In vitro mammalian mutagenesis as a model for genetic lesions in human cancer

Recently in vitro assays of mutagenesis have been criticized as being poorly predictive of long-term in vivo rodent assays of carcinogenicity. Questions have also been raised concerning the relevance of rodent assays to human risk. In vitro assays using mammalian cells can detect most types of genetic lesions thought to be important in human malignant disease. Molecular and cytogenetic analyses of mutations induced by a variety of genotoxic compounds at the heterozygous thymidine kinase locus in mouse lymphoma cells indicate that this in vitro assay does indeed register the range of genetic lesions recently found in a wide variety of human tumors. The types and complexity of the induced lesions are reflected in mutant colony phenotype in a compound-specific fashion. These studies point to the use of appropriate in vitro mammalian mutagenesis assays as new model systems for dissecting the genetic lesions important in human carcinogenesis, and as a means of determining the potential for compounds to induce such lesions.     

In vitro assessment of the toxicity of metal compounds

A review of the activity of metal compounds in mammalian cell transformation assays has been completed. Results from these assays appear to correlate well with the known carcinogenic activity displayed by specific metal compounds in vivo. Studies of cell transformation in vitro may provide information pertaining to the mechanism of the induction of carcinogenesis by certain metals.     

Measurement of DNA-excision repair in suspensions of freshly isolated rat hepatocytes after exposure to some carcinogenic compounds: its possible use in carcinogenicity screening.

When suspensions of freshly isolated rat hepatocytes were exposed to a number of carcinogenic compounds, it was possible to measure an increased UDS by a rapid procedure via liquid-scintillation counting. For a number of carcinogenic compounds and some of their non-carcinogenic structural analogues a good correlation between the carcinogenic property and the ability to induce UDS was demonstrable. Out of 12 carcinogenic compounds, belonging to several different chemical classes, 10 gave rise to an increased UDS, whereas only 2 compounds, the polycyclic aromatic hydrocarbons benzo[alpha]pyrene and benz[alpha]anthracene, did not. All 4 noncarcinogenic compounds tested were negative. Possibly this method can be of value as a routine screening test, in combination with other short-term test systems, thus improving the predictive value of screening in vitro with respect to carcinogenicity.     

用于蛋白质体外分子进化研究的DNA随机突变技术

蛋白质体外分子进化是模拟自然的进化过程,利用基因随机突变和定向筛选（选择）技术,以获得具有预期新功能的突变体分子。虽然体外进化近几年才产生,但已成为医药和工业领域中筛选具有特殊催化性质的酶的最重要的方法之一。ＤＮＡ随机突变技术是蛋白质体外分子进化研究的基础,本文将对几种最重要的突变方法：倾向错误的ＰＣＲ、ＤＮＡ重排、模板交错延伸反应和随机延伸突变的原理和应用等加以介绍。     

Mutagenicity of N-nitrosopiperazine derivatives in Salmonella typhimurium.

Mutagenicity of several nitroso derivatives of piperazine was assayed using histidine auxotrophic strains of Salmonella typhimurium. Nitroso derivatives of piperazine required metabolic activation with preference to phenobarbital induced rat-liver microsomal enzymes. We observed a good correlation between a positive effect in the mutation assay and the carcinogenic potency of the compound. Even though our results are not in complete agreement with earlier published work using several microbial mutation assay systems, the differences we observed demonstrate the predictive value of an in vitro activation system using S. typhimurium to detect carcinogenic compounds as mutagens.     

斑马鱼反转录病毒插入突变技术的发展及其在基因饱和突变与筛选中的应用

用于大规模基因突变与筛选的主要策略有化学诱变、插入突变、基因诱捕。插入突变是一种通过外源DNA整合的方式来获得突变体,并克隆得到对应突变基因的方法。运用反转录病毒介导的插入突变技术,在脊椎动物斑马鱼中已经获得了许多影响胚胎发育和细胞生长过程的突变体,并找到了对应的基因。基因诱捕技术也被运用于反转录病毒载体的构建。这套系统的建立使斑马鱼成为第一个有可能达到基因饱和突变和筛选的脊椎动物。     

