A system for the rapid determination of the mutation spectrum in Escherichia coli

The first step toward elucidating the mutagenic effects of chemicals and pathways is to determine the specificity of the mutations generated spontaneously or in response to treatment with mutagens. We constructed a set of plasmid-encoded probes for the specific detection of each type of base substitution mutation. Using these probes, we were able to quickly determine both the mutation rate and the specificity of the mutations caused by different types of mutagens and mutagenic conditions. We also developed a PCR-based method to rapidly and robustly determine the mutation spectrum in response to various mutagenic samples in parallel. This system allows one to not only analyze the mutation specificity of various chemicals, but also to search for novel genetic elements that promote the specific mutation events.     

Hereditary DNA alterations

Mutations and recombinations are hereditary alterations of DNA, which is found mainly in the chromosomes of cells. They can occur spontaneously and they can be induced by high energy radiation or by chemicals. This article considers the developments following the discovery of the DNA structure that have led to a molecular explanation of point mutations, of large chromosome alterations, and of recombination. In conclusion, consequences for human society are drawn from the knowledge gained.     

化学品安全性快速评价方法

简要评述了化学品安全性的传统评价方法。详细介绍了一种快速评价化学品安全性的重要方法——诱变性测试法。该方法选用几种标准(或特定)的鼠伤寒沙门氏菌(Salmonella typhimurium)作为观察对象来研究待测化学品对菌株的诱变情况，以菌株回复突变的数目表征待测化学品的诱变性。当回复突变数目为零剂量(化学品)的2倍以上时，所测定化学品具有诱变性。具有诱变性的化学品为不安全化学品。     

Ultraviolet action spectra in perspective: with special reference to mutation

Abstract— Problems of determining action spectra are considered as well as various types of action spectra for U.V. action upon cell activities. U.V. is an effective mutagenic agent producing point mutations and chromosomal changes. U.V. is readily absorbed by superficial layers of cells in tissues; therefore, special experimental procedures are necessary for induction of mutations in animals or plants. U.V. is, however, suitable for mutagenesis in microorganisms because their cells are small, permitting the radiation to reach the nuclei. Action spectrum studies reveal that u.v. mutagenesis results from absorption of the radiation by nucleic acid. The most prominent alteration in DNA following absorption of u.v. is dimerization of pyrimidines, chiefly thymine. Such a change not only retards DNA replication but results in errors (mutations). U.V. mutagenesis therefore depends upon the conditions before, during and after irradiation. Thus immediate post-treatment with visible and long u.v. light splits pyrimidine dimers, thereby reversing impending u.v. mutagenesis. For cells kept in the dark, conditions which prevent DNA replication by interfering with the metabolism of the cell provide time for dark repair of the DNA lesion and so for reversal of the impending mutation.     

In silico exploratory study using structure–activity relationship models and metabolic information for prediction of mutagenicity based on the Ames test and rodent micronucleus assay

The mutagenic potential of chemicals is a cause of growing concern, due to the possible impact on human health. In this paper we have developed a knowledge-based approach, combining information from structure–activity relationship (SAR) and metabolic triggers generated from the metabolic fate of chemicals in biological systems for prediction of mutagenicity in vitro based on the Ames test and in vivo based on the rodent micronucleus assay. In the first part of the work, a model was developed, which comprises newly generated SAR rules and a set of metabolic triggers. These SAR rules and metabolic triggers were further externally validated to predict mutagenicity in vitro, with metabolic triggers being used only to predict mutagenicity of chemicals, which were predicted unknown, by SARpy. Hence, this model has a higher accuracy than the SAR model, with an accuracy of 89% for the training set and 75% for the external validation set. Subsequently, the results of the second part of this work enlist a set of metabolic triggers for prediction of mutagenicity in vivo, based on the rodent micronucleus assay. Finally, the results of the third part enlist a list of metabolic triggers to find similarities and differences in the mutagenic response of chemicals in vitro and in vivo.     

