spontaneously or as a consequence of the action of some genotoxic agent. The mutagenic properties of a unique abasic site replicated in mammalian cells have been studied using a shuttle vector. A plasmid, able to replicate both in mammalian cells and in bacteria, carrying a unique abasic site chemically synthesized has been constructed. After replication in mammalian cells, plasmid DNA was recovered and used to transform bacteria. Mutants were screened without selection pressure by differential hybridization with a labelled oligonucleotide and their DNA was sequenced. A mutation frequency ranging from 1% to 3% was found, depending on the base originally inserted during the vector construction, opposite the abasic site. All the sequenced mutants correspond to single base-pair substitutions targeted at the abasic site. We observed a deficit in guanine incorporation opposite the abasic site, while the three other bases were incorporated with a similar efficiency. The mutational potency of abasic sites was observed without any voluntary preconditioning treatment of mammalian cells in order to induce "SOS" like conditions.     

Mutagenicity of cadmium in mammalian cells: implication of oxidative DNA damage

Cadmium and cadmium compounds are well established human carcinogens and are ubiquitously present in the environment. The carcinogenic mechanism(s) of cadmium remains largely unknown since direct mutagenic effect is weak in bacterial and in standard mammalian cell mutation assays. In this study, we show that when evaluated using the human-hamster hybrid A(L) cell mutation assay in which both intragenic and multilocus deletions can readily be detected, CdCl(2) is a strong mutagen that induces predominantly large deletion mutations. Concurrent treatment of A(L) cells with the oxyradical scavenger dimethyl sulfoxide significantly reduced the number of cadmium-induced mutations. In contrast, pre-treatment of cells with buthionine sulfoximine that depletes intracellular glutathione, increased cytotoxicity and mutagenicity of cadmium. These results demonstrate that reactive oxygen species mediate cadmium induced mutations in A(L) cells. With laser scanning confocal microscopy and the fluorescent probe 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, we demonstrated that cadmium induced a dose and time dependent formation of intracellular oxyradicals. Using immunoperoxidase staining coupled with a monoclonal antibody-specific for 8-OHdG adducts in DNA, we demonstrated that cadmium induced a dose dependent increase of 8-OHdG adducts, which accumulated with prolonged exposure. Furthermore, we showed that at low concentration, cadmium, attenuated removal of hydrogen peroxide induced 8-OHdG adducts. Thus, the carcinogenicity of cadmium can, in part, be explained by its mutagenic activity, which is mediated by reactive oxygen species induced DNA damage and by its interference with the repair of oxidative DNA damage.     

多聚磷酸盐在细菌和哺乳动物细胞中的作用

多聚磷酸盐(Poly P)广泛分布于真核生物和原核生物中,是由几十个到几百个无机磷酸盐单体通过高能磷酸键聚合而成的线性多聚体。Poly P能影响细菌的毒力,有助于细菌抵抗环境中的压力刺激。在真核细胞中,Poly P与核仁的转录相关,可促进凝血和细胞分化、调节促炎反应,并和骨的重构、矿化及去矿化相关。同时,Poly P也是线粒体通透性转换孔的激活物。本文综合阐述Poly P在微生物及哺乳动物中的作用及相关机制,同时结合我们的研究工作,分析Poly P对大肠杆菌致病性的影响,以期引起研究者对Poly P的关注。     

Mutagenicity of organophosphorus compounds in bacteria and drosophila

140 Organophosphorus compounds (OP's) have been tested for mutagenic activity in bacteria, principally by using two specially constructed sets of tester strains of the bacteria Salmonella typhimurium and Escherichia coli. It was found that 20% gave positive mutagenic responses and that this group of chemicals produce base substitutions rather than frame-shift mutations. In most cases the DNA repair genes exrA⁺ and recA⁺ were for mutagenic activity.Seven compounds were further tested in Drosophila melanogaster for the ability to induce recessive lethal mutations. In some of these cases the doses administered to the flies had to be very low due to the highly toxic nature of the compounds. To overcome this problem, the accumulation of recessive lethal mutations was measured in populations which were continually exposed to the compounds over a period of some 18 months. During this time the populations developed increased resistance to the compound and so the dose administered could gradually be increased. Six of the compounds were mutagenic.Of the compounds tested in both systems, those showing mutagenic activity in bacteria were also mutaganic in Drosophila, those mutagenic in bacteria were not mutagenic in Drosophila.     

Mutagenicity study of fried sausages in Salmonella, Drosophila and mammalian cells in vitro and in vivo

The basic extract of pan-fried sausages was studied for mutagenic potential in seven test systems. Mutagenic activity was high in the standard Ames assay in the Salmonella typhimurium strains TA1538 and TA98 in presence of S9 mix. In vivo, in the intrasanguine host-mediated assay with strain TA98 on Aroclor-pretreated mice, the mutagenic activity of the extract was low. A borderline activity was seen in the SCE assay in vitro with V79 Chinese hamster cells in presence of S9 mix. No significant mutagenic action was found in the gene-mutation assay for thioguanine resistance with V79 cells, the Drosophila sex-linked recessive lethal test, the micronucleus test and the mammalian spot test.     