Directed Evolution of an Enantioselective Bacillus subtilis Lipase

Chiral compounds are of steadily increasing importance to the chemical industry, in particular for the production of pharmaceuticals. Where do these compounds come from? Apart from natural resources, two synthetic strategies are available: asymmetric chemical catalysis using transition metal catalysts and biocatalysis using enzymes. In the latter case, screening programs have identified a number of enzymes. However, their enantioselectivity is often not high enough for a desired reaction. This problem can be solved by applying directed evolution to create enantioselective enzymes as shown here for a lipase from Bacillus subtilis. The reaction studied was the asymmetric hydrolysis of meso-1,4-diacetoxy-Zcyclopentene with the formation of chiral alcohols which were detected by electrospray ionization mass spectrometry. Iterative cycles of random mutagenesis and screening allowed the identification of several variants with improved enantioselectivities. In parallel, we have started to use X-ray structural data to simulate the Bacillus subtilis lipase A-catalyzed substrate hydrolysis by using quantum mechanical and molecular mechanical calculations. This combined approach should finally enable us to devise more efficient strategies for the directed evolution of enantioselective enzymes.     

Apparent changes in structure-activity relationships for antimitochondrial effects of 9-anilinoacridines according to Saccharomyces cerevisiae strain and methodology

Sensitivity of detection of antimitochondrial effects in S. cerevisiae as measured by the induction of 'petite' mutants, has been investigated in a closely related series of 9-anilinoacridines, using a new microtitre test which has been compared to a range of other techniques. Drugs were chosen to span antimitochondrial activity between the inactive compounds 9-amino- or 3-amino-acridine and the moderately active proflavine, also between proflavine and the strong antimitochondrial agent, ethidium bromide. As previously reported using other techniques, no compound without an amino substituent caused antimitochondrial effects, whereas all 9 anilinoacridines with a 1'-substituted anilino group and 3,6-diamino-substituted acridine ring acted like ethidium in causing strong 'petite' mutagenesis. Compounds with a single acridine 3-amino group, together with proflavine, might or might not be scored as an antimitochondrial agent depending on the time and conditions of drug exposure and, more importantly, on the selection of yeast strain used in the screening. Measurement of 'petite' mutagenesis in strain 5178B, using the microtitre assay, provided the most sensitive and efficient means of detection of antimitochondrial effects for all physical DNA-binding agents. Detailed interpretation of structure-activity relationships and prediction of carcinogenic activity based upon induction of 'petite' mutagenesis would vary considerably if this procedure is not followed.     

In vitro setting of dose-effect relationships of 32 metal compounds in the Balb/3T3 cell line, as a basis for predicting their carcinogenic potential

The results are reported of the second stage in a programme for a systematic in vitro study on the carcinogenic potential of metal compounds with Balb/3T3 clone A31-1-1 mouse fibroblasts. Nineteen metal compounds that exhibited a strong cytotoxic effect during a previous screening run with a 100 microM fixed dose were tested with a 72-hour exposure over a wide range of concentrations (from 0.1 microM to 1000 microM), to produce dose-effect curves to permit extrapolation of the 50% inhibition concentration (IC50) values for each metal compound. This allows the establishment of a suitable range of doses for individual metal species, for use in the subsequent Balb/3T3 assay based on a two-stage concurrent cytotoxicity and morphological transformation protocol. Another 13 metal compounds were also tested, to determine whether the Balb/3T3 cell transformation assay is really a valuable in vitro model in relation to the problem of metal speciation. Of the metal compounds assayed, 26 showed a dose related cytotoxic response with calculated IC50 values ranging from 0.25 microM (CH3HgCl) to 140 microM [(C5H5)2TiCl2], whereas six metal compounds, namely (NH4)6Mo7O24*4H2O, CH3AsO(OH)2, C2H6AsNaO2(3H2O, KBr, CrCl3*6H2O and (NH4)2[TiO(C2O4)2]*H2O, displayed no observable cytotoxicity or low cytotoxicity at all the doses tested. The determination of IC50 values permits a ranking of the cytotoxicity responses of metal compounds with the highest cytotoxicities. Dose-effect curves and IC50 values of different chemical forms of individual metal compounds of As, Br, Cr, Hg, Ir, Pt, Te, Ti and V (cationic/anionic inorganic or organometallic species) showed clearly how the chemical nature of the metal strongly influences the toxic response. This confirms that the Balb/3T3 cell line is a valuable in vitro model with respect to the problem of metal speciation. This is a fundamental aspect to be considered when incorporating the results from in vitro cell transformation assays of the carcinogenic potential of metal compounds into regulatory testing schemes. In this context, the choice of test metal species for the development and validation of such assays cannot disregard the possibility that humans will be exposed to specific chemical forms of individual metal compounds (different oxidation states, and inorganic or organometallic natures) that can profoundly affect their toxicity.     