Searching for an enhanced predictive tool for mutagenicity

The Multiple Computer Automated Structure Evaluation (MCASE) program was used to evaluate the mutagenic potential of organic compounds. The experimental Ames test mutagenic activities for 2513 chemicals were collected from various literature sources. All chemicals have experimental results in one or more Salmonella tester strains. A general mutagenicity data set and fifteen individual Salmonella test strain data sets were compiled. Analysis of the learning sets by the MCASE program resulted in the derivation of good correlations between chemical structure and mutagenic activity. Significant improvement was obtained as more data was added to the learning databases when compared with the results of our previous reports. Several biophores were identified as being responsible for the mutagenic activity of the majority of active chemicals in each individual mutagenicity module. It was shown that the multiple-database mutagenicity model showed a clear advantage over normally used single-database models. The expertise produced by this analysis can be used to predict the mutagenic potential of new compounds.     

Influence of structural and functional modifications of selected genotoxic carcinogens on metabolism and mutagenicity - a review

Alterations in molecular structure are responsible for the differential biological response(s) of a chemical inside a biosystem. Structural and functional parameters that govern a chemical's metabolic course and determine its ultimate outcome in terms of mutagenic/carcinogenic potential are extensively reviewed here. A large number of environmentally-significant organic chemicals are addressed under one or more broadly classified groups each representing one or more characteristic structural feature. Numerous examples are cited to illustrate the influence of key structural and functional parameters on the metabolism and DNA adduction properties of different chemicals. It is hoped that, in the event of limited experimental data on a chemical's bioactivity, such knowledge of the likely roles played by key molecular features should provide preliminary information regarding its bioactivation, detoxification and/or mutagenic potential and aid the process of screening and prioritising chemicals for further testing.     

Influence of structural and functional modifications of selected genotoxic carcinogens on metabolism and mutagenicity – a review

Alterations in molecular structure are responsible for the differential biological response(s) of a chemical inside a biosystem. Structural and functional parameters that govern a chemical's metabolic course and determine its ultimate outcome in terms of mutagenic/carcinogenic potential are extensively reviewed here. A large number of environmentally-significant organic chemicals are addressed under one or more broadly classified groups each representing one or more characteristic structural feature. Numerous examples are cited to illustrate the influence of key structural and functional parameters on the metabolism and DNA adduction properties of different chemicals. It is hoped that, in the event of limited experimental data on a chemical's bioactivity, such knowledge of the likely roles played by key molecular features should provide preliminary information regarding its bioactivation, detoxification and/or mutagenic potential and aid the process of screening and prioritising chemicals for further testing.     

Screening for genetic mutations. A review

A point mutation of a nucleotide within a single gene can have a profound effect on a specific organ and/or the entire human body. DNA sequences associated with human diseases may differ from the corresponding normal sequences by single nucleotide mutations or by large alterations such as deletions, insertions, duplications, or translocations of DNA segments or entire chromosomes. As a result of the heterogeneity of DNA alterations and genetic mutations, various screening approaches are required to detect these alterations. However, methods which facilitate the detection of large mutations in the genome are typically insensitive to point mutations, whereas methods which detect point mutations are not appropriate to detect large alterations within the genome. Since there is no single perfect method to screen for unknown mutations, combinations of these methods may be necessary for accurate genetic diagnosis. The applications of polymerase chain reaction (PCR) technology to genomic screening have made rapid and accurate genetical diagnosis possible. Furthermore, recent developments in the technology of DNA microarrays have opened the way for high throughput sequence analysis by hybridization, which shows great potential in both molecular biology and medicine in the near future.     

Increased level of oxidative stress in genomically unstable cell clones

Recently, we reported that ultraviolet radiation induces delayed mutations in mammalian cells. At the same level of cell death the oxidative component of sunlight (ultraviolet A radiation) was as potent in inducing this kind of genomic instability as ultraviolet B radiation. Ultraviolet B radiation predominantly harms cells by direct damage to DNA and thus is much more mutagenic than ultraviolet A radiation. From that study, clones with a significantly increased mutation rate in the hypoxanthine phosphoribosyl transferase gene were obtained. These genomically unstable clones were also found to have a higher variance in the number of chromosomes than the unirradiated control cells, indicating chromosomal instability. The mechanisms for induction and maintenance of radiation induced genomic instability are not known, but some studies suggest that reactive oxygen species might be involved. In the present study, we have measured the level of potentially mutagenic peroxides in the genomically unstable clones. The levels of intracellular peroxides and lipid peroxides were measured using the probes dihydrorhodamine 123 and diphenyl-1-pyrenyl-phosphine, respectively. The unstable clones had elevated levels of oxidants, supporting the hypothesis that intermediate reactive oxygen species might have a role in the maintenance of genomic instability induced by ultraviolet radiation.     