Wild-type intracellular bacteria deliver DNA into mammalian cells

Gene transfer in vitro from intracellular bacteria to mammalian phagocytic and non-phagocytic cells and in vivo in mice has been reported. The bacteria used as DNA delivery vectors were engineered to lyze upon entry in the cell due to impaired cell wall synthesis for Shigella flexneri and invasive Escherichia coli , or production of a phage lysin for Listeria mono- cytogenes . In vivo gene transfer was obtained with attenuated Salmonella typhimurium and resulted in stimulation of mucosal immunity. We report that wild-type intracellular human pathogens, such as L. monocytogenes EGD or LO28 and S. flexneri M90T, mediate efficient in vitro transfer of functional genes into epithelial and macrophage cell lines. A low- efficiency transfer was obtained from strain EGD to mouse peritoneal macrophages. DNA transfer with S. typhimurium was observed only from atten-uated aroA strain SL7207 into COS-1 cell line. As demonstrated by the study of listeriolysin-defective L. monocytogenes or of S. typhimurium SL7207 aroA engineered to secrete listeriolysin, escape of bacteria or of plasmid DNA from the intracytoplasmic vacuole is required for transfer of genetic information to occur.     

Mutagenicity testing of dichloromethane in short-term mammalian test systems

Several short-term mammalian test systems were used for mutagenicity testing of the organic solvent dichloromethane. The compound was negative in the forward mutation test on the HGPRT locus in Chinese hamster cells and the unscheduled DNA synthesis test in both human and hamster cells. In the test on DNA synthesis inhibition, dichloromethane caused an aspecific inhibition in both human and hamster cells, but in this test the effect did not indicate a DNA-damaging action. A weak positive effect was found in the test on sister-chromatid exchanges in hamster cells.     

Mutagenicity studies on coffee. The influence of different factors on the mutagenic activity in the Salmonella/mammalian microsome assay

Recently, mutagenic activity on several strains of Salmonella typhimurium has been found in many heat-processed foodstuffs. The previously reported direct-acting mutagenic activity of coffee in Salmonella typhimurium TA100 (Ames assay) was confirmed in our study. In addition to TA100, a mutagenic effect of coffee was also found by using the newly developed strain TA102. The mutagenic activity was abolished by the addition of rat-liver homogenate. 10% S9 mix completely eliminated the mutagenic activity of 30 mg of coffee per plate. The addition of reduced glutathione to active S9 further decreased the mutagenic activity and also reduced the mutagenicity together with inactivated S9. The compound or compounds responsible for this inactivation are heat-labile and seem to be located in the cytosol fraction of the S9. Part of the mutagenicity of coffee was also lost spontaneously upon incubation at temperatures between 0 degrees and 50 degrees C. The loss of activity was dependent on temperature, being more pronounced at 50 degrees C compared to 0 degrees C (at 50 degrees C approximately 50% of the mutagenic activity was lost after 6 h). As anaerobic conditions prevented this loss of mutagenicity almost totally, oxidative processes are probably responsible for the inactivation. The stability of the mutagen was not influenced by incubation at low pH values (pH 1-3), with or without the addition of pepsinogen. The mutagenic properties of methylglyoxal, which to some extent could be responsible for the mutagenic activity of coffee, were compared with those of coffee. Methylglyoxal was strongly mutagenic towards Salmonella typhimurium TA100 and TA102. Its mutagenic activity was partially inactivated by the addition of 10% S9. Glyoxalase I and II together with reduced glutathione abolished the mutagenic activity of methylglyoxal but reduced the mutagenicity of coffee by only 80%. Since these enzymes occur in mammalian cells, the mutagenic compound(s) of coffee could also be degraded in vivo. This conclusion is supported by the fact that a long-term carcinogenicity study with rats was negative. These results clearly demonstrate that the effects observed in vitro do not necessarily also occur in vivo, but that in vitro experiments may contribute to the understanding of fundamental mechanisms of chemical carcinogenesis.     

Improved green and blue fluorescent proteins for expression in bacteria and mammalian cells

Fluorescent proteins have become an invaluable tool in cell biology. The green fluorescent protein variant EGFP is especially widely applied. Use of fluorescent proteins, including EGFP, however can be hindered by inefficient protein folding, resulting in protein aggregation and reduced fluorescence. This is especially profound in prokaryotic cells. Furthermore, EBFP, a blue fluorescent variant of EGFP, is rarely used because of its dim fluorescence and fast photobleaching. Thus, efforts to improve properties such as protein folding, fluorescence brightness, and photostability are important. Strongly enhanced green fluorescent (SGFP2) and strongly enhanced blue fluorescent (SBFP2) proteins were created, based on EGFP and EBFP, respectively. We used site-directed mutagenesis to introduce several mutations, which were recently shown to improve the fluorescent proteins EYFP and ECFP. SGFP2 and SBFP2 exhibit faster and more efficient protein folding and accelerated chromophore oxidation in vitro. For both strongly enhanced fluorescent proteins, the photostability was improved 2-fold and the quantum yield of SBFP2 was increased 3-fold. The improved folding efficiency reduced the extent of protein aggregation in Escherichia coli, thereby increasing the brightness of bacteria expressing SGFP2 7-fold compared to the brightness of those expressing EGFP. Bacteria expressing SBFP2 were 16-fold more fluorescent than those expressing EBFP. In mammalian cells, the improvements were less pronounced. Cells expressing SGFP2 were 1.7-fold brighter than those expressing EGFP, which was apparently due to more efficient protein expression and/or chromophore maturation. Mammalian cells expressing SBFP2 were 3.7-fold brighter than cells expressing EBFP. This increase in brightness closely resembled the increase in intrinsic brightness observed for the purified recombinant protein. The increased maturation efficiency and photostability of SGFP2 and SBFP2 facilitate detection and extend the maximum duration of fluorescence imaging.