Chemical mutagenesis--a promising technique to increase metal concentration and extraction in sunflowers

Since most of the metal-hyperaccumulating wild plants only produce very low biomass and many high-yielding crops accumulate only moderate amounts of metals, the current research is mainly focused on overcoming these limitations and the optimization of metal phytoextraction. The main goal of the present study was the improvement of metal concentration and extraction properties of Helianthus annuus L by chemical mutagenesis (the non-GMO approach). Sunflowers--hybrid cultivar Salut and inbred lines-were treated with the chemical mutagen ethyl methanesulfonate (EMS). The effect of chemical mutagenesis on metal concentration in and extraction by new sunflower M1 and M2 mutants was directly assessed on a metal-contaminated field in Raft, Switzerland. Mutants of the M2 generation showed a 2-3 times higher metal shoot concentration than the control plants. The best M2 sunflower "giant mutant" 14/185/04 showed a significantly enhanced metal extraction ability: 7.5 times for Cd, 9.2 times for Zn, and 8.2 times for Pb in aboveground parts, as compared to the control plants. Theoretical calculations for the phytoextraction potential of new sunflower variants note that the best sunflower mutant can produce up to 26 t dry matter per hectare and remove 13.3 kg Zn per hectare and year at the sewage sludge contaminated site of Raft; that is a gain factor of 9 compared to Zn extraction by sunflower controls. Furthermore, the use of sunflower oil and biomass for technical purposes (lubricants, biodiesel, biogas) should produce an additional value and improve the economical balance of phytoextraction.     

Contribution of serum protein association to discrepancy between the in vivo and in vitro UDS results for 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidine (U-89843)

U-89843 has been shown to undergo biotransformation, both in vitro and in vivo, to form U-97924 as a major primary metabolite. U-89843 was found to be positive in an in vitro UDS mutagenesis screen conducted with primary rat hepatocytes in serum-free media. In contrast to in vitro results, no evidence of genetic toxicity of U-89843 was observed in rats in the in vivo/in vitro version of the UDS test with single oral doses up to 1400 mg/kg. The negative results may be related to more robust in vivo detoxification mechanisms or relatively lower exposure to reactive metabolites formed by bioactivation of U-89843 as compared to that observed in the serum-free in vitro hepatocyte test system. Further studies showed rat serum suppressed the in vitro metabolism of U-89843 as well as the formation of the corresponding hydroxylated metabolite, U-97924, the putative precursor of proposed reactive electrophilic metabolite. The measured in vivo systemic clearance of U-89843 (0.53 l/h/kg) in rats was about 1000-fold slower than the in vitro intrinsic clearance (606 l/h/kg) estimated by measuring the formation of U-97924 in rat liver microsomal incubations. Since U-89843 is extensively associated with serum proteins a poor extraction ratio into the liver may account for the slower biotransformation of U-89843 in vivo as compared to that exhibited in in vitro serum-free hepatocyte incubations. Addition of bovine serum albumin (1–40 mg/ml) to the in vitro UDS assay medium decreased the UDS mean net grains per nucleus response of U-89843. These results suggest that the effect of serum protein should be considered when comparing serum-free in vitro UDS and in vivo UDS results for highly serum protein bound compounds.     

Site-saturation mutagenesis is more efficient than DNA shuffling for the directed evolution of beta-fucosidase from beta-galactosidase

Protein engineers use a variety of mutagenic strategies to adapt enzymes to novel substrates. Directed evolution techniques (random mutagenesis and high-throughput screening) offer a systematic approach to the management of protein complexity. This sub-discipline was galvanized by the invention of DNA shuffling, a procedure that randomly recombines point mutations in vitro. In one influential study, Escherichia coli beta-galactosidase (BGAL) variants with enhanced beta-fucosidase activity (tenfold increase in k(cat)/K(M) in reactions with the novel para-nitrophenyl-beta-d-fucopyranoside substrate; 39-fold decrease in reactivity with the "native"para-nitrophenyl-beta-d-galactopyranoside substrate) were evolved in seven rounds of DNA shuffling and screening. Here, we show that a single round of site-saturation mutagenesis and screening enabled the identification of beta-fucosidases that are significantly more active (180-fold increase in k(cat)/K(M) in reactions with the novel substrate) and specific (700,000-fold inversion of specificity) than the best variants in the previous study. Site-saturation mutagenesis thus proved faster, less resource-intensive and more effective than DNA shuffling for this particular evolutionary pathway.     