Evaluation of Critical Body Residue QSARS for Predicting Organic Chemical Toxicity to Aquatic Organisms

Abstract

The critical body residue (CBR) is the concentration of chemical bioaccumulated in an aquatic organism that corresponds to a defined measure of toxicity (e.g., mortality). The CBR can provide an alternative measure of toxicity to traditional waterborne concentration measurements (e.g., concentration in water causing 50% mortality). The CBR has been suggested as a better estimator of dose than the external water concentration and has been postulated to be constant for chemicals with the same mode of action. CBR QSARs have both theoretical and experimental support, developed primarily from studies on the acute toxicity of narcotic chemicals to small fish. CBR QSARs are less well developed for the aquatic toxicity of non-narcotic chemicals. CBRs vary substantially with the mode of action and toxicity endpoint, and may be affected by genetic, hormonal or environmental variation. CBR QSARs may not be applicable to very hydrophobic chemicals, chemicals with specific modes of action, or those with toxicity controlled by kinetic processes such as biotransformation. CBRs models have not been developed or evaluated for sediment and dietary exposure routes. Application of CBR QSARs to contaminated site assessments will require further research and development.     

Toxicological problems relating to changes in the environment

Assessment of the toxicological risk from foreign substances in the environment is based on the determination of action thresholds below which effects injurious to health no longer occur, even with life-long intake. According to the latest theories it is likely that such thresholds exist even for “irreversible” (carcinogenic, mutagenic) effects. However, the difficulties besetting the determination of threshold values by experimental or epidemiological methods still appear to be virtually insuperable. The problems are both quantitative (excessive numbers of animals and observations required) and qualitative (poor predictability of effects owing to substantial differences in biological reactivity between species). Neither animal experiments nor experience with human subjects can guarantee total security from toxic effects. Legislative measures designed to restrict the introduction of chemical substances into the environment must therefore be based on compromise. Scientists must be consulted, but ultimately the decisions must be political, and politicians have to assume the responsibility for them.     

GENETIC TOXICOLOGY OF THE PHOTOSENSITIZATION OF CHINESE HAMSTER CELLS BY PHTHALOCYANINES

Chloroaluminum phthalocyanine (CAPC) was recently shown to photosensitize cell killing in culture and tumor destruction in vivo. Because this compound is potentially useful in the photodynamic therapy of cancer, its properties as a genotoxic agent were evaluated. Applying the technique of alkaline elution to study DNA integrity, it was found that CAPC could produce single-strand breaks in the DNA of Chinese hamster cells after exposure to white fluorescent light. At equicytotoxic doses, the number of DNA strand breaks produced by CAPC photosensitization was about three times lower than that induced by X-irradiation. During incubation in growth medium after exposure to CAPC-plus-fluorescent light, cells rejoined DNA strand breaks at a rate similar to that observed after X-irradiation. Resistance to 6-thioguanine (6-TG') or to ouabain (OUA') were used as end points of mutagenic potential. Following a treatment that caused -90% cell killing, there was a slight mutagenic effect, i.e. the frequencies were increased by -40% above the background or spontaneous mutations. However, this enhancement was not statistically significant. Taken together, the foregoing, plus an earlier observation that there is no variation in the sensitivity of cells to CAPC + light through the cell cycle, lead to the inferences that DNA damage does not play a major role in cell killing and that the mutagenic potential of this treatment is small.     

Activation and detection of (Pro)mutagenic chemicals using recombinant strains ofStreptomyces griseus

Two recombinant strains ofStreptomyces griseus have been developed to report on the activation of promutagenic° chemicals. This activation is monitored by reversion of the bacterial test strains to a kana-mycin-resistant phenotype. Strain H69 detects point mutations and was reverted at an increased frequency by acetonitrile, 2-aminoanthracene, 1,2-benzanthracene, benzidine, benzo(a)pyrene, 9,10-dimethyl-1,2-benzanthracene, and glycine. The second strain, FS2, detects frame shift mutations and was reverted at an increased frequency by 1,2-benzanthracene, benzidine, and glycine. Compounds such as butylated hydroxytoluene, catechol, chlorobenzene, hydroquinone, potassium chloride, phenol,cis-stilbene,trans-stilbene, and toluene did not elicit positive responses in either strain. In addition, these strains are capable of detecting direct-acting mutagens such asN-methyl-N’-nitrosoguanidine and ICR-191, providing further evidence of their promise for detecting a wider range of mutagens. To our knowledge, this is the first report of bacterial strains capable of activating promutagenic compounds and detecting their mutagenic metabolites without the benefit of an exogenous activation system such as the rodent liver homogenate (S9).     

Effects of chlorinated aliphatic hydrocarbons on the fidelity of cell division in human CYP2E1 expressing cells

Chlorinated organic chemicals are widely used in industry and are present in the environment. Five chlorinated aliphatic hydrocarbons, namely 1-2-dichloroethane, 1,1,2-trichloroethane, trichloroethylene, 2,3-dichlorobutane and 1-chlorohexane were investigated to determine their influence upon the fidelity of cell division in cultured mammalian cells. In order to determine the influence of these chemical compounds upon the fidelity of cell division, a technique known as differential staining of chromosomes and spindle was performed with one genetically engineered cell line and its parental cell line. The genetically engineered cell line used in this study expressed a human P450 enzyme, CYP2E1. Four chemicals, 1-2-dichloroethane, trichloroethylene, 2,3-dichlorobutane and 1-chlorohexane required metabolic bioactivations in order to induce spindle damage in cultured mammalian cells whereas 1,1,2-trichloroethane was a direct-acting spindle poison.     

In-silico screening of high production volume chemicals for mutagenicity using the MCASE QSAR expert system

Computational screening is suggested as a way to set priorities for further testing of high production volume (HPV) chemicals for mutagenicity and other toxic endpoints. Results are presented for batch screening of 2484 HPV chemicals to predict their mutagenicity in Salmonella typhimurium (Ames test). The chemicals were tested against 15 databases for Salmonella strains TA100, TA1535, TA1537, TA97 and TA98, both with metabolic activation (using rat liver and hamster liver S9 mix test) and without metabolic activation. Of the 2484 chemicals, 1868 are predicted to be completely nonmutagenic in all of the 15 data modules and 39 chemicals were found to contain structural fragments outside the knowledge of the expert system and therefore suggested for further evaluation. The remaining 616 chemicals were found to contain different biophores (structural alerts) believed to be linked to mutagenicity. The chemicals were ranked indescending order according to their predicted mutagenic potential and the first 100 chemicals with highest mutagenicity scores are presented. The screening result offers hope that rapid and inexpensive computational methods can aid in prioritizing the testing of HPV chemicals, save time and animals and help to avoid needless expense.     

UV mutagenic photoproducts in Escherichia coli and human cells: a molecular genetics perspective on human skin cancer

Abstract— The relevance of photoproducts produced by 254 nm irradiation to human skin cancer is first critically evaluated. Experiments identifying the mutagenic photoproducts at 254 nm are then described. Mutations are primarily due to the(6–4) photoproduct and the cyclobutane pyrimidine dimer, both in E. coli and in human cells. The(6–4) photoproduct may be more important in E. coli and the cyclobutane dimer more important in mammalian cells. In human cells, mutations occur at the C of a TC, CT, or CC cyclobutane dimer, but not at TT cyclobutane dimers, and also appear to occur, less frequently, at the C of TC and CC(6–4) photoproducts. The local structure of DNA is more important in determining the frequency of mutation at a site than is the photoproduct frequency at that site. The effect of DNA structure appears to be due to site-specific lethality.     

环境水体中酚类EDCs前处理技术及分析方法研究进展

环境内分泌干扰物(EDCs)是指干扰生物体内保持自身平衡和调节发育过程中天然激素的合成、分泌、运输、代谢、结合、反应、消除等生物过程的外源性化学物质,这类物质的存在会干扰人类和野生动物的内分泌系统,带来生殖障碍、发育异常、免疫功能减弱等问题。EDCs,尤其是使用最为广泛的酚类EDCs,在水环境中的污染特征研究已是当前科学界和公众共同关注的热点问题之一。环境样品基质非常复杂,使得痕量酚类EDCs的分析检测难度较大。该文对近年来环境水体中酚类EDCs的分析方法进行了综述,分别对样品前处理与检测分析技术进行了介绍,其中前处理技术包括样品萃取、样品净化和样品衍生化,检测分析技术包括化学分析和仪器分析。最后对酚类分析方法进行了展望。