Current aspects in metal genotoxicity

While carcinogenic metal ions are mostly non-mutagenic in bacteria, different types of cellular damage have been observed in mammalian cells, which may account for their carcinogenic potential. Two modes of action seem to be predominant: the induction of oxidative DNA damage, best established for chromium compounds, and the interaction with DNA repair processes, leading to an enhancement of genotoxicity in combination with a variety of DNA damaging agents. In the case of Cd(II), Ni(II), Co(II), Pb(II) and As(III), DNA repair processes are disturbed at low, non-cytotoxic concentrations of the respective metal compounds. Even though different steps in DNA repair are affected by the diverse metals, one common mechanism might be the competition with essential metal ions.     

The Size of Cells and Organisms in Relation to the Evolution of Embryophytes

Abstract: The earliest O₂-_-evolvers were marine cyanobacteria (3.5 billion years ago) with marine eukaryotic phototrophs from 2.0 billion years ago. These organisms were, and are, poikilo-hydric, i.e., cannot remain hydrated when exposed to a desiccating atmosphere (as can occur for intertidal benthic algae and cy anobacteria at low tide). The smallest marine primarily poikilo-hydric O₂-_-evolvers are close to the lower size limit imposed by non-scaleable components such as minimum genome size and constant membrane thickness, with cyanobacterial unicells 0.65 μn in diameter and eukaryotic unicells 0.95 μm in diameter. The largest (multicellular) marine primarily aquatic poikilohydric O₂-_-evolvers are brown algae at least 60 m long and over 100 kg fresh mass; there are no obvious constraints on the max imum size of such organisms. In freshwaters the size range for primarily poikilohydric O₂-_-evolving organisms is smaller, due to the absence of very large organisms. An even smaller size range characterizes terrestrial algae and cyanobacteria which have occurred for about 1 billion years. Desiccation-tolerant cyanobacterium and algae (intertidal, freshwater, terrestrial) are at the lower end of the size ranges. Embryophytic terrestrial O₂-_-evolvers arose some 450 million years ago and were than all poikilohydric and (probably) desiccation-tolerant. Embryophytic defining structural features re quire organisms of at least 100 μm equivalent spherical diameter for both gametophyte and sporophyte phases. Primarily poi kilohydric embryophytes are not more than 1 m tall as a result of a mechanistically mysterious size limit for desiccation-tolerant organisms. Homoiohydric embryophytes evolved some 420 mil lion years ago in the sporophyte phase (later to become the dominant terrestrial vegetation) and possibly in the gameto phyte phase (although no such homoiohydric gametophytes are known today). The homoiohydric features of gas spaces, stomata, cuticle, endohydric water conducting system and water and nutrient uptake structures require an organism at least 5 mm high; this has implications for the minimum size of mega-spores and seeds. The tallest homoiohydric plants are (or were within historic times) 130 m high, with height constrained by re source costs of the synthesis and maintenance of the mechanical and water conduction systems, andbr of xylem water trans port. Secondarily poikilohydric embryophytes in aquatic, or very damp terrestrial, habitats are derived from homoiohydric plants; they retain most homoiohydric features but are not functionally homoiohydric. The smaller secondarily poikilohydric plants are less than one tenth of the size of the smallest functionally homoiohydric plants.     

The beneficial and hazardous effects of simple phenolic compounds

The current emphasis on screening the environment for man-made genotoxic and carcinogenic compounds detracts from studies on the possible health hazard or beneficial effects of naturally occurring agents to which humans are exposed daily. The simple phenolics, which are ubiquitous among plants, used as food additives, and ingested daily in milligram quantities, belong to this category of compounds. They induce double-strand DNA breaks. DNA adducts, mutations and chromosome aberrations in a great variety of test systems. However, they can suppress the genotoxic activity of numerous carcinogenic compounds in both in vitro and in vivo assays. This dual function of dietary phenolics also becomes evident when their carcinogenic or anticarcinogenic potential is examined. Some, but not all, phenolics induce precancerous lesions, papillomas and cancers, act as cocarcinogens, and exert a promoting effect in various rodent assays. On the other hand, phenolics have proved to be potent inhibitors of carcinogenesis at the initiation and promotion stages induced by carcinogens and promoters of different molecular structures. The extent to which a health hazard or protective activity of complex dietary mixtures is due to their phenolic content remains an unresolved issue. In addition, these multiple, occasionally contradictory functions of simple phenolics make it difficult to propose their use as chemopreventive agents.     

A review of the mutagenicity and rodent carcinogenicity of ambient air

Although ambient air was first shown to be carcinogenic in 1947 and mutagenic in 1975, no overarching review of the subsequent literature has been produced. Recently, Claxton et al. [L.D. Claxton, P.P. Matthews, S.H. Warren, The genotoxicity of ambient outdoor air, a review: Salmonella mutagenicity, Mutat. Res./Rev. Mutat. Res. 567 (2004) 347-399] reviewed the literature on the mutagenicity of urban air in the Salmonella mutagenicity assay. Here, we review the literature on the mutagenicity of urban air in other test systems and review the carcinogenicity of urban air in experimental systems. Urban air was carcinogenic in most of the reports involving rodents. Studies ascribed carcinogenic activity primarily to PAHs, nitroarenes, and other aromatic compounds. Atmospheric conditions, along with the levels and types of pollutants, contributed to the variations in carcinogenic and mutagenic activity of air from different metropolitan areas. The majority of the mutagenesis literature was in the Salmonella assay (50%), with plant systems accounting for most of the rest (31%). The present data give little support to the use of plant systems to compare air mutagenicity among multiple sites or studies. Studies in mice have shown that particulate air pollution causes germ-cell mutations. Air sheds contain similar types and classes of mutagens; however, the levels of these compounds vary considerably among air sheds. Combustion emissions were associated with much of the mutagenicity and carcinogenicity of urban air. Most studies focused on the particulate fraction; thus, additional work is needed on the volatile and semi-volatile fractions, metals, and atmospheric transformation. Smaller particles have greater percentages of extractable organic material and are more mutagenic than larger particles. Although hundreds of genotoxic compounds have been identified in ambient air, only a few (<25) are routinely monitored, emphasizing the value of coupling bioassay with chemistry in the monitoring of air for carcinogenic and mutagenic activities and compounds.     

Mutagenicity, carcinogenicity and teratogenicity of beryllium

The carcinogenicity of a number of beryllium compounds has been confirmed in experiments on laboratory animals and this metal has to be treated as a possible carcinogenic threat to man. These carcinogenic properties are associated with mutagenic activity as shown by the results of short-term tests performed in vitro with beryllium chloride and beryllium sulfate. These soluble beryllium compounds can produce some infidelity of in vitro synthesis, forward gene mutations in microorganisms and in mammalian cells. They are also able to induce cell transformation. In addition to the positive results obtained in several short-term assays beryllium compounds have been found to bind to nucleoproteins, to inhibit certain enzymes needed for DNA synthesis, to bind nucleic acids to cell membranes and to inhibit microtubule polymerization. The teratogenicity of beryllium salts is relatively unknown and needs additional investigation.     

Hydrazines as mutagens in a histidine-requiring auxotroph of Salmonella typhimurium.

Hydrazines have been found naturally in tobacco and mushrooms. Other hydrazines are used in industry, medicine, and agriculture. Although about 38 hydrazines are carcinogenic, few, if any, have been tested successfully in rapid bacterial mutagenesis assays. We have utilized a tester strain of Salmonella typhimurium (TA1530) in order to determine the mutagenic activity of a number of hydrazines and related compounds. This strain is thus shown to be effective as a tester organism for the facile detection of hydrazines as mutagens.     

枯草杆菌蛋白酶基因工程的研究进展

本文介绍了枯草杆菌蛋白酶(Subtilisin)的研究现状,即利用定位诱变和体外重组等技术改变酶的性质,包括催化活性、底物特异性、稳定性、低温适应性以及酶在有机相中的性能等。对枯草杆菌蛋白酶的成功改造不仅有可观的商业价值,而且为蛋白质工程的发展作出了重要的贡献 。     

Identification and iterative combinatorial mutagenesis of a new naringinase-producing strain,Aspergillus tubingensis MN589840

Naringinase was mainly obtained by microbial fermentation, and mutagenesis was a major way for obtaining excellent mutants. The aim of this study was to screen out a high naringinase yielding mutant to enhance the potential application value of its industrialization and compare the effects of different mutagenic methods on the enzyme activity of the strain. A novel producing naringinase strain, Aspergillus tubingensis MN589840, was isolated from mildewed pomelo peel, later subjected to mutagenesis including UV, ARTP and UV-ARTP. After five rounds iterative mutagenesis, the mutants U1, A6 and UA13 were screened out with 1448·49, 1848·71, 2475·16 U mg⁻¹ enzyme activity, the naringinase productivity raised by 79·08, 123·56 and 206%, respectively. In addition, the naringinase activity of three mutants rose after each round of iterative mutagenesis. These results indicated that the mutagenesis efficiency of UV-ARTP was higher than that of single ARTP, and both are better than UV. In summary, the iterative UV-ARTP mutagenesis is an effective strategy for screening high naringinase-producing strains.